Planta (Berl.) 124, 297--302 (1975) 9 by Springer-Verlag 1975
Studies of Seed Development in Pisum sativum I. Seed Size in R e c i p r o c a l Crosses D. l~oy Davies John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. Received 13 March; accepted 2 April 1975
Summary. Variation in cell population size and cell weight in the cotyledons are important factors in determining seed weight in Pisum sativum. The regulation of these parameters as well as that of growth rate has been examined. The use of reciprocal crosses between varieties of contrasting seed size has allowed the recognition of two systems of control--an intrinsic one dependent on the seed's own genotype, and the extrinsic control of the maternal environment. It is shown that the use of particular kinds of crosses can aid in discriminating the separate roles of sinks and sources as determinants of seed size. Introduction A l t h o u g h t h e seed is t h e e c o n o m i c a l l y significant c o m p o n e n t of m a n y of our m o s t i m p o r t a n t a g r i c u l t u r a l crops l i t t l e is k n o w n r e g a r d i n g t h e factors which r e g u l a t e its d e v e l o p m e n t a n d which limit progress in increasing its size. The m o v e m e n t of assimilate into seed has been i n v e s t i g a t e d in m a n y s y s t e m s t h o u g h even here t h e r e l a t i v e roles of t h e sinks in t h e seed, a n d of t h e sources of assimilate, in r e g u l a t i n g seed g r o w t h need to he clarified. B u t t h e n a t u r e of t h e factors which are i m p o r t a n t in d e t e r m i n i n g size a n d r a t e of increase in g r o w t h of t h e seed have y e t to be i n v e s t i g a t e d in cellular a n d molecular biological terms. The e x t e n t of t h e v a r i a t i o n in cell p o p u l a t i o n size, in D N A a n d R N A c o n t e n t s p e r cell, in R N A activities, in e n z y m e activities a n d in cell size need to be r e l a t e d to seed or sink size, a n d t h e control of these p a r a m e t e r s u n d e r s t o o d . Such studies are n e c e s s a r y if t h e p l a n t b r e e d e r is to recognise some of t h e m o s t i m p o r t a n t c o n s t r a i n t s t h a t p r e s e n t l y limit progress in increasing seed size, a n d if he is to define his objectives m o r e clearly. Gcnetical a n a l y s e s of t h e control of seed d e v e l o p m e n t a n d size, a n d i n d e e d of m a n y seed c h a r a c t e r s can be difficult in m a n y genera because of t h e occurrence of m a t e r n a l effects. These are seen n o t o n l y for size b u t also for such other i m p o r t a n t economic c h a r a c t e r s as p r o t e i n (Leleji et al., 1972) oil, (Singh a n d H a d l e y , 1968) a n d m e t h i o n i n e (Porter, 1972) contents. B u t a l t h o u g h t h e y m a y complicate m a t t e r s on t h e one hand, a n e x p e r i m e n t a l l y useful consequence of m a t e r n a l effects is t h a t in m a n y genera, a n d t h e Leguminosae are a good e x a m p l e of this, t h e seed which arise from reciprocal crosses can h a v e c o m p l e t e l y different p h e n o t y p e s in spite of h a v i n g i d e n t i c a l genotypes. (In such genera t h e y have t h e same g e n o t y p e because t h e m a t u r e seed a p a r t from t h e coat is c o m p o s e d e n t i r e l y of ~F1 tissue.) This difference in p h e n o t y p e is i m p o r t a n t because it should lead to a b e t t e r u n d e r s t a n d i n g of t h e factors which control seed d e v e l o p m e n t , - - t h o s e which are intrinsic of t h e seed a n d its own g e n o t y p c on t h e one h a n d a n d t h o s e t h a t are r e l a t e d to t h e m a t e r n a l p a r e n t on t h e other. Conversely a c o m p a r i s o n of selfed a n d crossed seed on a
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given p l a n t is also instructive, because if m a t e r n a l effects are present, t h e n b o t h t y p e s of seed should h a v e a similar p h e n o t y p e a l t h o u g h d e r i v e d from dissimilar genotypes. W e are e x p l o i t i n g reciprocal crosses as e x p e r i m e n t a l systems for s t u d y ing t h e d e v e l o p m e n t of t h e seed in t h e pea, Pisum sativum. I n t h e p r e s e n t p a p e r this a p p r o a c h is i l l u s t r a t e d using d a t a on t h e g r o w t h of cotyledons, a n d in t h e following p a p e r (Davies, 1975) using d a t a on r i b o s o m a l R N A contents of c o t y l e d o n cells. Materials and Methods The following six genotypes of peas were intercrossed in all possible combinations, including reciprocals. J I 261 (a primitive form collected in Turkey); J I 181 (Keeran Pea from Nepal); JI281 (a primitive form collected in Sudan); JI 407 (ev Victory Freezer); JI430 (cv Greenshaft) ; J I 774 (a selection from Daehnfeldt's line Da 1608). Cell numbers were scored alter hydrolysing cotyledons of a particular developmental age in 5N HC1 at 20~ for 60 rain and washing them three times in water. After this the tissues readily gave a single cell suspension. Twelve replicate aliquots (each of 100-300 cells) per variety were counted. A stage was chosen at which cell division had been completed and before the seed had become too mature for cell separation.
Results The d r y seed weights after equilibration, of p a r e n t s a n d h y b r i d s are given in Table 1. Since t h e r e can be a m b i g u i t y a b o u t seed generations, t h e y will be defined here: F 1 seed is t h a t which has an F 1 g e n o t y p e a n d is p r o d u c e d on t h e p a r e n t a l plant. Table 1. Mean dry weight (in g) per seed of parental and F 1 seed parent 261
407
181
430
281
774
0.041
0.068
0.063
0.049
0.051
0.076
407
0.258
0.280
0.269
0.230
0.267
0.231
181
0.071
0.089
0.073
0.111
0.077
0.104
430
0.228
0.234
0.246
0.224
0.280
0.306
281
0.106
0.174
0.161
0.105
0.195
774
0.308
0.238
0.236
0.269
0.194
9 parent 261
0.256
The clear influence of t h e m a t e r n a l p a r e n t on t h e d r y weight of a n y F 1 seed is seen in all i n s t a n c e s ; t h i s gives rise to a s i m i l a r i t y of all offspring of a n y given m a t e r n a l p a r e n t , a n d to p r o n o u n c e d reciprocal differences (see Fig. 1). There is nevertheless a n influence of t h e seed's own g e n o t y p e ; e.g. J I 774 • gives a significantly larger seed t h a n J I 774 selfed. As a first step t o w a r d s u n d e r s t a n d i n g t h e n a t u r e of t h e differences which underlie v a r i a t i o n in seed weight, t h e r e l a t i o n s h i p between t h e n u m b e r of ceils in t h e cotyledons a n d m a t u r e d r y weight was d e t e r m i n e d for n i n e t e e n varieties. There was a clear linear r e l a t i o n s h i p ( P < 0 . 0 0 1 ) b e t w e e n t h e m (Fig. 2 ) , - - a v a r i e t y w i t h larger seed has a larger cell p o p u l a t i o n in t h e cotyledons K n o w i n g t h e cell n u m b e r a n d t h e d r y weight of t h e seed, t h e average d r y weigt p e r cell
Seed Development in Pisum I.
299
Fig. 1. Seed size in variety J I 430 (P1), J I 181 (P2) and in their reciprocal F 1 hybrids. J I 430 • 181 (left) and J I 181 • 430 (right) o
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Fig. 2. Relationship between number of cells in the cotyledon and dry seed weight for nineteen varieties of peas could be calculated. A significant ( P ~ 0.001) r e l a t i o n s h i p was f o u n d b e t w e e n t h e m e a n d r y cell weight a n d seed size (Fig. 3). Thus t h e larger seeded varieties show a n increase b o t h in cell p o p u l a t i o n a n d in t h e a v e r a g e d r y weight of t h e cells in t h e cotyledons. 20
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D.R. Davies 20 --~15 o •
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I I I 0.2 0 i3 0.4 0.5 Dry wt./seed (g)
Fig. 3. Relationship between mean dry weight of cotyledon cells and dry seed weight for nineteen varieties of peas Table 2 Genotype
Cell no. per pair of cotyledons
Peak wet wt. per cell ( • 10-~ g) a
Dry wt. per cell at maturity ( • 10-6 g)
JI JI JI JI
1.79 • 1.97 • 1.42 • 1.26 •
0.2961 0.1878 0.1479 0.0953
0.0811 0.0888 0.0704 0.0555
430 430 • 181 181 • 430 181
106 106 106 106
a Derived from data in Fig. 4. The n u m b e r s a n d weight of cells in t h e seed of reciprocal crosses were n e x t e x a m i n e d , The two v a r i e t i e s used were J I 430 a n d J I 181 a n d t h e values for p a r e n t s a n d h y b r i d s are given in Table 2. There was a difference in cell n u m b e r p e r cotyledon between reciprocal F 1 h y b r i d s i n d i c a t i n g a n effect of t h e m a t e r n a l p a r e n t ; however n e i t h e r h y b r i d e x a c t l y r e s e m b l e d its m a t e r n a l p a r e n t , showing t h a t t h e genetic c o n s t i t u t i o n of t h e seed also h a d a role in d e t e r m i n i n g cell p o p u l a t i o n size. P e a k wet weight p e r cell d i d n o t show as m a r k e d a m a t e r n a l s i m i l a r i t y ~ h e r e t h e seeds' own g e n o t y p e was t h e p r i m a r y d e t e r m i n a n t . A l t h o u g h values for d r y weight p e r cell showed some r e s e m b l a n c e to t h e m a t e r n a l p a r e n t , t h e role of t h e seed's own g e n o t y p e was a g a i n a p p a r e n t . The d a t a from one of four e x p e r i m e n t s on t h e r a t e of c o t y l e d o n d e v e l o p m e n t in these same p a r e n t s a n d h y b r i d s are shown in Fig. 4. The p e r i o d s t u d i e d r e p r e s e n t s t h a t in which cell division has ceased. Much m o r e r a p i d increases in seed wet weight occurred in J I 430 t h a n J I 181; t h e t o t a l t i m e of d e v e l o p m e n t in t h e former was also p o s s i b l y a few d a y s longer t h o u g h t h e effect of this was negligible in c o m p a r i s o n w i t h t h e effect of t h e difference in g r o w t h rate. I n o t h e r g e n o t y p e s t h e p e r i o d of d e v e l o p m e n t is a n i m p o r t a n t d e t e r m i n a n t of seed size (D. R. Davies unpubl.). I n all experiments, J I 430 has shown t h e g r e a t e s t increase in seed wet weight of all four g e n o t y p e s b u t this is l a t e r followed b y a larger loss of w a t e r The r a t i o of wet weights p e r cell for J I 430: J I 181 was 3.10, whereas for d r y weights i t was 1.46 (Table 2). This a b i l i t y of J I 430 cells to a c c u m u l a t e large q u a n t i t i e s of w a t e r was n o t as p r o n o u n c e d in t h e J I 430 • 181
Seed Development in Pisum I.
301
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0.5 0.4 ",u 0"3
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9
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22
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30 34 38 Time after flowering
r
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Fig. 4. Mean weight of cotyledons (wet A, dry B) at different developmental stages. JI 430 o; J I 430 • 181 []; J I 181@; J I 181 X430 9
seed, indicating that this character is more influenced by the cells' own genotype than by the parental plant on which the seeds are borne A difference between reciprocal crosses in growth rate, and in wet and dry seed weight was observed (Fig. 4) indicating again the role of the maternal parent, but the lack of identity between maternal parent and offspring also signified the role of the seed's own genotype. Discussion The present data indicate that the pathway to the evolution of large seeds in P i s u m has involved an increase in both cell number and weight. This could have been achieved through a change in the seed's own (intrinsic) regulatory system such t h a t its genotype now specifies more and larger cells. Alternatively or additionally the increase in number and weight of cells has occurred because of extrinsic factors such as greater photosynthetic efficiency, different partitioning of photosynthate, and extrinsic (hormonal) regulation of cell division and growth. I t is in helping to elucidate these two regulatory systems that the reciprocal crosses prove useful. I n the crosses of J I 430 and J I 181 there was a marked difference in the wet weight per cell of maternal and F 1 seed and it is a reasonable conclusion therefore that this character is primarily regulated by the seed's own genotype (intrinsic control) : for cell dry weight and cell number on the other hand there was a greater similarity between maternal parent and F1, and an influence of both maternal tissue (extrinsic control) and the seed's own genotype has to be assumed. However the relative roles of these two regulatory systems does differ in different 20*
302
D. t~. Davies
instances and shows itself as a greater or lesser similarity of a given F 1 seed to its maternal parent. For example if we examine the dry seed weights of all progeny of one maternal parent, J I 774, (see table 1) then we see t h a t the J I 774• genotype showed a very marked positive deviation from t h a t of the maternal parent, and this deviation was greater than t h a t shown by the other progenies of J I 774. Fls of this kind, showing a marked positive deviation from the maternal parent value will be those which have a superior intrinsic system, since all the Fls on a common maternal parent plant have a common source of photosynthate and common hormonal regulatory systems. Such comparisons of a range of Fls from a common maternal parent m a y provide a method of selecting for improved intrinsic "sink capacity", although it is possible that particular sink genotypes would perform optimally in combination with particular source genotypes. I n other words specific sink-source genotype interactions m a y occur; this is the subject of further investigation. Turning now to the extrinsic control, the evolutionary advantage of its existence and of the occurrence of maternal effects is readily understood; the greater the similarity between an F 1 seed and its maternal parent, the greater the likelihood that the F 1 seed will succeed in the environment in which its parent is growing and to which it is presumably adapted. In an inbreeding species such as P. sativum this of course is not relevant, and the maternal effect is presumably a relic of an outbreeding ancestry. In genera such as the Gramineae, a similarity of F1 seed to t h a t of its maternal parent is achieved through the role of the triploid endosperm, with its double genomie contribution from the maternal parent. But in the diploid cotyledon tissues which comprise most of the seed in the Leguminoseae this maternal similarity has to be achieved by some other method. The basis of the maternal effect has been tacitly interpreted in the past in nutritional terms; here two other alternatives are proposed. I t could be due to a regulation of gene activity by the maternal tissues or it could be due to a selective activation of alleles derived from the maternal parent (Davies, 1973). Data obtained from studies of a number of parameters important in seed development will be used to t r y and discriminate between these hypotheses; the following paper will consider t h a t on ribosomal RNA. I t is hoped that this illustration of the exploitation of reciprocal crosses will be of value to physiologists studying sink-source relationships in other crops. I am grateful to Miss Y. Allen for technical assistance.
References :Davies, I). P~. : I)ifferential activation of maternal and paternal loci in seed development. Nature (Lond.) New Biol. 245, 30-32 (1973) Davies, I). R. : Studies of seed development in Pisum sativum. II. Ribosomal t~NA contents in reciprocal crosses. Planta (Berl.) 124, 303-309 (1975) Leleji, 0. I., I)ickson, M. H., Crowder, L. V., Bourke, J. B. : Inheritance of crude protein percentage and its correlation with seed yield in beans, Pha~eolus vulgaris L. Crop. Sci. 12, 168-171 (1972) Porter, W. M. : Genetic control of protein and sulfur contents in dry bean, Phaseolu8 vulgaris. Phi) thesis. Purdue University (1972) Singh, B. B., Hadley, H. It. : Maternal control of oil synthesis in soybeans, Glycine max (L.) Crop Sci. 8, 622-625 (1968)