Plant Cell Reports
Plant Cell Reports (]989) 8:379 382
© Springer-Verlag 1989
Plant regeneration from pea protoplasts via somatic embyogenesis Renate Lehminger-Mertens and Hans-J6rg Jacobsen Institut ffir Genetik, Universit/it Bonn, Kirschallee 1, D-5300 Bonn I, Federal Republic of Germany Received May 12, 1989/Revised version received July 8, 1989 - Communicated by I. Potrykus
ABSTRACT
Plant r e g e n e r a t i o n via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and inc r e a s e d o s m o l a r i t y of the m e d i u m were necessary for e m b r y o induction. R e l a t i v e l y high am o u n t s of e m b r y o g e n i c calli c o u l d be o b t a i n e d in 2 genotypes. A f t e r a p e r i o d on hormonefree medium, a s e c o n d i n d u c t i o n of somatic e m b r y o s was p o s s i b l e . F u r t h e r d e v e l o p m e n t of somatic embryos was accomplished on GA3c o n t a i n i n g medium.
ABBREVIATIONS
ABA, a b s c i s i c acid; BA, 6 - b e n z y l a m i n o p u r ine; 2.4-D, 2 . 4 - d i c h l o r o p h e n o x y a c e t i c acid; GA3 , g i b b e r e l l i c acid; Kin, kinetin; NAA, naphthaleneacetic acid; Pic, Picloram, 4a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c acid;
INTRODUCTION
In r e c e n t years, i n t e n s i v e s t u d i e s on protoplast regeneration in the large seeded l e g u m e s have led to some s u c c e s s (Shekhawat & G a l s t o n 1983, H a m m a t t et al. 1987, P o l a n c o et al. 1988). Up to now, p r o t o p l a s t s from peas ( P i s u m s a t i v u m L.) could s u c c e s s f u l l y be reg e n e r a t e d to callus and shoots (Puonti-Kaerlas & E r i k s s o n 1988, L e h m i n g e r - M e r t e n s & Jacobsen 1989), but o r g a n o g e n e s i s took place only after a r e l a t i v e l y long p e r i o d of time (3 -9 months) and at r a t h e r low frequencies (1.3 -10 %). In no case could r e g e n e r a t i o n of whole p l a n t s be o b t a i n e d from these shoots, because r o o t i n g did not occur. Somatic embryogenesis w o u l d avoid these difficulties, since e l o n g a t i o n of r a d i c l e s in s o m a t i c embryos could e a s i l y be obtained. On the other hand, somatic embryogenesis and plant r e g e n e r a t i o n in peas are already well-established and deal w i t h complex explants like shoot apex or immature zygotic embryos as source m a t e r i a l . With both of these, a c c e p t a b l e a m o u n t s of e m b r y o s c o u l d be induced on m e d i u m c o n t a i n i n g h i g h levels of NH4 + (like MS medium) and a s t r o n g auxin o n l y (Kysely et al. 1987, K y s e l y & J a c o b s e n 1989).
Offprintrequeststo: H.-J. Jacobsen
A g a i n s t this b a c k g r o u n d , it was p o s s i b l e to d e v e l o p a r e g e n e r a t i o n scheme for pea p r o t o p l a s t s to whole p l a n t s via s o m a t i c embryogenesis.
METHODS
Six d i f f e r e n t g e n o t y p e s of the species P i sum sativum ssp. s a t i v u m were tested for their c a p a b i l i t y to form e m b r y o s on protop l a s t d e r i v e d callus. All b r e e d e r lines u s e d t h r o u g h o u t in this study (Belman, Birte, Bodil, Finale, Solara) were k i n d l y p r o v i d e d by M. F r a u e n ( N o r d d e u t s c h e P f l a n z e n z u c h t , H o h e n lieth) and P i s u m s a t i v u m ssp. a r v e n s e was prop a g a t e d at the I n s t i t u t f~r Genetik, Universit~t Bonn. P r e c u l t u r e s of c o t y l e d o n - f r e e e m b r y o axes from m a t u r e seeds were kept on B5 hormonefree m e d i u m (Gamborg et al. 1968). E v e r y 3 -4 days, their apices or later on, their l a t e r a l shoot buds could be u s e d as source material for p r o t o p l a s t isolation. The f r e s h l y isolated p r o t o p l a s t s were c u l t u r e d in LP* m e d i u m up to calli of ca. 2 mm in diameter, as described elsewhere (Lehminger-Mertens & Jacobsen 1989). For e m b r y o induction, the two auxins picloram and 2.4-D were added to d i f f e r e n t basal m e d i a in the o v e r a l l range of 4 -15 ~M. The basal m e d i a u s e d w e r e e i t h e r MS- (Murashige & Skoog 1962) or B5 salts w i t h 2 % s u c r o s e and 0.56 % P h y t a g a r (GIBCO). MS salts always were e n r i c h e d w i t h B5 v i t a m i n e s (MS') and w i t h casein h y d r o l y s a t e (MSC) or 3-5 % mannitol, respectively. For m a t u r a t i o n , the MSC m e d i u m w i t h 0-5 % mannitol was u s e d w i t h o u t h o r m o n e s or with d i f f e r e n t h o r m o n e c o m b i n a t i o n s (see Table 3). G e r m i n a t i o n c o u l d be o b t a i n e d on MS or B5 med i u m c o n t a i n i n g 1.5 -2.9 ~M GAa. As soon as somatic seedlings developed primary leaves, they were t r a n s f e r r e d to s t e r i l e soil, covered w i t h c o t t o n wool (Fig. id).
RESULTS
Embryo
AND
DISCUSSION
Induction
Somatic embryogenesis,
i n d u c e d with high
380
Figure i: S o m a t i c e m b r y o g e n e s i s of pea in the g e n o t y p e B e l m a n a. somatic e m b r y o s on p r o t o p l a s t d e r i v e d callus b. g e r m i n a t i n g s o m a t i c embryo c. developing p l a n t l e t d. left: d e v e l o p i n g pea plant in a s t e r i l e glass v e s s e l c o v e r e d w i t h cotton, right: a d a p t a t i o n to u n s t e r i l e c o n d i t i o n s e. r e g e n e r a t e d pea p l a n t levels of strong auxins alone, has been r e a l i z e d w i t h i n the family of F a b a c e a e in the al. genus G l y c i n e (references in H a r t w e c k et 1988), V i c i a (Griga et al. 1987, P i c k a r d t et al. 1989), V i g n a (Kumar et al. 1988) and L e n s (Saxena & King 1987). Further experiments with i m m a t u r e zygotic embryo explants from pea also s h o w e d c l e a r l y the requirement of picloram or 2.4-D (Kysely et al. 1987) and the s u p e r i o r i t y of the MS- to the B5 basal m e d i u m for the i n d u c t i o n p r o c e s s (Kysely pers. communication). Although this p r o t o c o l was s t r i c t l y followed, not a single e m b r y o primordium could be i n d u c e d on protoplast derived callus from an e m b r y o g e n i c line of P.s. ssp. a r v e n s e (Table I, MS'). E x p e r i m e n t s w i t h empty pea ovules f i l l e d with isoosmotic sucrose solution (Wolswinkel & A m m e r l a a n 1984), embryo r e s c u e techniques (Cook et al. 1988) and o s m o l a r i t y measurements of pea e n d o s p e r m as d e s c r i b e d by Wang
et al. (1987) gave the d e c i s i v e indications for e n r i c h i n g the i n d u c t i o n m e d i u m in stages w i t h m a n n i t o l (Table I) and l o w e r i n g sucrose c o n c e n t r a t i o n to 2 % a c c o r d i n g to L a z z e r i et al. (1988). Due to this i n c r e a s e in o s m o l a r i ty, for the first time g l o b u l a r e m b r y o s were formed on MSC m e d i u m w i t h 4 % mannitol and 4 ~M 2.4-D (Kysely et al. 1988). In order to also v e r i f y these initial hints in the field of b r e e d e r lines, largescale tests w i t h v a r y i n g h o r m o n e concentrations and m e d i u m v a r i a t i o n s were c a r r i e d out (Table 2). Their results showed the same t e n d e n c i e s as d i s c u s s e d by K y s e l y & Jacobsen (1989). For the i n d u c t i o n p r o c e s s of protop l a s t - d e r i v e d calli, m a n n i t o l could not be substituted by s u c r o s e in whole (i.e. MSC 40(- F . . . . . . . . /
30-
Table
I:
E m b r y o i n d u c t i o n in p r o t o p l a s t deP. s. ssp.arvense r i v e d calli of on d i f f e r e n t basal m e d i a
/ / /
20-
10medium MS' MSC MSC + 3% mannitol MSC + 4% mannitol B5 B5 + 3% mannitol B5 + 4% mannitol
auxin 2.4-D 2.4-D picloram 2.4-D picloram 2.4-D 2.4-D )icloram 2.4-D ~icloram picloram
[uM] 4 i-5 6 4 6 4 5-8
5
5-8 4-8 5
-
/r~.///
//
somatic embryos
t/t
:yy
~o --1. induction
o Figure
2~5
~'2. induction ~hormone- free
~-hormone-free
s~o
7's
100
1:25
150
175 days
2: i st and 2 nd i n d u c t i o n of s o m a t i c embryos on p r o t o p l a s t - d e r i v e d callus in the g e n o t y p e B e l m a n (~ of all calli tested) on: : MSC + 5 % m a n n i t o l + 7 uM p i c l o r a m : MSC + 5 % m a n n i t o l + 7 uM 2.4-D
381 Table
2: E m b r y o g e n e s i s in the g e n o t y p e B e l m a n (% of all c a l l i t e s t e d ) ; c a l l i w e r e d i f f e r e n t i n d u c t i o n m e d i a for 40 days, t h e r e a f t e r on h o r m o n e - f r e e m e d i u m
MSC + 4 % mannitol auxins [UM] n ~ { 4 0 Picloram 5 I001 6 i00{ 7 i001 8 i001 9 i00 { i0 i00 15 40 2.4-D 5 601 6 1001 7 i201 8 1001 9 301 i0 6o I 15 201
MSC + 5 % mannitol
days 75 days 105 days 8.0 20.0 21.0 4.0 22.0 24.0 5.0 15.0 17.0 7.0 25.0 25.0 4.0 20.0 20.0 6.0 16.0 18.0 2.5 2.5 2.5
5.0 3.3 0.0 8.0 3.3
3.3 0
11.7 11.0 7.5 12.0
n.7 12.0 zo.8 12.0
io.o 6.7
io.o 6.7 0
0
B5 + 4 % mannitol
[n~{40 days 75 days 105 days 80{ 5.0 17.5 17.5 I001 3.0 22.0 23.0 I00] 4.0 13.0 19.0 i00] 4.0 14.0 15.0 90] 5.5 23.3 24.4 80 I 1.3 15.0 18.8 201 5.0 i0.0 i0.0 ~ 6.7 17.8 18.9 5.0 17.5 27.5 i 901 5.5 33.3 34.4 i001 6.0 19.0 21.0 1001 5.0 22.0 23.0 1001 2.0 28.0 32.0 25 I 8.0 12.0 12.0
w i t h 6 %) or in p a r t (MSC w i t h 3.5 % sucrose a n d 2.5 % m a n n i t o l , d a t a n o t shown) a l t h o u g h m a n n i t o l s h o u l d be d e g r a d e d l i k e o t h e r s u g a r s b y the c a l l u s ( T h o m p s o n et el. 1986). From all the protoplast-derived calli t e s t e d for e m b r y o g e n e s i s , surprisingly high amounts were found to be embryogenic, normally bearing more than one embryo on the s u r f a c e (Fig. la). As s h o w n in T a b l e 2, 5 -i0 ~M p i c l o r a m or 5 -I0 ~M 2 . 4 - D yielded embryogenic c a l l u s in the r a n g e of 10 -30 % ( m a x i m u m 34 %). A f t e r a p e r i o d of 70 d a y s o n hormone-free medium, we s u c c e s s f u l l y i n d u c e d s o m a t i c e m b r y o f o r m a t i o n for a s e c o n d t i m e on the M S C D 7 - or M S C P7 m e d i u m w i t h 5 % mannitol (Fig.2). O n the M S C P7 medium, somatic embryos in c o m p a r a b l e a m o u n t s to the first induction c y c l e c o u l d be o b t a i n e d . This is t h e r e f o r e the f i r s t r e p o r t of s u c c e s s f u l embryogenesis on protoplast derived pea calli in t w o s u b s e q u e n t i n d u c t i o n c y c l e s . A l t h o u g h all g e n o t y p e s t e s t e d f o r m e d s o m a tic embryos, there still remained conside r a b l e g e n o t y p i c d i f f e r e n c e s in f r e q u e n c y . While Belman and Birte exhibited comparable r e g e n e r a t i o n a b i l i t i e s (Birte 2 0 - 3 0 %), c a l l i of B o d i l , Finale and Solara (semi-leafless) responded o n l y w i t h a f r e q u e n c y of approxim a t e l y 0.i -I %, s i m u l t a n o u s l y e x p r e s s i n g an u n e x p e c t e d h i g h s e n s i t i v i t y to 2.4-D. Whenever protoplast-derived c a l l i of t h e s e 3 g e notypes came into contact with a 2.4-D cont a i n i n g m e d i u m (4 -15 ~M), t h e y d i e d w i t h i n 3 weeks. S i m i l a r g e n o t y p i c e f f e c t s in penes for e m b r y o g e n e s i s or o r g a n o g e n e s i s h a v e b e e n p r e v i o u s l y r e p o r t e d ( R u b l u o et el. 1984, P u o n t i Kaerlas & E r i k s s o n 1988, Kysely & Jacobsen 1989).
and
2ol 3Ol 501 I _1
o
o
o o
o o
B5 + 5 % mannitol
o°
201
0
0
10,
0
0
80 I 101
0 0
1.3 0
I I I
in 1 s t e p f r o m the 4 -5 % in the induction m e d i u m to 0 % in the m a t u r a t i o n medium, nor s h o u l d it be i n c r e a s e d ( c o m p a r e hormone-free media, T a b l e 3). On m o s t of the h o r m o n e - f r e e media, globular embryos matured slightly, but stopped further development when they were not r e m o v e d f r o m the callus. We observed, however, c o n t i n u o u s f o r m a t i o n of n e w e m b r y o s on the i n d u c e d p r o t o p l a s t - d e r i v e d calli over a l o n g p e r i o d of t i m e (Fig. 2). K y s e l y et al. (1987) a n d K y s e l y & J a c o b s e n (1989) m a t u r e d s o m a t i c p e a e m b r y o s on BA containing medium, b u t w h e n e v e r any cytokinins for p r o t o p l a s t - d e r i v e d somatic embryos were used, the e m b r y o s recallussed (Table 3 ) . I n o r d e r to m a t u r e the e m b r y o s , the e f f e c t
Table
3: M a t u r a t i o n
medium composition MS
MS C MS C MS C + 3% mannitol MS C + 4% mannitol MS C + 4% mannitol
Germination
W h e n e v e r the e m b r y o g e n i c c a l l u s remained on the i n d u c t i o n m e d i u m for l o n g e r than 5 weeks, all s o m a t i c e m b r y o s f a i l e d to u n d e r g o further development and recallused under the strong auxin regime. F o r this reason, the c a l l i w e r e t r a n s f e r r e d to s e v e r a l maturation media w h i c h w e r e e i t h e r a u x i n - f r e e or which h a d a l o w e r h o r m o n e c o n t e n t (Table 3 ) . T h e u s e of n o r m a l s t r e n g t h or h a l f - s t r e n g t h MS salts seemed to have no effect. Besides that, m a n n i t o l c o n c e n t r a t i o n s h o u l d n o t be lowered
on
nil40 days 75 days n~_iI40 days 75 days 701 o o 0 901 0 90 I 3.3 4.4 I
MS + 4.5% mannitol Mat ura tion
cultured
MS C + 5% mannitol MS C + 5% mannitol
media
tested
further develop[UM] hormone ment of embryoids +I) 2.9 GA 2.9 GA 3.0 ABA free +/free 4.7 Kin callus 2.9 GA 3.8 ABA free +/+/free 0.4 Pic +/0.48 KIZ 2~ callus 0.48 KIZ 2~ 0.4 ABA 0.2 Pic 0.2 KIZ 2~ callus 4.7 Kin callus 2.9 GA +/1.9 ABA 2.9 GA 3.0 ABA free +/+/free 0.48 KIZ 2 )
3.0
ABA
0.12 NAA + 1.5 GA 4.7 Kin callus MS C + 6% mannitol free 1}not longer than i0 days 2)mixture of 1/3 kinetin, 2iP and zeatin, respectively
382 of some different hormones and their combinations were studied. Adding ABA at the globular stage (in different concentrations) should be advantageous since ABA was also d e t e c t a b l e in zygotic pea embryos at this stage (Browning 1980). As discussed by Browning, we also found that external ABA inhibited further d e v e l o p m e n t of embryos. Finally, the A B A - t r e a t e d embryos turned brown on top and died. Optimum results were o b t a i n e d with combinations of GA3, which seems to promote embryo m a t u r a t i o n (1.5 ~M GA3, 0.12 ~M NAA) followed by g e r m i n a t i o n (2.9 ~M GA3) within a few days (Fig. Ib, Table 3). The d e p e n d e n c e on external GA3 for c o n t i n u i n g m a t u r a t i o n may be explained by the lack of testa in somatic embryos, since this tissue provides g i b b e r e l l i n catabolites and -products to the zygotic embryo during seed d e s i c c a t i o n {Sponsel 1983) as well as promotes the accumulation of osmotic solutes (Miyamoto & Kamisaka 1988). Additionally, growing seedlings are d e p e n d e n t on d e n o v o g i b b e r e l l i n biosynthesis, especially for their epicotyl elongation (Sponsel 1983) and the somatic embryos thus probably require external GA3 at high levels during germination. G e r m i n a t i n g somatic embryos m o s t l y had rapidly growing roots and w e l l - d e v e l o p e d shoot apices, but no typical cotyledons (Fig ib). However, typical storage proteins could be found in these somatic embryos (Lehminger-Mertens et al., in prep.). Nevertheless, transfer to h o r m o n e - f r e e h a l f - s t r e n g t h m e d i u m promoted their growth towards forming plantlets (Fig. ic). Later on, the r e g e n e r a t e d plantlets were potted in sterile soil, initially covered with a glass vessel, which could be taken off within a week (Fig. id). A d a p t a t i o n to u n s t e r i l e conditions took place in a growth chamber. Until now, these r e g e n e r a t e d plants show a quite normal m o r p h o l o g y (Fig. le), p r o d u c i n g flowers and setting seeds.
ACKNOWLEDGEMENTS
This work has been supported by a GFP/BMFT grant to H.-J. Jacobsen (A 24/87-ZF). The authors wish to thank Mr. Frazer Walker for critical reading of the manuscript.
REFERENCES
B r o w n i n g G (1980) J.Exp. Bot. 31: 185-197. Cook SK, Adama H, Hedley CL, Ambrose MJ, Wang TL (1988) Plant Cell, Tissue and Organ Culture 14: 89-101. Gamborg OL, Miller RA, Ojima K (1968) Exp. Cell Res. 50: 150-158. Griga M, K u b a l a k o v a M, T e j k l o v a E (1987) Plant Cell, Tissue and Organ Culture 9: 167-171. Hammatt N, Kim HI, Davey MR, N e l s o n RS, Cocking EC (1987) Plant Science 48: 129-135. H a r t w e c k LM, Lazzeri PA, Cui D, Collins GB, Williams EG (1988) In Vitro 24: 821-828. Kumar AS, G a m b o r g OL, Nabors MW (1988) Plant Cell Reports 7: 138-141. Kysely W, Myers JR, Lazzeri PA, Collins GB, J a c o b s e n HJ (1987) Plant Cell Reports 6: 305-308. Kysely W, L e h m i n g e r - M e r t e n s R, J a c o b s e n H-J (1988) Bio E n g e n e e r i n g 4: 32-34. Kysely W, J a c o b s e n HJ (1989) Plant Cell, Tissue and Organ Culture: in press Lazzeri PA, H i l d e b r a n d DF, Sunega J (1988) Plant Cell Reports 7 : 5 1 7 - 5 2 0 L e h m i n g e r - M e r t e n s R, J a c o b s e n HJ (1989) In Vitro: in press M i y a m o t o K, Kamisaka S (1988) Physiologia P l a n t a r u m 74: 457-466. M u r a s h i g e T, Skoog F (1962) Physiol. Plant. 15: 473-497. Pickardt T, H u a n c a r u n a Peralef E, Schieder O (1989) P r o t o p l a s m a 149: 5-10. Polanco MC, Pel~ez MI, Riuz ML (1988) Plant Cell Tissue and Organ Culture 15: 175182. P u o n t i - K a e r l a s J, Erikson T(1988) Plant Cell Reports 7: 242-245. Rubluo A, Kartha KK, M r o g i n s k i LA, Dyck J (1984) J.Plant. Physiol. 117: 119-130. S a x e n a PK, King J (1987) Plant Science 52: 223-227. Shekhawat NS, G a l s t o n AW (1983) Plant Science Letters 32: 43-51. Sponsel VM (1983) Planta 159: 454-468. T h o m p s o n MR, Douglas TJ, O b a t a - S a s a m o t o H, Thorpe TA (1986) Physiol. Plant. 67: 365369. Wang TL, Cook SK, Francis RJ, Ambrose MJ, Hedley CL (1987) J.Exp. Bot. 38: 19211932. W o l s w i n k e l P, A m m e r l a a n A (1984) Physiol.Plant. 61: 172-182.
Plant Cell Reports (]989) 8:379 382
© Springer-Verlag 1989
Plant regeneration from pea protoplasts via somatic embyogenesis Renate Lehminger-Mertens and Hans-J6rg Jacobsen Institut ffir Genetik, Universit/it Bonn, Kirschallee 1, D-5300 Bonn I, Federal Republic of Germany Received May 12, 1989/Revised version received July 8, 1989 - Communicated by I. Potrykus
ABSTRACT
Plant r e g e n e r a t i o n via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and inc r e a s e d o s m o l a r i t y of the m e d i u m were necessary for e m b r y o induction. R e l a t i v e l y high am o u n t s of e m b r y o g e n i c calli c o u l d be o b t a i n e d in 2 genotypes. A f t e r a p e r i o d on hormonefree medium, a s e c o n d i n d u c t i o n of somatic e m b r y o s was p o s s i b l e . F u r t h e r d e v e l o p m e n t of somatic embryos was accomplished on GA3c o n t a i n i n g medium.
ABBREVIATIONS
ABA, a b s c i s i c acid; BA, 6 - b e n z y l a m i n o p u r ine; 2.4-D, 2 . 4 - d i c h l o r o p h e n o x y a c e t i c acid; GA3 , g i b b e r e l l i c acid; Kin, kinetin; NAA, naphthaleneacetic acid; Pic, Picloram, 4a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c acid;
INTRODUCTION
In r e c e n t years, i n t e n s i v e s t u d i e s on protoplast regeneration in the large seeded l e g u m e s have led to some s u c c e s s (Shekhawat & G a l s t o n 1983, H a m m a t t et al. 1987, P o l a n c o et al. 1988). Up to now, p r o t o p l a s t s from peas ( P i s u m s a t i v u m L.) could s u c c e s s f u l l y be reg e n e r a t e d to callus and shoots (Puonti-Kaerlas & E r i k s s o n 1988, L e h m i n g e r - M e r t e n s & Jacobsen 1989), but o r g a n o g e n e s i s took place only after a r e l a t i v e l y long p e r i o d of time (3 -9 months) and at r a t h e r low frequencies (1.3 -10 %). In no case could r e g e n e r a t i o n of whole p l a n t s be o b t a i n e d from these shoots, because r o o t i n g did not occur. Somatic embryogenesis w o u l d avoid these difficulties, since e l o n g a t i o n of r a d i c l e s in s o m a t i c embryos could e a s i l y be obtained. On the other hand, somatic embryogenesis and plant r e g e n e r a t i o n in peas are already well-established and deal w i t h complex explants like shoot apex or immature zygotic embryos as source m a t e r i a l . With both of these, a c c e p t a b l e a m o u n t s of e m b r y o s c o u l d be induced on m e d i u m c o n t a i n i n g h i g h levels of NH4 + (like MS medium) and a s t r o n g auxin o n l y (Kysely et al. 1987, K y s e l y & J a c o b s e n 1989).
Offprintrequeststo: H.-J. Jacobsen
A g a i n s t this b a c k g r o u n d , it was p o s s i b l e to d e v e l o p a r e g e n e r a t i o n scheme for pea p r o t o p l a s t s to whole p l a n t s via s o m a t i c embryogenesis.
METHODS
Six d i f f e r e n t g e n o t y p e s of the species P i sum sativum ssp. s a t i v u m were tested for their c a p a b i l i t y to form e m b r y o s on protop l a s t d e r i v e d callus. All b r e e d e r lines u s e d t h r o u g h o u t in this study (Belman, Birte, Bodil, Finale, Solara) were k i n d l y p r o v i d e d by M. F r a u e n ( N o r d d e u t s c h e P f l a n z e n z u c h t , H o h e n lieth) and P i s u m s a t i v u m ssp. a r v e n s e was prop a g a t e d at the I n s t i t u t f~r Genetik, Universit~t Bonn. P r e c u l t u r e s of c o t y l e d o n - f r e e e m b r y o axes from m a t u r e seeds were kept on B5 hormonefree m e d i u m (Gamborg et al. 1968). E v e r y 3 -4 days, their apices or later on, their l a t e r a l shoot buds could be u s e d as source material for p r o t o p l a s t isolation. The f r e s h l y isolated p r o t o p l a s t s were c u l t u r e d in LP* m e d i u m up to calli of ca. 2 mm in diameter, as described elsewhere (Lehminger-Mertens & Jacobsen 1989). For e m b r y o induction, the two auxins picloram and 2.4-D were added to d i f f e r e n t basal m e d i a in the o v e r a l l range of 4 -15 ~M. The basal m e d i a u s e d w e r e e i t h e r MS- (Murashige & Skoog 1962) or B5 salts w i t h 2 % s u c r o s e and 0.56 % P h y t a g a r (GIBCO). MS salts always were e n r i c h e d w i t h B5 v i t a m i n e s (MS') and w i t h casein h y d r o l y s a t e (MSC) or 3-5 % mannitol, respectively. For m a t u r a t i o n , the MSC m e d i u m w i t h 0-5 % mannitol was u s e d w i t h o u t h o r m o n e s or with d i f f e r e n t h o r m o n e c o m b i n a t i o n s (see Table 3). G e r m i n a t i o n c o u l d be o b t a i n e d on MS or B5 med i u m c o n t a i n i n g 1.5 -2.9 ~M GAa. As soon as somatic seedlings developed primary leaves, they were t r a n s f e r r e d to s t e r i l e soil, covered w i t h c o t t o n wool (Fig. id).
RESULTS
Embryo
AND
DISCUSSION
Induction
Somatic embryogenesis,
i n d u c e d with high
380
Figure i: S o m a t i c e m b r y o g e n e s i s of pea in the g e n o t y p e B e l m a n a. somatic e m b r y o s on p r o t o p l a s t d e r i v e d callus b. g e r m i n a t i n g s o m a t i c embryo c. developing p l a n t l e t d. left: d e v e l o p i n g pea plant in a s t e r i l e glass v e s s e l c o v e r e d w i t h cotton, right: a d a p t a t i o n to u n s t e r i l e c o n d i t i o n s e. r e g e n e r a t e d pea p l a n t levels of strong auxins alone, has been r e a l i z e d w i t h i n the family of F a b a c e a e in the al. genus G l y c i n e (references in H a r t w e c k et 1988), V i c i a (Griga et al. 1987, P i c k a r d t et al. 1989), V i g n a (Kumar et al. 1988) and L e n s (Saxena & King 1987). Further experiments with i m m a t u r e zygotic embryo explants from pea also s h o w e d c l e a r l y the requirement of picloram or 2.4-D (Kysely et al. 1987) and the s u p e r i o r i t y of the MS- to the B5 basal m e d i u m for the i n d u c t i o n p r o c e s s (Kysely pers. communication). Although this p r o t o c o l was s t r i c t l y followed, not a single e m b r y o primordium could be i n d u c e d on protoplast derived callus from an e m b r y o g e n i c line of P.s. ssp. a r v e n s e (Table I, MS'). E x p e r i m e n t s w i t h empty pea ovules f i l l e d with isoosmotic sucrose solution (Wolswinkel & A m m e r l a a n 1984), embryo r e s c u e techniques (Cook et al. 1988) and o s m o l a r i t y measurements of pea e n d o s p e r m as d e s c r i b e d by Wang
et al. (1987) gave the d e c i s i v e indications for e n r i c h i n g the i n d u c t i o n m e d i u m in stages w i t h m a n n i t o l (Table I) and l o w e r i n g sucrose c o n c e n t r a t i o n to 2 % a c c o r d i n g to L a z z e r i et al. (1988). Due to this i n c r e a s e in o s m o l a r i ty, for the first time g l o b u l a r e m b r y o s were formed on MSC m e d i u m w i t h 4 % mannitol and 4 ~M 2.4-D (Kysely et al. 1988). In order to also v e r i f y these initial hints in the field of b r e e d e r lines, largescale tests w i t h v a r y i n g h o r m o n e concentrations and m e d i u m v a r i a t i o n s were c a r r i e d out (Table 2). Their results showed the same t e n d e n c i e s as d i s c u s s e d by K y s e l y & Jacobsen (1989). For the i n d u c t i o n p r o c e s s of protop l a s t - d e r i v e d calli, m a n n i t o l could not be substituted by s u c r o s e in whole (i.e. MSC 40(- F . . . . . . . . /
30-
Table
I:
E m b r y o i n d u c t i o n in p r o t o p l a s t deP. s. ssp.arvense r i v e d calli of on d i f f e r e n t basal m e d i a
/ / /
20-
10medium MS' MSC MSC + 3% mannitol MSC + 4% mannitol B5 B5 + 3% mannitol B5 + 4% mannitol
auxin 2.4-D 2.4-D picloram 2.4-D picloram 2.4-D 2.4-D )icloram 2.4-D ~icloram picloram
[uM] 4 i-5 6 4 6 4 5-8
5
5-8 4-8 5
-
/r~.///
//
somatic embryos
t/t
:yy
~o --1. induction
o Figure
2~5
~'2. induction ~hormone- free
~-hormone-free
s~o
7's
100
1:25
150
175 days
2: i st and 2 nd i n d u c t i o n of s o m a t i c embryos on p r o t o p l a s t - d e r i v e d callus in the g e n o t y p e B e l m a n (~ of all calli tested) on: : MSC + 5 % m a n n i t o l + 7 uM p i c l o r a m : MSC + 5 % m a n n i t o l + 7 uM 2.4-D
381 Table
2: E m b r y o g e n e s i s in the g e n o t y p e B e l m a n (% of all c a l l i t e s t e d ) ; c a l l i w e r e d i f f e r e n t i n d u c t i o n m e d i a for 40 days, t h e r e a f t e r on h o r m o n e - f r e e m e d i u m
MSC + 4 % mannitol auxins [UM] n ~ { 4 0 Picloram 5 I001 6 i00{ 7 i001 8 i001 9 i00 { i0 i00 15 40 2.4-D 5 601 6 1001 7 i201 8 1001 9 301 i0 6o I 15 201
MSC + 5 % mannitol
days 75 days 105 days 8.0 20.0 21.0 4.0 22.0 24.0 5.0 15.0 17.0 7.0 25.0 25.0 4.0 20.0 20.0 6.0 16.0 18.0 2.5 2.5 2.5
5.0 3.3 0.0 8.0 3.3
3.3 0
11.7 11.0 7.5 12.0
n.7 12.0 zo.8 12.0
io.o 6.7
io.o 6.7 0
0
B5 + 4 % mannitol
[n~{40 days 75 days 105 days 80{ 5.0 17.5 17.5 I001 3.0 22.0 23.0 I00] 4.0 13.0 19.0 i00] 4.0 14.0 15.0 90] 5.5 23.3 24.4 80 I 1.3 15.0 18.8 201 5.0 i0.0 i0.0 ~ 6.7 17.8 18.9 5.0 17.5 27.5 i 901 5.5 33.3 34.4 i001 6.0 19.0 21.0 1001 5.0 22.0 23.0 1001 2.0 28.0 32.0 25 I 8.0 12.0 12.0
w i t h 6 %) or in p a r t (MSC w i t h 3.5 % sucrose a n d 2.5 % m a n n i t o l , d a t a n o t shown) a l t h o u g h m a n n i t o l s h o u l d be d e g r a d e d l i k e o t h e r s u g a r s b y the c a l l u s ( T h o m p s o n et el. 1986). From all the protoplast-derived calli t e s t e d for e m b r y o g e n e s i s , surprisingly high amounts were found to be embryogenic, normally bearing more than one embryo on the s u r f a c e (Fig. la). As s h o w n in T a b l e 2, 5 -i0 ~M p i c l o r a m or 5 -I0 ~M 2 . 4 - D yielded embryogenic c a l l u s in the r a n g e of 10 -30 % ( m a x i m u m 34 %). A f t e r a p e r i o d of 70 d a y s o n hormone-free medium, we s u c c e s s f u l l y i n d u c e d s o m a t i c e m b r y o f o r m a t i o n for a s e c o n d t i m e on the M S C D 7 - or M S C P7 m e d i u m w i t h 5 % mannitol (Fig.2). O n the M S C P7 medium, somatic embryos in c o m p a r a b l e a m o u n t s to the first induction c y c l e c o u l d be o b t a i n e d . This is t h e r e f o r e the f i r s t r e p o r t of s u c c e s s f u l embryogenesis on protoplast derived pea calli in t w o s u b s e q u e n t i n d u c t i o n c y c l e s . A l t h o u g h all g e n o t y p e s t e s t e d f o r m e d s o m a tic embryos, there still remained conside r a b l e g e n o t y p i c d i f f e r e n c e s in f r e q u e n c y . While Belman and Birte exhibited comparable r e g e n e r a t i o n a b i l i t i e s (Birte 2 0 - 3 0 %), c a l l i of B o d i l , Finale and Solara (semi-leafless) responded o n l y w i t h a f r e q u e n c y of approxim a t e l y 0.i -I %, s i m u l t a n o u s l y e x p r e s s i n g an u n e x p e c t e d h i g h s e n s i t i v i t y to 2.4-D. Whenever protoplast-derived c a l l i of t h e s e 3 g e notypes came into contact with a 2.4-D cont a i n i n g m e d i u m (4 -15 ~M), t h e y d i e d w i t h i n 3 weeks. S i m i l a r g e n o t y p i c e f f e c t s in penes for e m b r y o g e n e s i s or o r g a n o g e n e s i s h a v e b e e n p r e v i o u s l y r e p o r t e d ( R u b l u o et el. 1984, P u o n t i Kaerlas & E r i k s s o n 1988, Kysely & Jacobsen 1989).
and
2ol 3Ol 501 I _1
o
o
o o
o o
B5 + 5 % mannitol
o°
201
0
0
10,
0
0
80 I 101
0 0
1.3 0
I I I
in 1 s t e p f r o m the 4 -5 % in the induction m e d i u m to 0 % in the m a t u r a t i o n medium, nor s h o u l d it be i n c r e a s e d ( c o m p a r e hormone-free media, T a b l e 3). On m o s t of the h o r m o n e - f r e e media, globular embryos matured slightly, but stopped further development when they were not r e m o v e d f r o m the callus. We observed, however, c o n t i n u o u s f o r m a t i o n of n e w e m b r y o s on the i n d u c e d p r o t o p l a s t - d e r i v e d calli over a l o n g p e r i o d of t i m e (Fig. 2). K y s e l y et al. (1987) a n d K y s e l y & J a c o b s e n (1989) m a t u r e d s o m a t i c p e a e m b r y o s on BA containing medium, b u t w h e n e v e r any cytokinins for p r o t o p l a s t - d e r i v e d somatic embryos were used, the e m b r y o s recallussed (Table 3 ) . I n o r d e r to m a t u r e the e m b r y o s , the e f f e c t
Table
3: M a t u r a t i o n
medium composition MS
MS C MS C MS C + 3% mannitol MS C + 4% mannitol MS C + 4% mannitol
Germination
W h e n e v e r the e m b r y o g e n i c c a l l u s remained on the i n d u c t i o n m e d i u m for l o n g e r than 5 weeks, all s o m a t i c e m b r y o s f a i l e d to u n d e r g o further development and recallused under the strong auxin regime. F o r this reason, the c a l l i w e r e t r a n s f e r r e d to s e v e r a l maturation media w h i c h w e r e e i t h e r a u x i n - f r e e or which h a d a l o w e r h o r m o n e c o n t e n t (Table 3 ) . T h e u s e of n o r m a l s t r e n g t h or h a l f - s t r e n g t h MS salts seemed to have no effect. Besides that, m a n n i t o l c o n c e n t r a t i o n s h o u l d n o t be lowered
on
nil40 days 75 days n~_iI40 days 75 days 701 o o 0 901 0 90 I 3.3 4.4 I
MS + 4.5% mannitol Mat ura tion
cultured
MS C + 5% mannitol MS C + 5% mannitol
media
tested
further develop[UM] hormone ment of embryoids +I) 2.9 GA 2.9 GA 3.0 ABA free +/free 4.7 Kin callus 2.9 GA 3.8 ABA free +/+/free 0.4 Pic +/0.48 KIZ 2~ callus 0.48 KIZ 2~ 0.4 ABA 0.2 Pic 0.2 KIZ 2~ callus 4.7 Kin callus 2.9 GA +/1.9 ABA 2.9 GA 3.0 ABA free +/+/free 0.48 KIZ 2 )
3.0
ABA
0.12 NAA + 1.5 GA 4.7 Kin callus MS C + 6% mannitol free 1}not longer than i0 days 2)mixture of 1/3 kinetin, 2iP and zeatin, respectively
382 of some different hormones and their combinations were studied. Adding ABA at the globular stage (in different concentrations) should be advantageous since ABA was also d e t e c t a b l e in zygotic pea embryos at this stage (Browning 1980). As discussed by Browning, we also found that external ABA inhibited further d e v e l o p m e n t of embryos. Finally, the A B A - t r e a t e d embryos turned brown on top and died. Optimum results were o b t a i n e d with combinations of GA3, which seems to promote embryo m a t u r a t i o n (1.5 ~M GA3, 0.12 ~M NAA) followed by g e r m i n a t i o n (2.9 ~M GA3) within a few days (Fig. Ib, Table 3). The d e p e n d e n c e on external GA3 for c o n t i n u i n g m a t u r a t i o n may be explained by the lack of testa in somatic embryos, since this tissue provides g i b b e r e l l i n catabolites and -products to the zygotic embryo during seed d e s i c c a t i o n {Sponsel 1983) as well as promotes the accumulation of osmotic solutes (Miyamoto & Kamisaka 1988). Additionally, growing seedlings are d e p e n d e n t on d e n o v o g i b b e r e l l i n biosynthesis, especially for their epicotyl elongation (Sponsel 1983) and the somatic embryos thus probably require external GA3 at high levels during germination. G e r m i n a t i n g somatic embryos m o s t l y had rapidly growing roots and w e l l - d e v e l o p e d shoot apices, but no typical cotyledons (Fig ib). However, typical storage proteins could be found in these somatic embryos (Lehminger-Mertens et al., in prep.). Nevertheless, transfer to h o r m o n e - f r e e h a l f - s t r e n g t h m e d i u m promoted their growth towards forming plantlets (Fig. ic). Later on, the r e g e n e r a t e d plantlets were potted in sterile soil, initially covered with a glass vessel, which could be taken off within a week (Fig. id). A d a p t a t i o n to u n s t e r i l e conditions took place in a growth chamber. Until now, these r e g e n e r a t e d plants show a quite normal m o r p h o l o g y (Fig. le), p r o d u c i n g flowers and setting seeds.
ACKNOWLEDGEMENTS
This work has been supported by a GFP/BMFT grant to H.-J. Jacobsen (A 24/87-ZF). The authors wish to thank Mr. Frazer Walker for critical reading of the manuscript.
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