Plant Cell Reports (1984) 3:146-148

Plant Cell Reports © Springer-Verlag 1984

In vitro culture of pods from annual and perennial Medicago species J. W. Wang, E. L. Sorensen, and G. H. Liang Kansas Agricultural Experiment Station, Department of Agronomy and the U.S. Department of Agriculture, Agricultural Research Service, Kansas State University, Manhattan, KS 66506, USA Received February 16, 1984 / Revised version received May 31, 1984 - Communicated by J. Widholm

Abstract Because most i n t e r s p e c i f i c Medica9o embryos abort b e f o r e t h e y can be e x c i s e d and c u l t u r e d , our objective was to grow young pods in v i t r o . V a r i o u s media were used to grow three-day-old pods of annuals [diploids, M. b l a n c h e a n a B o i s s . , ivi. d i s c i f o r m i s DC., t e t r a p l o i d M. s c u t e l l a t a (L.) Mill.] and p e r e n n i a l s ( d i p l o i d M. f a l c a t a L., t e t r a p l o i d Wl. sativa L.). Few pods o f p e r e n n i a l species grew to maturity on media containing modified Hoagland's plus 1% glucose or sucrose w i t h or w i t h o u t 5% p o t a t o e x t r a c t . I n c r e a s i n g sucrose to 6% increased the percentage of M. s a t i v a pods t h a t produced mature seeds. On DM ( d i f f e r e n t i a t i o n medium), the best medium, the p e r c e n t a g e of pods p r o d u c i n g v i a b l e seeds was: iv[. b l a n c h e a n a (82), M. d i s c i f o r m i s (81), E. scutellata (48), ~. sativa (63), M. f--alcata ( 1 5 ) . 0M p l u s i ppm i n d o l e a c e t i c ~-r g i b b e r e l l i c acid did not enhance seed production. A b b r e v i a t i o n s : D i f f e r e n t i a t i o n medium (DM), DM plus i ppm i n d o l e a c e t i c a c i o (DMI), DM p l u s 1 ppm g i b b e r e l l i c acid (DMG). Introduction Post-fertilization o v u l e a b o r t i o n was the main b a r r i e r in i n t e r s p e c i f i c crosses between perennial and annual ~vJedicago s p e c i e s (Sangduen et a l . , 1983b). Ovule a b o r t i o n a l s o caused low s e l f fertility (Sayers and ivlurphy, 1966, Cooper and d r i n k , 1940). Sangduen et al. (1983a) determined that insufficient n u t r i e n t breakdown and transport in a l l n u t r i t i v e tissues at an early developmental stage of the embryo c o n t r i b u t e d to abortion of ovules in wide crosses. R e a r i n g the o v u l e or e n t i r e o v a r y in v i t r o may be more s u c c e s s f u l than embryo culture. Rao and Rangaswamy (1972) suggested that p i s t i l culture may prevent p r e c o c i o u s a b s c i s s i o n of the o v a r y . R i c h a r d s and Rupert (1980) s t a t e d that removing r e p r o d u c t i v e organs from the parent plant could aid in avoiding p o s t - f e r t i l i z a t i o n barriers. Maheshwari and R a n g a s w a m y (1963) r e v i e w e d examples of s u c c e s s f u l embryo c u l t u r e in a number of plant species. They indicated that the degree of success increased directly w i t h the amount of maternal tissue retained.

Rao and Rangaswamy (1972) suggested two conditions n e c e s s a r y f o r successful p i s t i l culture: ( i ) the a p p r o p r i a t e n u t r i e n t medium and (2) retention of f l o r a l parts that envelop the p i s t i l . The effect of the c a l y x on f r u i t development was investigated in Hyoscyamus niger L. ( B a j a j , 1 9 6 6 ) , A l l i u m cepa L. (Guha and d o h r i , 1 9 6 6 ) , I b e r i s amara L . ( M a h e s h w a r i and L a l , 1961), Sisymbrium i r i o L. (Pareek et a l . , 1 9 8 0 ) , Nicotiana rustica L. (Rao and Rangaswamy, 1972) and some T r i f o l i u m species ( R i c h a r d s and R u p e r t , 1980). The contribution of the c a l y x was presumed to be n u t r i t i v e rather than s t i m u l a t o r y (Maheshwari and Lal, 1961). The removal of the c a l y x r e s u l t e d in impaired growth, such as smaller fruits, decreased seed number, or seedless fruits t h a t m i g h t have been caused by shortage of some n i t r o g e n o u s compounds e s s e n t i a l to f r u i t d e v e l o p m e n t ( N i t s c h , 1963; Guha and Johri, 1966). The a d d i t i o n of a u x i n and cytokinin to the medium induced v a r i o u s e f f e c t s . W i t h g i b b e r e l l i c acid, i n d o l e a c e t i c a c i d , and k i n e t i n , A l l i u m cepa L. developed larger fruits i n v i t r o than In s i t u (Guha and J o h r i , i 9 6 6 ) . Ovary growth of Iberis a m a r a L. and H y o s c y a m u s n i g e r L. improved with indoleacetic acid (Maheshwari and Lal, 1961; Bajaj, 1966). In T r i f o l i u m s p e c i e s , growth regulators enhanced embryo growth in culture, though requirements varied f o r each s p e c i e s and t h e i r crosses (Richards and R u p e r t , 1980). Yeh and Bingham (1969) indicated that gibberellic acid might be useful in enhancing growth and seed p r o d u c t i o n of s p e c i f i c a l f a l f a stocks in vivo. The o b j e c t i v e s of our study were: (1) to determine the f e a s i b i l i t y of in v i t r o c u l t u r e of IvJedica~ pods w i t h a t t a c h e d c a l y x at an early development s t a g e , and (2) to investigate effects of g i b b e r e l l i c acid and i n d o l e a c e t i c acid additions to DM medium (Liang et al., 1982) on in v i t r o growth of e x c i s e d pods from annual and perennial Medicago species. Materials and Methods Pods from f i v e Medicago s p e c i e s 3 were used for in v i t r o culture: 3Plants of each s p e c i e s used in experiments were g r o w n f r o m seed produced in a g r e e n h o u s e at Manhattan, KS.

147 M. b l a n c h e a n a B o i s s . - annual d i p l o i d (i) (2n=2x=16). (2) !vl. d i s c i f o r m i s DC. annual d i p l o i d (2n=2x=16). annual (3) ~. s c u t e l l a t a (L.) Mill. t e t r a p l o i d (2n=4x=32). (4) M. f a l c a t a L. e x p e r i m e n t a l s y n t h e t i c KS94 - perennial d i p l o i d (2n=2x=16). (5) ~. s a t i v a L. e x p e r i m e n t a l s y n t h e t i c s perennial KS11 and KSi53 anO c l o n e SP9-16 tetraploiOs (2n=4x=32). For a l l t r i a l s , annual species were s e l f - p o l l i n a t e d by g e n t l y s q u e e z i n g f l o w e r s between f i n g e r s . P e r e n n i a l s w e r e c r o s s - p o l l i n a t e d by hand using a toothpick to t r i p flowers and transfer pollen. Pods were s u r f a c e s t e r i l i z e d as follows: (I) the faded c o r o l l a was removed, (2) the pod, calyx, and p e d i c e l were p l a c e d in 10% Clorox (0.525% sodium hypochlorite) s o l u t i o n f o r lO rain and agitated occasionally, (3) the m a t e r i a l was r i n s e d in autoclaved d i s t i l l e d water for I rain, and (4) excess w a t e r was removed w i t h s t e r i l i z e d f i l t e r paper b e f o r e t r a n s p l a n t i n g the pods to media. Each petri d i s h (100x15 ml) contained 25 ml of medium. Six or less pods, w i t h t h e i r p e d i c e l s inserted into the medium, were p l a c e d in each petri dish, which was s e a l e d w i t h Parafilm. Petri dishes w~re maintained in an i n c u b a t o r at approximately 25~C with a 16-h photoperiod at 0.94 umol m-2s -I . A l l media c o n t a i n e d 0.8% O i f c o agar and were autoclaved at i21~C under 15 psi p r e s s u r e f o r 20 min. The pH was a d j u s t e d to 6.0 w i t h sodium h y a r o x i d e and hydrochloric acid. E f f e c t of i ~ i n e r a l Salts, Sugar, and Potato Extract on G r o w t h of Medicaqo Pods i n v i t r o : FI. s a t i v a (KS11) and d i p l o i d M. f a l c a t a were grown in a g r e e n h o u s e . Pods were excised three t h r o u g h seven days after p o l l i n a t i o n . Media tested were: (a) m o d i f i e d Hoagland's solution (Hoagland and Arnon, 1950; Johnson et a l . , 1957; Harper and P a u l s e n , 1 9 6 9 a , b ) p l u s 1% glucose; (b) modified H o a g l a n d ' s s o l u t i o n plus 1% glucose and 5% potato extract; (c) m o d i f i e d Hoagland's solution plus 1% s u c r o s e ; and (d) modified Hoagland's solution plus 1% sucrose and 5% p o t a t o extract. The number of pods studied exceeded 300 for each medium. E f f e c t o f Sucrose C o n c e n t r a t i o n on Growth of Medica.qo sativa ( K S 1 5 3 ) Pods i n v i t r o : T h r e e - d a y - o l d excised pods from KS153 were cultured on m o d i f i e d H o a g l a n d ' s c o n t a i n i n g 2, 4, or 6% sucrose. F i f t y pods were studied for each medium. E f f e c t s o f Three Growth Media on Seed Production of in V i t r o Grown Pods from Annual and Perennial Medicago S p e c i e s : M. blancheana, M. disciformis, M. s c u t e l l a t a , M. f a l c a t a , and M. s a t i v a ~ l o n e SP9-16 and experimental synthetic KS79-20 were maintained in a growth chamber w i t h an 18-h p h o t o p e r i o d of O. 4 ~mol m-2 s -I oalnd d a constant t e m p e r a t u r e of 24~C. Three-dayannual and p e r e n n i a l pods were e x c i s e d and i m p l a n t e d in vitro. Media used were: (1) DM (Liang et a l . , 1982), (2) DMI = DM plus 1 ppm of indoleacetic acid, (3) DMG = OM plus l ppm of g i b b e r e l l i c acid. The number of pods testea for each species varied, but a v e r a g e d 26 grown in v i t r o for each species per r e p l i c a t i o n . The number of seeds per pod was determined at maturity. Treatments were included in

a completely randomized design with 13 r e p l i c a t i o n s , except only 5 replications f o r M. sativa SP9-16 x M. sativa KS79-20. Results Pods of a l l s p e c i e s tested developed s i m i l a r l y i__nn v i t r o and in v i v o a t t a c h e d to the p l a n t . They were c o i l e d f o r a l l species except M. f a l c a t a on which t h e y were f a l c a t e . They were green for a p p r o x i m a t e l y 30 days and then gradually changed to the mature c o l o r b l a c k f o r M. blancheana and l i g h t tan to brown f o r the other species. Plants from seeds we germinated appeared normal. Few pods o f Niedicago s a t i v a (3%, n=1444) or M. f a l c a t a (0.5%, n=1076) produced viable seeds on media containing modified Hoagland's plus i% glucose or sucrose w i t h or w i t h o u t 5~ p o t a t o e x t r a c t . I n c r e a s i n g sucrose to 2, 4, or 6% sucrose resulted in 20, 40 and 40%, r e s p e c t i v e l y , of the M. sativa pods (n=50) producing seeds in v i t r o . C u l t u r e of pods on two media (DM and DMI) was highly s u c c e s s f u l f o r the annual d i p l o i d s . Over 807o of M. b l a n c h e a n a and M. d i s c i f o r m i s (Table i) p-ods produced seeds o~ DM. The average number of seeds per pod was less in culture (Table 1) than on the plant (4), but three or more seeds were produced in many pods. Although UM was less suitable for the annual tetraploid M. s c u t e l l a t a than f o r the diploids, 48% o f the pods produced one seed. As e x p e c t e d , the success r a t e of the culturing pods o b t a i n e d from s e l f i n g a perennial t e t r a p l o i d clone (30%) was only half that for crossing clones of the same s p e c i e s (62%). Only 15% of d i p l o i d perennial M. f a l c a t a pods produced seeds on DM and they contained only one seed. UMG was less s u i t a b l e than DM or DMI f o r in v i t r o growth of pods from the three annual specie-s(Table 1 ) . H o w e v e r , 66% o f ~vi. b l a n c h e a n a produced seeds and 12% contained thr-ee seeds. Only 2% of M. s c u t e l l a t a pods produced seeds; 16% of the p e d i c e l s induced callus, followed by formation of t r i f o l i o l a t e leaves and coil-shaped tissue. Very little c a l l u s d e v e l o p e d on media w i t h i ppm indoleacetic acid. In v i t r o seed p r o d u c t i o n in pods of M. f a l c a t a and M. s a t i v a was not a f f e c t e d by a d d i t i o n of indoleacetic acid or g i b b e r e l l i c acid to DM. Discussion In v i t r o c u l t u r e of p a r e n t a l Medica9o species may be a p r e r e q u i s i t e to success with i n t e r s p e c i f i c h y b r i d s s i n c e o n l y one successful cross has been r e p o r t e d between annual and p e r e n n i a l Medica9o s p e c i e s (Sangduen et a l . , 1982). F e r t i l i z a t i o n o c c u r r e d but embryos a b o r t e d at an e a r l y age (Sangduen et a l . , 1983b). P l a n t l e t s have been o b t a i n e d from 8- and 12-day-old embryos of annual and p e r e n n i a l Medica9o s p e c i e s , r e s p e c t i v e l y , c u l t u r e d on a m o d i f i e d L i n s m a i e r - B e d n a r growth medium (Krause et a l . , 1979). Unfortunately most h y b r i d embryos a b o r t p r i o r to t h e s e ages. We s u c c e s s f u l l y cultured pods that were excised three days a f t e r p o l l i n a t i o n so this technique might be used to s u c c e s s f u l l y r e s c u e the embryo p r i o r to abortion.

148 Krause (1980) o b t a i n e d in v i t r o b e t t e r results w i t h d i p l o i d annual M. d i s c i f o r m i s than w i t h p e r e n n i a l M. s a t i v a ~ b r y o s . Our r e s u l t s with pods were s i m i l a r . Although DM c o n t a i n e d most of the n e c e s s a r y n u t r i e n t s for the d i p l o i d annuals, requirements for Medicago s p e c i e s neea a d d i t i o n a l i n v e s t i g a t i o n . In g e n e r a l , pod s i z e in v i t r o was smaller than in vivoo I n s t e a d o f f i v e to seven or e i g h t c o i l s per pod (Heyn, i963), M. s c u t e l l a t a in v i t r o had f o u r c o i l s at most and the diameter was reduced by 25-30%. Other s p e c i e s we s t u d i e d produced: Table I.

(1) s l i g h t l y s m a l l e r pods than those in vivo, (2) a p p a r e n t l y normal seedless pods, and (3) occasional small pods with one or two well developea seeds. A l t h o u g h we i d e n t i f i e d differences in requirements f o r pod c u l t u r e media between annual and perennial s p e c i e s and between d i p l o i d s and t e t r a p l o i d s , these d i f f e r e n c e s should not be i n t e r p r e t e d as c h a r a c t e r i s t i c of the Genus as a whole. Additional s t u d i e s o f o t h e r Medicago species are necessary to document differences in requirements for pod culture media between IV~edicago species of varying ploidy levels and between annuals and perennials.

Effect of media on seed production of in vi tro grown pods from f i v e Medicago species.

Species

Medium

Total number Pods Seeds

Average seeds/ pod

Percentaqe of pods with 1 to 6 seeds 1 2 3 4 5 6

Total percentage of pods with seeds

M. disciformis

DM DMI DMG

338 338 338

453 716 151

1.3 2.1 0°4

42 33 26

27 28 8

I0 12 1

1 1

M. blancheana

DM DMI DMG

338 338 338

535 409 372

1.6 1.2 I.I

43 47 36

17 19 18

13 6 12

7 4 1

M. s c u t e l l a t a

DM DMI DMG

338 338 338

161 131 7

0.5 0°4 0.0

48 39 2

48 39 2

M. falcata

DM DMI DMG

338 338 338

50 34 55

0.2 0oi 0°2

15 8 16

15 9 16

M. sativa SP9-16

DM DMI DMG

338 338 338

116 114 115

0.3 0.3 0°3

27 28 29

3 3 1

M. sativa SP9-16 x M. sativa K79-20"

DM DMI DMG

65 65 65

51 72 44

0.8 Ioi 0°7

49 31 43

12 15 9

2 17 2

14

9

7

L.S.D.

(5%)

81 74 35 3 1

83 77 67

30 31 31 63 63 54 2

1

14

*M. sativa (SP9-16 x KS79-20) data were not included in the s t a t i s t i c a l analysis°

References Bajaj YPS (1966) Phyton; Int J Exp Bot 23:57-62 Cooper DC, Brink RA (1940) J Agr Res 60:453-472 Guha S, Johri BM (1966) Phytomorphology 16:353-364 Harper JE, Paulsen GM (1969a) Plant Physiol 44:69-74 H a r p e r , JE, Paulsen GM (1969b) P l a n t P h y s i o l 44 : 636 -640 Heyn, CC (1963) The annual species of Medicago, Magnes P r e s s ; Hebrew U n i v e r s i t y , Jerusalem, Israel. Hoagland DR, Arnon DI (1950) C a l i f Agr Exp Sta Cir 347 Johnson CM, Stout PR, Broyer TC, Carlton AB (1957) Plant Soil 8:337-353 Krause JW, Sorensen EL, Lian9 GH (1979) Agron Abstr. P. 66 Krause, JW (1980) M.S. T h e s i s ; Kansas S t a t e University, Manhattan, Kansas L i a n g , GH, Sangduen N, Heyne EG, Sears RG (1982) J Hered 73:360-364 Maheshwari N, Lal M (1961) Phytomorphology 11:17-23

Maheshwari P, Rangaswamy NS (1963) In: Maheshwari P, Rangaswamy NS (eds) P l a n t t i s s u e and organ c u l t u r e - A symposium, Int Soc Plant Morphol, Delhi, India, pp 390-419 N i t s c h JP (1963) I n : Mahewhwari P, RangaswamyNS (eds) P l a n t t i s s u e and organ c u l t u r e A symposium, Int Soc Plant Morphol, Delhi, India, pp 199-211 Pareek LK, G o v i l CM, Chandra N (1980) In: Sala F, P a r i s i B, C e l l a R, C i f e r r i 0 (eds) Plant c e l l cultures: results and p e r s p e c t i v e s , E l s e v i e r / N o r t h H o l l a n d Biomedical Press, New York, pp 79-86 Rao PS, Rangaswamy NS (1972) Bot Gaz 133:350-355 Richards KW, Rupert EA (1980) In Vitro 16:925-931 Sangduen N, K r e i t n e r GL, Sorensen EL (1983a) Can J Bot 61 : 1241-1257 Sangduen N, Sorensen EL, Liang GH (1983b) Euphytica 32:527-534 Sangduen N, Sorensen EL, Liang GH (1982) Can J Genet Cytol 24:361-365 Sayers ER, Murphy RP (1966) Crop Sci 6:365-368 Yeh KJ, Bingham ET (1969) Crop Sci 9:835-837