Estimating the number of meristem initials after seed irradiation: A method, applied to flax stems

Radiation Botany, 1970, Vol. 10, pp. 47 to 57. Pergamon Press. Printed in Great Britain.

ESTIMATING THE N U M B E R OF MERISTEM INITIALS AFTER SEED IRRADIATION: A METHOD, APPLIED TO FLAX STEMS* 18. H, B E A R D Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture, Brawley, California, U.S.A.

(Received 18 January 1969) A b s t r a c t - - F l a x seeds (Linum usitatissimum L.) were irradiated with 30 kR of X-rays before sowing. If possible, seeds from 5 basal branches (secondary stems) were harvested separately from each of the Mx plants. The Mz seedlings from 47,452 branches were observed for chlorophyll mutants. I f mutants were found, all seedlings in that branch progeny were classified, and the M~ mutant frequency was calculated. Normal appearing M s plants were harvested and the M3 mutant frequencies were determined from the progeny of heterozygous M s plants. If the M , segregation was not significantly different from a 3 : 1 ratio, or if the M 2 and M 3 paired ratios were similar according to Z 2 and heterogeneity ?(~ calculations, respectively, the M1 branch was considered the product of a single initial. From a total of 65 branches with chlorophyll mutants, 45 or 69 per cent were considered to be division products of a single cell initial. If the M 2 and Mz paired mutant frequencies were significantly different, the number of cell initials contributing to the MI branch was estimated from a specially constructed graph. The graph was made on log-log paper by plotting the theoretical mutation frequency from a single mutated cell on the abscissa and number of cells contributing division products on the ordinate. Only 3 per cent of the branches were descended from five or more cell initials. R ~ s u m ~ - - D e s graines de lin (Linum usitatissimumL.) ont dtfi irradides par 30 k R de rayons X. avant d'etre sem6es. Les graincs des cinq branches basales (tiges secondaires) ont dt6, autant que possible, rficoltdes sfipardment pour chaque plante M1. Les mutants chlorophylliens ont dtd observfis parmi les plantules M2 provenant de 47.452 branches. Lorsque l'on reneontre des mutants, on classe sdpardment toutes les plantules constituant la progfiniture de eette branche et l'on calcule la frfiquence de mutants M 2. Les plantes M2 apparemment normales sont rdcolt~es et les frdquences de mutants M3 sont ddtermindes ~t partir de la progdniture des plantes hdt~rozygotes M 2, Lorsque la sdgrdgation M~ ne diff~re pas significativement d'une ratio 3 : 1 ou bien si les ratios M2 et M3 apparides sont semblables d'apr~s le chi-carr~ ainsi que les calculs du chi-carrd d'hdt~rogdndit~, on consid~re la branche M 1 comme produite par une seule initiale. Sur un total de 65 branches avec mutants chlorophylliens 45 ou 69°./o ont ~td eonsiddr~s eomme dtant les produits de division d'une seule cellule initiale. Lorsque les frdquences apparides des mutants M 2 et M 3 sont significativement diffdrentes, le nombre de cellules initiales qui ont contribu~ ~ la formation d'une branche M1 est estimde ~t partir d ' u n graphique spdcial. Le graphique a dtd rdalisd sur papier log-log en exprirnant * Cooperative investigations of the Crops Research Division, Agricultural Research Service, U.S. Department of Agrieulture, and the Department of Agronomy and Range Science, University of California, Davis, California. This investigation was supported in part by Atomic Energy Commission Contract AT(49-7)-202 I. 47

B. H. BEARD

48

en abciss¢la fr~quence th~orique de mutations provenant d'une seule cellule mut~e en fonction, en ordonn~e du nombre de cellules contribuant aux produits de division. Trois pour cent des branches seulement descendent de 5 initiales ou davantage. Zus-mmemfassimg--Flachssamen (Linum usitatissimum L.) wurden mit 30 kl~ R6ntgenstrahlung vor dem Ass~en behandelt. Soweit mOglich, wurden Samen von fiinf Basalzweigen (sekund~re Triebe) yon jeder Mz-Pflanze getrennt geerntet. Die Mz-Keimlige yon 47 452 Zweigen wurden auf Chlorophyllmutationen hin untersucht. Wurden Mutanten gefunden, so wurden alle Keimlinge, die sich von dem gleichen Zweig ableiteten, ldassifiziert und die H~ufigkeit der Ms-Mutanten errechnet. Ms-Pflanzen, die als normal angesehen wurden, wurden geerntet und die H~ufigkeit der Ms-lVfutanten wurde auf Grund der Nachkommen heterozygoter Ms-Pflanzen besfimmt. Wenn die M2-Segregation nicht signifikant yon einem 3 :I Verh~Itnis abwich, oder wenn die gepaarten Verh~ltnisse der M, und M3 ~hnllch waren entsprechend dem x*-Test und der x*-Berechnung der Heterogenit~t respektive, so wurde angenommen, dass der M1-Zweig das Produkt einer einzigen InitialzeUe war. Von insgesamt 65 Zweigen mit Chlorophyllmutanten waren 45, das entspricht 69 Prozent, Teilungsprodukte einer einzigen Initialzelle. Werm die gepaarten Mutafionsh~ufigkeiten von M,. und Ms signifikant verschieden waren, wurde die Zahl der Initialen, die zu dem M1-Zweig beitrugen, fla'ch einer speziell kormtruierten Zeichnung gesch~tzt, Fiir diese Zeichnung wurde auf doppelt 10garithmischem Papier die theorefische Mutatlonsh~ufigkeit einer einzelnen mutierten Zelle auf der Abszisse und dim Zahl der Teilungsprodukte beisteuernden Zellen auf der Ordinate aufgetragen. Nur 3 Prozent der Zweige leiteten sich von fiinf oder mehr Initialzellen ab. INTRODUCTION

HISTORICALLY. the number of cell initials in a plant meristem has been mainly of academic interest, although they probably influence the organization and differentiation of tissues in a stem or root. Detailed knowledge about the meristernafic region in mature embryos is of practical significance for estimates of mutation frequencies following seed irradiation, or for duplication studies. (s) STADLER('61) recognized that the frequency of mutants fi'om M s barley spikes gave an indication of the number of initial cells involved. Since then there have been many studies of various plant species in which the number of initial cells have been estimated from genetic data. This paper develops additional details in the use of mutation data for anatomical inferences and furnishes estimates of the number of cell initials contributing products to the basal branches of flax plants.

LITERATURE

REVIEW

Studies ofmeristem organization and development are numerous and three broad categories of techniques have been used. These are: (1) histological and morphological studies,

(2) marking and mutilation studieS, and (3) determining the limits of sectors by use of chimeras. Only the last of these methods will be considered here although the histological and morphological techniques have been the classical method of studying the meristems of growing plants. There are several reviews of these studies. (S,s,xs,sx-ss,es, 6~) The marking and mutilation techniques are more recent but also are not strictly pertinent to this study (see BALL(S) and SussEx(6s) for reviews). An understanding of the classical studies is necessary because these lead to the different but related conceptsregarding the organization of plant meristems. Even though these studies of older growing plant meristems would not necessarily be expected to give the same or similar results as those obtained from studies of the number of initials in the matt~re embryo of a dormant seed, the two periods of development are mutually dependent, all part of the same process. The simplest hypothesis from the classical studies is the apical cell concept; a condition found only in some pteridophytes. Many other hypotheses have been advanced which helped to develop the latest and at present the most useful, the tunica-corpus concept. Discussions a n d reviews

NUMBER OF MERISTEM INITIALS AFTER SEED IRRADIATION of these and other hypotheses have been published by BUVAT,(~) CLO~VES,(11,12), CROCKETT,(14) FOSTER,(21) GIFFORD,CSl)POPHAM(54)and others. The determination of sectors or tissues by studying chimeras has been helpful. CLOWES(1°) has explained their use in determining the organization of meristems. Theoretically, the chimeral sector can comprise the total organ, one-half the organ, one-third the organ, etc., showing that one, two, three, etc. initial cells were involved in furnishing division products to that organ of the plant. Chimeras are either naturally caused or result from treating the seed or other plant stage with a mutagen. The naturally caused frequency is usually too low to be of much use, so treated materials are generally studied. There are basically two methods of determining chimeral sector sizes. The first method is dependent on visually determining the area occupied by the chimeral tissue. There are various ways this can be done. BRUMFIELD(5) determined sector size in Vica faba and Crepis capillaris roots by cytologically determining the kind and extent of tissues produced from a single cell tagged with a chromosomal aberration from X-irradiation of primary root meristems. He estimated that three cell initials produced all the cells in the primary roots. Cytological observations on X - r a y induced chromosomal aberrancies in different spikelets on the same barley spike led CALDECOTT and SMITH(s) to conclude that a barley spike developed from a single initial cell in the irradiated dormant seed, but GAUL (z6) using the same method concluded that some spikes were from a single initial cell, and others were descendents of two or more initials. ANDERSONet al. (1) and ERIKSSON(9"°)determined sector limits by staining the pollen of maize and barley, respectively. ANDERSONet al. (1) concluded that 7 or 8 cells in the apical meristem of the dormant seed are represented in the maize tassel. ERIKSSON(2°) treated dormant barley seed with g a m m a rays or ethylmethane-sulfonate. The chimeral sector sizes varied from only four spikelets to an entire spike in the y-ray treated material and mostly small sectors not exceeding one side of the spike after the ethylmethanesulfonate treatment.

49

Another way of visually determining sector size is to cause mutations or deletions of a dominant gene in a plant that is heterozygous for a chlorophyll deficiency. A mutation or deletion will be visible as a chlorophyll deficient area in a lead or stem. The number of sectors and their relative size can be used to estimate the number of initial cells. NISHIYAMA, ICHIKAWA and AMANO,(4s) NISHI'YAMA, IKUSHIMA and

ICHIKAWA(49) and ICHIKAWA and IKUSHIMA(84) used this method to study oat leaf development. They determined that the first two leaves were preformed in the dormant seed, the third leaf developed from three initial cells, and leaves four through six were formed from three to five initial cells. Soaking the seeds in water before mutagen treatment increased the sensitivity of the initial cells that produced the second leaf. Periclinal chimeras from colchicine (SATINA, BLAKESLEE and AVERY(57)) or X-ray (PRATT,(55) DOMMEROUES and GILLOT(ls)) treatment have been used to show the number of tissues in a stem, but treatment was not applied to seeds and number of initial cells was not determined. There are also cases in which chimeras are never found, which leads to the conclusion that only one initial cell contributes division products to these stems. Moll(4~) using coffee plants and SPARROW, SPARROW and SCHAIRER(60) using Saintpaulia concluded that only one initial cell contributed to the formation of shoots and plantlets, respectively. NAYLOR and JOHNSON(45) using histological techniques came to the same conclusion for Saintpaulia. T h e second method of obtaining chimeral sector size information is based on genetics. Mutation ratios from main stem progenies, secondary stem progenies, tertiary stem progenies, or inflorescence progenies have been used to determine the number of cell initials represented. STADLER(61) developed the principles of this method and it has been used and elaborated m a n y times. The number of cell initials contributing to the sporophytic tissue in barley spikes has been determined by CALDECOTT and SMITH,(s)

GAUL,(z5-a°) SARVELLA,NILAN and KONZAK(56) and STADLER.(el) They all agree that some spikes originate from a single cell in the dormant seed. T h e y disagree on the n u m b e r of initials that

50

B. H. BEARD

can be included in a single spike. JACOBSEN(s0) compared spike progeny mutant data from single plants to determine the number of mutually exclusive meristematic regions in the embryo of a dormant seed. He concluded that in large barley seeds there are always nine meristems that can produce nine mutually exclusive sectors in the plant. U p to seven additional meristems can be present, but usually will not produce sectors. There are six spikes that are established in the mature embryo. These six have developed from one or two functional initial cells. The rest of the spikes usually belong in six groups and any two spikes within a group can arise from one initial cell in the embryo. GAUL(ss) and others report that the first five tillers are partially formed in the mature embryo, and SARVELLA et a/.(5e) state there are at least three partially formed tillers in the mature embryo. GLA~STO~nZ(3s) concluded that most branch inflorescences of blue lupin (Lupinus digitatus Forsk.) were descended from a single cell, but the apical inflorescences were often descended from two or more cells. Pea seeds subjected to treatments of ethylene oxide, diethylsulphate, or X-rays have been genetically analysed for estimates of initial cells. BL1XT,F,I-IRENBEROand GELIN(4) compared M s mutant frequencies with M s progeny tests and concluded that individual branches were usually descended from a single cell. WI~XLINO and GOTTSCHALK(65) reported 19-5 per cent of the primary stems of a cooking pea (Pisum) were from one initial cell, and MONTI(48) reported 50 per cent of the primary stems were descended from one initial. MONTI(43) concluded that five was the highest number of cell initials in an apical meristem of a primary stem, and four for a secondary stem. KAUKIS and REITZ(se) determined the distribution of M S chlorophyll mutants in sorghum inflorescences after irradiating seeds with X-rays or thermal neutrons. They did not estimate number of initial cells but the limited data indicate some inflorescences were the product of a single cell while others were the product of two cells. Their diagrams seem to indicate that from one to m a n y initial cells m a y be involved. Apparently some rice panicles are descendants

of a single initial cell, but others contain descendants of m a n y initials (OsONE,(50) MATSUO, YAMAGUCH! and ANDO,(By) NISHIYAMA and KURAKAMI(4v) and YAMAOUCHI(6e)). Fujn(23, 24) did not estimate the number of cell initials but his data show that in m a n y cases einkorn wheat spikes are descended from more than one initial cell. MONTI(44) using Triticum durum and a mutation that segregated 3:1 found 41 per cent of the spikes were from one initial, 20 per cent from two initials and 38 per cent from more than two initial cells. With another mutation that segregated only 20 per cent mutants, he estimated only 32 per cent of the spikes were descended from one initial cell. The results reported by MONTI(44) and m a n y others illustrate some of the problems associated with these studies. The mode of inheritance of the mutation,(44) deleterious changes that may accompany the mutation,(~7, so) the effects of post-irradiation treatments,(19) the morphology of the Mx plant, (2~.,2s,~.~,6e)the physiological condition of the seed or plant during irradiation,0S) and radiation sensitivity changes(SS, 5~) m a y modify the results from different studies. N E W M A N ( 46} has shown that Tropaelum and Coleus plants have certain definite meristematic regions under normal conditions, but if these are damaged or destroyed, the cells in other areas and other tissues can become meristematic and actually m a n y cells m a y be initials under certain conditions. Finally another technique was developed by MERICLE and MERICLE(38) and MERICLE, MERICLE and CAMPBELL.(40) T h e y irradiated developing barley embryos and determined the number of cell initials. T h e y report that after the eight-celled stage, more than one cell usually give rise to the germinal tissue of the first five spikes. With irradiation exposure at certain embryo stages as m a n y as 20 cells m a y initiate the first five spikes. However, if all of these are killed a single cell can take over and m a y contribute to the entire above ground portion of the plant. The only estimate of number of cell initials for flax was given by CROOKS.(15) He removed the original apex and reported only one cell of the epidermis eventually contributes the axis of the adventitious shoot.

NUMBER OF MERISTEM INITIALS AFTER SEED IRRADIATION MATERIALS AND METHODS

The hypothesis The embryo of a seed is a population of cells. Some of these through continued division form the axis of the mature plant. These original meristematic initials are present if the seed is exposed to irradiation. A recessive mutation in one of these cells, that contributes to the sporogenous tissue, will produce a heterozygous condition that is similar to a hybrid zygote. Derivatives from this cell that take part in gametogenesis will produce two kinds of gametes, and these gametes will combine to produce a segregating population. This assumes that the male and female gametes are formed from the same tissue, and this assumption appears valid for perfect flowers that are self pollinating. The same principles apply with cross pollinated species or if male and female gametes are from different tissue, but the procedures are necessarily more complicated. The segregation rates from the perfect selfed flower will again be 75 per cent normal and 25 per cent mutants. Throughout this paper, mutation will be used to denote the event or condition that caused a different phenotype. Mutant denotes the seedling or plant that phenotypically expresses the mutation. Thus, a 75:25 per cent ratio of normal to mutant progeny would be expected from seeds harvested from a plant organ that is composed entirely from division products of a single, mutated, initial cell. I f two initial cells are present at the time of irradiation, a mutation can occur in one, both or neither. I f neither cell is mutated all progeny of both cells will be normal and no segregation occurs. This possibility will not be considered further. I f a recessive mutation occurs in one cell, and the division rate is not changed, the particular plant organ will consist of two cell populations, actually two undetected sectorial chimeras, because the plant appears normal. I f both cells have mutations or there are mutations at two different loci in the same cell, the situation is more complicated, but the probability of this happening is small because the single mutation frequencies are low. Sectorial chimeras show that derivatives from an original initial occupy a contiguous area of

51

the plant organ. Thus, it is reasonable to assume that the stamens and pistil of a perfect flower have a common ancestry. Because of this assumption it would be possible to distinguish between two mutations in a single initial cell and mutations in each of two initials. The number of cell initials determines the frequency of mutants. If the progeny from seeds produced on a plant organ are all from division products of a single mutated initial cell, 25 per cent will be mutants. If there are two cell initials and only one with a mutation, one population of seedlings will segregate 25 per cent mutants, the other will be normal. The normal progeny cannot be separated into their respective populations. There will be 12"5 per cent mutants in the total population. I f there are three original meristematic initials, one with a mutation, seedlings will be normal in two populations. The third population will have 25 per cent mutants. There will be a mutant frequency of 8.3 per cent for the total of all seedlings. Similar calculations can be made assuming any number of cell initials contributing division products to the plant organ under consideration. The segregation ratio of some mutations may vary from the expected 25 per cent mutants. Mort and SMITHC42) studied a large number of barley mutations and found the average segregation frequency was only 20 per cent. These data were obtained from segregating populations; number of cell initials was not involved. Thus, GAUL(25,2e) compared Iris individual M 2 mutant frequencies with 20 per cent expected in estimating the number of cell initials in barley spikes. In this report the M 2 mutant frequencies are compared to the M a mutant frequencies for the same mutation. A graph showing the number of initial cells on the abscissa and expected frequency of mutants on the ordinate was prepared for a 3 : 1 segregation. The line connecting the points was a typical asymptotic curve. The data, plotted on log-log paper, gave a straight fine. Figure 1 was prepared to show the expected mutant frequency with segregation percentages ranging from 5 to 30 per cent, and the progeny descended from 1 to I0 initial cells.

B. H. BEARD

52 30

3O

25

20

\\

20

,0 \ \ \

18

I,-10

0

10

"\ \'k

a.

'-,.j ,,,j ,q

?.g5

Iz uJ o ¢

a.

5~g

w

iN I',L I 2

:5

4

5

6789

NUMBER OF CELLS

FIO. 1. Expected segregation frequencies if one cell initial carries a mutation and 1 - 1 0 cell initials contribute equally to the division products of the organ producing the seed.

PTocgduTg I n this study, each cycle consists of all treatments and collection of data during the following chronological periods: (1) control and irradiated seed, (2) growing the M 1 plants, (3) selecting seed for the next cycle, (4) growing the M s plants, (5) selecting M s plants from progenies with mutants, and (6) growing the M a progenies. T h e next cycle starts with the seeds that were selected from the M 1 plants (step 3). Different cycles were in different chronological periods at any given time. (1) Lots of 1000 seeds of each of 7 flax varieties were irradiated; similar non-irradiated lots of 500 seeds served as controls. T h e flax seeds were allowed to reach equilibrium over saturated N a C I O a (the whole seed contained approximately 10 per cent water), and were then X-rayed with 3 0 k R at an exposure rate of 3150 R/min. T h e G.E. maxitron X - r a y machine was operated at 1 0 0 k V p and 7 m A with a target distance of 24.6 cm. There was no added filtration.

(2) T h e seeds were held at room temperature and were sown approximately 5 cm apart, on beds 100 cm apart, a week or more after the irradiation. T h e control and M 1 plants were covered with cheese cloth to eliminate bees; the major cause of outcrossing. (3) W h e n the M 1 plants were mature, seeds for the next cycle were obtained. O n e thousand bolls were selected at r a n d o m from the surviving plants in each irradiated varietal population and 500 from each control. O n e seed was selected from each boll, and these were bulked in treatment-variety seed lots for the next cycle. (4) Five branches (primary or secondary stems) were selected from each M x plant. T h e seeds from each b r a n c h were sown in rows 60 cm long. A b o u t one m o n t h after sowing the seeds the M s plants in the b r a n c h progeny rows containing chlorophyll mutants were cotmted and classified. These counts were used to calculate the M s m u t a n t frequencies. (5) At least 20 normal appearing plants were harvested from each Ms b r a n c h progeny row containing mutants. (6) T h e seeds from each of these plants were sown in rows 300 cm long. T h e M z progenies from heterozygous M2 plants were counted and classified. These counts were used to estimate the M 3 segregation frequencies for each mutation. T h e five M s b r a n c h progenies from each M I plant were identifiable until harvest time. I f mutants were found in 1, 2, 3 or 4 of the progenies from a single M1 plant, they were counted as one m u t a t i o n unless the mutants were obviously different. T h e m u t a t i o n was assumed to be a carry-over from the previous cycle if all b r a n c h progenies contained mutants. Thus, only the mutations from the most recent irradiation were used in this study. T h e r e were three steps in estimating the n u m b e r of cell initials for the M 1 branch. First, Z2 values for a 3 : 1 ratio were calculated for each M 2 n o r m a l : m u t a n t association. I f Z2 probabilities were higher than 0"05, one initial cell was assumed, a n d these data are not considered further. Second, if the n o r m a l : m u t a n t ratio was significantly different from 3: 1, heterogeneity Z2 was calculated for M ~ - M a data. I f heterogeneity X~ indicated the M s and M a ratios werc not significantly different, the M 1 b r a n c h was

NUMBER OF MERISTEM INITIALS AFTER SEED IRRADIATION assumed to have been derived from a single initial cell. Third, if heterogeneity ) 2 calculations indicated that M 2 and M 3 ratios were significantly different, more than one cell initial was assumed to have caused the discrepancy. The number of initials was determined fi'om Fig. 1. When using Fig. 1, the numbers below the point of intersection of the M 8 and M 2frequencies indicate the number of cell initials involved. For example, suppose the M 3 mutant frequency was 20 per cent and the M S frequency was 10 per cent, and the number of plants in each generation was large enough for heterogeneity Z* to show a significant difference between the two ratios. First, find the M 3 or expected frequency (20 per cent) on the left side of Fig. 1. Second, find the observed or Ms frequency (10 per cent) on the right side of the graph. Follow the diagonal line from the left side and the horizontal line from the right side to a point of intersection. The number on the abscissa directly below this point indicates the number of cell initials involved. In this example the number of cell initials is two. Theoretically, all points of intersection should be directly above an abscissa number. Actually, random chance of gamete recombination and unequal division rates of normal and mutated cells can cause intersection points at other locations. I f the seedling populations are large the deviations due to gamete recombination would be expected to be small. A decrease in the division rate of the mutated cell could result in overestimation of numbers of cell initials. The magnitude of overestimation increases with a larger number of cells. See Discussion for additional details. RESULTS

Earlier descriptions of flax plant morphology have been of the fiber varieties. Genetic and cultural control of plant type produces long slender stems with branches near the top (see HAYWORTH(33)). Seed flax, on the other hand, has a slightly different morphology. The stems are short, coarse and extensively branched. Basal branches as used in this paper means secondary stems that originate at the cotyledonary node. After the primary stem has elongated 10-15 era, two basal branches appear, one in the axil of each cotyledon. Later, basal branches may

53

originate at any point around the cotyledonary node. The number of basal branches is dependent on plant spacing, but usually two and as many as 10-15 may develop. The primary stem and basal branches (secondary stems) usually develop another level of branches (tertiary stems). Flower buds usually develop on the tertiary stems. Flax is self pollinated, with from 0.3 to 2.0 per cent outcrossing (DmLMAN(16)). Normally a boll has a maximum of 10 seeds, although occasionally there may be 12-15 seeds. The average is seven or eight mature seeds per boll. Flax seeds are resistant to X-ray effects. In other studies not reported here, seeds exposed to 100-120 kR of X-rays have germinated and produced mature plants. Plant height and seed production are reduced following these treatments. The cotyledons from irradiated seeds show minute necrotic spots but the true leaves exhibit no visible damage. During irradiation experiments with flax, I have observed approximately 50,000 M t plants and have detected only three visible chimeras. Two of these chimeras were on the primary stem and in each case comprised approximately half of the stem, indicating two initial cells in the original meristem. The third chimera was a yellow chlorophyll mutation of a basal branch. The rest of the plant was normal and only normal seedlings grew in the next generation. This chimera would indicate that only a single cell initial was involved in the development of the chlorophyll deficient branch. Occasionally mutant seedlings were found in two or three branch progenies from a single M 1 plant, indicating that one initial cell may furnish division products to more than one basal branch. Data from three cycles of a recurrent irradiation study were available for estimating the number of cell initials in flax branches. The M2 frequencies were determined from 47,452 branch progenies, and ranged from 4 to 30 per cent. I f Z~ calculations indicated no significant difference from a 3:1 ratio, the M 1 branch was considered to be descended from a single initial cell. The M 3 mutant frequencies varied from 8 to 28 per cent with the average for all mutations at 20 per cent. In most cases 400-500 seedlings

B. H. BEARD

54

were classified for each m u t a t i o n . These d a t a are similar to those for b a r l e y r e p o r t e d by M o l l and SMITH. {42)

T h e r e were 45 basal branches that d e v e l o p e d from one initial ( T a b l e 1). T h e m u t a n t ratios for 20 M 1 basal branches were significantly different from a 3:1 ratio a n d were significantly different from the corresponding M3 ratio. Figure 1 was used to estimate the n u m b e r of cell initials furnishing division products to these branches. Eight branches h a d d e v e l o p e d from two cells, a n d three cells c o n t r i b u t e d to the d e v e l o p m e n t o f seven other branches. O n l y two branches were descended from five or m o r e cell initials. T h e r e were 28 b r a n c h progenies with a larger m u t a n t frequency in M 2 t h a n in M 3.

t h a n n o r m a l cells, the result could be a n overestimation of the n u m b e r of initials. T h e m a g n i tude of the o v e r e s t i m a t i o n increases w i t h a larger n u m b e r of a c t u a l initials. F o r example, if there are a c t u a l l y two initial cells, b u t the cells w i t h the m u t a t i o n divide at only h a l f the n o r m a l rate, the estimate will be three initial cells. I f there are a c t u a l l y four initial cells a n d the cells with the m u t a t i o n d i v i d e at h a l f the n o r m a l rate, seven initial cells will be estimated. These calculations show the m a g n i t u d e of errors that could occur from u n e q u a l cell division rates. H o w e v e r , it is possible that such differences in division rate could cause twining or o t h e r noticeable m a l f o r m a t i o n o f the M 1 stem. W h e t h e r these errors are real or i m a g i n a r y is of m i n o r i m p o r t a n c e with the d i s t r i b u t i o n shown

Table 1..Number and percentage of flax branches that originate from 1-5 or more cell initials

Number meristematic cells

Cycle 1, No.

Cycle 2, No.

Cycle 3, No.

1

19

22

4

2 3 4 5+

8 1 3 1 32 5

0 3 0 0 25 22

0 3 0 1 8 1

Total Not usable*

Total No.

%

45 8 7 3 2 65 28

69 12 11 5 3

* The M 2 mutant frequency was higher than the M 3 mutant frequency. DISCUSSION

T h e technique described in this p a p e r a p p e a r s to be m o r e a c c u r a t e t h a n the m e t h o d used b y GAUL,C25,26) because the M 2 m u t a n t frequency is c o m p a r e d with the M 3 frequency of the same m u t a t i o n , not with the average frequency for a n u m b e r of mutations. T h e r e is little d o u b t t h a t some m u t a t i o n s segregate at frequencies other t h a n 20 or 25 p e r cent. F r o m the time STADLER (61) r e p o r t e d his results to the present, all of the mutagenic-statistical procedures for estimating n u m b e r of meristematic initial cells d e p e n d on the assumption that the cells c a r r y i n g the m u t a t i o n a n d the n o r m a l cells divide at the same average rate. I f the cells with the m u t a t i o n divide less frequently

in T a b l e 1. Meristems t h a t a c t u a l l y have only one cell initial are not subject to error due to division rate. These a c c o u n t for 69 p e r cent of the b r a n c h e s in this study. Estimates of meristems w i t h two initial cells a r e not subject to this error either which accounts for a n o t h e r 12 p e r cent or a total of 81 p e r cent of the estimates not subject to errors due to division rate. Thus, 19 p e r cent of the estimates could be subject to error, b u t in each case the original m e r i s t e m m u s t have h a d at least two initial cells. Thus, a n a d d i t i o n a l 11 per cent of the estimates are at most only one cell too high. O n l y 3 p e r cent of the estimates could be three or m o r e cells too high d u e to u n e q u a l cell division rates. T h e d a t a from 28 M x b r a n c h e s show a higher

NUMBER OF M E R I S T E M I N I T I A L S A F T E R SEED I R R A D I A T I O N m u t a n t f r e q u e n c y in the M 2 t h a n in the M s. OSONE (50) r e p o r t e d M s a n d M3 ratios a n d some of these were also larger in M 2 t h a n M s. Fujn(2a) contends it is possible for a c h r o m o s o m a l a b e r r a tion in a n o n - m u t a t e d cell to slow d o w n the division rate of the n o r m a l cells. This w o u l d cause the M~. frequency to be high. Fujn's(2s) hypothesis m a y e x p l a i n the discrepancies in this study. H o w e v e r , the M 3 p r o g e n y tests were sown on soil that was l a t e r found to be quite salty. G e n e r a l l y c h l o r o p h y l l m u t a n t s are less resistant to stress conditions t h a n n o r m a l seedlings, a n d the M 3 frequencies m a y have been low due to a differential m o r t a l i t y b e t w e e n m u t a n t a n d n o r m a l seedlings. Some b r a n c h e s seem to have arisen from as m a n y as five or m o r e initial cells, a n d others a r e c o m p o s e d o f division p r o d u c t s from only one initial. These results suggest t h a t either there is considerable cell e l i m i n a t i o n b y the level of i r r a d i a t i o n used, or the m a t e r i a l is genetically heterogeneous. T h e i r r a d i a t i o n level used in this s t u d y is low for flax. A n exposure to 30 k R causes less t h a n 3 p e r cent r e d u c t i o n in seedling height. O n the o t h e r h a n d , the seed used in these experiments was selected because the seed lots h a d b e e n purified. As p o i n t e d out above, r e d u c e d cell division rates with some m u t a t i o n s a n d not with others could also cause this effect. JACOBSEN (aS) found b a r l e y seed size influenced the n u m b e r of m e r i s t e m a t i c regions. P e r h a p s m e r i s t e m d e v e l o p m e n t in flax seeds is associated w i t h seed size. P r i m a r y stems a n d basal b r a n c h e s were not s e p a r a t e d in this study. W i t h the m e t h o d o f selecting b r a n c h e s at harvest, it is certain t h a t a m a j o r i t y o f basal b r a n c h e s were chosen. I t seems p r o b a b l e t h a t the p r i m a r y stem m a y be c o m p o s e d o f division p r o d u c t s o f m o r e cell initials t h a n w o u l d be true for the m a j o r i t y of basal branches. A d d i t i o n a l e x p e r i m e n t s in which the level o f i r r a d i a t i o n is v a r i e d a b o v e a n d below 30 k R , seed size is carefully controlled, a n d p r i m a r y stems are s e p a r a t e d from basal branches, should p r o d u c e d a t a t h a t could be used to distinguish b e t w e e n these alternatives. Acknowledgements--I wish to thank Dr. R. S. CALDECOTT and Mr. DAVID T. NORTH for X-ray treatment of the seeds. Dr. D. M. YERMANOS for furnishing some of the original purified seed. Miss

55

SUE TILSWORTH for technical assistance during this study. Dr. WESLEY BONN and Dr. T. W. WHITAKER for criticism and suggestions during the preparation of the manuscript.

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