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Anethum graveolens: Polyploidy and Pollen variability
r Flora. Abt. B, Bd. 157, S. 179-189 (1967) Cytogenetics Laboratory Department of Botany, University of Lucknow, Lucknow (India)
Anethum graveolens: Polyploidy and Pollen variability By
SATENDRA. S. RAGHUVANSHI
and
SHEILA JOSHI
With 51 figures in the text (Received February 9, 1967)
Introduction
Artificial induction of variants and polyploids with various chemicals is under investigation to explore the possibilities of producing improved high yielding vigorous plants. The present investigation deals with the morphological and cytological study of the variants in Anethum graveolens. The induction of polyploidy in this species with colchicine was an utter failure although all the possible timings as well as concentrations were tried on shoot as well as seed treatment. However, in the following year when the experiment was repeated by using the new technique of combinations developed in this laboratory (RAGHUVANSHI and JOSHI 1965a) a tetraploid (E. 225) was produced which exhibited pollen variability while the controls had normal bipolar radio symmetric pollen (RAGHUVANSHI and JOSHI 1965b). Besides this tetraploid among the treated plants a diploid (E. 210) also had pollen grains of different shapes. It may however be added that gammexane treatment alone did not have any effect on the plants and these were perfectly normal as the controls. Material and Methods Seedlings of Anethum graveolens were treated with colchicine-gammexane solution. In saturated solution of gammexane 0.2 % solution of colchicine was prepared. The most suitable and effective treatment was that of intermit ant 6 hours daily repeated for 3 days in succession. The plants were raised in separate pots along with suitable controls. Fixation for cytological studies was made in 1 : 3 acetic alcohol and stored in 70 percent alcohol under refrigration. For meiotic study young anthers were squashed in acetocarmine and preparations were made permanent by the ethanol-butanol schedule. Ontogeny of the pollen grains was traced by placing an anther in a drop of carmine under a cover glass, then the coverslip was tapped to release the sporads without rupturing their cell walls. Acetolysed slides of pollen grains were prepared following ERDTMAN'S technique (1952).
Observations
Morphological The tetraploid (E. 225) and E. 210 were smaller than the controls (Fig. 1). The tetraploid was compact and bushy in habit with stout short stem and leaves dark
180
SATE;\DRA S. RAGHUVAXSHI
und
SHEIL>\ JOSH!
r 1
In
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Fig. 1. General habit of E. 225 (tetraploid), control and E. 210.
green, thick and distorted while in E. 210 the leaves were larger and thinner. Both these plants had extended flowering period.
Cytological Cytological studies revealed the diploid chromosome number of Anethum graveolens to be 22 which confirms the observations of TAMASCHJAN (1933). The controls had regular meiosis. However, the variants exhibited meiotic instability toa considerable extent as is evident from the following description.
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Tetraploid (E. 225) Configurations observed in the tetraploid were univalents, chains and rings of different size except trivalents (Fig. 2). Univalents were observed in 2 % cells and their number never exceeded 2. The chain and ring quadrivalents were present ranging from 6 to 1 (Fig. 3) in practically every cell. Bivalents displayed a range between 20-10 per pollen mother cell (PMC). The absence of trivalents and rare occurrence of univalent indicates complete pairing at pachytene. The number of chiasmata per PMC ranges from 32-46, the most frequant numbers being 34, 35, 36, 37 and 38.
Ta
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Anethum graveolens: Polyploidy and Pollen variability
Fig. 2. Diakinesis in a tetraploid PMC.
In a few PMCs the chromosomes instead of being oriented on one equatorial plate were distributed on 2 to 3 equal or unequal plates forming separate spindles. Further division stages clearly show the independent functioning of these groups. At anaphase (a.) lout of 806 PMCs 582 were normal, the rest showed anomalies like stray chromosomes (Fig. 5), bridges (Fig. 7), breakdown of spindle (Fig. 4), tripolar spindles (Fig. 6) and laggards (Table I). At telophase (t.) I the strays and laggards were excluded from the nuclei. In some cells there were more than two nuclei which at further division stages created more complex situations. Restitution nucleus resulting from breakdown of the spindle mechanism was observed in few cells. At metaphase (m.) II there were multiple and multipolar spindles. The number of chromosomes at m. II plates did not show any relation to the genomic number but showed random separation varying from equal to very unequal. In a few cases the entire chromosome complement was oriented on a single plate instead of the normal 2, probably the result of failure of spindle mechanism at a. I. Laggards and stray chromosomes were observed at this stage. In some cells lobed metaphase plates were noted, perhaps, indicating the multipolar organisation of the spindles. Fig. 11 shows combination of a bipolar and a tripolar spindles in the same cell while in Fig. 9 both the spindles are tripolar. Three independently functioning bipolar spindles are seen in Fig. 10. Breakdown of the spindle was also noted at this stage (Fig. 8). Table I Frequency of various anomalies observed in the tetraploid (E. 225) at first anaphase Normal
One stray ch.
Two stray chs.
One laggard
Two laggards
One bridge
Two bridges
Break down of the spindles
Tripolar Total spindle No. of Cells studied
582
47
21
89
14
37
4
9
3
13 Flora, Aht. B, Bd. 157
806
F 182
SATB:-iDRA S. RAGHl:VANRHl
and
14
SHEILA JORHI
Due to these meiotic irregularities the number of nuclei per PMC ranges from 1-9 with the majority of 4. The variable number of nuclei in different cells must have resulted due to multiple spindles at m. I, laggards and stray chromosomes at a. I and II, multipolar spindles at a. I and II, nondisjunction at m. I and II and formation of restitution nuclei. Table II presents analysis of 1137 sporads.
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ti o tl Figs. 3-11: Tetraploid. Fig. 3. Diakinesis showing 6 quadrivalents. Fig. 4. Breakdown of the spindle at anaphase (a.) I. Figs. 5-7. a. I showing laggards, tripolar separations and chromatin bridges respectively. Fig. 8. Breakdown of spindles at a. II. Figs. 9-11. a. II: two tripolar spindles, three bipolar spindles and the combination of two in the same PMC respectively.
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Table II
Frequency of the number of spores per sporad in the tetraploid (E. 225)
g
o Number of spores per sporad 1
2
3
4
5
6
8
Total
2
146
80
674
144
79
12
1137
b a
t
F
14
Anethum graveolens: Polyploidy and Pollen variability
183
Experimental 210
Meiosis in this 2n plant was considerably irregular. Two univalents were observed in 22% of the cells. The chiasmata per PMC ranged from 15-19. At m. I the irregularities exhibited include bivalents being off the plate and univalents at the poles. These univalents were the result of precocious bivalent separation. Laggards, bridges, tripolar spindles, unequal anaphasic separation and stray chromosomes were common. Besides variable grains this plant had large united grains of different shapes. Non-dividing PMCs
The 2n plant (E. 210) had certain PMCs which had deeply stained comparatively large nuclei which did not show any sign of division while division did occur in normal PMCs surrounding them. Though the nuclei of such PMCs had lost the capacity to divide, yet, they were transformed into pollen grains along with the normal ones. The pollen grains thus produced were small of oblong shape having large deeply stained nucleus as was observed at PMC stage. The nuclear division takes place in the normal grains but even at this stage the nuclei of such aberrantly formed grains do not divide. The frequency of such PMCs in E. 210 was very low as compared to E. 67 of Coriandrum sativum (JOSHI and RAGHUVANSHI 1965a). Pollen One of the interesting observations was pollen variability in these treated plants while the control plants had only one type of grains. The various genera of Umbelliferae (Coriandrum, Foeniculum, Carum, Daucus, Pimpinella, Cuminum etc.) studied by the authors exhibit pollen variability in varying proportion when their young seedlings (2" - 4") were treated with colchicine and colchicine-gammexane solutions. Some of the pollen shapes are common in all the cases while others differ, so in order to have a comparative idea of the presence or absence of various shapes in treated plants of different genera they have been classified into types. The pollen of the variants was asymmetric, nonfixiform while controls have aU radio symmetric grains, 3 colporate, prolate-perprolate. The equatorial view of these grains exhibits different shapes. The equator was bulging or constricted. Polar view circular or angular. The grains were colporate, colpi usually long ectosexine tenuitegillate, in a few it was crassitegillate while in others it was an admixture of the two on one and the same grain. In some very conspicuously baculate, simp Ii or hetrobrochate, sculpture almost psilate. The apertures were nonoperculate and covered by a psilate membrane. Ontogenetic study of these pollen clearly revealed that the lobed grains are not the result of incomplete separation of individual spores, rather they are the outcome 13'
184
SATENDRA S. RAGHUVA,\,SHI
and
SHEILA JOSHI
of single spores which assume variable shapes. The pattern of development was the same as observed in Foeniculum vulgare (RAGHUVANSHI and JOSHI 1967) and 00riandrum sativum (JOSHI and RAGHUVANSHI 1965b). It was noted that the grains developing from the same sporad were not of the same shape but an admixture of normal and the variants in different proportions i.e. a single PMC produced both normal as well as variant grains. These variant grains showed random distribution in the plants, individual heads of a plant, buds on the same head, and different anthers in one and the same bud. These variant grains have been classified as types based on the study of treated plants of Foeniculum vulgare which had types A to M (RAGHUVANSHI and JOSHI 1967). In the present study Types A, B, E, F, G, N, 0, P and giants were metwith. The spectrum of pollen variability was broader in E. 210 than the tetraploid although the latter had higher frequency of variable grains (Table III). The various types of grains observed are described below and their development has been presented in figure 51. Table III The proportion of different types of pollen grains as observed in tetraploid and 2n plant No. of plant
Level of ploidy
E. 225 E. 210
Types of pollen grains A
B
E
F
G
N
0
P
4n
870
2
15
4
33
3
60
16
2n
932
14
12
13
17
3
Joined grains
Total 1003
30
1026
Type A (Fig. 12). This is the control type. It is bipolar radio symmetric grain. Type B (Figs. 13, 14). This is like type A with a partition wall in the equatorial
region which divides the grain into two equal compartments. Development of such grains was observed in Foeniculum vulgare. In these grains the nucleus divided in an abnormal manner and the two daughter nuclei were formed followed by wall formation at the equatorial region. Type E (Figs. 16, 17). A triradiate or trilobed grain in few cases the poles are flat while mostly they are rounded. Type F. The grains have triangular outline. Type G (Fig. 18). A tetralobed grain in which the arms may be of equal or unequal length. Type M (Fig. 50) these are polyads. Type N (Fig. 19). The development of a side protrusion in Type A grain results into L or J shaped grain. Type 0 (Figs. 20-22). This is also triradiate type but in this case two protrusions arise at one of the poles while in type E the third protrusion arises at the equatorial region. Type P (Fig. 15). It is a dumbell shaped grain with flat poles and a constriction at the equator. The bacula is equally developed on all the sides and is simplibrochate. Round thick walled giant pollen grains were also observed (Figs. 27, 28). Some of the rare occurring shapes are shown in Figs. 23, 26 and 29.
I I I
I!
III
I
i:
12
73
Figs. 12-50: Pollen grains. Fig. 12. Control type A. Figs. 13, 14. Bicelled type B. Fig. 15. Type P. Figs. 16, 17. Type E. Fig. 18. Type G. Fig. 19. Type N. Figs. 20-22. Type O. Fig. 23. A variant. Figs. 24, 26, 29. Variants. Figs. 27,28. Round giants. Fig. 30. Joined variants. Figs. 31-35. Two joined grains. Figs. 36-39, 40-44. Three and four joined grains at different positions. Fig. 45. Acetolysed joined four grains showing the pores of individual grains. Fig.46. Two separate nuclei in the joined grains. Fig. 47. Single nucleus in the joined grains. Fig. 48. One and two nuclei in the joined grains. Fig. 49. A single nucleus in a mass of four joined grains. Fig. 50. Polyad of variants.
iiaE
186
SATENDRA S. RAGHUVANSHI
and
SHEILA JOSHI
United grains. A unique feature of E. 210 was the presence of united pollen grains, which assume various shapes. The number of grains that have been found in this condition varies from 2 to 5. Two pollen grains showed connections at different positions either at the poles, equator or any where between the two (Figs. 31-35). In cartain cases the common wall between the grains was noted (Fig. 35) while in still others the passage between the two grains was direct (Fig. 31). In carmine preparations of the grains the nuclei were observed. In certain cases there was a single large nucleus in the centre whereas in others they were seen in individual grains (Figs. 46-49). In Fig. 49 a single nucleus was seen in a united mass of four grains This single nucleus could be the outcome of breakdown of spindles both at a. I and a. II followed by the transformation of such PMC into a single giant grain, or alternatively the individual nuclei of the grains may have fused to form a single nucleus. It may be mentioned that no breakdown of spindle mechanism either at a. I or a. II was ever encountered. United 2 or 4 nucleate round giant grains having cytoplasmic connections were observed in Foeniculumvulgare (RAGHUVANSHI and J oSHI1966 a, 1967). But these arose due to transformation of the whole PMC into a pollen grain after t. I {)f t. II. In one instance in three united giant grains the nuclei and cytoplasm of the lateral grains was transferred to the central grain. This situation was almost analogous to fertilization process. In Coriandrum sativum (JOSHI and RAGHUVANSHI 1965 b) the nucleus of type a pollen grain was under going transfer into another control type A grain. This situation resembles differential sexuality of two grains i.e. donar functioning as male while recepiant grain as female. These giant grains in Anethum assume the shape of types E, G and H, but close examination shows them to be a product of type A grains rather than a single giant grain (Figs. 38, 41-43). The pores on the individual grains were clearly noted at their respective equatorial regions (Figs. 35,36,41,44,45). Not only normal type A grains behave in this manner but the variable grains also showed the same condition (Fig. 30).
Discussion
Autopolyploidy is considered more conducive to lability than allopolyploidy and this lability increases with the increase in the level of ploidy. The Anethum tetraploid certainly provides no exception but the most unique feature of the variants is the presence of pollen variability when the controls have normal bipolar radiosymmetric grains (RAGHUVANSHI and JOSHI 1965 b). The low frequency of univalents and multivalents in autopolyploides appears to be a feature shared by Umbellifers under study (Coriandrum sativum, Pimpinella monoecia, Carum copticum, Foeniculum vulgare etc.). Though multivalents occur in every cell, yet, their average is fairly low (3-4 per cell). This tendency was still more marked in Foeniculum where not more than two quadrivalents were seen in any cell. In contrast to these in tetraploids of one variety
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Anethum graveolens: Polyploidy and Pollen variability
187
of Capsicum frutescens all the 48 chromosomes of the PMC formed 12 quadrivalents (unpublished). But mention may be made that in Carum copticum tetraploids also all the 36 chromosomes formed quadrivalents in some PMCs. The complete absence of trivalents is also conspicuous, probably this may explain to a certain extent the low frequency of univalents. Multipolar and multiple spindles have been observed in some ·cells. WALTERS (1958) has given a scheme of origin of different number of spindles due to anomalous division of spindle organisors. Some of the supernumary spindles in Anethum graveolens have very few chromosomes, this may be due to most unequal division of spindle organisors. Further, number of functional groups of chromosomes in such cells at later divisional stage depended on the position of the poles of different spindles with regard to each other. The presence of such aberrant spindles do not appear to have any specific relationship with the frequency of univalents. They were observed in colchiploids of Capsic'um fr'utescens having high univalent frequencyA (RAGHUVANsHIand JOSHI 1964a) and Anethumwhich have very low univalent frequency. Some genera of angiosperms are known to have dimorphic pollen grains (ERDTMAN 1952 p. 4) but artificially induced pollen variability of the magnitude reported in the present paper is unique. This pollen variability appears to be a peculiar feature of first generation of polyploids in Umbellifers and certain types of grains are common in different genera. CERCEAU (1959) and TING (1961) have studied the pollen of some Umbellifers. In variants of Foeniculum vulgare there were types A to M while Pimpinella !!I'onoecia had types A, E, D, F, G, and M. In Coriandrum sativum there were types A, E, F, G, Hand N besides type a, which was direct transformation of a PMC into a pollen grain without the intervention of meiosis. Though certain grains are common in the genera under study yet their frequency shows a great range of variation. The frequency of different grains is not high in Anethum, still, the range of variability is very wide. Regarding the frequency of different types it could be postulated that certain types like G and E are not only uniformaly present in the treated plant of different genera but are also dominant types. Type G (tetraradiate) occurs most frequently in tetraploids of Coriandrum sativum, Foeniculum vulgare and Pimpinella monoecia but in Anethum it appears to have suffered eclipse at the hands of type E (triradiate) which has been a close follower in the other genera. Our studies have shown that all these types can be derived from normal type A as presented in the scheme (Fig. 51). These various observations clearly indicate that pollen grains in Umbellifers with regard to their shape are not very stable and disturbances could be caused by judicious use of certain chemicals as shown in Pimpinella monoecia (unpublished). It is suggested that chemical treatment of young seedlings ultimately brings about a change in the expression of genes controlling the pollen shapes. Similar shapes observed in the treated plants of different genera further substantiated this conclusion.
188
S~\TENDRA S.
RAGHlJVA?'
Fig. 51. Diagrammatic representation of the development of different types of variable grains.
Summary Oolchicine-gammexane treatment of seedlings of Anethum graveolens resulted in the production of a tetraploid (E. 225) and a 2n plant (E. 210) which exhibited pollen variability while the controls had normal bipolar grains. The meiotic instability exhibited by E. 225 is classical for polyploids. The encountered variable grains are types A, B, E, F, G, N, 0, P and giant grains. The origin of these various types of grains has been proposed with the help of a scheme. It is suggested that this pollen variability is due to change in expression of genes controlling the pollen shape.
Literature OERCEAlJ, M. T., 1959. Ole de determination d'Ombellifers de France etv d'Afrique du Nord d'appears leurs grains de pollen. Pollen et Spores 4,145-190. DARLINGTON, O. D., and THOMAS, P. T., 1937. The breakdown of cell division in a Festuca Lolium derevative. Ann. Bot., N. S. 1, 747-762. ERDTMAN, G., 1952. Pollen morphology and plant taxonomy. Stockholm. JACOB, F., and MONOD, J., 1961. Genetic regulatory mechanisms in the Synthesis of proteins. (Review article.) J. Mol. BioI. 3, 318-356.
Anelhum graveolens: Polyploidy and Pollen variability
189
JOSHI, SHEILA, and RAGHUVANSHI, S. S., 1965a. Pollen formation without the intervention of meiosis in a variant (E. 67) of Coriandrum salivum and Pollen variability. Grana Palynologia. 6, 186-190. - - 1965 b. Coriandrum sativum: Mutation, Polyploidy, Nondividing pollen mother cells and pollen variability. Canadian s. Genet. Cytol. 7,223-236. RAGHUVANSHI, S. S., and SHEILA JOSHI, 1964. Cytomorphological studies on the colchiploids of Capsicum frutescens. Cytologia 29, 61-78. - - 1965a. Studies on the comparative effects of certain chemicals on the polyploidizing efficency of colchicine in Trigonella foenum-graecum. Caryologia 18, 69-84. - - 1965 b. Artificial induction of pollen variability in Anethum and Pimpinella. Naturwiss. 52,397. - - 1966. Foeniculum vulgare: Polyploidy, Translocation heterozygosity and Pollen variability. Part I. (CYTOLOGY) Cytologia 31,43-58. - - 1967. Foeniculum vulgare: Polyploidy, Translocation heterozygosity and Pollen variability. Part II (Pollen GRAINS). (In communication.) SWANSON, C. P., and NELSON, R., 1942. Spindle abnormalities in Mentha. Bot. Gaz. 104,273-280. TAMAMSCHJAN, S., 1933. Bull. Appl. Bot. Sci. 2, 137. TING, W. S., 1961. On some pollen of Californian Umbellifers. Pollen et Spores 3, 189-199. WALTERS, M. S., 1958. Aberrant chromosome movement and spindle formation in meiosis of Bromus hybrids. An interpretation of spindle organisation. Amer. Jour. Bot. 45, 271-289. Authors' address: Dr. Satendra S. RAGHUVANSHI and Dr. (Miss) SHEILA JOSHI, Cytogenetics Laboratory, Department of Botany, University of Lucknow, Lucknow (India).
Anethum graveolens: Polyploidy and Pollen variability By
SATENDRA. S. RAGHUVANSHI
and
SHEILA JOSHI
With 51 figures in the text (Received February 9, 1967)
Introduction
Artificial induction of variants and polyploids with various chemicals is under investigation to explore the possibilities of producing improved high yielding vigorous plants. The present investigation deals with the morphological and cytological study of the variants in Anethum graveolens. The induction of polyploidy in this species with colchicine was an utter failure although all the possible timings as well as concentrations were tried on shoot as well as seed treatment. However, in the following year when the experiment was repeated by using the new technique of combinations developed in this laboratory (RAGHUVANSHI and JOSHI 1965a) a tetraploid (E. 225) was produced which exhibited pollen variability while the controls had normal bipolar radio symmetric pollen (RAGHUVANSHI and JOSHI 1965b). Besides this tetraploid among the treated plants a diploid (E. 210) also had pollen grains of different shapes. It may however be added that gammexane treatment alone did not have any effect on the plants and these were perfectly normal as the controls. Material and Methods Seedlings of Anethum graveolens were treated with colchicine-gammexane solution. In saturated solution of gammexane 0.2 % solution of colchicine was prepared. The most suitable and effective treatment was that of intermit ant 6 hours daily repeated for 3 days in succession. The plants were raised in separate pots along with suitable controls. Fixation for cytological studies was made in 1 : 3 acetic alcohol and stored in 70 percent alcohol under refrigration. For meiotic study young anthers were squashed in acetocarmine and preparations were made permanent by the ethanol-butanol schedule. Ontogeny of the pollen grains was traced by placing an anther in a drop of carmine under a cover glass, then the coverslip was tapped to release the sporads without rupturing their cell walls. Acetolysed slides of pollen grains were prepared following ERDTMAN'S technique (1952).
Observations
Morphological The tetraploid (E. 225) and E. 210 were smaller than the controls (Fig. 1). The tetraploid was compact and bushy in habit with stout short stem and leaves dark
180
SATE;\DRA S. RAGHUVAXSHI
und
SHEIL>\ JOSH!
r 1
In
we div
Fig. 1. General habit of E. 225 (tetraploid), control and E. 210.
green, thick and distorted while in E. 210 the leaves were larger and thinner. Both these plants had extended flowering period.
Cytological Cytological studies revealed the diploid chromosome number of Anethum graveolens to be 22 which confirms the observations of TAMASCHJAN (1933). The controls had regular meiosis. However, the variants exhibited meiotic instability toa considerable extent as is evident from the following description.
lik po ga wh res me ch sh( en pr( rn no
co
th in
Tetraploid (E. 225) Configurations observed in the tetraploid were univalents, chains and rings of different size except trivalents (Fig. 2). Univalents were observed in 2 % cells and their number never exceeded 2. The chain and ring quadrivalents were present ranging from 6 to 1 (Fig. 3) in practically every cell. Bivalents displayed a range between 20-10 per pollen mother cell (PMC). The absence of trivalents and rare occurrence of univalent indicates complete pairing at pachytene. The number of chiasmata per PMC ranges from 32-46, the most frequant numbers being 34, 35, 36, 37 and 38.
Ta
No
58
;
i
Ii
181
Anethum graveolens: Polyploidy and Pollen variability
Fig. 2. Diakinesis in a tetraploid PMC.
In a few PMCs the chromosomes instead of being oriented on one equatorial plate were distributed on 2 to 3 equal or unequal plates forming separate spindles. Further division stages clearly show the independent functioning of these groups. At anaphase (a.) lout of 806 PMCs 582 were normal, the rest showed anomalies like stray chromosomes (Fig. 5), bridges (Fig. 7), breakdown of spindle (Fig. 4), tripolar spindles (Fig. 6) and laggards (Table I). At telophase (t.) I the strays and laggards were excluded from the nuclei. In some cells there were more than two nuclei which at further division stages created more complex situations. Restitution nucleus resulting from breakdown of the spindle mechanism was observed in few cells. At metaphase (m.) II there were multiple and multipolar spindles. The number of chromosomes at m. II plates did not show any relation to the genomic number but showed random separation varying from equal to very unequal. In a few cases the entire chromosome complement was oriented on a single plate instead of the normal 2, probably the result of failure of spindle mechanism at a. I. Laggards and stray chromosomes were observed at this stage. In some cells lobed metaphase plates were noted, perhaps, indicating the multipolar organisation of the spindles. Fig. 11 shows combination of a bipolar and a tripolar spindles in the same cell while in Fig. 9 both the spindles are tripolar. Three independently functioning bipolar spindles are seen in Fig. 10. Breakdown of the spindle was also noted at this stage (Fig. 8). Table I Frequency of various anomalies observed in the tetraploid (E. 225) at first anaphase Normal
One stray ch.
Two stray chs.
One laggard
Two laggards
One bridge
Two bridges
Break down of the spindles
Tripolar Total spindle No. of Cells studied
582
47
21
89
14
37
4
9
3
13 Flora, Aht. B, Bd. 157
806
F 182
SATB:-iDRA S. RAGHl:VANRHl
and
14
SHEILA JORHI
Due to these meiotic irregularities the number of nuclei per PMC ranges from 1-9 with the majority of 4. The variable number of nuclei in different cells must have resulted due to multiple spindles at m. I, laggards and stray chromosomes at a. I and II, multipolar spindles at a. I and II, nondisjunction at m. I and II and formation of restitution nuclei. Table II presents analysis of 1137 sporads.
...--...... .....
..
E SE
Ir
w sl
•
••• •• •• •••• •••• • ••
:: e.
la n: ci
4
OJ
s1 t]
d E
p
8
W
fE b Sf
ti o tl Figs. 3-11: Tetraploid. Fig. 3. Diakinesis showing 6 quadrivalents. Fig. 4. Breakdown of the spindle at anaphase (a.) I. Figs. 5-7. a. I showing laggards, tripolar separations and chromatin bridges respectively. Fig. 8. Breakdown of spindles at a. II. Figs. 9-11. a. II: two tripolar spindles, three bipolar spindles and the combination of two in the same PMC respectively.
r:
g C
Table II
Frequency of the number of spores per sporad in the tetraploid (E. 225)
g
o Number of spores per sporad 1
2
3
4
5
6
8
Total
2
146
80
674
144
79
12
1137
b a
t
F
14
Anethum graveolens: Polyploidy and Pollen variability
183
Experimental 210
Meiosis in this 2n plant was considerably irregular. Two univalents were observed in 22% of the cells. The chiasmata per PMC ranged from 15-19. At m. I the irregularities exhibited include bivalents being off the plate and univalents at the poles. These univalents were the result of precocious bivalent separation. Laggards, bridges, tripolar spindles, unequal anaphasic separation and stray chromosomes were common. Besides variable grains this plant had large united grains of different shapes. Non-dividing PMCs
The 2n plant (E. 210) had certain PMCs which had deeply stained comparatively large nuclei which did not show any sign of division while division did occur in normal PMCs surrounding them. Though the nuclei of such PMCs had lost the capacity to divide, yet, they were transformed into pollen grains along with the normal ones. The pollen grains thus produced were small of oblong shape having large deeply stained nucleus as was observed at PMC stage. The nuclear division takes place in the normal grains but even at this stage the nuclei of such aberrantly formed grains do not divide. The frequency of such PMCs in E. 210 was very low as compared to E. 67 of Coriandrum sativum (JOSHI and RAGHUVANSHI 1965a). Pollen One of the interesting observations was pollen variability in these treated plants while the control plants had only one type of grains. The various genera of Umbelliferae (Coriandrum, Foeniculum, Carum, Daucus, Pimpinella, Cuminum etc.) studied by the authors exhibit pollen variability in varying proportion when their young seedlings (2" - 4") were treated with colchicine and colchicine-gammexane solutions. Some of the pollen shapes are common in all the cases while others differ, so in order to have a comparative idea of the presence or absence of various shapes in treated plants of different genera they have been classified into types. The pollen of the variants was asymmetric, nonfixiform while controls have aU radio symmetric grains, 3 colporate, prolate-perprolate. The equatorial view of these grains exhibits different shapes. The equator was bulging or constricted. Polar view circular or angular. The grains were colporate, colpi usually long ectosexine tenuitegillate, in a few it was crassitegillate while in others it was an admixture of the two on one and the same grain. In some very conspicuously baculate, simp Ii or hetrobrochate, sculpture almost psilate. The apertures were nonoperculate and covered by a psilate membrane. Ontogenetic study of these pollen clearly revealed that the lobed grains are not the result of incomplete separation of individual spores, rather they are the outcome 13'
184
SATENDRA S. RAGHUVA,\,SHI
and
SHEILA JOSHI
of single spores which assume variable shapes. The pattern of development was the same as observed in Foeniculum vulgare (RAGHUVANSHI and JOSHI 1967) and 00riandrum sativum (JOSHI and RAGHUVANSHI 1965b). It was noted that the grains developing from the same sporad were not of the same shape but an admixture of normal and the variants in different proportions i.e. a single PMC produced both normal as well as variant grains. These variant grains showed random distribution in the plants, individual heads of a plant, buds on the same head, and different anthers in one and the same bud. These variant grains have been classified as types based on the study of treated plants of Foeniculum vulgare which had types A to M (RAGHUVANSHI and JOSHI 1967). In the present study Types A, B, E, F, G, N, 0, P and giants were metwith. The spectrum of pollen variability was broader in E. 210 than the tetraploid although the latter had higher frequency of variable grains (Table III). The various types of grains observed are described below and their development has been presented in figure 51. Table III The proportion of different types of pollen grains as observed in tetraploid and 2n plant No. of plant
Level of ploidy
E. 225 E. 210
Types of pollen grains A
B
E
F
G
N
0
P
4n
870
2
15
4
33
3
60
16
2n
932
14
12
13
17
3
Joined grains
Total 1003
30
1026
Type A (Fig. 12). This is the control type. It is bipolar radio symmetric grain. Type B (Figs. 13, 14). This is like type A with a partition wall in the equatorial
region which divides the grain into two equal compartments. Development of such grains was observed in Foeniculum vulgare. In these grains the nucleus divided in an abnormal manner and the two daughter nuclei were formed followed by wall formation at the equatorial region. Type E (Figs. 16, 17). A triradiate or trilobed grain in few cases the poles are flat while mostly they are rounded. Type F. The grains have triangular outline. Type G (Fig. 18). A tetralobed grain in which the arms may be of equal or unequal length. Type M (Fig. 50) these are polyads. Type N (Fig. 19). The development of a side protrusion in Type A grain results into L or J shaped grain. Type 0 (Figs. 20-22). This is also triradiate type but in this case two protrusions arise at one of the poles while in type E the third protrusion arises at the equatorial region. Type P (Fig. 15). It is a dumbell shaped grain with flat poles and a constriction at the equator. The bacula is equally developed on all the sides and is simplibrochate. Round thick walled giant pollen grains were also observed (Figs. 27, 28). Some of the rare occurring shapes are shown in Figs. 23, 26 and 29.
I I I
I!
III
I
i:
12
73
Figs. 12-50: Pollen grains. Fig. 12. Control type A. Figs. 13, 14. Bicelled type B. Fig. 15. Type P. Figs. 16, 17. Type E. Fig. 18. Type G. Fig. 19. Type N. Figs. 20-22. Type O. Fig. 23. A variant. Figs. 24, 26, 29. Variants. Figs. 27,28. Round giants. Fig. 30. Joined variants. Figs. 31-35. Two joined grains. Figs. 36-39, 40-44. Three and four joined grains at different positions. Fig. 45. Acetolysed joined four grains showing the pores of individual grains. Fig.46. Two separate nuclei in the joined grains. Fig. 47. Single nucleus in the joined grains. Fig. 48. One and two nuclei in the joined grains. Fig. 49. A single nucleus in a mass of four joined grains. Fig. 50. Polyad of variants.
iiaE
186
SATENDRA S. RAGHUVANSHI
and
SHEILA JOSHI
United grains. A unique feature of E. 210 was the presence of united pollen grains, which assume various shapes. The number of grains that have been found in this condition varies from 2 to 5. Two pollen grains showed connections at different positions either at the poles, equator or any where between the two (Figs. 31-35). In cartain cases the common wall between the grains was noted (Fig. 35) while in still others the passage between the two grains was direct (Fig. 31). In carmine preparations of the grains the nuclei were observed. In certain cases there was a single large nucleus in the centre whereas in others they were seen in individual grains (Figs. 46-49). In Fig. 49 a single nucleus was seen in a united mass of four grains This single nucleus could be the outcome of breakdown of spindles both at a. I and a. II followed by the transformation of such PMC into a single giant grain, or alternatively the individual nuclei of the grains may have fused to form a single nucleus. It may be mentioned that no breakdown of spindle mechanism either at a. I or a. II was ever encountered. United 2 or 4 nucleate round giant grains having cytoplasmic connections were observed in Foeniculumvulgare (RAGHUVANSHI and J oSHI1966 a, 1967). But these arose due to transformation of the whole PMC into a pollen grain after t. I {)f t. II. In one instance in three united giant grains the nuclei and cytoplasm of the lateral grains was transferred to the central grain. This situation was almost analogous to fertilization process. In Coriandrum sativum (JOSHI and RAGHUVANSHI 1965 b) the nucleus of type a pollen grain was under going transfer into another control type A grain. This situation resembles differential sexuality of two grains i.e. donar functioning as male while recepiant grain as female. These giant grains in Anethum assume the shape of types E, G and H, but close examination shows them to be a product of type A grains rather than a single giant grain (Figs. 38, 41-43). The pores on the individual grains were clearly noted at their respective equatorial regions (Figs. 35,36,41,44,45). Not only normal type A grains behave in this manner but the variable grains also showed the same condition (Fig. 30).
Discussion
Autopolyploidy is considered more conducive to lability than allopolyploidy and this lability increases with the increase in the level of ploidy. The Anethum tetraploid certainly provides no exception but the most unique feature of the variants is the presence of pollen variability when the controls have normal bipolar radiosymmetric grains (RAGHUVANSHI and JOSHI 1965 b). The low frequency of univalents and multivalents in autopolyploides appears to be a feature shared by Umbellifers under study (Coriandrum sativum, Pimpinella monoecia, Carum copticum, Foeniculum vulgare etc.). Though multivalents occur in every cell, yet, their average is fairly low (3-4 per cell). This tendency was still more marked in Foeniculum where not more than two quadrivalents were seen in any cell. In contrast to these in tetraploids of one variety
o
a o I
liP ,
r
Anethum graveolens: Polyploidy and Pollen variability
187
of Capsicum frutescens all the 48 chromosomes of the PMC formed 12 quadrivalents (unpublished). But mention may be made that in Carum copticum tetraploids also all the 36 chromosomes formed quadrivalents in some PMCs. The complete absence of trivalents is also conspicuous, probably this may explain to a certain extent the low frequency of univalents. Multipolar and multiple spindles have been observed in some ·cells. WALTERS (1958) has given a scheme of origin of different number of spindles due to anomalous division of spindle organisors. Some of the supernumary spindles in Anethum graveolens have very few chromosomes, this may be due to most unequal division of spindle organisors. Further, number of functional groups of chromosomes in such cells at later divisional stage depended on the position of the poles of different spindles with regard to each other. The presence of such aberrant spindles do not appear to have any specific relationship with the frequency of univalents. They were observed in colchiploids of Capsic'um fr'utescens having high univalent frequencyA (RAGHUVANsHIand JOSHI 1964a) and Anethumwhich have very low univalent frequency. Some genera of angiosperms are known to have dimorphic pollen grains (ERDTMAN 1952 p. 4) but artificially induced pollen variability of the magnitude reported in the present paper is unique. This pollen variability appears to be a peculiar feature of first generation of polyploids in Umbellifers and certain types of grains are common in different genera. CERCEAU (1959) and TING (1961) have studied the pollen of some Umbellifers. In variants of Foeniculum vulgare there were types A to M while Pimpinella !!I'onoecia had types A, E, D, F, G, and M. In Coriandrum sativum there were types A, E, F, G, Hand N besides type a, which was direct transformation of a PMC into a pollen grain without the intervention of meiosis. Though certain grains are common in the genera under study yet their frequency shows a great range of variation. The frequency of different grains is not high in Anethum, still, the range of variability is very wide. Regarding the frequency of different types it could be postulated that certain types like G and E are not only uniformaly present in the treated plant of different genera but are also dominant types. Type G (tetraradiate) occurs most frequently in tetraploids of Coriandrum sativum, Foeniculum vulgare and Pimpinella monoecia but in Anethum it appears to have suffered eclipse at the hands of type E (triradiate) which has been a close follower in the other genera. Our studies have shown that all these types can be derived from normal type A as presented in the scheme (Fig. 51). These various observations clearly indicate that pollen grains in Umbellifers with regard to their shape are not very stable and disturbances could be caused by judicious use of certain chemicals as shown in Pimpinella monoecia (unpublished). It is suggested that chemical treatment of young seedlings ultimately brings about a change in the expression of genes controlling the pollen shapes. Similar shapes observed in the treated plants of different genera further substantiated this conclusion.
188
S~\TENDRA S.
RAGHlJVA?'
Fig. 51. Diagrammatic representation of the development of different types of variable grains.
Summary Oolchicine-gammexane treatment of seedlings of Anethum graveolens resulted in the production of a tetraploid (E. 225) and a 2n plant (E. 210) which exhibited pollen variability while the controls had normal bipolar grains. The meiotic instability exhibited by E. 225 is classical for polyploids. The encountered variable grains are types A, B, E, F, G, N, 0, P and giant grains. The origin of these various types of grains has been proposed with the help of a scheme. It is suggested that this pollen variability is due to change in expression of genes controlling the pollen shape.
Literature OERCEAlJ, M. T., 1959. Ole de determination d'Ombellifers de France etv d'Afrique du Nord d'appears leurs grains de pollen. Pollen et Spores 4,145-190. DARLINGTON, O. D., and THOMAS, P. T., 1937. The breakdown of cell division in a Festuca Lolium derevative. Ann. Bot., N. S. 1, 747-762. ERDTMAN, G., 1952. Pollen morphology and plant taxonomy. Stockholm. JACOB, F., and MONOD, J., 1961. Genetic regulatory mechanisms in the Synthesis of proteins. (Review article.) J. Mol. BioI. 3, 318-356.
Anelhum graveolens: Polyploidy and Pollen variability
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JOSHI, SHEILA, and RAGHUVANSHI, S. S., 1965a. Pollen formation without the intervention of meiosis in a variant (E. 67) of Coriandrum salivum and Pollen variability. Grana Palynologia. 6, 186-190. - - 1965 b. Coriandrum sativum: Mutation, Polyploidy, Nondividing pollen mother cells and pollen variability. Canadian s. Genet. Cytol. 7,223-236. RAGHUVANSHI, S. S., and SHEILA JOSHI, 1964. Cytomorphological studies on the colchiploids of Capsicum frutescens. Cytologia 29, 61-78. - - 1965a. Studies on the comparative effects of certain chemicals on the polyploidizing efficency of colchicine in Trigonella foenum-graecum. Caryologia 18, 69-84. - - 1965 b. Artificial induction of pollen variability in Anethum and Pimpinella. Naturwiss. 52,397. - - 1966. Foeniculum vulgare: Polyploidy, Translocation heterozygosity and Pollen variability. Part I. (CYTOLOGY) Cytologia 31,43-58. - - 1967. Foeniculum vulgare: Polyploidy, Translocation heterozygosity and Pollen variability. Part II (Pollen GRAINS). (In communication.) SWANSON, C. P., and NELSON, R., 1942. Spindle abnormalities in Mentha. Bot. Gaz. 104,273-280. TAMAMSCHJAN, S., 1933. Bull. Appl. Bot. Sci. 2, 137. TING, W. S., 1961. On some pollen of Californian Umbellifers. Pollen et Spores 3, 189-199. WALTERS, M. S., 1958. Aberrant chromosome movement and spindle formation in meiosis of Bromus hybrids. An interpretation of spindle organisation. Amer. Jour. Bot. 45, 271-289. Authors' address: Dr. Satendra S. RAGHUVANSHI and Dr. (Miss) SHEILA JOSHI, Cytogenetics Laboratory, Department of Botany, University of Lucknow, Lucknow (India).