Cytogenetics of Pearl Millet*

ADVANCES IN AGRONOMY, VOL. 34

CYTOGENETICS OF PEARL MILLET* Prem P. Jauhar-t. Department of Botany and Plant Sciences, University of California-Riverside, Riverside, California

.................... I . Introduction . . . . . . . . . . . . 11. Karyotypic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karyomorphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Meiosis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........ A. P . typhoides ( B u m . ) Stapf et Hubb. ( 2 n = 2x = 14). . . . . . . . . . . . . . . . . . . . . . . B. P . purpureutn Schumach. (2n = 28, 56). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I V . Abnormal Meiosis and Its Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Desynapsis and Its Genetic Basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effect of Nutrients on Desynapsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Effect of Ploidy on Desynapsis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Effect of Desynaptic Gene on B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Experimental Induction of Desynapsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Inbreeding and Disruption of Chromosome Pairing: Heterozygosis and Heterosis for Chromosome Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Haploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spontaneous and Induced Haploids ................................... ................................... B. Chromosome Pairing in Haploids . . C. Haploids i n Plant Breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V1. Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spontaneous Occurrence . . . . . . . . . . . . . . . . . B. Factors Favoring Spontaneous Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Induction of Polyploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Polyploidy and Plant Breedin VII. Aneuploids . . . . . . . . . . . . . . . . . . A. Spontaneous Occurrence . . . . B. Synthesis of Aneuploids of Pe ................................... C. Trisomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Tetrasomics VIII. Structural Changes in Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spontaneous Rearrangements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................. B. Induced Translocations C. Building Up a Complete Interchange Ring . . . . D. Karyomorphology and the Incidence of Interchanges . . . . . . . . . . . . . . . . . . . . . . . .

408 415 415 417 417

422 422 423 424 424 424 427

429 430 432 434 434 435 436 441 442 442 442

444

*This article is dedicated with admiration to Dr. Glenn W. Burton, whose pioneering work has contributed significantly to the genetic improvement of pearl millet. ?Present address: Division of Cytogenetics and Cytology, City of Hope National Medical Center, Duarte. California 91010. 407

Copyright 0 1981 by Academic Press. Inc. All nghts of repduction in any form resewed ISBN 0-12-000734-7

408

PREM P. JAUHAR E. Identification of Translocated Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Disjunction of Interchange Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Interchange Heterozygosity and Plant Breeding . . . . . . . . . . . . . . . .

444 445

IX. B Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B Chromosomes as Indicators of the Origin of Pearl Millet. . . . . . . . . . . . . . . . . . . 447

B . Mode of Pollination.. . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . 454

XI. Hybridization and Chromosome Relationships . . . . . . . . . . . . . . . . . .................................... A. lntraspecific Hybrids B. Interspecific Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Intergeneric Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Conclusion . ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

458 472 473

I. INTRODUCTION Pennisetum is one of the most important genera of the tribe Paniceae of the grass family. Pearl millet [Pennisetum ryphoides (Burm.) Stapf et Hubb.] is the most important constituent of this genus. It is a dual-purpose crop: its grain is used for human consumption and its fodder serves as feed for cattle. In Asia and Africa, however, it is grown primarily as a grain crop on an estimated 60 million acres of relatively poor land. It has remarkable ability to grow in areas of low rainfall. In sub-Saharan Africa harvests of pearl millet are obtained with as little as 250 mm of annual rainfall (Brunken, 1977). Its grain is traditionally considered to be nutritious and is put to a variety of uses. As poor man’s bread, it sustains a large proportion of the populace of Africa and Asia. It also contributes to the economy of countries like the United States, where it is grown as a forage crop on an estimated 1 million acres. Pearl millet is also grown as a forage crop in the tropical and warm-temperate regions of Australia and several other countries. Pearl millet originated in West Africa. Selection exercised by the early cultivators under a myriad of cultural contexts led to the development of several morphologically diverse forms. Its protogynous nature facilitated the introgression of characters from other wild and cultivated annual species of the section Penicillaria. It is now widely cultivated in different parts of the world. In terms of annual production, pearl millet is the sixth most important cereal crop in the world, following wheat, rice, maize, barley, and sorghum. Among the millets it is second only to sorghum. In India, it is the fourth most important cereal after rice, wheat, and sorghum.

CYTOGENETICS OF PEARL MILLET

409

Pearl millet is a favorable organism for genetic research. Its chromosome number, 2n = 14, was determined more than 50 years ago by Rau (1929). Several favorable features of the chromosome complement, e.g., small number and large size of chromosomes with one distinctive pair of nucleolar organizers, make pearl millet a suitable organism for cytogenetic studies. Moreover, its protogynous flowers and outbreeding system make it ideal for interspecific hybridization and for breeding work. It is indeed ideally suited for heterosis breeding. Although pearl millet has a remarkable ability to grow on soils of marginal fertility, it responds very well to proper fertilization, which helps in realizing the high yield potential of its hybrids. The hybrids’ greater N use efficiency (biomass production per unit of N in the plant) is probably attributable to the highly efficient (C,) photosynthetic pathway of this crop. Although pearl millet has great agricultural importance, is a very favorable organism for cytogenetic studies and breeding work, and has a low chromosome number that was also determined at about the same time as those of most other crops, the information available on its genetics and cytogenetics is much less than that known for other important crops. There are several reasons why this crop has been largely overlooked as a genetic and cytogenetic tool:

1. It has long been considered to be a crop of secondary importance and, thus, could not compete for research funding with other crops like wheat and corn. 2 . It has a restricted area of use, being a food for the poor only, although it is also an excellent fodder crop. 3. Its potential as a research tool was not appreciated until recently. 4. The existence of long-standing nomenclatural controversies (in the postLinnaean period from 1753 to 1759, pearl millet has been treated as a member of at least six different genera, viz., Panicum, Holcus, Alopecurus, Cenchrus, Penicillaria, and Pennisetum, and has been given different botanical names; see Jauhar, 1981a) could also have had an adverse impact on research. Studies on chromosome pairing in interspecific hybrids-with pearl millet as one of the parents-have contributed to our understanding of phylogenetic relationships between different P ennisetum species and pearl millet. In these studies, the large size of the pearl millet chromosomes has been helpful in ascertaining chromosome relationships. Because of its low chromosome number, pearl millet also offers a particularly favorable material for aneuploid analyses, which should be helpful in the elucidation of its cytogenetic architecture. Primary trisomics constitute a valuable tool for locating genes on different chromosomes and for assigning them to linkage groups. Although considerable progress has been made in developing a set of primary trisomics in pearl millet, the establishment of linkage groups awaits completion. A good deal of information is available on certain other cytogenetic aspects, e.g., polyploidy, interchange heterozygosity , haploidy, and B chromosomes. All these studies should contribute to the improvement programs of pearl millet.

410

PREM P. JAUHAR

The purpose of this article is to summarize and integrate the available information on different aspects of pearl millet cytogenetics. It is hoped that this article will provide useful information to cytogeneticists and breeders engaged in the improvement of pearl millet and other forage species of Penniseturn. This article may also be of interest to a spectrum of other workers engaged in basic research.

II. KARYOTYPIC ANALYSIS Karyotypic analysis includes the study of the number, size, and morphology of chromosomes. Total length and arm ratios of chymosomes are helpful in systematic and phylogenetic investigations. Levitskii (193 1) and Avdulov (1 93 1) pioneered the use of cytological features as aids in establishing taxonomic and phylogenetic relationships among species and genera. Although basic number, size, and morphology of the chromosomes can indeed be useful in taxonomic classification (Hunter, 1934; Constance, 1957), these parameters should be subsidiary to morphological characters in any taxonomic treatment (Pilger, 1954). Modern cytological techniques, e.g., the banding of chromosomes with Giemsa (Vosa and Marchi, 1972; Vosa, 1973, 1975), and staining heterochromatic patterns with fluorochromes like quinacrine mustard (Vosa, 1970) can provide information of phylogenetic value. The occurrence of cytotypes or chromosomal races (intraspecific polyploid series) is a characteristic feature of the perennial species of Penniseturn. However, no such cytotypes exist in the annual cultivated or wild pearl millets, which all have 2 n = 14 chromosomes (Table I); in fact, all these taxa belong to the species P . ryphoides. There is a report of 2 n = 36 chromosomes for a Nigerian collection of “ P . violaceurn (Lam.) L . Rich.” (Olorode, 1975), but this could be an incorrect identification. Since the material classified as P . violaceurn forms fully fertile hybrids with pearl millet (2n = 14), the former must have 2 n = 14 chromosomes (see Section XI,A). KARYOMORPHOLOGY

Chromosomes are generally measured at somatic metaphase after pretreatments that condense and spread them. The main drawback inherent in these studies is that the magnitude of error in the measurements of condensed chromosomes is high. Therefore relatively small size differences among chromosomes of a species, of infraspecific categories, or of different species cannot be resolved accurately. However, karyomorphological studies can be done more precisely on pachytene chromosomes in taxa with low chromosome numbers, e.g., P .

41 1

CYTOGENETICS OF PEARL MILLET

TABLE I Chromosome Numbers of Different Taxa in the Section Penicillaria of Genus Penniserurn Taxa Cultivated pearl millet P. typhoides (Burm.) Stapf et Hubb. [Syn. P. tvphoideutn Rich. P. spicarurn (L.) Koern. P. glaucum (L.) R. Br. P. amerironum ( L . ) K. Schum.] Annual relatives of pearl millet" P. alhicauda Stapf et Hubb. P. rmcvhchaele Stapf et Hubb. P. rinereum Stapf et Hubb. P. dalzielii Stapf et Hubb. P. echinurus (K. Schum.) Stapf et Hubb. P. fullax (Fig. & De Not.) Stapf et Hubb. P. gambiense Stapf et Hubb. P. leanis Stapf et Hubb. P. tnuiwa Stapf et Hubb. P. nigrilarurn Schlecht. P. mollissimum Hochst. P. perrottetii (Klotzch ex A.Br.)K. Schum P. pyrnostachyum (Steud.) Stapf et Hubb. P. pynostachvunr var. gambia P. versicolor Schrad. P. violareum (Lam.) L. Rich Perennial relative of pearl millet P. purpureutn Schum.

2n

14

14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 I4? 28

56

References

Rau (1929)

Thevenin (1952) Krishnaswamy (1951) Krishnaswamy (1951) Krishnaswamy (1951);Thevenin (1952) Krishnaswamy (1951) Thevenin (1952) Thevenin (1952)

Thevenin (1952) Krishnaswamy (1951) Bilquez and Lecomte (1969) Mehra et ul. (1968) Burton (1942); Nishiyama and Kondo ( 1942) Krishnaswamy and Raman (1948); Gadella and Kliphuis (1964)

"These and other annual, cultivated, or wild relatives of pearl millet have 2n = 14 chromosomes. They are not reproductively isolated from the cultivated species-P. fyphoides-and in fact do not deserve specific ranks.

ramosum ( 2 n = 10) and P . typhoides ( 2 n = 14). For critical comparisons, the DNA content of chromosomes can also be measured. The genus Pennisetum is a heterogeneous assemblage of species with chromosome numbers ranging from 2 n = 10 to 2 n = 7 2 , being multiples of 5, 7, 8, and 9. Their chromosome morphology is also very diverse, with tremendous size differences; a noteworthy feature is that the species with lower numbers have the larger sizes. Thus, pearl millet ( P . ryphoides) has only 2 n = 14, but relatively very large chromosomes. Avdulov (1931) noted that pearl millet had 14 large chromosomes, larger than those of any other member of the tribe Paniceae.

412

PREM P. JAUHAR

However, I think that the annual (or rarely biennial) species P . ramosum (2n = 10) has the largest chromosomes in the genus Pennisetum and probably in the entire tribe Paniceae. The chromosomes of P . ramosum are approximately 5% larger than those of P . typhoides. Thus, in the genus Pennisetum, the species with the lowest chromosome number (2n = 10) has the largest chromosomes. In contrast, the species with higher chromosome numbers (e.g., P . orientale, 2n = 18, 36, 54) have strikingly smaller chromosomes than those of P . ramosum or P . typhoides. The trend of species with low chromosome numbers to have much larger chromosomes is evident in several other plant groups. In Sorghum, for example, the average lengths of chromosomes of S. versicolor (2n = lo), S . vulgare (2n = 20), and S . halepense (2n = 40) were 4.86, 2.24, and 1.98 p m , respectively (Karper and Chisholm, 1936). 1 . P . typhoides (2n = 1 4 )

Rau (1929) determined from root tips the chromosome number of pearl millet as 2n = 14. Moreover, he mentioned that “the chromosomes are very large” and that the homologous pairs could be easily distinguished. Avdulov (1931) studied the chromosomes of pearl millet, which was at that time classified as Penicillaria spicata Willd. His drawing shows 14 chromosomes with median to submedian centromeres, the shortest chromosome being the satellited one. It is interesting to note that as early as 1931 when cytological techniques were not perfected, Avdulov noticed one pair of satellited chromosomes; this observation has been confirmed by numerous workers. The small nucleolar bivalent is clearly observed to be associated with the nucleolus (Fig. 1 ) . Pantulu (1 958) examined the chromosomes at pachytene and grouped them into four classes on the basis of relative length and position of centromere: ( 1 ) two large pairs (chromosomes 1 and 2) with median centromeres; ( 2 ) two somewhat shorter pairs (chromosomes 3 and 4) with median to submedian centromeres; (3) two medium-sized pairs (chromosomes 5 and 6) with submedian centromeres; and (4)the shortest pair (chromosome 7) with the nucleolus organizer. Later, essentially similar results were obtained on the analysis of karyotype at pachytene (Venkateswarlu and Pantulu, 1968; and Lobana and Gill, 1973), at pollen mitosis (Krishnaswamy and Raman, 1953a), and at somatic metaphase (Burton and Powell, 1968). Virmani and Gill (1972) and Tyagi (1975a) karyotyped the somatic chromosomes and classified them as follows: chromosomes 1 , 2, 3, and 5 as metacentric; chromosomes 4 and 6 as submetacentric; and chromosome 7 as subterminal. Thus, there are minor disagreements among different workers as to the position of centromere. Looking at a condensed chromosome at somatic metaphase, it is not unexpected that one worker locates the centromere as median, whereas another classifies it as submedian. The same workers, looking at pachytene and somatic chromosomes, can also arrive at different conclusions. For example,

CYTOGENETICS OF PEARL MILLET

413

FIG. 1. Diakinesis in pearl millet ( 2 n = 14) showing one nucleolar bivalent; note small rod associated with the nucleolus. Also note chiasma terminahation. [ X 12601

Virmani and Gill (1972) studied somatic chromosomes and classified chromosome 1 as metacentric; whereas, based on pachytene analysis, Lobana and Gill (1973) considered it to be submetacentric. There is no doubt that pearl millet has a fairly symmetrical karyotype. It is certainly not very easy to identify all of the seven chromosomes by the techniques currently used; therefore, Giemsa banding (see Vosa, 1973, 1975; Zelleret al., 1977; Filion and Blakey, 1979) of somatic prometaphase chromosomes must be tried to identify individual members of the complement. It has mostly metacentric or submetacentric chromosomes, the longest being approximately 1.5 times the shortest; both these features are indices of symmetry of karyotype. Under Stebbins’ (1958) classification of types of asymmetry, pearl millet will fit best in the class la, i.e., the most symmetrical of the 12 karyotypes described. The shortest chromosome pair is somewhat subterminal with the satellite on its short arm. It can be identified in somatic plates as well as at pachytene and diakinesis, where, as a small bivalent, it is associated with the nucleolus (Fig. 1). Chromosomes of some diploid taxa of the section Penicillaria, which are annual relatives of pearl millet, have been observed. The materials classified as Pennisetum cinereum, P. echinurus, P . gumbiense, P. leonis, and P. pycnosruchyum had 2n = 14 chromosomes, as in cultivated pearl millet (Krishnaswamy, 1951). Veyret (1957) found that P. ancylochaete, P . gambiense, P . maiwa, and P. nigritarum had 2n = 14 chromosomes, and their chromosome morphologies were similar to one another and also to that of cultivated pearl millet. Genetic studies by Bilquez and Lecomte (1969) and Brunken (1977)

414

PREM P. JAUHAR

have shown that P . violuceum and P . fullux-two of the important wild, annual relatives of pearl millet-are not reproductively isolated from it; their hybrids with pearl millet were highly fertile. Although these workers have not mentioned the chromosome number of these wild taxa, they obviously have 2n = 14 chromosomes in order to form fertile hybrids with pearl millet.

2. P . purpureum (2n

= 4x =

28)

Napier grass, an allotetraploid and a relative of pearl millet, has a somewhat asymmetrical karyotype consisting of chromosomes with median, submedian, and subterminal centromeres. On the basis of pachytene studies, Pantulu and Venkateswarlu (1968) reported that the longest chromosome of the complement (chromosome 1) was 2.7 times the length of the shortest (chromosome 14), thus making the karyotype asymmetrical. Based on these observations, the karyotype of P . purpureum will fall in the category 2b in Stebbins’ (1958) classification of types of asymmetry. Pantulu and Venkateswarlu (1968) reported that chromosomes 1 and 14 have nucleolus organizers. The largest chromosome of the complement (chromosome 1) is certainly satellited, as evidenced by the association of the largest bivalent with the nucleolus (Fig. 2 ) . The other nucleolar bivalent is one of the smallest, if not the smallest, in the complement. If chromosome 14 is indeed satellited, then

FIG.2. Diakinesis in napier grass ( 2 n = 4x = 28) showing 14 bivalents (9 rings and 5 rods) with terminalized chiasmata. Note one large bivalent (the largest in the complement) and one relatively small bivalent associated with the nucleolus. Also note an additional small nucleolus (marked with arrow). [ x 12601

CYTOGENETICS OF PEARL MILLET

415

P. purpureum shares an important karyotypic feature with P. typhoides, i.e., the shortest chromosomes of both the species are satellited. Moreover, during meiotic prophase both typhoides and purpureurn show rapid terminalization of chiasmata (see Section 111). They also seem to have similar patterns of centromeric heterochromatin. Thus, P. typhoides and P. purpureum seem to share some important karyological features of phyletic value.

Ill. MEIOSIS All penicillarias fall into the x = 7 group (see Table I). They have conspicuously penicillate anther tips (see Fig. 14a,b). Of these, only one species ( P . purpureum) is a perennial tetraploid. All other taxa are annual and diploid with 2n = 2x = 14 chromosomes. The annual, semiwild taxa are not reproductively isolated from the cultivated pearl millet and must be considered as infraspecific categories within P . typhoides. They have regular meiosis with 7,,, as in P. typhoides. A . P . ryphoides ( B u R M . )STAPFET HUBB.( 2 n = 2x = 14)

Rau (1929) determined the chromosome number of pearl millet as 2 n = 14. Rangaswamy (1935) studied meiosis and found at diakinesis mostly seven ringshaped bivalents having two terminalized chiasmata each. In different populations of pearl millet, mostly ring bivalents with two chiasmata each are observed at diakinesis, but the nucleolar bivalent is generally a small rod with one chiasma (Figs. 1 and 3b). The rapid terminalization of chiasmata seems to be a characteristic feature, so that at diakinesis the bivalents generally appear loose and dissociated (Figs. 1 and 3b,c). At metaphase, both ring and rod bivalents are observed. In some cultivated varieties in India, the mean chiasma frequency at metaphase was found to be 12.10 per cell and 0.86 per paired chromosome; this means that ring bivalents are preponderant (see Fig. 3d). Some populations of pearl millet show secondary associations of bivalents. Two groups of two bivalents each were clearly observed (Fig. 3c) in some cells. Although the phyletic significance of secondary associations in diploid species remains controversial, such associations cannot be entirely meaningless. In hexaploid wheat, such associations are known to take place between genetically and evolutionarily related chromosomes (Riley, 1960; Kempanna and Riley, 1964). In pearl millet, the secondarily associated bivalents look very similar to each other, although their genetic and phyletic relatedness cannot be determined. In the haploid complement when their homologous partners are missing, the chromosomes involved in these secondary associations probably form bivalents.

416

PREM P. JAUHAR

FIG. 3. Meiotic stages in pearl millet. (a) Late diplotene showing 7 bivalents (711).(b) Diakinesis with 711with teminalized chiasmata. Note 6 ring bivalents and the small, nucleolar rod bivalent. (c) Diakinesis with 711.Note the secondary associations of two pairs of bivalents; the associated bivalents look similar in size and shape. (d) Metaphase 1 with 611.[(a, d) X ca. 2050; (b, c) x ca. 21501

CYTOGENETICS OF PEARL MILLET

417

It is interesting to note that two bivalents have been reported in haploids studied by different workers (see Section V,B, Table 11; Fig. 6c). These observations lend favor to the suggestion that the complement of typhoides has been derived from a basic set of x = 5 chromosomes (see Jauhar, 1968, 1970b; Sections V,B and XI,B,l,c). B . P . purpureum SCHUMACH. ( 2 n = 28, 56)

Elephant or napier grass ( P . purpureum) is a perennial relative of pearl millet and is native to Africa. Burton (1942) and Nishiyama and Kondo (1942) determined its somatic chromosome number as 2n = 28, which is tetraploid based on x = 7. It shows diploid-like meiosis, 14,, being regularly formed at diplotene, diakinesis, and metaphase (Fig. 4a-c). No multivalents or univalents are generally formed. The occasional occurrence of a quadrivalent (Olorode, 1974) can be attributed to a floating interchange in certain populations. At diakinesis, there is a rapid terminalization of chiasmata (Figs. 2 and 4b)-a feature also characteristic of pearl millet. Two bivalents are generally associated with the nucleolus (Fig. 4a,b). One of the nucleolar bivalents is the largest in the complement, whereas the other is a small one (see also Section 11,2). Occasionally, additional nucleolar material is organized (see Fig. 2). At metaphase, there are noticeable size differences among bivalents; the smaller ones are generally rod-shaped with one chiasma each, whereas the majority of the large ones are ring-shaped with mostly two chiasmata each (Fig. 4c). Chiasma frequency per cell and per paired chromosome was found to be 18.9 and 0.68, respectively, in some collections. Several factors speak for the allotetraploid nature of elephant grass: ( I ) its 2n = 28 chromosome number; (2) the regular bivalent formation and high pollen fertility; and (3) the noticeable size differences among bivalents. This is further borne out by studies on chromosome pairing in its hybrids with pearl millet (see Section XI,B,2,d). This allotetraploid can be genomically represented as A'A'BB. The A' genome is homoeologous with the A genome of pearl millet (which is genomically AA). The large bivalents observed at metaphase in P . purpureum are evidently formed by the A'A' genome, whereas the small ones belong to the BB genome, the donor of which is not yet known.

IV. ABNORMAL MEIOSIS AND ITS GENETICS The nature of events that lead to synapsis and crossing over during the meiotic prophase remains one of the most intriguing problems in cytogenetics today. It is

418

PREM P. JAUHAR

FIG. 4. Meiotic stages in napier grass ( 2 n = 4x = 28). (a) Early diakinesis with 14 bivalents (l4,,). Note that two bivalents (one the largest in the complement and one much smaller) are associated with the nucleolus. (b) Diakinesis with 14,: Due to terminalization of chiasmata, most bivalents appear to be loose and dissociated. Note that 2,, are associated with the nucleolus; the large bivalent-largest in the complement-is lying on the nucleolus. (c) Metaphase I with 14,,; I,, is separated. [ x 14801

CYTOGENETICS OF PEARL MILLET

419

known, however, that meiosis is an integrated process consisting of a series of sequential events that are under the control of specific genes. Genotypes that deviate from the normal course of meiosis have been described in numerous organisms, including some species of Pennisetum, and their study has led to a better understanding of this complex process. Several major genes that, in homozygous recessive condition, bring about failure or disruption of chromosome pairing are now known (see Riley and Law, 1965; Jauhar and Singh, 1969a; Gottschalk and Klein, 1976; Golubovskaya, 1979). Genes that influence the very initiation and, hence, bring about complete failure of pairing at meiotic prophase are referred to as asynaptic ( a s ) , whereas those that disrupt the maintenance of pairing between initially synapsed chromosomes are termed desynaptic ( d s ) . It is, however, very difficult to distinguish between the phenomena of asynapsis and desynapsis, distinction being all the more difficult between partial asynapsis and partial desynapsis. Studies on the pairing variants have nevertheless helped in the elucidation of the genetic control of synapsis and its concomitant process of recombination. A . DESYNAPSIS A N D ITS GENETIC BASIS

Generally, desynapsis is of more common occurrence than asynapsis. Although both these phenomena have been described in pearl millet, the former is certainly much more common. Krishnaswamy et a f . (1949) reported on a desynaptic plant derived from X-ray-treated seeds. At pachytene, synapsis was noted in one or two pairs, but at diplotene-diakinesis there was almost complete disruption of pairing, resulting in 14 univalents. Irregular and random anaphasic separation of univalents resulted in 98.5% sterility. Later, Patil and Vohra (1962), Minocha et al. (1968), Jauhar (1969), Dhesi et al. (1973), and Singh et a / . (1977a) reported cases of partial desynapsis. Figure 5b, for example, shows a cell with 211 10,; two of the newly desynapsed chromosomes lie juxtaposed. More recently, Rao and Koduru (1978a) observed that failure of pairing (Fig. 5c,d) was associated with syncyte formation. The desynaptic condition was attributed to the double recessive condition of the gene ds (Minocha et al., 1975; Pantulu and Rao, 1976). In the same variety (T55) of pearl millet, Patil and Vohra (1962) and Jauhar (1969) reported different degrees of desynapsis at diplotene, diakinesis, and metaphase I. At metaphase I , for example, Jauhar (1969) observed a mean of 3.46,,, whereas Patil and Vohra (1962) had recorded only 0.3 1 in their desynaptic. Although the genetics of desynapsis was not determined, it was probably under monogenic recessive control as in other desynaptic stocks of pearl millet. Different degrees of desynapsis could be due to different environmental conditions. Like other mutant genes that are generally less buffered against environmental fluctuations (Darlington, 1958), the degree of manifestation of a desynaptic gene can be affected

+

,,

420

PREM P. JAUHAR

FIG.5. Meiotic stages i n normal, partially desynaptic, and desynaptic plants of pearl millet. (a) Diakinesis in a normal plant showing 7 bivalents (7,,). (b) Diakinesis in a partial desynaptic showing 2 + 10,. Note that two of the desynapsed homologs lie juxtaposed. (c) A PMC (at a stage comparable to diakinesis) in a desynaptic showing 14,. (d). Meta-anaphase I in the desynaptic showing 14,. Note that some univalents are arranged on the metaphase plate, while others are randomly scattered. [(a, b) x ca. 1860; (c) X ca. 2120; (d) X ca. 16201 Negatives of c and d were kindly supplied by Dr. P. R. K . Koduru.

,,

CYTOGENETICS OF PEARL MILLET

42 1

by temperature, e.g., in wheat (Li et al., 1945) or by other environmental conditions (Gottschalk, 1976). Another possibility that the degree of expression of the desynaptic gene in pearl millet is controlled by a set of modifiers (or perhaps even polygenes), which tend to make desynapsis a sort of quantitative trait, cannot be excluded (Jauhar, 1969). Rao’s (1980) work also suggests that some modifying genes may be responsible for the differential expressivity of the ds gene. With the failure of pairing, the activity of the gene responsible for normal disjunction is also probably hampered. Anaphasic separation is therefore very irregular, the univalents moving to the poles like “unguided missiles’’ (see Fig. 5d). Thus, desynapsis generally results in high to complete male sterility.

B. EFFECTOF NUTRIENTS O N DESYNAPSIS It is well known that chiasma frequency is under genetic control and that it can be influenced easily by various environmental factors like temperature. It has been claimed that different nutrients can alter chiasma frequency. Thus, Dhesi er al. (1975) reported that the application of phosphate and potash resulted in an increase in chiasma frequency of pearl millet desynaptics. In other words, these treatments reduced the strength of desynapsis. Phosphate treatments were reported to increase chiasma frequency in desynaptic strains of rye (Bennett and Rees, 1970) and barley (Fedak, 1973). In desynaptic barley, Fedak (1973) observed “a strong positive relationship between phosphate treatment and chiasma frequency. ” It is difficult to explain how nutritional status of the soil significantly alters chiasma frequency of normal or desynaptic plants. If a higher mineral status of soil does increase chiasma frequency in desynaptic plants, mineral starvation should accentuate the effect of the desynaptic genes. Lakshmi er al. (1979) recently estimated the phosphate and potassium contents in the flag leaves of normal pearl millet plants, and in desynaptics with 2-6 univalents, 2-10 univalents, and 10-14 univalents per cell, but they did not detect any significant differences in mean values. More studies are needed therefore before any definite conclusions can be derived regarding the effect of mineral nutrition on chiasma frequency. C. EFFECTO F PLOIDY O N DESYNAPSIS

A desynaptic tetraploid was observed by Rao (1978) in the open-pollinated progeny of some desynaptic diploids. It showed a mean chromosome configura-

422

PREM P. JAUHAR

+

tion 1.02,, + 1.30,,, + 7.37,1 5.28, per pollen mother cell (PMC), suggesting an improvement in pairing apparently resulting from chromosome doubling. Its mean chiasma frequency was 15.12, whereas the parental desynaptic diploids had mean frequencies of 0.2-8.4. Thus, four doses of the desynaptic ( d s ) gene seemed to increase chiasma frequency in the desynaptic tetraploid over that in the desynaptic diploid, which had two doses of the ds gene. Since desynapsis in pearl millet is due to the double recessive (dsds) condition of the ds gene, the desynaptic tetraploid should have the nulliplex condition (dsdsdsds) for this gene in order for this character to be expressed. D . EFFECTO F DESYNAPTIC GENEO N B CHROMOSOMES

Pantulu and Rao (1976) found that in a desynaptic stock with B chromosomes, the desynaptic gene brought about a reduction in synapsis and chiasma frequency of the A as well as B chromosomes. This is the only report of the effect of a ds gene on B chromosomes. However, it has been observed that the diploidizing genes controlling diploid-like pairing of A chromosomes in hexaploid tall fescue (Jauhar, 1975a,c) seem to regulate pairing of B chromosomes also to bivalent level even when up to 10 B chromosomes are present (Jauhar, 1980). That a particular gene(s) should coordinately control the behavior of A and B chromosomes is quite understandable. E. EXPERIMENTAL INDUCTION OF DESYNAPSIS

Since desynapsis and asynapsis are mostly under genetic control, they can be artificially induced like any other mutation. The first case of desynapsis in pearl millet recorded by Krishnaswamy et a / . (1949) was probably a mutant induced as a result of X irradiation. Later, some desynaptic mutants of pearl millet were produced by both physical and chemical mutagens (Singh e t a / . , 1977a; Lakshmi and Yacob, 1978). Colchicine, which is primarily an effective polyploidizing agent, has been reported to induce a desynaptic mutant in pearl millet (Rao, 1980). It is now becoming evident that the whole meiotic sequence from premeiotic mitosis to spore formation is controlled by different genes that, acting in a coherent fashion, produce normal meiosis and, hence, male and female fertility. Mutation of any of these genetic determinants can disrupt, distort, or even block a particular stage and with it the subsequent stages in this integrated and ordered sequence of events.

CYTOGENETICS OF PEARL MILLET

423

F. INBREEDINGA N D DISRUPTION O F CHROMOSOME PAIRING: HETEROZYGOSIS A N D HETEROSIS FOR CHROMOSOME BEHAVIOR

Pearl millet is an allogamous crop. When subjected to enforced selfing, it shows inbreeding depression, which is associated with several meiotic abnormalities, like reduction in chiasma frequency and desynapsis (Pantulu and Manga, 1972). In an inbred line that was maintained by selfing, some desynaptics were obtained (Koduru and Rao, 1978). In another inbred line, partial “asynapsis” was accompanied by chromosome breakages that seemed to be localized near the centromeric regions (Rao and Koduru, 1978b). Hybrids between inbred lines have been reported to show heterosis for chiasma frequency and meiotic behavior. Thus, Mahadevappa and Ponnaiya (1 967) observed that a hybrid had higher chiasma frequency than that of the parental inbreds. Pantulu and Manga (1972) found that all the F, hybrids obtained by intercrossing inbred lines had mean chiasma frequencies higher than those of the parents involved. When tested under stress and nonstress conditions, the heterozygotes showed consistently higher chiasma frequencies and less variation in their means compared to the parental inbreds (Manga and Pantulu, 1974). Harinarayana and Murty (1971), however, reported that pearl millet inbreds had higher chiasma frequencies than did the parental outbred populations. This increase in chiasma frequency under enforced selfing was attributed to a “buffering mechanism of chiasma frequency under inbreeding, referred to as cytological homeostasis. ” Other studies have not confirmed the presence of the so-called cytological homeostasis in pearl millet. Recently, Srivastava and Balyan (1977) also reported heterosis for chiasma frequency and chromosome behavior. The pearl millet hybrids had 15-47% higher chiasma frequencies and also showed fewer meiotic abnormalities than the parental inbreds. Some of the deleterious genes, which otherwise remain masked in the heterozygous condition, are uncovered by inbreeding, and they adversely affect meiotic pairing and chiasma frequency in pearl millet inbreds. A similar decline in chiasma frequency also results from enforced inbreeding of other outbreeders, e.g., rye (Secale cereale), but on intercrossing of inbreds a positive heterosis for chiasma frequency is obtained (see Rees, 1961a). The results obtained by Srivastava and Balyan (1977) suggested a positive association of increased chiasma frequency with desirable quantitative characters like grain yields. They noted that the highest-yielding pearl millet hybrid, NHB-5, also had the highest chiasma frequency. Since the hybrids show heterosis for chiasma frequency, and heterosis for yields also closely parallels the heterozygosis of the breeding material (Burton, 1968), it is not surprising that some hybrids with high chiasma frequencies should also have high grain yields. Further studies along these lines may prove rewarding.

424

PREM P. JAUHAR

V. HAPLOIDY Haploid sporophytes, with half the chromosome complement, are useful cytogenetic tools. They offer an opportunity for the study of chromosome relationships within species because the internal homologies, which generally remain masked in the diploid, can be revealed in the complement in haploid state. Haploids can be usefully employed in several other areas of fundamental research, such as studies on induced mutagenesis, gene dosage effects, interaction of genes, and linkage analyses. Haploids can also help accelerate a breeding program. Haploids of polyploids are more appropriately termed polyhaploids, whereas haploids of diploid species are referred to as rnonoploids, rnonohaploids, or simply haploids. However, I feel that the term “monoploid” should be used for the haploids of true diploids with no history of polyploidy, whereas haploids of such doubtful cases as corn (see Section VI1,B) should, preferably, not be called monoploids, but simply haploids. A. SPONTANEOUS A N D INDUCED HAPLOIDS

Haploids of pearl millet (2n = x = 7) have been reported to occur spontaneously (Pantulu and Manga, 1969; Jauhar, 1970b; Powell et al., 1975). They have also been isolated in the progeny of trisomics, as well as induced artificially by treating pearl millet seeds with methyl methane sulfonate (Gill et al., 1973). B. CHROMOSOME PAIRINGI N HAPLOIDS

In the pearl millet haploids studied by different workers, mostly univalents were observed at stages comparable to diplotene (Fig. 6a), diakinesis (Fig. 6b), and metaphase I (Fig. 6c), and the subsequent meiotic stages were highly disorganized. Anaphase I disjunction led to 4 : 3, 5 : 2, 6 : I , or 7 : 0 distribution of chromosomes to the poles (Fig. 6d-g). Thus, as a result of 7 : 0 distribution, some balanced male (and also perhaps female) gametes were formed that fused to produce some viable seeds on the haploid earhead studied by Powell et al. (1975). This provided direct evidence of the formation of unreduced gametes in haploid pearl millet. The frequencies of bivalents observed at diakinesis and metaphase were very low (Table 11). A significant feature, however, was the occasional formation of some chiasmate bivalents. Jauhar (1970b) observed both ring and rod bivalents,

FIG.6 . Meiotic stages in haploid pearl millet (2n = x = 7). (a) Late diplotene with 7 univalents (71). (b) Diakinesis with 111 + 51. Note that the chiasma in the bivalent is completely terminalized and two univalents are showing end-to-side association. (c) Metaphase I with 2" 3,. (d-g) Anaphase I stages showing 4 : 3,5 : 2 , 6 : l , and 7 : 0 distributions to poles. [(a) X ca. 1970; (b) x ca. 1836; (c-g) x ca. 14221. Negatives of a, c-g were kindly supplied by Dr. J. B. Powell.

+

426

PREM P. JAUHAR

Table I1 Range and Mean of Bivalents Observed in Different Haploids of Pearl Millet References 0-2 0-2 0-2 0-2 0-2 0-2 0-2

0.57 0.40 0.20 1.13 0.46 0.58 0.87

Pantulu and Manga ( 1 969) Manga and Pantulu (1971) Jauhar (1970b) Gill et a / . (1973) Powell el a / . (1975) Powell et a / . ( I 975) Powell el a / . (1975)

but the chiasma terminalization was very rapid, which is a characteristic feature of diploid pearl millet (see Section III,A and Figs. 1 and 3b,c). Aside from chiasmate pairing, secondary associations of univalents were also observed. More significant among these were the s-s (side-to-side) associations, which are generally considered to result from residual homology (Person, 1955; Riley and Chapman, 1957). The realization of a maximum of two bivalents per cell, as also a maximum of two s-s pairs, was attributed to homologies between four members of the complement that may have resulted from duplication (Jauhar, 1970b). It was inferred therefore that the complement (x = 7) of pearl millet was derived from a basic set of x = 5 chromosomes. Intrahaploid pairing in the typhoides complement in P . typhoides x P . purpureutn and P . ryphoides x P . orientale hybrids lends support to this view (see Section XI,B.) If the x = 7 complement of pearl millet has indeed arisen from a basic set of x = 5 chromosomes, the duplicated chromosomes must have undergone sufficient differentiation during the course of evolution so that they pair as bivalents in the diploid pearl millet, although they occasionally show conspicuous secondary associations (see Section III,A and Fig. 3c). Gill et al. (1973) studied different haploids of pearl millet and reached the same conclusions as Jauhar (1970b). Manga and Pantulu (1971) also made essentially similar observations, but they did not support the view that the complement of pearl millet has been derived from a lower basic number. Haploid meiosis may not provide unequivocal evidence in regard to the phyletically basic chromosome number of a species. It is, nevertheless, interesting to note that different workers, studying different haploids of pearl millet from diverse sources, observed a maximum of two bivalents per cell (Table 11; Fig. 6c). The mean bivalent frequency, however, varied from 0.20 to 1.13. Haploids from different genetic backgrounds are expected to show such a variation in chromosome pairing.

CYTOGENETICS OF PEARL MILLET

427

It is noteworthy that apparently normal synaptinemal complexes (SCs) have been observed in haploids of maize (Ting, 1971, 1973) and barley (Gillies, 1974). Maize does not seem to be a true diploid (see Section VII,B) and some pairing accompanied by the formation of SCs is not unexpected. Also, extensive pachytene pairing in haploid barley (Sadasivaiah and Kasha, 1971) coupled with the formation of SCs-which are structurally similar to those in diploid barley (Gillies, 1974)-may not be without phylogenetic significance. However, it is not known whether pairing in pearl millet haploids is accompanied by the formation of SCs. Such studies should of course yield valuable information. C . HAPLOIDS I N PLANTBREEDING

If haploids of an open-pollinated crop like pearl millet could be produced on a large scale, they would help produce homozygous lines in one step and, thus, accelerate a breeding program. Such a haploidy method of breeding diploid species has great potential. It involves the following main steps: (1) production of haploids in the desired material of diverse origin; (2) selection, if any, at the haploid level; (3) induction of chromosome doubling in selected haploids to produce homozygous, diploid lines; and (4) the usual testing and selection in the homozygous lines before using them in heterosis breeding or production of synthetics. It is interesting to note that doubled haploids of maize have been utilized in the production of commercial hybrids (Chase, 1974). However, the frequency of the spontaneous occurrence of haploids in nature is very low. For example, the frequency of one haploid per 10,000 plants found in the pearl millet inbred Tift 23A (Powell et al., 1975) is far too low to be of any use in a breeding program. Therefore various methods of producing haploids by anther culture, including the anther-panicle culture technique of Kasperbauer et al. (1980), should be tried. It is noteworthy that ability to produce haploids is genetically controlled. The production of haploids in corn, for example, is under genetic control, different strains apparently having distinct rates of haploid frequency (see Carlson, 1977). Coe (1959) identified a high haploidy line, Stock 6, which on self-pollination produces more than 3% haploids. More recently, the high haploidy-inducing potential of some inducer lines was reported to be a highly heritable trait not appreciably influenced by environment (Aman and Sarkar, 1978). This trait may therefore be transferred rather easily to other desirable lines. If similar genetic mechanisms of inducing haploidy are discovered in pearl millet, its breeding program could be accelerated, at least in some cases. In this context, it is interesting to note that haploid-derived hornozygous maize lines are being used profitably in breeding programs in the Soviet Union, where some superior single hybrids have been developed from these lines (Golovin, 1979).

428

PREM P. JAUHAR

VI. POLYPLOIDY The fact that a large proportion of the existing plant species-including many of our crop plants-are polyploids clearly shows the importance of polyploidy in evolution. Since plant breeding is, in essence, man-made evolution, induced polyploidy has been used as a tool in improving diverse types of plant species. Most of the artificially induced polyploids of grain crops have not proved to be of direct practical value, although a small number of species have responded favorably to chromosome doubling (Stebbins, 1956a). Thus, autotetraploid rye, further improved upon through hybridization and selection among the tetraploid strains, has shown some promise (Muntzing, 1951, 1954). Like rye, pearl millet has certain features considered desirable for a favorable response to induced polyploidy. It is a diploid with low chromosome number (2n = 14) and is typically outbreeding; hence, it offers promise for polyploidy breeding. A. SPONTANEOUS OCCURRENCE

Krishnaswamy and Ayyangar (1941b) reported a spontaneous autotriploid (2n = 3x = 21) in the selfed progeny of a partially sterile (and presumably partially desynaptic) diploid plant (2n = 2x = 14) of pearl millet. Since a partially desynaptic plant can produce a small proportion of both normally reduced and unreduced gametes, the triploid must have resulted from the fusion of an unreduced gamete with a normal gamete. During meiosis the triploid formed an average of 4.95111per cell, 6% of the PMCs having 7111.Thus, the triploid showed meiotic pairing typical of an autotriploid. Burton and Powell (1968) also observed spontaneously occurring autotriploids that were highly sterile, but were not studied meiotically . Recently, Koduru and Rao (1978) reported spontaneous triploid and tetraploid plants in the progeny of an inbred line. While the triploid showed chromosome pairing typical of an autotriploid, forming up to 7111in 2% of the cells with a mean frequency of 4.37111per cell, the tetraploids fell into two categories. Four of the tetraploids behaved more or less like autotetraploids, but one nonhairy tetraploid was reported to show reduced quadrivalent frequency and higher univalent frequency. The low chromosome pairing in the nonhairy tetraploid was attributed to partial desynapsis. However, differences in quadrivalent frequency can also be caused by different genotypes. In the selfed progeny of a doubletelotrisomic (2n = 13 2 telocentrics) that was also a translocation heterozygote, Pantulu and Rao (1977b) obtained a normal triploid (2n = 3x = 21) that showed meiotic pairing typical of an autotriploid.

+

CYTOGENETICS OF PEARL MILLET

429

Powell and Burton (1968) observed tetraploids among twin and triplet seedlings arising from polyembryonic caryopses of pearl millet. A later study by Hanna et al. (1976) showed that autotetraploids occurred at frequencies as high as 1 per 13 plants in polyembryonic and I per 292 plants in nonpolyembryonic material. At metaphase, the mean chromosome configurations per cell were 1.491v 0.38,,, + 8.97,, + 2.64,, which are far less than those in thecolchicineinduced tetraploids (see Section VI,C,2).

+

B . FACTORSFAVORING SPONTANEOUS

POLYPLOIDY

Spontaneous polyploidy appears to be common in the higher plants. The grass family is particularly characterized by the preponderance of polyploid taxa. Of the different Pennisetum species whose chromosome numbers are known, including the intraspecific cytotypes, nearly 76% are polyploids. There is no reason to believe that the proportion would be much different in the rest of the family. Spontaneous chromosome doubling seems to be common in grass species as well as hybrids. The functioning of unreduced gametes of either one or, rarely, both parents of interspecific, intergeneric, and even intervarietal hybrids has contributed significantly towards the creation of spontaneous polyploids in nature (see Harlan and de Wet, 1975; de Wet, 1980). In a synthetic interspecific hybrid (2n = 3x = 21) between P . typhoides and P . purpureum, Jauhar and Singh (1969b) recorded a case of spontaneous chromosome doubling resulting in amphidiploidy apparently induced by decapitation or severe pruning. This was a case of somatic doubling. Meiotic nonreduction, however, is more common in hybrids. Stebbins ( 1956b) has listed different factors responsible for the high frequency of amphiploidy in grasses: (1) the occurrence of several species together gives ample opportunities for hybridization; (2) the production of abundant wind-borne pollen and the existence of self-incompatibility systems promote crosspollination; and (3) most of the species and their hybrids are perennials and have efficient means of vegetative propagation. It may be pointed out that in the genus Pennisetum, polyploids occur only in perennial, vegetatively propagated species of Pennisetum. All these factors of course help in the production, survival, and successful establishment of new hybrid combinations, many of which colonize new ecological niches. In addition to these factors, I feel that through natural decapitation (for example, severe grazing by cattle or wild animals), amphiploids of natural interspecific hybrids may be produced, thus further contributing towards the abundance of allopolyploidy in grasses.

430

PREM P. JAUHAR C. INDUCTION OF POLYPLOIDY

1 . Triploidy

Autotriploids (2n = 3x = 21) of pearl millet have been synthesized by crossing colchicine-induced autotetraploids with diploids (Gill et al., 1969, 1970a; Jauhar, 1970a). In addition to triploids, some hypotriploids (2n = 20, 19) are also obtained by this method (see, for example, Fig. 7). Triploids have also been produced through gamma irradiation (Pantulu, 1968) as well as by combined treatments with gamma rays and ethyl methane sulfonate (Singh et al., 1977b). At diakinesis in the spontaneous autotriploids, mean trivalent frequencies of 4.95 (Krishnaswamy and Ayyangar, 1941b) and 4.37 (Koduru and Rao, 1978) were observed; however, most of the synthetic triploids had ironically lower trivalent frequencies: 2.62 at diakinesis and metaphase (Gill et ul., 1970a), 2.76 at metaphase (Jauhar, 1970a), 3.57 at diakinesis and 3.41 at metaphase (Singh et al., 1977b). Such a variation in trivalent frequencies is not unexpected in synthetic triploids that have been produced by different means and have different genetic backgrounds. While Jauhar (1970a) used nearly raw autotetraploids for making triploids, Gill et al. (1969) used relatively diploidized material. An unusually high trivalent frequency was recorded in a radiation-induced

FIG. 7. Metaphase I in a hypotriploid cell (2ri = 20) showing 6 trivalents and I bivalent. Note chain-, V-, and frying pan-shaped trivalents. [ x 18201

CYTOGENETICS OF PEARL MILLET

43 1

triploid, in which more than 50% of the PMCs were reported to have 7111 each, with a mean of 6.35111and 6.1811, per cell at diakinesis and metaphase, respectively (Pantulu, 1968). The autotriploids of pearl millet have proved very useful in the synthesis of a series of aneuploids (see Section VI1,B). 2 . Tetraploidy The first experimental induction of tetraploidy in pearl millet was accomplished by Krishnaswamy et al. (1950) by injecting aqueous solution of colchicine into shoots of young seedlings. Studies on the C, generation of this allotetraploid showed up to 71vin about 0.8% of the cells, the mean per cell for 15 plants being 2.61v (Raman et al., 1962). Later, several workers produced synthetic tetraploids by colchicine treatment (Gill et al., 1966, 1969, 1970a; Jauhar, 1970a; Minocha et al., 1972) and by X irradiation (Singh et al., 1977b). Varying numbers of multivalents were reported in the C, generation colchi-tetraploids produced in different materials, the means being 2.6,, (Raman el al., 1962), 3.011~+111 (Gill et al., 1969), and 4.311v+111 (Jauhar, 1970a). Raman et al. (1962) also observed some trivalents, but they did not report the mean trivalent frequency. Different multivalent frequencies in these synthetic tetraploids may be attributed to their different genotypic backgrounds. Minocha et al. (1972) studied meiotic pairing in the autotetraploids of the open-pollinated variety, T55, and its inbred, BIL-4. Whereas the autotetraploids of T55 showed a mean frequency of 2.541~+111 per cell, the autotetraploids derived from the inbred BIL-4 had a mean of 3.011~+111 per cell. This indicates that the degree of heterozygosity of parental material can affect the multivalent frequency of the derived autotetraploids, with homozygosity favoring higher multivalent frequency.

3 . Reversion to Diploidy The colchi-tetraploids showed considerable meiotic instability as evidenced by the recovery of diploid revertants in their progeny. In the C, population raised by selfing the C, plants, Raman et al. (1962) observed about 18% diploids, which they attributed to haploid parthenogenesis in the parental tetraploids. A similar reversion from tetraploidy to diploidy was observed by Gill et al. (1 969). In other synthetic tetraploids (maintained in the first two generations by sib-mating and later by open pollination among tetraploids) diploid revertants were recovered even up to the C6 generation (Jauhar, 1970a; Jauhar et al., 1976). Since the diploid revertants were rnatromorphic, they may have arisen from the unfertilized (and perhaps pseudogamous) egg through parthenogenesis. The

432

PREM P. JAUHAR

intra- andor extracellular factors contributing to stability or instability of synthetic polyploids are not well understood today. The occurrence of hypotetraploid aneuploids in the progeny of pearl millet tetraploids showed that imbalanced gametes were functional.

4 . Cytological Diploidization Gill et a / . (1969) and Jauhar (1970a) studied chromosome pairing in the raw (C,) and advanced generation tetraploids and noted a marked cytological diploidization in successive generations, i.e., a gradual shift from multivalent to bivalent type of association. This was probably due to natural selection of genes that condition regular meiosis with bivalent formation (Jauhar, 1970a). Equally interesting was the fact that an increase in bivalents was not associated with an increase in univalent frequency; hence, the fertility of the tetraploids improved in later generations. However, even in the evolved tetraploids with 1.841v+1,1 per cell, the seed set was much lower than in the parental material. It was inferred that meiotic abnormalities coupled with genetic factors were jointly responsible for sterility of the advanced generation tetraploids (Jauhar, 1970a) (see also Section VI,D). Synthetic pearl millet tetraploids had bolder grains compared to the parental diploids. However, for them to be of any practical use, their fertility must be improved by a program of intercrossing among the tetraploid strains and selecting for high seed fertility. Providing this kind of evolution would probably improve their meiotic behavior by bringing about cytological diploidization. D. POLYPLOIDY A N D PLANTBREEDING

Many of our crop plants, e.g., wheat, oats, cotton, tobacco, potato, peanut, alfalfa, tall fescue, and napier grass owe their success to polyploidy. Their diploid progenitors are either extinct or, if living, cannot compete with them. It was envisaged therefore that the existing diploid species could be improved by inducing polyploidy . Although initial expectations of the success of polyploidy as a tool in plant improvement have not been realized, it has contributed somewhat to the improvement of ornamental plants and forage crops. Other areas of its usefulness include the following: (1) bridging the ploidy gap between taxa to permit their hybridization; (2) overcoming interspecific sterility barriers and thereby facilitating gene transfers; and (3) applications in special breeding techniques like the haploidy method of breeding diploids. The genomic makeup of an organism is delicately balanced. Artificial chromosome doubling upsets this balance and produces sterility. At the chromosomal

CYTOGENETICS OF PEARL MILLET

433

level, the main problem is irregular meiotic behavior that results in imbalanced gametes and sterility. However, genetic factors can also contribute to sterility. Doggett (1 964) found that fertility of autotetraploid sorghums was genotypically controlled with apparently “fairly simple” inheritance. Moreover, high fertility proved to be dominant to low fertility and, hence, was rather easily transferable from one taxon to another. Doggett suggested therefore that there was no “barrier to the successful development of cultivated tetraploid sorghum as a grain crop. ’ ’ Since trivalents and univalents are the main causes of aneuploidy, a reduction in their frequency should improve fertility. In rye and Lolium tetraploids, Hazarika and Rees (1 967) and Crowley and Rees (1968) found that an increase in chiasma frequency was accompanied by a reduction in univalents and trivalents, an increase in quadrivalents, and an improvement in fertility. These workers (Hazarika and Rees, 1967; Rees and Jones, 1977, p. 63) have suggested, therefore, that improvement in fertility can be achieved by increasing the frequency of quadrivalents, although they feel that increase in bivalent frequency (cytological diploidization) would be an “equally acceptable’ ’ alternative. The question now arises as to which of the two alternatives provides a more realistic approach to improving fertility of synthetic tetraploids. In this connection, it is important to note that quadrivalent formation cannot be achieved with the same efficiency as bivalent formation, because the former can be easily affected by the availability and redistribution of chiasmata. Chiasma frequency, in turn, is easily influenced by environmental stress. In the event of a slight reduction in chiasmata, some quadrivalents (especially the chain-type) could easily be converted into trivalents plus univalents. It has indeed been observed that the frequency of trivalents and univalents increases with decrease in chiasma frequency (see Rees and Jones, 1977). Moreover, chiasmata can be redistributed in favor of bivalents (Hossain, 1978). Thus, complete quadrivalent formation cannot be achieved. In that case, a combination of quadrivalents and bivalents would be a good compromise. Even so, the regular disjunction of all quadrivalents cannot be precisely achieved. On the other hand, complete bivalent formation ensures regular disjunction and balanced gamete formation. There are several reports that suggest that bivalents, but not quadrivalents, are associated with increased fertility (Miintzing, 1954; Hilpert, 1957; see McCollum, 1958; Aastveit, 1968; Gill er al., 1969; Jauhar 1970a). All these factors weaken the argument of Rees and Jones (1977) that increasing quadrivalent frequency by increasing chiasma frequency is an effective means of improving fertility of autotetraploids. It may be concluded therefore that cytological diploidization offers the best means of improving fertility of synthetic tetraploids. After all, this is the route nature has taken by genetically diploidizing polyploid crop plants such as wheat and oats.

434

PREM P. JAUHAR

VII. ANEUPLOIDS The aneuploids of a crop plant are helpful in assigning genes to particular chromosomes and for establishing linkage groups. By virtue of its low chromosome number, pearl millet offers a particularly favorable material for aneuploid analyses. Some progress has been made in building up a trisomic series in pearl millet, but the establishment of linkage groups still awaits completion. In describing different aneuploids in this article, the terminology used by Khush (1973, pp. 3-5) is adopted with minor modifications. An individual with an extra chromosome is referred to as a trisomic (2n + l), whereas the extra chromosome itself is designated as a trisome. Similarly, whereas an individual deficient for a chromosome is termed a monosomic (2n - I ) , the chromosome involved is referred to as a monosome. A telocentric chromosome will be referred to as a telosome or simply as a telo and will be symbolized by the letter t. A few monorelodisomic (2n - 1 + I t ) plants which are deficient for one chromosome arm are also reported in pearl millet; thus, they have 2n = 13 It. Some new terms have been coined, e.g., doubletelotetrasomic (2n = 14 2t), tripletelotetrasomic (2n = 13 + 3t), and quadruplerelotetrasomic ( 2 n = 12 4t) to describe adequately the newly isolated aneuploids of pearl millet (see Section VI1,D).

+ +

+

A. SFONTANEOUS OCCURRENCE

In pearl millet some spontaneous aneuploids, mostly trisomics, have been reported. Li and Li (1943) described four dwarf, weak plants that presumably arose in the progeny of a triploid. Two of the plants were primary trisomics (2n 1 = 15) and formed one trivalent each. The third plant was a double trisomic (2n + 1 + 1 = 16) involving one large and one small chromosomes, which formed two trivalents. This plant was very weak with no tillers and had indehiscent anthers. The fourth plant was extremely weak and sterile and had three extra chromosomes (2n 3 = 17), two medium-sized and one large. From the figures given by Li and Li (1943, p. 141), it appears that this plant formed a chain quadrivalent and a chain trivalent, indicating that it was probably tetrasomic for a medium-sized chromosome and trisomic for a large chromosome. This plant can, therefore, be represented as (2n + 2 + 1 = 17). The fact that the 17-chromosome plant was extremely weak and completely sterile shows that there is a limit to the tolerance of extra chromosomes in pearl millet, as in most other diploids. However, corn is an exception in this respect and has a tremendous tolerance for extra chromosomes. From a triploid x

+

+

435

CYTOGENETICS OF PEARL MILLET

diploid cross, Punyasingh (1947) obtained plants having up to eight extra chromosomes (2n 8 = 28). Of the trisomics of pearl millet observed by other workers, one was from the progeny of a desynaptic plant (Krishnaswamy et al., 1949), another was found in the progeny of a spontaneous triploid (Koduru and Rao, 1978), whereas yet another occurred spontaneously (Burton and Powell, 1968). Meiotic studies in these trisomics were, however, not reported.

+

B. SYNTHESIS OF ANEUPLOIDS OF PEARL MILLET

Of all the aneuploids, the trisomic condition for different chromosomes is best tolerated in pearl millet. Moreover, trisomics, particularly primary trisomics, are the easiest to obtain. However, there is one isolated report of the occurrence of a monosomic (2n - 1 = 13) in the progeny of triploid X diploid crosses (Jauhar, 1970a). The monosomic that survived until maturity was a diminutive plant with thin, grassy leaves and a highly sterile, globose head. It generally formed 6,, lI; rarely, 511 3, were observed. Detailed studies on the meiotic behavior and transmission of monosome could not be done. It is nevertheless interesting that, unlike most diploids, a monosomic condition in pearl millet is tolerated so that it grows until maturity. Some monotelodisomic plants with 2n = 13 1 telocentric, i.e., monosomic for one chromosome arm, have also been produced (Pantulu et al., 1976) (see Section VII,C,S). Recently, Koduru et al. (1980) have reported a case of interchange monosomy in pearl millet. In corn (2n = 20), however, 9 of the possible 10 monosomics (2n - 1 = 19) have been produced along with some occasional double monosomics (2n - 1 1 = 18) and even triple monosomics (2n - 1 - 1 - 1 = 17) (Weber, 1970, 1973). Corn is again an exception in this regard, because no other diploid species is known to withstand a triply rnonosomic condition. In theory, of course, haploids of diploid species are monosomic for n chromosomes. Haploids of corn and pearl millet, for example, are essentially monosomics for 10 and 7 chromosomes, respectively. But in haploids the complement is in a balanced state, even though in half a dose, and is better tolerated. Such an ability to tolerate extensive whole chromosome deficiencies as well as whole extra chromosomes would cast doubt on corn being a truly diploid species. I would imagine that corn is an ancient, secondarily balanced species with an extensive duplication (and probably redundance) of genetic information in the form of whole chromosomes as well as large segments of chromosomes. There is some evidence in favor of pearl millet also being a secondarily balanced species (Jauhar, 1968, 1970b; Minocha and Singh, 1971a,b; Gill et al., 1973). (See also Sections III,A, V,B, and XI,B,l,c.)

+

+

+

436

PREM P. JAUHAR

C. TRISOMICS

+

Since trisomics (2n 1 = IS) carry an extra chromosome, the phenotypic ratios for the genes located on this chromosome are modified in segregating progenies, thus helping in assigning genes to particular chromosomes. Khush (1973) has listed numerous types of trisomics reported in various plant groups. Several types of trisomics are reported in pearl millet.

I . Primary Trisomics A trisomic in which the extra chromosome is one of the normal chromosomes of the complement is referred to as primary single trisomic, or primary trisomic, or simply trisomic (2n + 1); such a trisomic may form one trivalent per cell. Similarly, a plant having an extra dose each of two different chromosomes of the complement can be referred to as primary double trisomic, or simply double trisomic (2n + 1 + l), and may form a maximum of two trivalents per cell. Likewise, a plant with one extra dose of three different chromosomes of the complement, i.e., having three different chromosomes in triple dose, can be referred to as primary triple trisomic or triple trisomic (2n + 1 + 1 + 1). Such a trisomic should ordinarily form a maximum of three trivalents per cell. a . Single Trisomics. Primary single trisomics constitute a particularly valuable tool for locating genes on different chromosomes and for establishing linkage groups. Jauhar (1970a) isolated two trisomics in the progeny of triploid X diploid crosses. These trisomics showed 611 + I,,, (Fig. 8a,b) or 711 1, (Fig. 8c). A systematic attempt at synthesizing a series of trisomics in pearl millet was, however, made by Gill et al. (1970b,c), Gill and Minocha (1971), Virmani and Gill (1971), and Minocha and Gill (1974), who isolated a set of primary trisomics from the progenies of triploid plants as well as from triploid x diploid crosses. Manga (1976) isolated seven trisomics from the selfed progeny of a triploid. Later, some primary trisomics were obtained from the progeny of a desynaptic (Tyagi, 1976c) and from the selfed progeny of primary interchange heterozygotes (Tyagi, 1977). i . Assigning morphological types to trisomes. The primary trisomics isolated by Gill et al. (1970~)were distinguishable from related diploids by their relatively poor vigor, shorter height, narrower leaves, and later flowering. They were also distinguishable from one another and were classified into seven morphological types designated as Tiny, Dark Green, Lax, Slender, Spindle, Broad, and Pseudonormal. The extra chromosome involved in the trisomics was identified by Gill et al. (1970~)at somatic metaphase on the basis of total length, arm ratios, and presence or absence of satellite. Thus, they found that Tiny was trisomic for chromo-

+

437

CYTOGENETICS OF PEARL MILLET

FIG. 8. Chromosome pairing in some primary trisornics ( 2 n + 1 = 15) of pearl millet. (a) Metaphase 1 with I,,, 6,,;the trivalent i s marked with arrow. (b) Metaphase 1 with l,,, 6,,;note the frying-pan trivalent marked with an arrow. ( c ) Metaphase I showing 7,, I,; the univalent is marked with an arrow. [ X 2 3 9 0 ]

+

+

+

438

PREM P. JAUHAR

some 1, Dark Green for chromosome 2 , Lax for chromosome 3, Slender for chromosome 4, Spindle for chromosome 5 , Broad for chromosome 6; the chromosome involved in Pseudonormal was identified at diakinesis as (nucleolar) chromosome 7 (Gill et al., 1970c; Gill and Minocha, 1971). Manga (1976) also described the morphology of seven primary trisomics developed by her and designated them as Dwarf, Bush, Slender, Semidwarf, Purple, Robust, and Pseudonormal. Thus, some of the primary trisomics described by her differ in their morphological features from those reported by Gill et a l . (1970~). As pointed out in Section II,l , it is difficult to identify some chromosomes of pearl millet at somatic metaphase. Extreme caution must be exercised therefore before assigning morphological types to trisomes. The information on somatic karyotype should be supplemented by karyomorphological studies at pachytene. Differential Giemsa banding patterns, when established (see Section 11, I ) , may prove extremely helpful in the identification of individual trisomes. ii. Translocatioii testers and identification of trisomes. Tyagi ( 1 9 7 6 ~1977) employed translocation-tester stocks for the identification of extra chromosomes involved in primary trisomics of pearl millet. This identification is based on the assumption that, in a trisomic F, hybrid between a primary trisomic type and a translocation tester, a configuration of l v + 511 indicates that the extra chromosome in the trisomic is one of those involved in the translocation, whereas the formation of lIv + 1111+ 411 implies its noninvolvement. iii. Trivalent frequency. Virmani and Gill (1 97 1) found a positive correlation between trivalent frequency and the length of the extra chromosome in different pearl millet trisomics. Thus, the trisomic for chromosome 1 showed 84% of the PMCs with a trivalent, whereas in the trisomic for chromosome 7 only 24% of the PMCs had a trivalent. Manga (1976), on the other hand, did not observe any correlation between chromosome length and trivalent frequency, except in the trisomic for chromosome 7. It is somewhat surprising that, barring the trisomic for chromosome 7, she observed higher trivalent frequencies in the trisomics involving shorter chromosomes than in the trisomic for the longest chromosome. Since one of the important factors governing chiasma formation is the length of the chromosome (or the length of a particular arm) in which chiasma formation takes place, the trisomics for the longer chromosomes generally form trivalents at a higher frequency, for example in maize (Einset, 1943) and tomato (Rick and Barton, 1954). In the trisomics for the shorter chromosomes, the opportunity for chiasma formation to bind the extra member is lower probably because of spatial limitation and, hence, the trivalent frequency should be lower. In any case, trivalent frequency is a variable feature easily influenced by genetic and environmental factors, and must never be used for identification of trisomics for different chromosomes as has been done by Virmani and Gill (1971) and Gill and Minocha (1971). Trivalent frequency probably would also be affected by chiasma frequency.

439

CYTOGENETICS OF PEARL MILLET

Manga (1976) reported that the trivalent frequency and chiasma frequency were “correlated” in the trisomics studied by her. She recorded a higher chiasma frequency in the trisomic for chromosome 7 than in the trisomic for chromosome 1; the latter also had the lowest frequency of all the trisomics as well as the diploid sib. The differences in chiasma frequencies among trisomics, and between the trisomics and their diploid sibs can probably be attributed to genotypic differences. A primary trisomic of tall fescue was also found to have significantly higher chiasma frequency per cell and per paired chromosome than its sister disomics (Jauhar, 1978). Minocha and Gill (1974) and Minocha et al. (1976) studied the transmission frequency of the extra chromosomes involved in the seven primary trisomics and found that it was significantly higher through the egg than through the pollen. In the selfed progeny of trisomics, only 3.4% were trisomics and such a low recovery was a major impediment in the maintenance of trisomics. b. Double and Triple Trisomics. Some double and triple trisomics have been isolated in pearl millet (Gill et al., 1970b). In the selfed progenies of triploids and from triploids x diploid crosses, a total of 169 aneuploids were isolated of which 96% were primary trisomics (2n + 1 = 15), while the remainder consisted of double trisomics (2n 1 + 1 = 16), triple trisomics (2n 1 + 1 + I = 17) and tetrasomics (2n + 2 = 16). The double trisomics, like the primary trisomics, had a reduced vigor and were sterile. They showed at diakinesis 2,,, + 511, or 1111 + 611 + l,, or 711 + 21,in 21, 5 5 , and 24% of the cells, respectively (Gill and Minocha, 1971).

+

+

2 . Secondary Trisomics In pearl millet there is one isolated example of gamma-ray-induced trisomic plant (Pantulu, 1967a). The extra chromosome involved in this trisomic was probably an “isochromosome,” as it formed a “ring trivalent” in two cells (Pantulu, 1967a), and apparently gave an indication of secondary trisomic condition. However, since this plant was a translocation heterozygote, a ring trivalent could also have formed in other ways. There is therefore no clear report of secondary trisomy in pearl millet. Rajhathy and Fedak (1970) described a clear case of secondary trisomy (2n = 14 1 isochromosome) in the diploid species Avena strigosa, in which the isochromosome formed a ring trivalent, or an isoring by interarm pairing. This plant was male- and female-sterile presumably because of the imbalance caused by the tetrasomic condition of the genes on the quadruplicated arm. It would be interesting to know the effect of secondary trisomy in pearl millet.

+

3 . Tertiary Trisomics

Since a tertiary trisomic has only one extra a m , or a part of an arm, of a particular chromosome, the genetic ratios are modified only for the genes located

440

PREM P.JAUHAR

on that arm or segment of the arm.Tertiary trisomics are therefore very useful for assigning genes to a particular chromosome arm and for the location of the position of centromere. Tertiary trisomics of pearl millet have been produced from selfed interchange heterozygotes and their crosses (Minocha et al., 1974; Tyagi, 1975b; Venkateswarlu and Mani, 1978), selfed interchange trisomics (Murthy et al., 1979), and from the selfed progeny of a triploid (Venkateswarlu and Mani, 1978). The expected pentavalent configuration was observed in 15% of the cells, and it was either chain-shaped or dumbbell-shaped (Minocha et al., 1974). Recently, Murthy et al. (1979) observed that of all the different pentavalent configurations formed in two tertiary trisomics, only 10% were dumbbell-shaped. Such differences in chromosome pairing can be caused by different factors, genotype being an important one. 4 . Interchange Trisomics

In pearl millet, interchange trisomics were reported from crosses between two interchange heterozygotes (Minocha and Brar, 1976), in the progeny of interchange heterozygotes (Manga, 1977; Venkateswarlu and Mani, 1978), in the selfed progeny of a triploid (Venkateswarlu and Mani, 1978), and in the progeny of primary trisomics (Rao and Rao, 1977). Tertiary trisomics are also formed as a result of translocation, but they can be distinguished from interchange trisomics. In the tertiaries, the trisome is normally involved in the dumbbell-shaped pentavalent configuration, or in other multivalent associations, like trivalents. Moreover, when the trisome remains as a univalent, the other chromosomes should generally form bivalents, but no multivalents. In the interchange trisomics, however, the trisome may be present as a univalent in addition to an interchange configuration. For example, the occurrence of a ring quadrivalent and a univalent may provide a crucial test of the presence of interchange heterozygosity coupled with trisomy . Thus, in an interchange trisomic a ring hexavalent configuration plus 41, l1 were observed in the majority of the cells (Minocha and Brar, 1976). Manga (1977), Rao and Rao (1977), and Venkateswarlu and Mani (1978) also found a ring quadrivalent plus a univalent in the interchange trisomics studied by them.

+

5 . Telotrisomics and Other Aneuploids with Telocentrics

Some telotrisomics and other aneuploids with telocentric chromosomes have been reported in pearl millet. Pantulu et al. (1976) described a plant with 2 n = 13 2 telocentrics. The telocentrics (t) had arisen from one of the chromosomes as a result of misdivision of its centromere, and they paired with the normal homolog to form a trivalent. Such a trisomic can be technically described as doubletelotrisomic, but is essentially a pseudotrisomic because no new genetic

+

441

CYTOGENETICS OF PEARL MILLET

material is added. In the progeny of a doubletelotrisomic, Pantulu et al. (1976) observed monotelodisomic (2n = 13 I t ) and telotrisomic (2n = 14 It) plants. From crosses between doubletelotrisomic (2n = 13 + 2t) and normal disomic plants, the authors isolated two monotelodisomics (2n = 13 + I t ) that were deficient for one chromosome arm and were also heterozygous for an interchange. Doubletelotrisomic plants ( 2 = ~ 13 + 2t) for different chromosomes of pearl millet have been isolated: for chromosome 1 , in the progeny of an autotriploid (Pantulu and Rao, 1977a); for one of the metacentric chromosomes, in triploid X diploid crosses (Rao et al., 1977); and for chromosome 7, in the progeny of a primary trisomic for chromosome 3 (Rao, 1977).

+

D.

+

TETRASOMICS

By selfing triploids and from triploid x diploid crosses, Gill et a/. (1970b) isolated a few tetrasomic (2n 2 = 16) plants of pearl millet. However, the cytology of these plants was not reported. Some aneuploids with tetrasomic number (2n = 16) but having varying number of telocentrics (t) have also been described. Thus, in the progeny of a doubletelotrisomic (2n = 13 2t), Pantulu et al. (1976) isolated some plants with 2n = 14 2t that can be termed doubletelotetrasomics. From the progeny of another doubletelotrisomic (2n = 13 2t), Rao e t a / . (1977) observed a plant with 2n = 13 3t. This plant with three telocentrics, thus making up a tetrasomic chromosome number (2n = 16), can be described as tripletelotetrasomic. Similarly, on selfing a doubletelotrisomic for chromosome 1, plants with 2n = 12 4t were obtained (Pantulu and Rao, 1977a). These plants with 4 telos making a tetrasomic chromosome number (2n = 16) can be most appropriately described as quadrupletelotetrasomics.

+

+

+

+

+

+

VIII. STRUCTURAL CHANGES IN CHROMOSOMES The preponderance of interchange heterozygotes in several natural populations is a clear indication of the adaptive value of certain chromosomal rearrangements (Bumham, 1956; Darlington, 1956; Rees, 1961b). Chromosomal hybridity confers adaptive advantage because it helps conserve adapted gene complexes. Interchanges, being good cytogenetic markers, are useful in basic studies in cytology and genetics, and offer potential for chromosome engineering (see Bumham, 1962; Ramage, 1964, 1970; Ramage and Wiebe, 1969). Thus, the study of the course and consequences of structural alterations is important to our understanding of many cytogenetic and evolutionary phenomena.

442

PREM P. JAUHAR A. SWNTANEOUS REARRANGEMENTS

There are a few reports of spontaneously occurring chromosomal rearrangements in pearl millet. Pantulu (1958) observed a reciprocal translocation in a first-generation synthetic, Gahi-1, evolved by Burton in the United States. During meiosis it showed, in addition to five bivalents, a ring or chain of four chromosomes that resulted in 40% pollen sterility. Krishnaswamy (1962) noted some partially sterile plants in selfed lines that formed a configuration of four chromosomes, thus indicating heterozygosity for an interchange. Later, Powell and Burton (1 969) reported on some plants that were heterozygous for one or two interchanges. Heterozygosity for a single interchange in the inbred lines Tift 13 and Tift 239 formed a configuration of four chromosomes, whereas an African introduction had a rearranged chromosome common to both interchanges, as evidenced by the formation of a hexavalent configuration. Plants heterozygous for one and two interchanges had, respectively, about 67 and 48% pollen fertility. The present author has observed spontaneous chromosomal interchanges in several populations of pearl millet. B. INDUCEDTRANSLOCATIONS

Chromosomal interchanges have been induced in pearl millet by a variety of physical and chemical mutagens. On the whole, the physical mutagens seem to induce more translocations than the chemical mutagens. Thus, the 90-min treatment with thermal neutrons produced four times as many plants (28%) with chromosome interchanges as with the highest concentration (0.6% aqueous solution) of ethyl methane sulfonate (EMS) (Burton and Powell, 1966). Similar results were reported by La1 and Srinivasachar (1979). Using X-ray treatments, Krishnaswamy and Ayyangar (1941a) induced interchange heterozygosity for one, two, or three reciprocal translocations, which resulted in the formation of quadrivalent, hexavalent, and octavalent configurations, and semisterility of the plants. Later, Pantulu (1967a) and Jauhar (1972, 1974) induced several multiple interchanges by treating seeds with gamma rays. Figure 9b,c shows PMCs of interchange heterozygotes for one reciprocal translocation. C. BUILDING UP A COMPLETE INTERCHANGERING

Through recurrent irradiation coupled with intercrossing of cytologically established interchange heterozygotes, Jauhar (1972, 1974) obtained several com-

CYTOGENETICS OF PEARL MILLET

443

FIG.9. Diakinesis in normal plants (a) and interchange heterozygotes (b-d) of pearl millet produced by gamma irradiation. (a) Seven bivalents (711)in a normal plant. (b,c) Early and late diakinesis with one ring configuration of 4 chromosomes (0') 5". (d) A multiple interchange of 10 chromosomes (partly broken) + 211in a complex interchange heterozygote. [(a) X 1460. (b-d) x ca. 19451

+

444

PREM P. JAUHAR

plex stocks (see, for example, Fig. 9d). However, interchange heterozygotes involving more than 12 chromosomes could not be obtained even by further cycles of irradiation. Irradiations probably broke higher interchange multiples in some cases. Moreover, sterility problems in the interchange stocks involving several chromosomes impeded their successful intercrossing. Interchange heterozygotes involving several chromosomes are difficult to obtain probably because of the limitations imposed on viability of the gametes and/or seeds. Jauhar (1974) believed that the somatic and gametic sieves operated more rigorously with the involvement of more chromosomes in interchanges. Brar et al. (1973), Tyagi and Singh (1974), and Tyagi (1976a), however, were successful in synthesizing interchange stocks involving all 14 chromosomes of pearl millet; these stocks were, of course, almost completely sterile. D. KARYOMORPHOLOGY A N D T H E INCIDENCE O F ~ N T E R C H A N G E S

The response of a species to induced interchange heterozygosity is probably conditioned by certain chromosomal attributes. James (1965, 1970) concluded that lsotoma petraea is cytologically preadapted to the occurrence and maintenance of interchange hybndity. It is interesting to note that karyomorphology and meiotic features, including the mode of chiasma formation, in pearl millet are essentially similar to those of Isotoma petraea. As described in Section I I , l , the chromosomes of pearl millet are mostly isobrachial with median and submedian centromeres, making the karyotype symmetrical. Mean chiasma frequency per bivalent (mean taken of several populations) is 1.84 ? 0.085, so that most chromosomes associate as ring bivalents (Fig. 9a). Moreover, the chiasmata are mostly terminally localized, and at diakinesis their terminalization is nearly complete (see Figs. 1 , 3b,c, and 5a). The favorable response of pearl millet to initial induction of interchanges may be, at least partly, a function of its symmetrical karyotype (Jauhar, 1974). The heterochromatic regions flanking the centromeres may also be helpful in the induction of breakages, because the break points are reported to be localized in the heterochromatic regions (see Evans and Bigger, 1961). E. IDENTIFICATION

OF

TRANSLOCATED CHROMOSOMES

The availability of translocation testers can greatly help in the identification of the chromosomes involved in different interchange stocks. In a cross between a stock and an established tester with known chromosome, the formation of 2,"

+

CYTOGENETICS OF PEARL MILLET

445

311 would suggest that the translocated chromosomes in the unknown stock and the tester are different, whereas l,, + 41, would show that the unknown stock has one chromosome in common with the tester stock. Thus, Tyagi (1976b) was able to identify the chromosomes involved in some translocation stocks by crossing them with tester stocks. F.

DISJUNCTION O F I N T E R C H A N G E CONFIGURATIONS

In a structural heterozygote, the shape and orientation of the quadrivalent configuration depend upon (1) the site of the occurrence of crossing over, and ( 2 ) the co-orientation of the four centromeres involved in the quadrivalent. Thus, two basic types of orientation may be obtained: (1) adjacent, and ( 2 ) “zigzug ” or alternate. Only combinations of chromosomes resulting from alternate orientation, however, result in genetically balanced gametes. Cytological and genetic studies by Burnham (1934) proved the existence of two types of adjacent orientation: adjacent I , in which homologous centromeres co-orientate and thus move to opposite poles; and adjacent 2 , in which nonhomologous centromeres co-orientate and thereby move to opposite poles. Using interchanges T ( 1 , 3) and T (3, 6) of pearl millet, Koduru (1979) studied at metaphase I the orientation types-alternate 1 and 2 , and adjacent 1 and 2 . He found that the relative frequencies of various orientation types were influenced by the genetic background. Frequency of alternate orientation was earlier found to be genotypically controlled in rye heterozygotes (Thompson, 1956), and selection for high and low frequencies of alternate co-orientations gave rise to distinct lines with high and low frequencies of such configurations (Sun and Rees, 1967). These studies provide evidence for the presence of a genetically regulated potential mechanism in meiosis that could be used to offset sterility due to translocations. By exercising selection for high seed fertility in pearl millet, it may be possible to increase the frequency of alternate orientations or vice versa. Such studies may prove useful from the breeding standpoint (see the following section). G. INTERCHANGEHETEROZYGOSITY A N D PLANT BREEDING

The advantage associated with chromosomal repatterning is evident from the fact that interchanges occur in high frequencies in several plant populations. Thus, their contribution to fitness must more than compensate for the infertility of the heterozygotes (Bailey er al., 1978). To explain the benefits of chromosomal hybridity, Darlington (1963) introduced the concept of hybridity

446

PREM P. JAUHAR

optimum, which implies that each species has an optimal level of heterozygosity to which it is accustomed, and that any departure from this level results in deleterious effects. The occurrence of interchanges in several populations of pearl millet suggests that they confer some adaptive advantage. It is likely that heterozygosity for certain interchanges locks up favorable gene arrays (“super genes” of Darlington and Mather, 1952). Interchange hybridity probably helps preserve heterosis associated with certain segments. Recent observations on the possible association of heterosis for yield with a chromosome segment in some hybrids between radiation-induced mutants and the parental line Tift 23 (Burton and Hanna, 1977) suggest the possibility of inducing translocation heterozygotes or certain duplications of value in plant breeding. Since chromosomal hybridity does confer a certain amount of heterosis in the outbreeder Isotoma petraea (James, 1970), it is logical to deduce correspondence between interchange heterozygosity and heterosis in pearl millet. By irradiating interspecific hybrids of Pennisetum, chromosome segments of one species can be transferred into the chromosome complement of the other. Induced translocations may thus be helpful in the interspecific gene transfers (see also Section XI,B,S).

IX. B CHROMOSOMES The occurrence of a special category of chromosomes, in addition to the normal complement, has been reported in numerous plant and animal species. Such extra chromosomes-which are usually smaller than normal chromosomes, do not have synaptic homology with the normal chromosomes, are mostly heterochromatic and genetically inert, and generally vary in number from organism to organism or even in different meiocytes within an organism-are referred to as B, accessory, supernumerary, diminutive, inert, parasitic, or even ghost chromosomes. The nuclear-genetic makeup of an organism is delicately balanced. Whereas each normal chromosome and, indeed, every segment is essential for normal development, B chromosomes are nonessential-if not detrimental-to organisms possessing them. Rhoades and Dempsey (1972) stated that B-chromosome DNA is specialized and shows little transcription or translation. Miintzing (1974) considers the term “accessory” to be more appropriate to describe these chromosomes. However, for the sake of simplicity and brevity, it is preferable to call them uniformly as B chromosomes (the normal chromosomes being A chromosomes). Considerable controversy hangs over the question of the origin of B chromosomes. Although no definite answer has been found so far,

CYTOGENETICS OF PEARL MILLET

447

there is substantial-albeit circumstantial-evidence to support the theory that they originated from normal chromosomes and that they are the by-products of karyotypic stabilization (see Jones, 1975). B chromosomes have been reported in only a few species of the genus Pennisetum, including pearl millet. When chromosome numbers of other species are known, Bs would probably be found in several of them. A. B CHROMOSOMES AS INDICATORS OF T H E ORIGIN OF P E A R L

MILLET

Based on the more frequent occurrence of B chromosomes in primitive varieties than in selected, commercially bred cultivars, Muntzing (1958) suggested that their occurrence might be used as an indicator of a crop’s center of origin. On the basis of the occurrence of B chromosomes in the pearl millet collections, the Sudan (Pantulu, 1960) and Nigeria (Powell and Burton, 1966a; Burton and Powell, 1968) were considered as this crop’s centers of origin. Drawing conclusions on this basis, however, is fraught with danger, because there are several different ecological and edaphic factors that influence the occurrence of B chromosomes. The number of Bs, for example, is reported to be higher in meadow fescue (Fesruca pratensis) growing in areas with clay soil than in areas with lighter soil (Bosemark, 1956); and in rye (Secale cereale) the frequency of Bs is higher in the material growing on acidic than on basic soils (Lee, 1966). Still more striking are the observations of Kishikawa (1970), who worked on clonal plants of rye grown under different regimes of temperature, soil, and moisture. He found that the frequency of Bs was lower in the progeny populations derived from plants grown under higher temperature or drier soil conditions. Such a preferential occurrence of B chromosomes in certain ecological niches would suggest a better selective advantage of Bs in certain conditions than in others. In view of these considerations, great caution should be exercised before drawing conclusions about the center of origin of a crop plant based on the presence or absence of Bs. Moreover, before using any such criteria several collections of a crop plant from different environments should be analyzed. The occurrence of B chromosomes in certain pearl millet collections from the Sudan and Nigeria does not indicate that pearl millet originated at these places. Recent evidence suggests that the Sahel region of West Africa is, in fact, the center of origin of pearl millet (Brunken er al., 1977). B. MEIOTICBEHAVIOR A N D POLYMORPHISM

Pantulu (1 960) reported 1-3 B chromosomes in a population of pearl millet from Sudan. The Bs were predominantly heterochromatic with subterminal cen-

448

PREM P. JAUHAR

tromeres, and there were no morphological differences between plants with Bs and those without. When 3 Bs were present, they paired either as a trivalent or as 111 + 11,or they remained unpaired in a small proportion of PMCs. Similarly, the presence of 4 Bs resulted in the formation 211,1111+ l,, 4,,or occasionally a quadrivalent (Venkateswarlu and Pantulu, 1970). Recently, Rao et al. (1979) studied the effect of 3 and 5 B chromosomes on the duration of mitotic cycle of pearl millet. They found that 3 Bs had little effect, whereas 5 Bs extended the duration by about 39%. Powell and Burton (1966a) reported 1-5 B chromosomes, which they considered euchromatic, in an inbred line derived from seed collected in Nigeria. The Bs either remained unpaired or formed bivalents (Fig. 10). The authors reported a somewhat novel property of the Bs in the Nigerian material, i.e., their ability to organize nucleoli. There is no clear report of the presence of a nucleolusorganizing region in B chromosomes of any species, although Flavell and Rimpau (1975) found evidence of genes coding for ribosomal RNA in the B chromosomes of rye. Additional nucleolar material, as observed by Powell and Burton (1966a), can also be organized by the normal chromosomes. The Bs in the Nigerian material (Powell and Burton, 1966a) showed a size variation among themselves; they were small, intermediate, or large (Fig. 10.). The interarm pairing in the large B coupled with its median centromere suggested that it arose as an isochromosome from the large arm of the intermediate-sized B. The small Bs must also have arisen as telocentrics and/or as isochromosomes from other Bs as a result of misdivision of the centromere. In any case, the Bs in pearl millet show polymorphism with respect to their morphology, if not function. Polymorphic Bs have been reported, among others, in maize (see Jones, 1975). C. EFFECTS O N NORMALCHROMOSOME PAIRING

In the course of this discussion, the terms “association,” “pairing,” and “synapsis” have been used interchangeably. There are varied reports of the effect of B chromosomes on pairing of normal or A chromosomes. Powell and Burton (1966a) observed that the number of B chromosomes per cell had no effect on pairing behavior of A chromosomes. However, the association of Bs among themselves and the A chromosome pairing seemed to be related. Thus, the microsporocytes with the least amount of B chromosome association also seemed to have the least A chromosome pairing. The pattern of pairing in the B chromosomes, however, had no relation to the pairing behavior of the A chromosomes in the material of a different genetic background (Venkateswarlu and Pantulu, 1970). Pantulu and Manga (1975) found that up to 4 Bs did not affect the mean chiasrna frequency of A chromo-

CYTOGENETICS OF PEARL MILLET

449

FIG. 10. Meiotic behavior and polymorphism of B chromosomes. Note that B bivalents (B,,)are orientated on the metaphase plate. (a) Metaphase I with I B,,+ I B,(marked with mows) in addition to 711 of normal chromosomes. (b) Metaphase I with 2B,, (marked with mows). (c) Metaphase I showing 4 B, lying off the metaphase plate. Note the size differences of the B univalents. [ x ca. 20001

450

PREM P. JAUHAR

somes; however, when the number of Bs exceeded 4 and went up to 8, they appeared to have a depressing effect on mean chiasma frequency, although the variance increased with increasing number of Bs. In rye (Secale cereale), Jones and Rees (1967) also reported that 0-8 Bs did not have a significant effect on the mean A-chromosome chiasma frequency; but they did find increase in variance between PMCs within plants, as well as an increase in variance between bivalents within cells. Thus, Bs could generate additional variability by increasing the amount of recombination. More recently, Rao and Pantulu (1978) studied the effects of the standard Bs and their derivatives-standard, deficient, and iso-Bs-on meiosis. With regard to their effect on A-chromosome chiasma frequency, the deficient Bs apparently had a depressing effect and the iso-Bs appeared to have an enhancing effect, whereas the standard Bs had no influence. Thus, the extra euchromatin present in the form of iso-Bs was interpreted to have an enhancing effect, whereas the extra heterochromatin in the form of the deficient Bs was considered to have a depressing effect. However, it is difficult to understand why the standard Bs and the deficient Bs apparently having the same amount of heterochromatin would have differential effect on chiasma frequency. That extra euchromatin in the form of trisomes can have an enhancing effect on chiasma frequency is known in pearl millet (Manga, 1976) and tall fescue (Jauhar, 1978). But the so-called depressing effects of heterochromatin and the enhancing effects of euchromatin (Pantulu and Manga, 1975, p. 244; Rao and Pantulu, 1978, pp. 125, 127) are much too low to be of any interest to a cytogeneticist or a breeder. Thus, in the report by Rao and Pantulu (1978, p. 125), for example, the means of the standard, deficient, and is0 1-4 B classes are 12.74, 12.55, and 13.41, respectively. These differences observed by Rao and Pantulu (1978) are so low that they could be resolved only by scoring a very large number of cells, i.e., 500 in each class. It is debatable whether the Bs, by lowering or promoting chiasma frequencies-if at all-are really regulating the release of variability, and whether or not such an additional variability supposedly released is of any essence to an outbreeder like pearl millet. D. B CHROMOSOMES-A BIOLOGIST'S DILEMMA

As mentioned earlier, B chromosomes generally have erratic meiotic behavior and do not seem to obey the normal laws of cytology and genetics, yet they persist in populations by some devious mechanisms, such as nondisjunction. In this respect, they constitute a cytologist's dilemma. Their genetic organization is not known. No major genes have been located on the B chromosomes, yet they are well known to influence several gene-controlled phenomena, e.g., chiasma

CYTOGENETICS OF PEARL MILLET

45 1

frequency of normal chromosomes. Thus, they also constitute a geneticist’s dilemma. One of the phenomena-nondisjunction-controlled by the B chromosome of maize has nevertheless proved to be a useful genetic tool (Carlson, 1978). Recently, they have been implicated in the suppression of homoeologous chromosome pairing. A report by Evans and Macefield (1972) that B chromosomes strongly suppress homoeologous pairing in a Lolium hybrid and their suggestion that ‘‘this would give, in terms of chromosome behavior, wheatlike (stable) amphidiploids . . . . has aroused considerable interest in breeding circles. Later work has shown, however, that the diploidizing effect of Bs, if any, is not consistent in several Lolium-Festuca hybrids and amphiploids and, hence, not dependable from the breeding standpoint (Jauhar, 1976, 1977a). After all, in nature the well-established diploidizing mechanisms operative in polyploid crop plants (see Jauhar, 1977b) are not related to any B chromosome activity. B chromosomes do not even occur in commercial varieties of crop plants. Moreover, as stated above, B chromosomes have arisen from normal chromosomes and as such must owe their ability to suppress homoeologous pairing, if any, to normal chromosomes. It is doubtful that the incorporation of B chromosomes into the complement of interspecific hybrids of Pennisetum, Lolium-Festuca complex, or any other plant group would help stabilize their meiosis. Moreover, the B chromosome DNA is specialized with no functional cistrons and may not even be transcribable (Rhoades and Dempsey, 1972). Rhoades and Dempsey (1972) further report that B chromosomes normally exist as “an unwanted guest who feasts on the cell’s resources. Thus, depending on B chromosomes for such an important function as stabilization of meiosis would seem fruitless. ”

”

X. FLORAL BIOLOGY AND HYBRIDIZATION Information on a crop plant’s floral biology is directly useful in its breeding program. Knowledge of floral characteristics, anthesis, mode of pollination and pollen viability is necessary for controlled matings. The inflorescence of pearl millet is a compound, cylindrical, unbranched, tapering spike (Fig. 11). The spikelets are commonly borne in clusters of two on rachillae that are seriately arranged on the main axis. Each spikelet has two florets, one bisexual and the other staminate (Fig. 12). The bisexual or hermaphrodite floret is pedicellate and consists of a single pistil with two feathery stigmas (see Fig. 13a), and three anthers. Borne below the hermaphrodite floret is a sessile, staminate (male) floret with three anthers.

452

PREM P. JAUHAR

FIG. 11. A spike of pearl millet showing protogyny. Note stigmas have emerged all over the earhead and anthers are emerging toward the tip.

CYTOGENETICS OF PEARL MILLET

453

FIG. 12. A scanning electron micrograph of a pearl millet spikelet with two florets; the larger one with protruding feathery stigma is hermaphrodite and the smaller one is staminate. [ ~ 2 4 1

454

PREM P.JAUHAR

A . PROTOGYNY A N D ANTHESIS

An important feature of Pennisetum species is their protogynous (profos = before or ahead) nature, which means that the carpels emerge and mature before the stamens. Consequently, anthesis starts only after most or all stigmas have emerged. Protogyny is particularly conspicuous in pearl millet (Fig. 11). It is, of course, a welcome feature from the evolutionary and breeding standpoints. It facilitates the introgression of characters from wild or semiwild, annual penicillarias into pearl millet and, thus, has helped in the genetic enrichment of this species. The emergence of stigmas generally starts near the tip of the partially exserted spikes and proceeds downward. Sometimes the spikes still inside the boot leaf have fully exserted stigmas. Pearl millet, like most other species of Pennisktum, has bifid, feathery stigmas (Fig. 13a). The two stigmatic branches provide enough surface for effective pollinations. The fully emerged stigmatic branches of pearl millet are glistening white with a bluish tinge. They usually remain receptive for three days. Since pollination is accomplished by wind (anemophily), the stigmatic surface-a reticulum of feathery hairs (Fig. 13b)-plays an important role in bringing about effective trapping of pollen. Generally, one day after the process of the emergence of stigmas is completed on a particular head, the anthers start emerging toward the tip (Fig. 11) and work their way down the head. The time lag between the two processes depends primarily on the temperature conditions. Warmer temperatures are conducive to earlier emergence of anthers and their dehiscence. The species of the section Penicillaria (all cultivated and semiwild or wild pearl millets, and napier grass) are characterized by the presence of conspicuously penicillate anther tips, i.e., they end in a tuft of fine hairs (Fig. 14), whereas most other species of Pennisetum have glabrous anther tips. However, the function of these cilia or trichomes is not known to date. B . MODEOF POLLINATION

Although anemophily-or wind pollination-is the rule in pearl millet, insects are reported to effect occasional cross-pollination (Leuck and Burton, 1966). Using a marker gene, Rao ef al. (1949) estimated 77.8% natural crossing in pearl millet. Of course, the extent of cross-pollination varies with different factors, such as the time of flowering of the parental plants, spacing between plants, and wind velocity and direction. In a uniformly flowering germplasm pool, Burton (1974) estimated natural crossing of 69 and 82% in two consecutive years. In controlled matings, glassine bags must be used.

CYTOGENETICS OF PEARL MILLET

455

FIG. 13. Scanning electron micrographs of feathery stigmas of pearl millet. (a) A bifid stigma; note numerous stigmatic hairs. (b) A portion of stigma enlarged to show a reticulum of feathery hairs that help in effectively trapping wind-borne pollen. [(a) ~ 2 1 (b) ; ~451

C. POLLENMORPHOLOGY AND VIABILITY

The pearl millet pollen grains are generally spheroidal (Fig. 15a). They are uniaperturate (monoporate) with nearly isodiametric pore (porus) (Fig. 15b), 2.5-4 k m in diameter. The nexine (the inner unsculptured layer of exine) is thickened around the porus to form a costa. There is slight variation in the costae of different cultivars; in some it is more pronounced than in others. Under humid conditions, the fresh pollen grains are generally inflated to give a spheroidal appearance. Under dry and hot conditions, however, they shrink to varying degrees. Shrinkage probably makes them lighter and more buoyant so that they can be carried long distances. Thus, this feature seems to have an adaptive value in wind-pollinated species. With the moisture from the stigmatic surface, the shrunken pollen grains swell up and this is probably the first step toward germination. The pearl millet pollen normally remains viable for a few hours, although it

456

PREM P. JAUHAR

FIG. 14. Scanning electron micrographs of pearl millet anthers showing penicillate anther tips. (a) Complete anther. (b) Anther tip magnified to show the tuft o f hairs. [(a) X28; (b) X2401

can be preserved under suitable conditions for future crossing. Cooper and Burton (1965) reported that pollen stored at 4.5"C for 3 weeks gave 80% as good seed set as fresh pollen, whereas that stored for 4 weeks was inviable. Cryopreservation of pollen should be tried with a view to preserving pearl millet germplasm for future use. The hybridization work is best carried out in the mornings between 7 A . M . and 9 A . M . under the Indian conditions. However, Cooper and Burton (1965) have found that hybrids may be made at any time of the day, but those made at midday generally set the least amount of seed per centimeter of spike.

XI. HYBRIDIZATION AND CHROMOSOME RELATIONSHIPS Although mutations have played a significant role in bringing about diversity in the biological world and, hence, are a major force in organic evolution, the catalytic effect of hybridization upon evolution should not be underestimated.

FIG. 15. Scanning electron micrographs of a pollen grain of pearl millet. (a) A spheroidal grain; note a single germ pore (potus) with cmfa around it. (b) A portion of pollen grain magnified to show the porus with costa (C)around i t . [(a) X2600; (b) x5400l

458

PREM P. JAUHAR

Natural hybridization between related species followed by natural polyploidy has in fact given rise to our most important food, fiber, oilseed, and fodder crops. The catalytic effect of hybridization on evolution lies primarily in enlarging the size of the gene pool that would facilitate a favorable response of a population to a changing environment (Grant, 1963; Stebbins, 1969, 1974). The study of chromosome pairing in hybrids has facilitated genome analyses and, thus, helped in the elucidation of phylogenetic relationships between different taxa. Meiotic pairing in hybrids, of course, depends upon the nature and extent of differentiation among the parental genomes. Moreover, different types of genetic control of chromosome pairing can complicate the pairing patterns in intergeneric, interspecific, and even intervarietal hybrids between geographically diverse ecotypes, especially of polyploid species (see Jauhar, 1975c, 1977b). In view of these restrictions, de Wet and Harlan (1972) have questioned the value of chromosome pairing data in interpreting phylogenetic affinities. However, I do feel that, in spite of the aforesaid limitations, chiasmate pairing has provided and will continue to provide useful information on the nature of ploidy of a species and on phylogenetic affinities between species. Chiasmate pairing is a specific process generally confined to chromosomes (or segments) of corresponding genetic similarities. Homologous or homoeologous segments are probably able to recognize each other based on congruence of pairing sites (recognition is probably based on similarity in nucleotide sequences). Information on chromosome pairing in hybrids coupled with that in their amphiploids should therefore give a realistic picture of genomic relationships of the parental species. In the genus Pennisetum, some interspecific, intergeneric, and even intertribal (Penniserum X Oryza) hybrids have been reported. The interspecific hybrids involved mostly pearl millet as one of the parents, and have helped in the study of chromosome relationships between pearl millet and several other species of Pennisetum. A . INTRASPECIFIC HYBRIDS

There are several semiwild, annual, diploid races in the section Penicillaria (Table I) with which the cultivated pearl millet is interfertile and essentially forms a single, composite reproductive unit. So the hybridization between pearl millet and other annual penicillarias will, in effect, be intraspecific hybridization. Pennisetum typhoides is a polymorphic species. Its polymorphism could be due primarily to its hybridization with the taxa in the section Penicillaria. The wild, annual relatives of pearl millet have been treated as separate species by Stapf and Hubbard (1933, 1934) and Clayton (1972), although the latter suggested their merger into single species with pearl millet. They are interfertile with pearl millet and have contributed to its genetic enrichment. Such a process

CYTOGENETICS OF PEARL MILLET

459

of hybridization and intefflow of genes is facilitated by the protogynous nature of the species. Chromosome studies by Krishnaswamy (1951), Thevenin (1952), and Veyret (1957) have shown that the annual relatives of pearl millet-viz., P . ancylochaete, P . cinereum, P . echinurus, P . gambiense, P . leonis, P . maiwa, P . nigritarum, and P . pycnostachyum'-are diploids with 2n = 14 chromosomes like the cultivated pearl millet (see Table I). Moreover, the fact that the chromosome morphology of several of these taxa was similar to one another and also to that of pearl millet (Veyret, 1957) further indicated their affinity with pearl millet. P . violaceurn and P . fallax are two of the important wild, annual relatives of pearl millet. Genetic studies by Bilquez and Lecomte (1969) and by Brunken (1977) have shown that these taxa form fully fertile hybrids with pearl millet and, hence, are not reproductively isolated from it. They must obviously have 2 n = 14 chromosomes in order to form fertile hybrids with pearl millet. The chromosomal, hybridization and genetic studies showed conclusively that the wild or semiwild annual relatives of pearl millet are not sufficiently isolated from it to deserve specific ranks. Brunken (1977) has therefore merged all annual penicillarias with pearl millet (Pennisefum typhoides), which he calls P . americanum. For the sake of convenience, he has subdivided the species into three subspecies: (1) subsp. americanum, which includes all the cultivated races of pearl millet; (2) subsp. monodii, which encompasses all the wild and semiwild races of pearl millet; and (3) subsp. stenostachyum, which is morphologically intermediate between subspecies americanum and monodii, and includes all mimetic weeds associated with the cultivation of pearl millet. B.

INTERSPECIFIC

HYBRIDS

Several hybrids between pearl millet and other species of Pennisetum have been made by different workers. In some cases amphidiploids have also been produced by colchicine-induced chromosome doubling of the interspecific hybrids or by natural meiotic nonreduction.

1 . P . typhoides x P . purpureum Hybrids Interspecific hybrids between P. typhoides ( 2 n = 14) and P . purpureum (2n = 4x = 28) are the most widely studied in the genus Pennisetum and probably

also in the entire tribe Paniceae. These species of the section Penicillaria are reported to cross in nature to produce spontaneous hybrids (Stapf and Hubbard, 'These are all infraspecific categories within the species P . ryphoides

460

PREM P. JAUHAR

1934). They have also been hybridized by numerous workers using either one of them as the female parent. However, using pearl millet as the female parent has several advantages: (1) its protogynous nature generally eliminates the need for emasculation; (2) seed shattering is absent or minimal; and (3) it is easier to identify hybrids at seedling stage. Burton (1944), working in the United States, was the first to make interspecific hybrids, which were later produced in India (Krishnaswamy and Raman, 1949, 1953a,b; Krishnaswamy, 1951), South Africa (Gildenhuys, 1950; Gildenhuys and Brix, 1958, 1964), Pakistan (Khan and Rahman, 1963), Australia (Pritchard, 1971; Muldoon and Pearson, 1977), Sri Lanka (Dhanapala et a l . , 1972), Nigeria (Aken’Ova and Chheda, 1973), and other countries. The main objective of hybridizing these species was to produce a high-quality, highyielding, perennial fodder plant that would inherit pearl millet’s forage quality, nonshattering nature, and capacity to establish readily, and also have the perennial, aggressive nature and rust resistance of napier grass. In South Africa, the main objective was to breed a large-seeded perennial for use in ley farming (Gildenhuys, 1950). The hybrids produced in different countries are generally high-yielding and more acceptable as fodder plants than napier grass. They exhibit considerable heterosis for both fodder yield and quality (Burton, 1944; Krishnaswamy and Raman, 1949; Patil, 1963; Khan and Rahman, 1963; Hussain et al., 1968; Pritchard, 1971; Aken’Ova et a f . , 1974; Gupta, 1974; Muldoon and Pearson, 1977). Powell and Burton (1966b) described a commercial method of producing interspecific hybrids by using a male-sterile line of pearl millet (Tift 23A) as the female parent; the hybrid thus produced was described as the highest-yielding forage millet grown in the United States. However, the hybrids’ complete seed sterility (they can be propagated only by vegetative means) has restricted their adoption on a large scale. While these hybrids are widely grown in some countries, particularly in the Indian subcontinent, they are being grown in trials in several other countries (see Muldoon and Pearson, 1979; see also Section XI ,B,2 ,e) . a. Gross Morphology. With respect to several vegetative characters such as panicle morphology, internode length, leaf and ligule size, the hybrid is either intermediate between the parents or more often approaches the purpureum parent. This is to be expected in view of the greater contribution of genetic material from the purpureum parent. However, depending on the genotype of the parental species used, there is a considerable variation in expression of heterosis for different vegetative characters like height, stem thickness, tillering, and leaf size. b. Chromosome Pairing. The hybrid is a triploid with 2n = 3x = 21 chromosomes (Fig. 16a), 7 contributed by diploid (2x) typhoides and 14 by tetraploid (4x) purpureum. As discussed in Section I1,1, pearl millet has very large chromosomes, larger than those known for any other species of Pen-

CYTOGENETICS OF PEARL MILLET

46 1

nisetum, except P . ramosum. Burton (1944) could identify the 7 large chromosomes of pearl millet ir. the interspecific hybrids, but he did not study chromosome pairing. Krishnaswamy (1951) and Krishnaswamy and Raman (1953a, 1954) studied chromosome pairing in the triploid hybrids. On the basis of the formation of 711 + 7, in most of the cells and the absence of trivalents, these authors concluded that the genome of typhoides was homologous to one of the genomes of purpureum. Khan and Rahman (1963), Ramulu (1968), Sethi et al. (1970), and Rangaswamy (1972) made essentially similar observations on chromosome pairing in the hybrids and reached the same conclusions regarding the genomic makeup of the parental species. Raman (196% reviewing the earlier work of himself, Krishnaswamy, and co-workers, designated the genomic constitution of P . ryphoides as AA, and that of P . purpureum as A'A'BB. The formation of 711in the triploid hybrid (AA'B) was attributed to synapsis between the A genome of typhoides and A' of purpureum. It was evident, however, that A and A' were not completely homologous. c . Analysis of Inter- and Intragenomal Chromosome Pairing. In the hybrids studied by Jauhar (1968), the easily recognizable size differences of the parental chromosomes (Fig. 16a-c) permitted a detailed analysis of inter- and intragenomal chromosome pairing. A range of 0-9 bivalents was observed, the mean per cell being 5.3. Whereas the majority of bivalents were formed between A and A' genomes and, thus, were clearly heteromorphic (Fig. 16b,c), some bivalents resulted from intragenomal pairing. Up to five heteromorphic AA' bivalents were observed per cell, which suggested that the two genomes are evolutionarily related, and that they probably arose from a common progenitor with x = 5 chromosomes. All three genomes-A, A', and B-showed intragenomal (autosyndetic) chromosome pairing. Such bivalents were almost homomorphic and hence easily distinguishable from the heteromorphic, intergenomal (A-A') bivalents described above. The bivalents formed by the A genome chromosomes were the largest (Fig. 16b), whereas those of A' and B genomes were, respectively, intermediate and the smallest in size. The intragenomal pairing appeared to be limited to a maximum of two bivalents, further suggesting x = 5 as the phyletically basic number from which, probably, the genomes with x = 7 were subsequently derived. Thus, it was inferred that x = 5 is the original basic number of the genus Pennisetum and that P . typhoides is a secondarily balanced species (Jauhar, 1968). Later studies by Jauhar (1970b), Minocha and M . Singh (1971b), and Minocha and A. Singh (1971a) provided corroborative evidence favoring these conclusions. It may be pointed out that as early as 1951, Krishnaswamy had suggested that intragenomal pairing occurred within the B genome. On the basis of pachytene pairing in the hybrid, Pantulu (1967b) reported that the chromosomes 1-5 of typhoides were homologous with chromosomes 1-5 of

462

PREM P. JAUHAR

Flc. 16. Meiotic stages in the Penniserurn ryphoides X P . purpureum ( 2 n = 3 x = 21). Note clear size differences of parental chromosomes. [Small arrow, wphoides univalents; medium arrows, intragenomal bivalents within ryphoides complement; thick arrows, intergenomal bivalents formed by A genome of typhoides and A ' of purpureurn.] (a) Metaphase I showing 21 chromosomes, 7 large ryphoides chromosomes ( A genome) and 14 small purpureum chromosomes (A'B genomes). (b) 7, that comprise 1 heteromorphic intergenomal bivalent (thick m o w ) , 2 large Metaphase I with 7,,

+

CYTOGENETICS OF PEARL MILLET

463

purpureum, and that chromosomes 6 and 7 of typhoides were homologous with chromosomes 8 and 14 of purpureum, respectively. However, in view of the markedly larger chromosomes of typhoides, a part of its chromosome should remain unpaired with the corresponding purpureum chromosome. Consequently, terminal forks, terminal unpaired regions, or intercalary loops should be seen at pachytene. Pantulu (1967b) did not, however, report any such structures, as his drawings of different bivalents show almost perfect pairing.

2 . P . typhoides x P . purpureum Amphidiploids a . Chromosome Pairing and Fertility. Krishnaswamy and Raman (1949) produced amphidiploids by treating the P. typhoides X P . purpureum hybrid seedlings with 0.4% colchicine. Chromosome doubling largely restored the fertility in the synthetic amphidiploids (AAA'A'BB; 2n = 6x = 42). During meiosis they generally formed 2 I,,, multivalents being either absent or infrequent (Krishnaswamy, 1951; Krishnaswamy and Raman, 1954; Ramulu, 1968, 1971; Jauhar and Singh, 1969b; and Jauhar, unpublished results). Some univalents observed at metaphase resulted from precocious separation of bivalents and caused disjunctional abnormalities. In view of the formation of 711 + 71 in the triploid hybrid (AA'B), some quadrivalents or trivalents should be expected in the derived amphidiploids, but they are observed rarely, if at all. This is probably due to preferential pairing between the A-A, A'-A', and B-B genome chromosomes, resulting in 21,1. Although the A and A' genomes are somewhat differentiated, the corresponding chromosomes of these genomes can pair in the absence of their own homologous partners. On chromosome doubling, however, bivalent formation is probably brought about by strong preferential pairing. However, the possibility of some sort of genetic control of pairing cannot be ruled out. The diploidizing genes in a double dose probably bring about diploid-like (genetically enforced preferential) pairing in the allohexapioid. [Such diploidizing genes that are effective only in a double dose are known in polyploid species of Festuca (Jauhar, 1975a,c).] Thus, the synthetic amphidiploid behaves meiotically like a typical allohexaploid. b. Chromosomal Instability. Gildenhuys and Brix (1961) also produced an amphidiploid by colchicine treatment of the cuttings of triploid hybrid. Although largely fertile, the amphidiploid showed marked instability in somatic chromointrahaploid bivalents within typhoides complement (medium m o w ) , and 4 intragenomal bivalents within A ' and B genomes; 2 large univalents belong to A genome and the remaining 5 univalents belong to A' and B genomes. (c) Metaphase I with 6,, + 9,. Note clearly heteromorphic bivalents (thick arrows) and distinct size differences among univalents. (d) A cell at anaphase with 22 chromosomes showing 2 typhoides and 5 purpureum chromosomes going to one pole, 3 fyphoides and 9 purpureum chromosomes going to other pole, and 3 Wphoides chromosomes lagging. The ryphoides chromosomes are marked with m o w s . [(a-c) x ca. 1800; (d) x ca. 13501

464

PREM P. JAUHAR

some number within the plant; its number was in the range 2 n = 36-49, with 2 n = 42 occurring most frequently (Gildenhuys and Brix, 1964). The backcross progeny obtained from the cross 2x P. typhoides X 6x amphidiploid also showed intraplant variation in chromosome number. Gildenhuys and Brix ( 1 964) concluded that this intraplant numerical mosaicism was under genetic control and that the genetic determinants expressed themselves only when present in a double or higher dose. Thus, it appears that these genes were hemizygous ineffective. Conversely, Krishnaswamy and Raman (1956) and Ramulu (1968, 1971) did not report any intraplant or even interplant numerical mosaicism in the amphidiploids or their derivatives. Because of the formation of univalents, however, some variation in chromosome numbers in the progenies of amphidiploids is inevitable and may be detected if a large population is scored. c . Phenotypic Manifestation of Different Genomes. The amphidiploids are vigorous in growth and show gigantic features typical of several polyploids. Generally, they have thicker stems, broader leaves, and larger panicles compared to the parental triploids. They show greater morphological resemblance to P. purpureum than to P . typhoides. This is expected in view of the greater genomic contribution from the tetraploid purpureum. Contrarily, if it is considered that A genome of typhoides and A‘ of purpureum are similar in genic content, then the amphidiploids have, in effect, four A genomes and should resemble the typhoides parent more closely. However, their greater resemblance to purpureum would show that either the A and A’ genomes are sufficiently differentiated and have different phenotypic expressions, or else it is more likely that the B genome dominates the A as well as A‘ genomes and masks their phenotypic manifestation. That the B genome is indeed “dominant” is borne out by the studies of Krishnaswamy and Raman (1949, 1953a, 1954, 1956), Raman and Krishnaswami (1961), Raman et al. (1963), and Raman and Nair (1964). All these workers have demonstrated that even when the ratio of A to B genomes is altered from 2 : 1 to 5 : 1, the phenotypic manifestation of the B genome is noticeably greater than that of A genomes combined. In an amphiploid derivative with AAAAA’B constitution (Raman and Krishnaswami, 1961), for example, there was only one dose of B genome, but the characters of the wild parent, P. purpureum, were still expressed. This indicated that one dose of B genome was dominant (or perhaps epistatic) over five doses of the A genome. Studying the morphology of selfed progenies of the amphidiploids by metroglyph analysis, Ramulu and Ponnaiya (1967) and Ramulu (1971) also found a distinct skewedness towards P . purpureum in the expression of several morphological features. d . Amphidiploid Derivatives. Synthetic amphidiploids have been successfully backcrossed to pearl millet to produce derivatives with different genomic constitutions. Thus, Krishnaswamy and Raman (1956) found that hybrids could be easily secured whichever way the cross was attempted, e.g., 2x x 4x, 2x x

CYTOGENETICS OF PEARL MILLET

465

6x, 4x x 2x,or 6x x 2x. All nine tetraploids produced from the cross 2n P. typhoides x 6x amphidiploid had 2n = 28 chromosomes, showing that the amphidiploids generally formed 2 1 -chromosome gametes or else such gametes were at a competitive advantage over the aneuploid gametes. This is in sharp contrast to the observations of Gildenhuys and Brix (1964), who reported that the compatibility of the cross improved when subhaploid pollen of the hexaploid fused with the haploid egg cell of P . typhoides. Gildenhuys and Brix ( 1 964) also found a high degree of incompatibility when amphidiploid was used as pollen parent in backcrosses to P . typhoides. Of the 31 offsprings thus obtained, only 4 had the expected number of 2n = 28 or less. The remainder had 2n = 35 or thereabout, and hence arose from the unreduced egg cells of P . typhoides. It appeared that the pollen from the hexaploid was more compatible with diploid rather than haploid eggs of typhoides. Conversely, the compatibility also seemed to be enhanced when the pollen from hexaploid contained less than the haploid set of chromosomes (Gildenhuys and Brix, 1964). From the standpoint of plant breeding, however, this is not a welcome situation. It was further inferred from these results as well as from the cross [2x P. typhoides x 6x (P.typhoides x P . purpureum)] x 2x P . typhoides, that incompatibility probably resided in the hybrid embryo itself and that its normal development (e.g., the success of the cross) was not dependent on the normal endosperm acting as a nurse tissue (Gildenhuys and Brix, 1964, 1965). Chromosome pairing was studied in several other amphiploid derivatives, with various genomic constitutions, obtained after a series of backcrossings of 6x amphidiploid to 2x and 4x P. typhoides, followed by selfing and further crossing of the derivatives (Raman and Krishnaswami, 1960, 1961; Raman et af., 1963; Nair et al., 1964; Raman and Nair, 1964). As expected, these derivatives formed different frequencies of multivalents, bivalents, and univalents, depending upon their genomic composition. They were sterile by varying degrees. From the studies on triploid hybrid (AA'B), the amphidiploids (AAA'A'BB), autoallotetraploids (AAA'B) and numerous other amphiploid derivatives with different genomic constitutions, the following conclusions can be made:

I . When present in duplicate, the genomes A, A', and B maintain their meiotic integrity, forming mostly bivalents between homologous partners; this is probably due to genetically enforced preferential pairing. 2. When present in a single dose or in more than two doses, there is intergenomal pairing between A and A'; however, A-B and A'-B synapsis is seemingly absent, showing thereby that A and A' are largely homologous to each other, whereas they both are nonhomologous to the B genome. 3. There is some amount of intragenomal pairing probably limited to a maximum of two bivalents within each of the three genomes. These studies have confirmed the allotetraploid nature of P . purpureum. It is a

466

PREM P. JAUHAR

genomic allotetraploid (A’A’BB) with one genome largely homologous to the typhoides (AA) genome at least in terms of its pairing behavior. The donor of B genome may be one of the diploid members of the section Penicillaria, which may exist somewhere in Africa or is probably extinct. e . Selection of Superior Forage Hybrids. As described above, several superior triploid hybrids between pearl millet and napier grass have been produced in different countries. However, they have not been adopted on a wide scale primarily because they are completely sterile and can be propagated only by vegetative means. From the point of view of easy distribution to farmers, superior, fertile amphidiploid hybrids or derivatives would need to be developed, so that seed can be supplied to farmers. Since the colchicine-induced amphidiploids are largely regular meiotically and their progenies show a wide range of pollen and seed fertility, it may be possible to select fertile, superior forage amphiploids which produce large amounts of good seed. f. Possible Synthesis of Perennial Pearl Millet. From the point of view of incorporating the desirable features of P . purpureum into P . vphoides, the triploid plant obtained by Raman and Krishnaswami (1960) is interesting. This plant was derived by crossing 2x typhoides (AA) with an autoallotetraploid (AAA’B). The derived triploid had four nucleolar chromosomes, and the pattern of pairing showed that it had mostly chromosomes of A and A’ genomes and probably none of the B genome. It is interesting that most, if not all, B genome chromosomes were selectively eliminated. Raman and Krishnaswami (1960) suggested that from such triploid plants it might be possible to secure fertile allodiploids with A and A’ genomes that would probably combine the desirable features of ryphoides and purpureum. It was observed earlier at the University of Hawaii Agricultural Experiment Station (Anonymous, 1947) that the 34 F , hybrids, obtained by crossing a selfed line of pearl millet (2n = 14) with an East African strain of napier grass (2n = 28), included plants with 2 n = 28,21, 18, 16, and 14. The occurrence of plants with 2 n = 14 suggests the possibility of selecting the desired “allodiploid” plants. In this way perhaps a perennial pearl millet can be produced. The selfed pearl millet line used in the above cross was probably desynaptic and produced imbalanced gametes and, hence, aneuploid hybrids. If a desynaptic purpureum line can be used as a male parent and crossed with a diploid typhoides with desirable features, different plants with a whole array of chromosome numbers can be obtained to exercise selection for the desired 2 n = 14, meiotically regular, fertile plants. In successive backcrosses of the 6x amphiploids to P . typhoides, Gildenhuys and Brix (1969) observed a selective elimination of imbalanced gametes and zygotes, particularly those with chromosomes of the B genome, and to a lesser extent, those of the A‘ genome (of purpureum). After three generations of backcrossing, all plants expressed the annual pearl millet habit. However, Gildenhuys and Brix (1969) could not combine the desirable features of typhoides

CYTOGENETICS OF PEARL MILLET

467

(viz., fertility, date of flowering, and grain size) with the perennial habit of purpureurn. If further attempts are made using gamma-ray treatments also, it may be possible to incorporate the desirable segments from purpureum into the typhoides complement. If a perennial pearl millet is indeed produced through the techniques described above, it would be useful in the drought-stricken, semiarid regions of Africa, tropical India, and other tropical and subtropical regions.

3 . P . typhoides x P . squamulatum These species have been hybridized with a view to evolving a grass combining the forage quality of pearl millet with the frost resistance and perenniality of P . squamulatum (2n = 6x = 54). Patil et al. (1961) and the present author (Jauhar, unpublished results) successfully obtained the hybrids. Taking advantage of the protogynous nature of pearl millet, we dusted profusely the pollen of squamulatum on a number of typhoides ears that had freshly emerged, receptive stigmas. From the progeny, Patil et al. (1961) picked up a single hybrid that had 2n = 41 chromosomes. The hybrid obviously arose from the union of an unreduced 14-chromosome egg of pearl millet with a haploid 27-chromosome male gamete of squamulatum. Morphologically, the hybrid resembled the squamulatum parent very closely although it had the leafiness of pearl millet. Patil et al. (1961) observed mostly 1611 9, in the hybrid. It appeared to the authors that seven bivalents (which must have been much larger than the rest) were formed by the typhoides complement, whereas the remaining nine bivalents (the small ones) and nine univalents resulted from pairing within the squamulatum complement. The small bivalents in the hybrid could have formed as a result of intergenomal pairing between the three genomes of squamulatum and some by intragenomal pairing.

+

4 . TrispeciJic Hybrids: ( P . typhoides x P . purpureum) x P . squamulatum

Several hybrids involving three distinct species have been made in grasses, e.g., Lolium-Festuca complex (Jauhar, 1975b, 1976). In the genus Pennisetum, there is one report of a trispecific hybrid obtained by crossing ( P . typhoides X P . purpureum) hybrid with P . squamulatum (Rangaswamy and Ponnaiya, 1963). The two hybrid plants thus obtained resembled the squamulutum parent in several features, including the panicle and spikelet characters. They also had penicillate anther tips, a typical character of typhoides (see Fig. 14) and purpureum. Rangaswamy and Ponnaiya (1963) found that the hybrid had 2n = 48 chromosomes, which showed that it had arisen from an unreduced 21-chromosome egg of the female parent (typhoides X purpureum hybrid) fertilized by the normal 27-chromosome male gamete from squamulatum. The formation of quadrivalents and pentavalents at meiosis suggested relationship of the genomes of the

468

PREM P. JAUHAR

three species (Rangaswamy and Ponnaiya, 1963; Menon and Devasahayam, 1964). 5 . P . typhoides

X

P . orientale

P . orientale is a valuable, perennial forage species consisting of different cytotypes with 2 n = 18, 27, 36,45, 54. It belongs to the section Heterostachya. With a view to evolving a perennial forage grass, we hybridized pearl millet and diploid P . orientale (2n = 18). The interspecific hybrids were first produced by Patil and Singh (1964) and later by Jauhar in 1966 and 1967 (unpublished results), using pearl millet as the female parent (Fig. 17). a. Gross Morphology and Chromosome Pairing. The hybrids showed considerable hybrid vigor. In gross morphology, they were intermediate between the parental species and easily recognizable even at seedling stage. All the hybrids were completely sterile and could be propagated only by vegetative means. However, some variation was noted in the clonal progeny raised from a single hybrid. For example, some clones produced spikes of varying compactness. It appears that there was “somatic segregation” for this character.

FIG. 17. Spikes of P ryphoides (a), P. orientale ( c ) , and their hybrid (b). Note intermediate morphology of the hybrid spike and its heterosis for size.

CYTOGENETICS OF PEARL MILLET

469

They hybrid had 2n = 16 chromosomes, 7 contributed by the female typhoides parent and 9 by orientale. There were very marked size differences among the parental chromosomes (see Fig. 18). Preliminary studies by Patil and Singh (1964) revealed some pairing between the typhoides and orientale chromosomes, suggesting their ancestral relationship. They also observed one or two bivalents as a result of pairing (autosyndesis) within the orientale complement. Taking advantage of the easily recognizable size differences (Fig. 18a), Jauhar (1973, 1981b) made a detailed analysis of the inter- and intragenomal chromosome pairing relationships. Association between typhoides and orientale chromosomes resulted in the formation of distinctly heteromorphic bivalents (Fig. 18b) or heteromorphic multivalents. Generally one or two heteromorphic bivalents were noticed, but a maximum of five was observed in one cell. Apart from intergenomal pairing, intracomplement associations within the typhoides and the orientale complements were also observed. Within the typhoides complement a maximum of 2 bivalents were noted in 6 cells, and 1 bivalent in 11 cells, out of the total of 50 analyzed. Within the orientale complement, autosyndetic bivalents (Fig. 18b,c) were observed; rarely, a loose trivalent was noticed in some cells (Fig. 18b). Of the 50 cells studied, 5 had an autosyndetic trivalent and 30 had 1-3 autosyndetic bivalents. A maximum of 4 autosyndetic bivalents were observed in 2 cells. From this trend of chromosome pairing, it was inferred that a part of the orientale genome is partially homololous (or homoeologous) to a part of the typhoides genome. The formation of a maximum of 5 intergenomal bivalents, 2 intragenomal bivalents within the typhoides complement, and 4 intragenomal bivalents within the orientale complement suggests that x = 5 is the phyletically basic number for the genus Pennisetum and that x = 7 and x = 9 have been derived from it subsequently during the course of evolution. This hypothesis is necessarily speculative and difficult to test experimentally. b. Developing Superior Hybrids. As mentioned, the typhoides x orientale hybrids are completely sterile. Colchicine-induced amphidiploids should be produced to restore fertility and seed set. These characters are important not only from the point of view of further breeding but also for easy distribution to farmers. Pairing between the parental chromosomes should facilitate interspecific transfer of desirable genes to produce superior hybrids combining the aggressiveness and tillering ability of P . orientale with the leafiness and forage quality of P. typhoides. Gamma irradiation may be tried to accelerate interspecific gene transfers. Following gamma irradiation of the vegetative tillers, desired somatic segregants (having the typhoides complement with segments of orientale chromosomes) may be picked up. If the vegetative segregants have 7 typhoides chromosomes, stable, homozygous diploids may be produced by colchicine treatment.

470

PREM P. JAUHAR

FIG. 18. Meiotic stages in P . ryphoides X P. orientale hybrids ( 2 n = 16). Note striking size differences between parental chromosomes. [Thin arrows, typhoides univalents; medium arrows, autosyndetic bivalents within the orientale complement; thick arrows, heteromorphic bivalents formed by parental chromosomes.] (a) Diakinesis with 16 univalents (2 associated with the nucleolus). Note 7 large typhoides chromosomes and 9 small orientale chromosomes. (b) A PMC with 2 heteromorphic bivalents + I autosyndetic bivalent within orientale complement. (c) Metaphase I showing 2 autosyndetic bivalents within orientale complement + 7 typhoides univalents + 5 orienlale univalents. (d) Early anaphase 1 showing asynchrony of behavior of parental chromosomes. Note the large typhoides chromosomes still on the metaphase plate, while the orientale chromosomes are moving to poles. [(a,b,d) X ca. 2360; (c) X ca. 19801

CYTOGENETICS OF PEARL MILLET

6. P . typhoides

X

47 1

P . setaceutn (Syn. P . ruppellii)

Fountain grass ( P . setaceurn) is a triploid ( 2 n = 3x = 27). Since it is an apomict, it must be used as male parent in any hybridization program. Using a pearl millet male-sterile line Tift 23 DA as female parent, Hanna (1979) produced an apomictic interspecific hybrid P . typhoides x P . setaceum. The hybrid closely resembled the polyploid seraceum parent. This is expected in view of greater contribution of chromatin from setaceum. Of the three hybrids produced, Hanna (1979) found that two had 2n = 25 (7 from pearl millet and 18 from fountain grass) and the third had 2n = 24 (7 from pearl millet and 17 from fountain grass). On the basis of size differences, the chromosomes of the parental species were readily distinguishable. During meiosis, up to 3 typhoides chromosomes associated with 3 setaceum chromosomes, and at least one such association was observed in 50% of the PMCs. The hybrids were male-sterile. Interestingly enough, Hanna (1979) found that in somatic divisions some of the setaceum chromosomes were eliminated and somatic segregation for parental characters occurred in the clonal progeny. Based on the somatic elimination of setaceum chromosomes, Hanna (1979) attempted to recover from the clonal progeny some plants with the typhoides genome incorporating some segments of setaceum chromosomes. If successful, this should be an elegant technique (see also Section XI,B,5) that may be applied to several other interspecific and intergeneric hybrids. C . INTERGENERIC HYBRIDIZATION

Numerous intergeneric hybrids have been reported in the grass family. Up until 1972, over 800 intergeneric hybrids had been reported in the Gramineae (Knobloch, 1972), and at least one (Tritium X Secale) has led to the evolution of a new man-made cereal-Triticale. A few intergeneric hybrids have been synthesized in Pennisetum. P . typhoides x Cenchrus ciliaris

Buffelgrass, Cenchrus ciliaris [Syn. Pennisetutn ciliare (L.) Link], is an excellent fodder grass that is highly nutritious before flowering. It is also recognized as one of the most important perennial pasture species in the semiarid regions of northern India (Whyte, 1957). It is an apomict (Bashaw, 1962; Taliaferro and Bashaw, 1966). Read and Bashaw (1974) made extensive efforts to cross a male-sterile line (Tift 23A) of P . typhoides ( 2 n = 14) with an apomictic cultivar of C.ciliaris (2n = 36) and

472

PREM P. JAUHAR

produced one F, hybrid. Morphologically, the hybrid resembled the polyploid male parent, C. ciliaris. This hybrid, in a way, is an intergeneric hybrid, although until recently C . ciliaris has been considered as a species of Pennisetum. In the somatic cells of the hybrid, Read and Bashaw (1974) observed 2 n = 25 chromosomes (7 large ones from pearl millet and 18 small ones from buffelgrass). However, chromosome pairing relationships could not be studied. The hybrid was completely sterile and did not produce any seed after pollinations with pollen of either parent. It appeared to have inherited the aposporous mechanism from the apomictic parent ( P . ciliaris), but did not produce any seed even by apomixis. It is nevertheless interesting from the breeder’s standpoint that apomixis can be transferred from the apomictic parent to its hybrid.

X I I . CONCLUSION

Meeting the ever-expanding demand for food for the ever-increasing world population is the biggest challenge confronting agricultural scientists. One way to meet this demand is to bring additional areas-for example, the dry and relatively infertile lands in the tropical and subtropical regions of the worldunder cultivation. Pearl millet has a remarkable ability to grow in some of the driest agricultural conditions. It already provides food for millions of poor people in Africa and Asia. In terms of annual production, it is the sixth most important cereal crop in the world. Because of its ability to provide feed for cattle, pearl millet acquires added importance. Therefore the need for the genetic improvement of this crop cannot be overstated. Fortunately, pearl millet is favorable for both basic studies as well as applied work. Pearl millet is a favorable organism for basic research in cytogenetics. Because of its small number but large size of chromosomes, it provides a suitable tool for studying chromosome pairing and chiasma frequencies and for understanding the factors controlling these intriguing phenomena. Some of the pairing variantsdesynaptics and partial desynaptics-can facilitate these studies. Pearl millet also lends itself for aneuploid analyses that should elucidate its cytogenetic architecture. Although considerable progress has been made in producing a set of trisomics, the establishment of linkage groups awaits completion. Basic studies on chromosomal rearrangements and induced mutagenesis can also be done in this crop. Pearl millet has an efficient photosynthetic ( C , ) pathway and responds very well to fertilizers. It also responds very well to heterosis breeding. Dwarf hybrids should therefore be evolved for maximum grain yields. The development of cytoplasmic male-sterile (cms) lines by Burton (1958, 1965) in the United States and later by Athwal(l965, 1966) in India has greatly facilitated the production of commercial

CYTOGENETICS OF PEARL MILLET

413

hybrids. The speed with which the Indian breeders accomplished the development of high-yielding grain hybrids using cms lines, particularly Burton's Tift 23A, is considered to be one of the most remarkable plant breeding success stories of all time. This should serve as a model for emulation by other Asian and African plant breeders. Although several commercial hybrids in India yield nearly twice as much as the best standard varieties, undoubtedly there is scope for further improvement. Genetic enrichment of their nutritional status-particularly the protein content and amino acid balance-also deserves greater attention, so that pearl millet can better feed the underprivileged man. Superior, high-yielding forage hybrids of pearl millet, pearl millet x napier grass, and pearl millet X other species should also be evolved to better feed our cattle. Cytogenetic studies could help stabilize the interspecific hybrids. Because of its distinctly protogynous nature, pearl millet is well suited for hybridization work. Distinct size differences between the chromosomes of pearl millet and other species permit a study of inter- and intragenomal pairing relationships and should help in elucidating phylogenetic trends in the polybasic, fascinating genus Pennisetum. Several heterotic hybrids combining the desirable characters of pearl millet and other forage species should be produced. Another area that merits special attention is the development of a perennial pearl millet that can yield some grain as well as forage for several years (see Section XI,B,2,f). A perennial strain of pearl millet should be very useful in arid and semiarid regions of Africa and Asia. A knowledge of different aspects of cytogenetics of pearl millet and related species should also help formulate further rational breeding programs. Evidently, pearl millet provides excellent opportunities for both fundamental and applied research. Such studies have already produced enough dividends to encourage further work. With more concerted research, pearl millet should emerge as a leading, economically viable crop that will play an ever-increasing role in the welfare of man. REFERENCES Aastveit, K . (1968). Hereditas 60, 294-315. Aken'Ova, M. E., and Chheda, H. R. (1973). Niger. Agric. J . 10, 82-90. Aken'Ova, M. E . , Crowder, L. V . , and Chheda, H. R . (1974). Agron. Abstr. p. 49. Madison, Wisconsin. Aman, M. A , , and Sarkar, K . R. (1978). Indian J . Genet. Plant Breed. 38, 452-457. Anonymous (1947). Forage crops. In Biennial Rep. Univ. Hawaii Agric. Exp. Sta. for the Biennium ending June 30, 1947. Athwal, D. S. (1965). lndian Farming 15(5), 6-7. Athwal, D. S . (1966). Indian J . Genet. Plant Breed. Symp. Suppl. 26A, 73-85. Avdulov, N. P. (1931). Bull. Appl. Eot. Genet. Plant Breed. Suppl. 43. [Russian]

474

PREM P. JAUHAR

Bailey, R. J., Rees. H., and Adena, M. A. (1978). Heredity 41, 1-12. Bashaw, E. C. (1962). Crop Sci. 2, 412-415. Bennett, M. D., and Rees, H. (1970). Gener. Res. 16, 325-331. Bilquez, A,-F., and Lecomte, J. (1969). Agron. Trop. 24, 249-257. [French]. Bosemark, N. 0. (1956). Heredifas 42, 189-210. Brar, D. S., Minocha, J. L.,and Gill, B. S. (1973). Curr. Sci. 42, 653-654. Brunken, J. N. (1977). Am. J. Bot. 64, 161-176. Brunken, J. N., de Wet, J. M. J., and Harlan, J . R. (1977). Econ. Eot. 31, 163-174. Burnham, C. R. (1934). Generics 19, 430-447. Burnham, C. R. (1956). Bot. Rev. 22, 419-552. Burnham, C. R. (1962). “Discussions in Cytogenetics. ” Burgess, Minneapolis, Minnesota. Burton, G . W. (1942). Am. J. Bor. 29, 355-359. Burton, G. W. (1944). J. Hered. 35, 227-232. Burton, G. W. (1958). Agron. J. 50, 230-231. Burton, G. W. (1965). Crops Soils 17(5), 19. Burton, G. W. (1968). Crop Sci. 8, 229-230. Burton, G. W. (1974). Crop Sci. 14, 802-805. Burton, G. W., and Hanna, W . W. (1977). Mutar. Breed. Newsleft. 9, 3. Bunon, G. W., and Powell, J. 9 . (1966). Crop Sci. 6 , 180-182. Burton, G. W., and Powell, J. 9 . (1968). Adv. Agron. 20, 49-89. Carlson, W. R. (1977). In “Corn and Corn Improvement” (G. F. Sprague, ed.), pp. 223-274. Amer. SOC.Agron., Madison, Wisconsin. Carlson, W. R. (1978). Annu. Rev. Genet. 16, 5-23. Chase, S . S. (1974). In “Haploids in Higher Plants: Advances and Potential’’ (K. J . Kasha, ed.), pp. 211-230. Univ. of Guelph, Canada. Clayton, W. D. (1972). Gramineae. In “Flora of West Tropical Africa” (F. N. Hepper, ed.), 2nd Ed., Vol. 3, Pt. 2., pp. 349-512. Crown, London. Coe, E. H. (1959). Am. Nut. 93, 381-382. Constance, L. (1957). Am. J. Bot. 44, 88-92. Cooper, R. B., and Burton, G . W. (1965). Crop Sci. 5 , 18-20. Crowley, J. G . , and Rees, H. (1968). Chromosoma 24, 300-308. Darlington, C. D. (1956). Proc. R. SOC. Ser. B 145, 350-364. Darlington, C. D. (1958). “Evolution of Genetic Systems.’’ Oliver & Boyd, Edinburgh. Darlington, C. D. (1963). “Chromosome Botany and the Origin of Cultivated Plants,’’ rev. 2nd Ed. Allen & Unwin, London. Darlington, C. D., and Mather, K . (1952). “The Elements of Genetics.” Allen & Unwin, London. de Wet, J . M. J. (1980). If1 “Polyploidy: Biological Relevance” (W. H. Lewis, ed.), pp. 3-15. Plenum, New York. de Wet, J. M. J., and Harlan, J . R. (1972). Taxon 21, 67-70. Dhanapala, S. B., Siriwardene, J. A. de S., and Pathirana, K. K. (1972). Ceylon Vet. J . 20, 77. Dhesi, J. S., Gill, 9 . S., and Sharma, H.L. (1973). Cytofogia 38, 311-316. Dhesi, J. S., Minocha, J. L., and Sidhu, J. S. (1975). Curr. Sci. 44, 862-863. Doggett, H. (1964). Heredify 19, 543-558. Einset, J. (1943). Genetics 28, 349-364. Evans, G. M.,and Macefield, A. J. (1972). Nature (London), New Biof. 236, 110-ill. Evans, H. J . , and Bigger, T. R. L. (1961). Genetics 46, 277-289. Fedak, G. (1973). Can. J. Gener. Cytol. 15, 647-649. Filion, W. G., and Blakey, D. H. (1979). Can. J. Genet. Cyfol.21, 373-378. Flavell, R. B., and Rimpau, J. (1975). Heredity 35, 127-131. Gadella, T. W.J., and Kliphuis, E. (1964). Acta Bot. Neerl. 13, 432-433. Gildenhuys, P. J. (1950). Farming S. Afr. 15, 189-191.

CYTOGENETICS OF PEARL MILLET

475

Gildenhuys, P. J . , and Brix. K . (1958). Heredity 12, 441-452. Gildenhuys, P. J., and Brix, K . (1961). Heredity 16, 358-363. Gildenhuys. P. J . , and Brix, K . (1964). Heredity 19, 533-542. Gildenhuys, P. J., and Brix, K . (1965). Ann. Bor. 29, 709-715. Gildenhuys. P. J . , and Brix, K . (1969). Agroplantae 1, 77-83. Gill, B. S . , and Minocha, J. L. (1971). J. Cytol. Genet. Congr. Suppl. pp. 33-36. Gill, B. S . , Sraon, H., and Minocha, J. L. (1966). J. Res. Punjab Agric. Univ. 3, 260-263. Gill, B. S., Minocha, J. L., Gupta, D . , and Kumar, D. (1969). Indian J. Genet. Plant Breed. 29, 462-467. Gill, B. S., Minocha, J . L., George, M. K., and Brar, D. S . (l970a). J . Res. Punjab Agric. Univ. 7 , 41 7-422. Gill, B. S., Virmani, S. S., and Minocha. J. L. (1970b). Experientia 26, 1021. Gill, B. S., Virmani, S. S., and Minocha, J. L. (1970~).Can. J. Genet. Cyrol 12, 474-483. Gill, B. S . , Sharma, H. L., and Dhesi, J. S. (1973). Cytologia 38, 411-416. Gillies. C. B . (1974). Chromosoma 48, 441-453. Golovin. V. P. (1979). Sel. Semenovod. 3, 13-14. [Russian]. Golubovskaya, I. N. (1979). Int. Rev. Cytol. 58, 247-290. Gottschalk, W . (1976). In “Induced Mutations in Cross-Breeding,’’ pp. 37-53. IAEA, Vienna. Gottschalk, W., and Klein, H. D. (1976). Theor. Appl. Genet. 48, 23-34. Grant, V. (1963). “The Origin of Adaptations.” Columbia Univ. Press, New York. Gupta, V. P. (1974). In “Breeding Researches in Asia and Oceania” (S. Rumanujam and R. D. Iyer, eds.). pp. 162-172. Indian SOC.Genet. & Plant Breeding, New Delhi. Hanna, W. W . (1979). J . Hered. 70, 425-427. Hanna, W . W . , Powell, J. B. and Burton, G . W. (1976). Can. J . Genet. Cytol. 18, 529-536. Harinarayana, G., and Mufly, B. R. (1971). Cytologia 36, 435-448. Harlan, J . R . , and de Wet, J. M. J. (1975). Dot. Rev. 41, 361-390. Hazarika, M. H., and Rees, H. (1967). Herediry 22, 317-332. Hilpen, G. (1957). Hereditas 43, 318-321. Hossain, M. G . (1978). Cytologia 43, 21-34. Hunter, A. W. S. (1934). Can. J . Res. Ser. C 11, 213-241. Hussain, A , , Ullah, M., and Ahmad, B. (1968). W. Pak. J . Agric. Res. 6(2), 69-72. James, S. H. (1965). Heredity 20, 341-353. James, S. H. (1970). Heredity 25, 53-77. Jauhar, P. P. (1968). Generica 39, 360-370. Jauhar, P. P. (1969). Natunvissenschafren 56, 571-572. Jauhar, P. P. (1970a). Generica 41, 407-424. Jauhar, P. P. (1970b). Genetica 41, 532-540. Jauhar, P. P. (1972). Experientia 28, 710. Jauhar, P. P. (1973). Proc. Inr. Congr. Genet., 13th, Gal$. (in Genetics 74, 126-127.) Jauhar, P. P. (1974). Theor. Appl. Genet. 44, 58-62. Jauhar, P. P. (1975a). Nature (London) 254, 595-597. Jauhar, P. P. (1975b). Chromosoma 52, 103-121. Jauhar, P. P. ( 1 9 7 5 ~ )Chromosoma . 52, 363-382. Jauhar, P. P. (1976). In “Chromosomes Today,” Vol. 5 (P. L. Pearson and K.R.Lewis, eds.), pp. 165-177. Wiley, New York. Jauhar, P. P. (1977a). Proc. Int. Congr. SABRAO. 3rd, (SOC.for the Adv. of Breed. Res. in Asia and Oceania) Canberra 14(b), 34-39. Jauhar, P. P. (1977b). Theor. Appl. Genet. 49, 287-295. Jauhar, P. P. (1978). J. Hered. 69, 217-223. Jauhar, P. P. (1980). Genetics 94 (Suppl.), 49. (Abstr.) Jauhar, P. P. (1981a). Indian J. Bot. 4, 1-4.

476

PREM P. JAUHAR

Jauhar, P. P. (1981b). (in press). Jauhar, P. P., and Singh, U. (1969a). Theor. Appl. Genet. 39, 315-319. Jauhar, P. P., and Singh, U. (1969b). Curr. Sci. 38, 420-421. Jauhar, P. P., Singh, U.,and Alice, C. J . (1976). Genet. Iber. 28, 15-20. Jones, R. N. (1975). f n t . Rev. Cytol. 40, 1-100. Jones, R. N., and Rees, H. (1967). Heredity 22, 333-347. Karper, R. E., and Chisholm, A. T. (1936). Am. J . Bot. 23, 369-374. Kasperbauer, M. J., Buckner, R. C., and Springer, W . D. (1980). Crop Sci. 20, 103-107. Kempanna, C., and Riley, R. (1964). Herediry 19, 289-299. Khan, M.-D., and Rahman, H. (1963). W . Pa&. J . Agric. Res. 1, 61-65. Khush, G. S. (1973). “Cytogenetics of Aneuploids.” Academic Press, New York. Kishikawa, H. (1970). Jpn. J . Breed. 20, 269-274. Knobloch, I. W . (1972). Taxon 21, 97-103. Koduru, P. R. K. (1979). Genet. Res. 34, 69-76. Koduru, P. R. K., and Rao, M. K. (1978). Cytologia 43, 445-452. Koduru, P. R. K., Murthy, T. G. K., and Lakshmi, K. V. (1980). Chromosoma 78, 365-370. Krishnaswamy, N. (1951). fndian J. Genet. Plant Breed. 11, 67-74. Krishnaswamy, N. (1962). “Bajra, Pennisetum typhoides S. Kt H.” Indian Council Agricultural Research, New Delhi. Krishnaswamy, N., and Ayyangar, G . N. R. (1941a). J . fndian Bot. SOC.20, I 1 1-1 17. Krishnaswamy, N., and Ayyangar, G. N. R. (1941b). Proc. fndian Acad. Sci. Ser. B 13, 9-23. Krishnaswamy, N., and Raman, V. S. (1948). Curr. Sci. 17, 153-154. Krishnaswamy, N., and Raman, V. S. (1949). Curr. Sci. 18, 15-16. Krishnaswamy, N., and Raman, V. S. (1953a). Proc. Sci. Workers Conf, f s t . Agric. Coll. Res. Inst., Coimbatore. pp. 43-71. Krishnaswamy, N., and Raman, V. S. (1953b). Cytologia 18, 305-321. Krishnaswamy, N., and Raman, V. S. (1954). Genetica 27, 253-272. Krishnaswamy, N., and Raman, V. S . (1956). Genetica 28, 345-360. Krishnaswamy, N., Raman, V. S., and Madhavamenon, P. (1949). Proc. fndian Acad. Sci. Ser. B 30, 195-206. Krishnaswamy, N., Raman, V. S . , and Nair, N. H. (1950). Curr. Sci. 19, 252-253. Lakshmi, K. V . , Murthy, T. G. K., and Koduru, P. R. K. (1979). Theor. Appl. Genet. 55, 189-190. Lakshmi, N., and Yacob, Z. (1978). Microbios Lett. 7, 75-82. Lal, J . , and Srinivasachar, D. (1979). Theor. Appl. Genet. 54, 27-32. Lee, W . J. (1966). Korean J . Eot. 9(3-4), 1-6. Leuck, ,D. B., and Burton, G. W . (1966). J . Econ. Entomol. 59, 1308-1309. Levitskii, G. A. (1931). Bull. Appl. Bot. Genet. Plant Breed. 27, 220-240. Li, C. H., and Li, H . W. (1943). Chin. J . Sci. Agric. 1, 139-141. Li, H . W . , Pao, W.K., and Li, C. H. (1945). Am. J . Bot. 32, 92-101. Lobana, K. S., and Gill, B. S. (1973). Cytologia 38, 713-717. Mahadevappa, M., and Ponnaiya, B. W . X. (1967). Madras Agric. J . 54, 85-88. McCoUum, G. D. (1958). Chromosoma 9, 571-605. Manga, V. (1976). Can. J . Genet. Cytol. 18, 11-15. Manga, V. (1977). Experientia 33, 1581-1582. Manga, V . , and Pantulu, J. V. (1971). Genetica 42, 319-328. Manga, V . , and Pantulu, J. V. (1974). Proc. fndian Acad. Sci. Ser. B 80, 105-120. Mehra, P. N., Khosla, P. K., Kohli, B. L., and Koonar, J . S. (1968). Res. Bull. Punjab Univ. 19, 157-230. Menon, P. M., and Devasahayam, P. (1964). Madras Agric. J . 51, 70. (Abstr.) Minocha, 1. L., and Brar, D. S . (1976). Indian J . Genet. Plant Breed. 36. 153-155.

CYTOGENETICS OF PEARL MILLET

477

Minocha, J. L., and Gill, B. S. (1974). In “Advancing Frontiers in Cytogenetics” (P. Kachroo, ed.), pp. 51-53. Hindustan Publ., Delhi. Minocha, J. L., and Singh, A. (1971a). Sci. Cult. 37, 198-199. Minocha, J. L., and Singh, kl. (1971b). J . Cvtol. Genet. Congr. Suppl. pp. 270-273. Minocha, J. L . , Gill, B. S., and Singh, S . (1968). J . Res. Punjab Agric. Univ. 5, 32-36. Minocha, J. L., Sharma, H. L., and Gill, B. S . (1972). Indian J . Genet. Plant Breed. 32,211-214. Minocha, J. L . , Brar, D. S . , and Gill, B. S. (1974). Experientia 30, 623-624. Minocha, J. L . , Dhesi, J. S., and Sidhu, J. S. (1975). Indian J . Genet. Plant Breed. 35, 470-471. Minocha, J. L . , Sharma, H. L., Sidhu, J. S . , and Gill, B. S. (1976). Indian J . Genet. Plant Breed. 36, 38-43. Muldoon, D. K . , and Pearson, C. J. (1977). Aust. J . Exp. Agric. Anim. Husb. 17, 277-283. Muldoon, D. K . , and Pearson, C. J. (1979). Herb. Absrr. 49, 189-199. Miintzing, A. (1951). Hereditas 27, 273-308. Miintzing, A. (1954). Proc. Indian Acad. Sci. Ser. E 34, 227-241. Miintzing, A. (1958). Trans. Bose. Res. Inst. (Calcuttu) 22, 1-15. Miintzing, A. (1974). Annu. Rev. Genet. 8, 243-266. Murthy, J. V. V. S. N., Sukhadev, P.,Rao, M. V . S., and Manga, V. (1979). Indian J . Eot. 2, 70-72. Nair, M. K., Raman, V. S . , and Ponnaiya, B. W. X . (1964). Madras Agric. J . 51, 356-357. Nishiyama, I . , and Kondo, N. (1942). Seiken Ziho 1, 26-28. [Japanese] Olorode, 0. (1974). Cytologia 39, 429-435. Olorode, 0. (1975). Brirtonia 27, 63-68. Pantulu, J. V. (1958). Curr. Sci. 27, 497-498. Pantulu, J. V. (1960). Curr. Sci. 29, 28-29. Pantulu, J. V. (1967a). Nature (London) 213, 101-102. Pantulu, J. V. (1967b). C.vtologia 32, 532-541. Pantulu, J. V. (1968). Curyologia 21, 11-15. Pantulu, J. V., and Manga, V. (1969). Curr. Sci. 38, 143-144. Pantulu, J. V., and Manga, V. (1972). Theor. Appl. Genet. 42, 68-74. Pantulu, J. V., and Manga, V. (1975). Genetica 45, 237-251. Pantulu, J. V., and Rao, M. V. S. (1976). Curr. Sci. 45, 418-420. Pantulu, J. V., and Rao, G. J. N. (1977a). Curr. Sci. 46, 390-392. Pantulu, J. V., and Rao, G. J . N. (1977b). Proc. Indian Acad. Sci. Ser. E 86, 15-22. Pantulu, J. V., and Venkateswarlu, J . (1968). Genetica 39, 41-44. Pantulu, J. V., Manga, V . , and Rao, M. V. S. (1976). Theor. Appl. Genet. 47, 85-86. Patil, B. D. (1963). Indian Farming 12(11), 20, 23. Patil, B. D., and Singh, A . (1964). Curr. Sci. 33, 255. Patil, B. D., and Vohra, S . K. (1962). Curr. Sci. 31, 345-346. Patil, B. D., Hardas, M. W., and Joshi, A. B. (1961). Nature (London) 189, 419-420. Person, C. (1955). Can. J . Eot. 33, 11-30. Pilger, R. (1954). Eor. Jahrb. 76, 281-384. Powell, J. B., and Burton, G . W. (1966a). Crop Sci. 6, 131-134. Powell, J. B., and Burton, G. W. (1966b). Crop Sci. 6, 378-379. Powell, J. B., and Burton, G. W. (1968). Crop Sci. 8, 771-773. Powell, J. B . , and Burton, G. W. (1969). Crop Sci. 9, 252-253. Powell, J. B., Hanna, W. W., and Burton, G . W. (1975). Crop Sci. 15, 389-392. Pritchard, A. J. (1971). Trop. Grassl. 6, 35-39. Punyasingh, K. (1947). Genetics 32, 541-554. Rajhathy, T., and Fedak, C. (1970). Can. J . Genet. Cytol. 12, 358-360. Ramage, R. T. (1964). Int. Barley Genet. Symp., Ist, Wageningen, pp. 99-115.

478

PREM P. JAUHAR

Ramage, R. T. (1970). Proc. Inr. Barley Genet. Symp.. 2nd. Pullman, Wash. pp. 89-93. Ramage. R. T . , and Wiebe, G. A. (1969). Proc. IAEA F A 0 S v r p . , Pullman, Wash. pp. 655-659. Raman, V. S. (1965). I n “Advances in Agricultural Sciences and Their Applications” (S. Krishnamurthi, ed.), pp. 122-143. Agric. Coll. Res. Inst., Coimbatore, India. Raman, V. S., and Krishnaswami, D. (1960). J . Indian Bot. Soc. 39, 382-385. Raman, V . S . , and Krishnaswami, D. (1961). J . Indian Bot. Soc. 40, 61-65. Raman, V . S., and Nair, M. K. (1964). J . Indian Bor. SOC. 63, 508-514. Raman, V . S., Krishnaswami, D.. and Nair, M. K. (1962). J . Indian Bor. SOC. 41, 268-270. Raman, V . S . , Nair, M. K . , and Krishnaswami, D. (1963). J . Indian Bor. SOC. 62, 469-473. Ramulu, K. S. (1968). Caryologia 21, 147-156. Ramulu, K. S. (1971). Cytologia 36, 652-668. Ramulu, K. S . , and Ponnaiya, B. W . X . (1967). Madras Agric. J . 54, 503-51 I . Rangaswamy, K. (1935). J . Indian Bar. Soc. 14, 125-131. Rangaswamy, S. R . S. (1972). Generica 43, 257-273. Rangaswamy, S. R. S., and Ponnaiya, B. W. X. (1963). Madras Agric. J . 50, 239-241. Rao, M. K., and Koduru, P. R. K. (1978a). Theor. Appl. Genet. 53, 1-7. Rao, M. K., and Koduru, P. R. K. (1978b). Proc. Indian Arad. Sci. Ser. B 87, 29-35. Rao, M. K., Pantulu, J. V., and Jayalakshmi, K. (1979). Experierzria 35, 1154. Rao, M. V. S. (1978). Proc. Indian Acad. Sci. Ser. B 87, 17-22. Rao, M. V. S. (1980). Theor. Appl. Genet. 56, 85-89. Rao, M. V . S . , and Pantulu, J. V. (1978). Chromosoma 69, 121-130. Rao, P. K., Nambiar, A. K., and Munhy, 1. V. G. K. (1949). Madras Agric. J . 36, 526-529. Rao, P. S. R . L. N. (1977). Curr. Sci. 46, 464. Rao, P. S. R. L. N., and Rao, I. N. (1977). Curr. Sci. 46, 314-315. Rao, P. S. R. L. N., Rao, M. V. S . . and Rao, 1. N. (1977). Experientia 33, 308-309. Rau, N . S. (1929). J . Indian Bor. SOC. 8, 126-128. Read, J. C . , and Bashaw, E. C. (1974). Crop Sci. 14, 401-403. Rees, H. (1961a). Bor. Rev. 27, 288-318. Rees, H . (1961b). Evolurion 15, 145-152. Rees. H.. and Jones, R. N . (1977). “Chromosome Genetics.” Univ. Park Press, Baltimore, Maryland. Rhoades, M. M., and Dempsey, E. (1972). Generics 71, 73-96. Rick, C. M., and Barton, D. W. (1954). Generics 39, 640-666. Riley, R. (1960). Nature (London) 185, 751-752. Riley, R., and Chapman, V. (1957). Heredify 11, 195-207. Riley, R., and Law, C. N. (1965). Adv. Genet. 13, 57-1 14. Sadasivaiah, R . S . , and Kasha, K. J. (1971). Chromosorna 35, 247-263. Sethi. G. S . , Kalia, H. R., and Ghai, B. S. (1970). Cyrologia 35, 96-101. Singh, R. B., Singh, B. D., Laxmi, V., and Singh, R . M. (1977a). Cyrologia 42, 41-47. Singh, R. B.. Singh, B. D., Singh, R. M., and Laxmi, V . (1977b). Cyrologia 42, 633-637. Srivastava, H . K., and Balyan, H. S. (1977). J . Hered. 68, 338-340. Stapf. O., and Hubbard, C. E. (1933). Kew Bull. pp. 269-302. Stapf, O., and Hubbard, C. E. (1934). I n “Flora of Tropical Africa” (D. Prain, ed.), Vol. 9, Part 6, pp. 954-1070, Reeve, Ashford, Kent, England. Stebbins, G . L. (1956a). Brookhaven Symp. Biol. 9, 27-52. Stebbins, G. L. (1956b). Am. J . Bot. 43, 890-905. Stebbins, G. L. (1958). Cold Spring Harbor Symp. Quant. Biol. 23, 365-378. Stebbins, G. L. (1969). Taxon 18, 26-35. Stebbins, G. L. (1974). “Flowering Plants: Evolution above the Species Level.” The Belknap Press of Harvard Univ. Press, Cambridge, Massachusetts.

CYTOGENETICS OF PEARL MILLET

479

Sun, S., and Rees, H. (1967). Heredity 22, 249-254. Taliaferro, C. M., and Bashaw, E. C. (1966). Crop Sci. 6, 473-476. Thevenin. L. (1952). Ann. C. R . A . Bambey (Senegal) 8, 102-105. [French] Thompson, J. B. (1956). Heredity 10, 99-108. Ting. Y . C. (1971). Genetics 68, Suppl., 67 (Abstr.) Ting, Y. C. (1973). Cytologia 38, 497-500. Tyagi, B. R. ( I 975a). Proc. Indian Natl. Sci. Acad. Ser. B 41, 462-465. Tyagi, 9.R. (1975b). Proc. Indian Natl. Sci. Acad. Ser. B 41, 545-549. Tyagi, 9.R. (1976a). Nucleus 19, 58-63. Tyagi, B. R. (1976b). Proc. Indian Natl. Sci. Acad. Ser. B 42, 117-121. Tyagi, 9.R. (1976~).Nucleus 19, 112-114. Tyagi, B. R. (1977). Proc. Indian Natl. Sci. Acad. Ser. B 43, 66-69. Tyagi, 9.R., and Singh, R. B. (1974). Proc. Indian Natl. Sci. Acad. Ser. B 40, 359-362. Venkateswarlu, J.. and Mani, J . N. R. (1978). Genetica 48, 145-149. Venkateswarlu, J., and Pantulu, J. V. (1968). J . Hered. 59, 69-70. Venkateswarlu, J., and Pantulu, J. V. (1970). Cytologia 35, 444-448. Veyret, Y. (1957). Agron. Trop. 12, 595-598. [French] Virmani, S. S., and Gill, B. S. (1971). Caryologia 24, 427-433. Virmani, S. S., and Gill, B. S. (1972). Cytologia 37, 257-260. Vosa, C. G. (1970). Chromosoma, 30, 366-372. Vosa, C. G. (1973). Chromosoma, 43, 269-278. Vosa, C. G. (1975). Curr. Adv. Plant Sci. 6, 495-510. Vosa, C. G., and Marchi, P. (1972). Nature (London), New Biol. 237, 191-192. Weber, D. F. (1970). Maize Genet. Coop. Newslett. 44, 203. Weber, D. F. (1973). Theor. Appl. Genet. 43, 167-173. Whyte, R. 0. (1957). "The Grassland and Fodder Resources of India." Scientific Monograph No. 22, Indian Council of Agricultural Research, New Delhi. Zeller, F. J., Kimber, G., and Gill, 9.S. (1977). Chromosoma 62, 279-289.