Compositional analysis of in vitro matured soybean seeds

Plant Science Letters, 27 (1982) 129--135 Elsevier Scientific Publishers Ireland Ltd.

129

COMPOSITIONAL A N A L Y S I S OF IN VITRO MATURED SOYBEAN SEEDS*

F.C. HSU** and R.L. OBENDORF***

Department of Agronomy, New York State College o f Agriculture and Life Sciences, Cornell University, Ithaca, N Y 14853 (U.S.A.) (Received December 23rd, 1981) (Revision received April 2nd, 1982) (Accepted April 12th, 1982)

SUMMARY

Soybean seeds at 50--70 mg and 3 0 0 - - 5 0 0 mg stages were excised and grown to maturity in a chemically defined medium. Total protein, lipid, starch, free amino acids, D N A and R N A contents were analyzed in seeds matured in vitro and in seeds matured on plants. The comparison of these seed constituents indicates that seed development in vitro simulates natural seed development.

INTRODUCTION

In contrast to the extensive research on seed germination, relatively little attention has been paid to various problems of seed development [1]. One major difficulty in studying seed development is the lack of techniques which allow the manipulation of various factors that may affect seed development. Seed development in higher plants is regulated by complex interactions between the maternal plant and the environment, the developing seed and the maternal plant, and among different tissues within the seed itself [2]. With developing seeds attached to the maternal plant, it is very difficult to manipulate various factors singly and over a long enough time period to observe their effects on general or specific aspects

*This work was supported by the United States Department of Agriculture-Science and Education Administration/Cooperative Research Special Grant 801-15-48. Agronomy Department Series Paper 1412, New York State College of Agriculture and Life Sciences, CorneU University, Ithaca, NY 14853, U.S.A. **Present address: Section of Plant Biology, Cornell University, Ithaca, NY 14853, U.S.A. ***To whom correspondence should be sent. 0304--4211/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.

130 of seed maturation. For example, manipulations of environmental parameters such as temperature [3] would affect all aspects of physiology and development of all parts of the plant. Effects of these manipulations on seed development are either mixed with, or even masked by, the indirect effects on the whole plant. A different technique which provides ready access to a wide range of easy and specific manipulations is needed. Aseptic plant tissue culture techniques have been successfully applied to a wide range of excised tissues and organs including immature embryos and fruits [4--7]. These techniques can provide good accessibility and specificity for many kinds of manipulations. In recent years, Obendorf et al. [8,9] have successfully developed such an in vitro culturing technique to grow immature soybean pods and seeds to maturity. Young seeds at 50 mg fresh weight or greater can be cultured to maturity in intact pods or free of pod tissues. After proper maturation in culture medium, seeds can be removed, air dried and subsequently germinated. Germinability is generally above 80%. Seeds matured in pod culture are visually indistinguishable from commercial seeds. However, in addition to the morphological resemblance to commercial seeds and high germinability, further evidence is needed to ascertain how closely the culture system in vitro simulates natural seed development. Unlike c o t t o n ovules which differentiate lint fibers as they mature in culture [10], soybean seeds do n o t produce morphologically distinct maturation structures so that other seed characteristics need to be examined. This paper reports a comparative analysis of protein, lipid, starch, free amino acids, DNA and R N A of soybean seeds matured in vitro and seeds grown on plants in the greenhouse. Seeds matured in vitro accumulated protein, lipid, D N A and R N A at concentrations comparable to seeds matured on plants. Starch declined to low levels at maturity in vitro as in normal seed maturation. Seeds matured in vitro contained higher concentrations of free amino acids. Culture of small seeds (initially at 50--70 mg fresh weight) to maturity in vitro resulted in less dry weight and DNA accumulation and a lower concentration of lipid than seeds matured on plants. MATERIALS A N D METHODS

Plant source and culturing techniques Soybean plants, Glycine max (L.) Merrill cultivar Chippewa 64, were grown in a greenhouse thermostated at 27°C day/21°C night with 14 h day -1 supplementary incandescent lighting at approx. 740 gE • m -2 • s -1 (approx. 225 W • m -2) [11]. Only three-seeded pods were used for culturing experiments. Pods were selected for uniformity either by the size of seed shadow or b y pod thickness. Seeds at 50--70 mg fresh weight would give a shadow about 7 m m in length which is clearly discernible by holding the p o d against a bright light. These seeds would shadow a b o u t one-half o f the p o d lumen width. Pods containing seeds of 300--350 mg fresh weight

131

each were usually at a thickness of around 8 mm. The two seed sizes occured at a b o u t 3 and 5 weeks after flowering. Pods with seeds of appropriate sizes were excised from the plant, brought back to the lab and used for culturing within 1 h. The pod surface was sterilized (20 min) with 1% NaOC1 solution containing 0.01% Tide (commercial detergent) as a surfactant. Aseptic transfers of sterilized pods or excised seeds into 125-ml Erlenmeyer culturing flasks with 20 ml of medium were performed in a laminar flow hood. A revised Linsmaier and Sk-5"6~-}'nedium [ 12] was used for all cultures. In addition to 50 g of sucrose and 9.13 g of glutamine, each liter of culture medium contained 746 mg KC1, 1850 mg MgSO4 • 7H20, 84.4 mg MnSO4 • H:O, 43 mg ZnSO4 • 7H20, 0.125 mg CuSO4 • 5H20, 440 mg CaC12 • 2H20, 0.83 mg KI, 0.025 mg CoCI: • 6H20, 170 mg KH:PO4, 6.2 mg H3BO3, 0.252 mg Na:MoO4 • 2H20, 27.85 mg FeSO4 • 7H20, 37.25 mg Na2EDTA, 2 mg glycine, 0.5 mg nicotinic acid, 0.4 mg thiamine • HC1, 0.1 mg pyridoxine • HCI and 100 mg myo-inositol. No growth regulators were included in this medium. Culture flasks containing pods or seeds in medium were placed on shakers under continuous fluorescent light at 350--400 gE • m -2 . s -! (approx. 82 W • m -s) for the entire culturing period. Temperature of the culture medium was maintained at 25°C. Seeds remained in the original medium until maturity.

Me thods o f seed analysis Seeds were harvested from culture flasks when the surface of the cotyledons became light yellow. Seeds from pods in culture were harvested when the pods were completely brown. Cotyledons were separated from the other seed parts, quickly frozen in liquid nitrogen and stored a t - 1 7 ° C . A sample of seeds from each batch was air dried for a germination test. Frozen cotyledon samples were pulverized with a mortar and pestle on dry ice. The pulverized samples were then homogenized in a Duall ground glass tissue grinder and processed for protein, lipid, starch, free amino acids, DNA and R N A quantifications according to Madison et al. [13]. A revised Lowry m e t h o d was used for protein assay [14]. Free amino acids were assayed b y a modified ninhydrin m e t h o d [15]. DNA analysis was b y a revised diphenylamine m e t h o d [16]. Methods for starch, lipid and R N A assays were the same as used b y Madison et al. [13]. Sugars and cell wall constituents were n o t analyzed. RESULTS AND DISCUSSION

Seeds reached 50--70 mg fresh weight a b o u t 300--350 mg fresh weight a b o u t 5 weeks after matured seeds were harvested 7--8 weeks after 50--70 mg fresh weight seed size, seeds matured

3 weeks after flowering, flowering and greenhouse flowering. Starting at the in p o d culture were harvest-

35 10.0 10.2 6.7 0.174 2.20

Protein Lipid Starch Amino acids DNA RNA 40

0.5 1.1 0.5 0.022 0.17

21.9 10.8 0.9 0.015 0.89

98 39.4 22 10.6 0.8 0.015 0.87

± ± ± ± ±

0.7 0.3 0.1 0.02 0.07 0.001 0.03

± 1

± ± ± ± ± + ±

± 0.6 ± 0.6 -+ 0 . 5 ÷ 0.001 ± 0.09

± 2

± 5 ± 0.5 ± 2 ± 0.1 ± 0.5 +- 0 . 0 0 1 ± 0.05

12.5 1.1 0.9 0.018 0.64

32

63 20 8 0.7 0.57 0.011 0.39

± ± ± ± ±

0.7 1.0 0.3 0.001 0.02

± 8

13.8 0.9 1.3 0.016 0.81

38

± ÷ ± ± ±

2.0 0.8 0.1 0.002 0.07

± 3

mg/2 cotyledons c + 11 93 ± 5 ± 4 36 ± 4 ± 1 13 ± 3 ± 0.7 0.8 ± 0.7 ± 0.07 1.2 ± 0.2 +- 0 . 0 0 5 0.015 ± 0.001 ± 0.01 0.76 ± 0.09 mg/100 mg dry weight c

Seed culture starting at 50--70 mg FW

51

29 17.7 1.0 0.6 0.007 0.49

325 95 58 3.2 1.9 0.024 1.58

+± ± ± ± ±

1 5.4 0.1 0.2 0.002 0.02

± 24 ± 9 ± 20 ± 0.2 ± 0.4 ± 0.007 ± 0.15

Seed culture starting at 300 mg FW

25

35 20.6 0.3 0.12 0.011 0.40

202 70 42 0.6 0.24 0.022 0.80

± + ± ± ± ±

3 0.3 0.1 0.01 0.001 0.02

± 14 ± 1 ± 4 ± 0.1 ± 0.01 ± 0.001 ± 0.02

Greenhouse matured seeds

40

apods containing immature seeds at 50--70 mg or 300--350 mg fresh weight each (approx. 3 and 5 weeks after flowering) were removed from greenhouse plants. Composition of these immature seeds was determined. b c o m p o s i t i o n w a s d e t e r m i n e d a f t e r s e e d m a t u r a t i o n u n d e r f o u r c o n d i t i o n s . I m m a t u r e s e e d s i n i t i a l l y at 5 0 - - 7 0 m g f r e s h w e i g h t w e r e g r o w n t o m a t u r i t y in v i t r o in d e t a c h e d p o d s f o r 4 2 d a y s o r as s i n g l e s e e d s f r e e o f p o d t i s s u e s f o r 2 4 d a y s . I m m a t u r e s e e d s i n i t i a l l y a t 3 0 0 - - 3 5 0 m g f r e s h w e i g h t w e r e g r o w n t o m a t u r i t y in v i t r o as s i n g l e s e e d s f r e e o f p o d t i s s u e s f o r 28 d a y s . O t h e r s e e d s r e m a i n e d o n greenhouse plants until seed maturity (7--8 weeks after flowering). CMean ± S.D. o f t h e p o p u l a t i o n .

5.4 1.9 0.5 0.54 0.36 0.009 0.12

Pod culture starting at 50--70 mg FW

300 mg FW

50--70 mg FW

86 Mature seeds b

41

Immature seeds from greenhouse a

234

Seed number

OF SOYBEAN SEEDS

Dry weight Protein Lipid Starch Amino acids DNA RNA

COMPOSITION

TABLE I

b~

133 ed after 42 days in culture and showed 75% germination while seeds matured in seed culture were harvested after 24 days in culture and yielded 100% germination. Starting at 300--350 mg fresh weight seed size, seeds matured in seed culture were harvested after 28 days in culture and showed 100% germination. Constituents analyzed in seeds matured in seed culture were comparable with those in seeds matured on plants in the greenhouse (Table I). Soybeans have the distinctive features of high protein and lipid c o n t e n t but very low starch levels at maturity. The starch c o n t e n t is maximum (about 10% of dry weight) in y o u n g seeds and declines to below 1% at seed maturity while protein and lipid accumulate to maturity (Table I). The pattern is the same in greenhouse or field environments [13,17]. These changes also occurred in the seeds matured in seed culture (Table I). Seeds initially at 50--70 mg fresh weight accumulated 18--34 mg of protein and 8--13 mg of lipid during pod or seed culture. Seeds initially at 300--350 mg fresh weight accumulated 55 mg of protein and 36 mg of lipid during seed culture. In all cases starch levels declined to a b o u t 1% of dry weight for seeds matured in culture (Table I) illustrating normal seed maturation events. Protein concentration (mg/100 mg dry weight) in seeds matured in vitro was comparable to that of seeds matured on plants. Lipid concentration in seeds matured in seed culture (initially at 300--350 mg fresh weight) was near that of seeds matured in the greenhouse. Although lipid concentration increased during pod or seed culture of 50--70 mg fresh weight seeds, the increase in lipid concentration was not as great as occurred on the plant despite a 16--26-fold increase in lipid accumulation (Table I). Similar patterns of protein and lipid accumulation were observed during culture o f soybean cotyledons [ 12]. Amino acids accumulated in cultured seeds to higher concentrations and amounts than in seeds on plants (Table I). This result is probably a reflection of the high glutamine concentration in the culture medium. DNA accumulated linearly throughout seed formation at 0.4 pg • d a y - ' • 2 c o t y l e d o n s - ' in the greenhouse (Table I). Cultured seeds also accumulated DNA. Seeds initially at 300---350 mg fresh weight accumulated DNA during seed culture at a b o u t the same rate as seeds growing on plants in the greenhouse while dry matter accumulated at a faster rate in the cultured seeds. Seeds initially at 50--70 mg fresh weight accumulated DNA in proportion to final seed weight. Averaging across cultivars, Egli et al. [18] estimated that the maximum cell number in soybean cotyledons occurred when seeds had attained 24% of m a x i m u m seed dry weight corresponding to the beginning of the phase of linear dry matter accumulation. By this guide, m a x i m u m cell numbers in our seeds would occur at 48 mg/seed dry weight or a b o u t 200 mg/seed fresh weight. Endoredupiication of nuclear DNA [19] therefore accounts for net DNA accumulation above approx. 13 pg/ seed in seeds on greenhouse-grown plants. Using data of Egli et al. [18], we calculate that our seeds at 50--70 mg fresh weight (just beginning cell

134 expansion) should contain 70--80% of the maximum number of cells. We do not know how long cell division continues in cultured seeds. Since cell division had ceased before the 300--350 mg seeds were placed in culture, the near normal accumulation of DNA during culture probably reflects accumulation by endoreduplication. DNA accumulation during culture of initially 50--70 mg fresh weight seeds may reflect cell division and/or endoreduplication. A general decrease of RNA concentration from early embryogeny toward seed maturity has been observed in four different legumes [ 13]. The same trend occurred in our cultured seeds (Table I). Accumulation of RNA by cultured seeds was greatest in seeds with the largest seed weight. The mature seed size attained in pod and seed cultures of initially 50--70 mg fresh weight seeds was much smaller than greenhouse-grown seeds. This general reduction of mature size in culture has been reported in many cases of ovule or fruit culture [7,20]. It appears that the complex requirements of nutrients and other growth factors of very young seeds are not totally satisfied by the culture medium or by transport of medium to the growing seed. On the other hand, older seeds at 300--350 mg fresh weight in this study seemed to have less stringent growth requirements, and were able to exploit the vast amount of nutrients in the medium to reach 325 mg dry weight at maturity. This study and two other reports on pea seed culture [20,21] all indicate that exogenous growth regulators are not required for seed maturation. This is in sharp contrast to the maturation of cotton ovules in vitro which requires exogenous growth regulators [ 10]. It appears that soybean and pea seeds either are capable of producing all growth regulators necessary for their development [20] or are relatively less responsive to exogenous growth regulators. REFERENCES

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