Plant Cell Reports
Plant Cell Reports (1982) 1 : 154-156
© Springer-Verlag 1982
In vitro Haploid Plantlet Induction in Physalis ixocarpa Brot. Through Microspore Embryogenesis V. A. Bapat* and G. Wenzel** Max-Planck-Institut fiir Zfichtungsforschung (Erwin-Baur-Institut) D-5000 K61n 30, Federal Republic of Germany Received March 12, 1982; April 26, 1982
Abstract Anthers of P h y s a l i s ixocarpa Brot. exised from 2-3 mm long flower buds were treated for 2 d at 3°C, and w e r e c u l t u r e d on the basal m e d i u m of NN (1969), supplemented w i t h p l a n t hormones. T h e y formed e m b r y o i d s from m i c r o s p o r e s w i t h i n 6 weeks. Upon transfer to a regener a t i o n medium, e m b r y o i d s g r e w into functional plants with the h a p l o i d number of c h r o m o s o m e s (n=12). Abbreviations CM - Coconut-milk, IAA - indole acetic acid, Kn - Kinetin, MS - M u r a s h i g e and Skoog, N N - N i t s c h and Nitsch, ZEA - Zeatin. Introduction D i f f e r e n t species of P h y s a l i s (Solanaceae) c o n t a i n steroids of m e d i c a l importance, some of w h i c h h a v e b e e n isolated and c h a r a c t e r i z e d (Glotter et al. 1978, A l l u r i et al. 1976). In the genus P h y s a l i s in v i t r o g r o w t h and o r g a n o g e n e s i s has b e e n s u c c e s s f u l l y achieved in the species Ph. m i n i m a Linn. (Bapat and Rao 1977). In further i n v e s t i g a t i o n s S i p a h i m a l a n i et al. (1981) d e m o n s t r a t e d the b i o s y n t h e t i c p o t e n t i a l i t y of d i p l o i d and t r i p l o i d callus tissues and r e g e n e r a n t s of Ph. minima. Hence, it m a y be feasible to improve the steroid c o n t e n t of P h y s a l i s specfes b y i n c o r p o r a t i o n of a h a p l o i d selection step. Such a t e c h n i q u e is a v a l u a b l e tool for i n c r e a s i n g the e f f i c i e n c y in the b r e e d i n g p r o g r a m s of n u t r i t i o n a l and m e d i c a l p l a n t s (Nitzsche and Wenzel 1977). The p r e s e n t c o m m u n i c a t i o n r e p o r t s the p r o d u c t i o n of h a p l o i d p l a n t l e t s v i a microspore c u l t u r e w i t h i n the anther sac of Ph. ixocarpa. Such h a p l o i d s or h o m o z y g o u s d o u b l e d h a p l o i d s respectively m a y be useful also for studies aiming at the e l u c i d a t i o n of genetic control for s e c o n d a r y b i o s y n thesis and as a s t a r t i n g m a t e r i a l for the isolation, r e g e n e r a t i o n and m a n i p u l a t i o n of a p r o t o p l a s t system. Material
and M e t h o d s
Flower buds of 2-10 m m were c o l l e c t e d from Ph. ixocarpa plants (2n=24) grown in g r e e n h o u s e s (day temperature 22 J 4°C). In 2-3 m m long buds, a h i g h propor ± tion of the m i c r o s p o r e s were in the late u n i n u c l e a t e stage. These buds were h a r v e s t e d and p r e c u l t u r e d in d r y test tubes at 3°C for two d in the dark. After surface s t e r i l i z a t i o n of the buds w i t h O.1% HgCl for 20 m i n and subsequent w a s h i n g w i t h the a u t o c l a v e d water, the anthers were d i s s e c t e d o u t and p l a t e d on
the culture m e d i u m of NN (1969) or on MS (1962) basal m e d i u m s u p p l e m e n t e d w i t h p l a n t hormones. Simultaneously anthers w e r e also c u l t u r e d in the liquid media as float cultures as d e s c r i b e d by S u n d e r l a n d and Roberts (1979). For e x p e r i m e n t s on the isolated p o l l e n culture, anthers w e r e crushed and m i c r o s p o r e s w e r e s e p a r a t e d first by filtration and later by sucrose gradients as d e s c r i b e d b y Wenzel et al. (1975). After w a s h i n g w i t h the w a s h i n g m e d i u m the m i c r o s p o r e s w e r e c u l t u r e d in the liquid m e d i a containing d i f f e r e n t comb i n a t i o n s and c o n c e n t r a t i o n s of auxins and cytokinins. D i f f e r e n t i a t e d embryoids were t r a n s f e r r e d to the r e g e n e r a t i o n medium, a m o d i f i e d MS medium, for plantlet development. P l o i d y level of the p l a n t l e t s was d e t e r m i n e d by c o u n t i n g c h r o m o s o m e s from y o u n g leaves. For each experiment, solid and liquid medium, 15 anthers w e r e c u l t u r e d per 6 cm petri d i s h e s and each t r e a t m e n t c o n s i s t e d of 5 to 8 replicates. Half the cultures w e r e k e p t in the d a r k and half in the 12 h light regime of 2,000 lx at t e m p e r a t u r e of 26 ~ l°C. Results Of the two basal m e d i a tested (NN and MS), embryoid f o r m a t i o n from the m i e r o s p o r e s was a c h i e v e d only on NN medium, c o n t a i n i n g 5 mgl- 1 IAA and 0.2 mgl- i Kn. Fig. la d e m o n s t r a t e s the first d i v i s i o n of m i c r o s p o r e s on this medium. In c o n t r a s t to several other r e p o r t s (Maheshwari et al. 1980), a c t i v a t e d charcoal at 0.5% did not enhance the e m b r y o i d formation. I l l u m i n a t i o n was also not e s s e n t i a l and there was even a t e n d e n c y that from anthers kept in the dark about 10% more macroscopic structures developed. After iO d of anther culture p r o e m b r y o i d s could be isolated, w h i c h were just b u r s t i n g t h r o u g h the exine (Fig. ib). On average 50 - 60% of the p l a t e d 2,O00 anthers showed p r o e m b r y old formation. These structures d e v e l o p e d r a p i d l y and r e a c h e d the typical e m b r y o i d structure w i t h i n 15 d (Fig. ic). Normally, the embryoids d e v e l o p i n g from the m i c r o s p o r e s broke through the anther wall 6 weeks after plating, b u t sometimes they d r o p p e d o u t m u c h earlier and d e v e l o p e d on the m e d i u m in isolation from the anther. This o b s e r v a t i o n together w i t h the fact that u s u a l l y only one m a c r o s c o p i c structure per anther grew into a plantlet, a l t h o u g h m a n y more m i c r o s p o r e s started showing androgenesis, m a d e it feasible to try isolated mic r o s p o r e culture. However, so far no m i c r o s p o r e s showe d dii~£sions of further d e v e l o p m e n t after early separation from the anther tissue. A l s o anthers c u l t u r e d in the liquid NN m e d i u m as float cultures showed no
* Present address: Bhabha Atomic Research Centre, Bio-Organic Division, Bombay, India ** Present and correspondence address:Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Institut fiir Resistenzgenetik,
D-8059 Griinbach, FRG
0721-7714/82/0001/0154/$ 01.00
155
Fig.
i: D e v e l o p m e n t
of P h y s a l i s
ixocarpa microspores
into h a p l o i d
plantlets.
a) I n i t i a t i o n of d i v i s i o n s on a microspore; b) d e v e l o p m e n t of an e m b r y o i d from a m i c r o s p o r e ; c) six celled embryoid; d) e m b r y o i d s g r o w i n g on MS r e g e n e r a t i o n medium; e) squash p r e p a r a t i o n of a young leaf s h o w i n g h a p l o i d number of c h r o m o s o m e s (n=12) ; f) p l a n t l e t s d e v e l o p i n g on G a m b o r g et al. B5 medium. e m b r y o i d formation. S h e d d i n g of the m i c r o s p o r e s was a slow process. C o m b i n a t i o n s of growth h o r m o n e s or the a d d i t i o n of O.O1% of c h a r c o a l failed to p r o v o k e any p o s i t i v e r e s p o n s e from such f l o a t i n g anthers. T h e y rem a i n e d green for m o r e than 6 weeks, p r o b a b l y d e m o n s t r a ting that the shift from the d e v e l o p m e n t a l route of p o l l e n grains to the s p o r o p h y t i c route was not induced successfully. The e m b r y o i d s o b t a i n e d from the m i c r o s p o r e s cultured w i t h i n an anther on the agar m e d i a w e r e isolated after 6 weeks f r o m the anther tissue and t r a n s f e r r e d to the r e g e n e r a t i o n medium, c o n t a i n i n g m a c r o - and micr o - n u t r i e n t s of MS medium, t o g e t h e r w i t h 10% CM and 0.3 mgl -I ZEA. On this m e d i u m w i t h i n 3 weeks m u l t i p l e bud f o r m a t i o n started (Fig. i d). F r o m these roots and normal p l a n t s w e r e d e v e l o p e d (Fig. i f] u p o n t r a n s f e r to the B5 m e d i u m of G a m b o r g et al. (1968) w h i c h g r e w to full m a t u r i t y in the greenhouse. S q u a s h p r e p a r a t ions f r o m y o u n g leaves showed the h a p l o i d n u m b e r of c h r o m o s o m e s (Fig. I e). Discussion The p r e s e n t i n v e s t i g a t i o n d e m o n s t r a t e s that the devel o p m e n t of e m b r y o i d s and h a p l o i d p l a n t l e t s (n=12) f r o m m i c r o s p o r e s of Ph. i x o c a r p a (2n=24) is possible; in c o n t r a s t to the a n d r o g e n e s i s of Ph. m i n i m a (2n-48) (George and Rao ~979), w h e r e all p l a n t s showed the t r i p l o i d n u m b e r of c h r o m o s o m e s . P r o b a b l y the .formation of 3n plants is m o r e f r e q u e n t f r o m p o l y p l o i d s as in Ph. m i n i m a than f r o m a true d i p l o i d species such as Ph. ixocarpa. For the r e g e n e r a t i o n e x p e r i m e n t s NN m e d i u m was s u p e r i o r o v e r the MS medium, but as in other species, c o c o n u t m i l k was e s s e n t i a l for further e m b r y o i d development. C o m p a r i s o n of d i f f e r e n t m i c r o spore c u l t u r e t e c h n i q u e s d e m o n s t r a t e d that the m o s t f r e q u e n t r e g e n e r a t i o n of h a p l o i d s was o b t a i n e d f r o m m i c r o s p o r e c u l t u r e w i t h i n the anther. E v e n b y o m i t t i n g
the agar, and c u l t u r i n g the anthers on identical med i u m in float c u l t u r e r n o r e s p o n s e was observed. Surp r i s i n g was the clear e m b r y o g e n i c c a p a c i t y of Ph. ixocarpa microspores. We did not o b s e r v e callus formation from the m i c r o s p o r e or as a s e c o n d a r y effect from an o r i g i n a l l y formed embryoid. This m i g h t be due to the low levels of sucrose in the medium. The r e l a t i v e h i g h c o n c e n t r a t i o n of the h e a t instable IAA was actually in the m e d i u m after a u t o c l a v i n g so low that it did n o t induce callus formation. The d i r e c t e m b r y o i d f o r m a t i o n h a s t e n e d p l a n t l e t f o r m a t i o n and, in addition, a v o i d e d the p o s s i b i l i t y for c h r o m o s o m e variations, w h i c h are f r e q u e n t d u r i n g longer callus stages. There are no data a v a i l a b l e yet in the steroid content of the h a p l o i d regenerants. A m o n g s t the d i f f e r e n t regenerants, a w i d e v a r i a b i l i t y is expected, as the donor m a t e r i a l will not be homozygous. It should be p o s s i b l e to increase v i a p o l y p l o i d i z a t i o n the secondary p r o d u c t formation, after simple s e l e c t i o n at the h a p l o i d level. In Ph. m i n i m a for example, the callus f r o m t r i p l o i d plants c o n % a i n e d a b o u t twice the steroid c o n c e n t r a t i o n c o m p a r e d to callus from d i p l o i d p l a n t s (Sipahimalani et al. 1981). A m o s t favorable a p p r o a c h for h a p l o i d genome c o m b i n a t i o n is via p r o t o p l a s t fusion. One p r e r e q u i s i t e for such e x p e r i m e n t s is the reg e n e r a t i o n of p r o t o p l a s t s from d i p l o i d s t a r t i n g material of Ph. i x o c a r p a which has b e e n a c c o m p l i s h e d (Bap a t and S c h i e d e r ~981). Acknowledgement We thank Mrs. A. T r u c k e n b r o d t and Mr. W. L e h m a n n for the t e c h n i c a l a s s i s t a n c e and Mr. D. B o c k for the photography. VAB wishes to thank B M F T / K F A J ~ l i c h for the f e l l o w s h i p and BARC, DEA, Bombay, India, for the deputation.
156 References Alluri RR, Miller RJ, Shelver WH, W e h b a k h a l i l SK (1976) Lloydia 3 9 : 4 0 5 Bapat VA, Rao PS (1977) Z P f l a n z e n p h y s i o l 8 5 : 4 0 3 - 4 1 6 Bapat VA, Sehieder O (1981) P l a n t Cell Reports i: 697O Gamborg OL, Miller RA, O j i m a K (1968) Exp Cell Res 50: 151-158 George L, Rao PS (1979) P r o t o p l a s m a iOO: 13-19 Glotter E, Kirson I, A b r a h a m A, Sethi PC, S u b r a m a n i a m SS (1975) J C h e m Soc I 1370
M a h e s h w a r i SC, Tyagi AK, M a l h o t r a K, Sopory SK (1980) Theor AppI Genet 58: 193-206 Murashige T, Skoog F (1962) Physiol P l a n t 1 5 : 4 7 3 - 4 9 7 Nitsch JP, N i t s c h C (1969) Science 163: 85-87 N i t z s c h e W, Wenzel G (1977) Haploids in p l a n t breeding, P Parey, Berlin und H a m b u r g Sipahimalani AT, B a p a t VA, Rao PS, Chadha MS (1981) J Nat Products 44: 114-118 S u n d e r l a n d N, Roberts M (1979) Ann Bot 4 3 : 4 0 5 - 4 1 4 Wenzel G, H o f f m a n n F, Potrykus I, Thomas E (1975) Mol gen G e n e t 138: 293-297
Plant Cell Reports (1982) 1 : 154-156
© Springer-Verlag 1982
In vitro Haploid Plantlet Induction in Physalis ixocarpa Brot. Through Microspore Embryogenesis V. A. Bapat* and G. Wenzel** Max-Planck-Institut fiir Zfichtungsforschung (Erwin-Baur-Institut) D-5000 K61n 30, Federal Republic of Germany Received March 12, 1982; April 26, 1982
Abstract Anthers of P h y s a l i s ixocarpa Brot. exised from 2-3 mm long flower buds were treated for 2 d at 3°C, and w e r e c u l t u r e d on the basal m e d i u m of NN (1969), supplemented w i t h p l a n t hormones. T h e y formed e m b r y o i d s from m i c r o s p o r e s w i t h i n 6 weeks. Upon transfer to a regener a t i o n medium, e m b r y o i d s g r e w into functional plants with the h a p l o i d number of c h r o m o s o m e s (n=12). Abbreviations CM - Coconut-milk, IAA - indole acetic acid, Kn - Kinetin, MS - M u r a s h i g e and Skoog, N N - N i t s c h and Nitsch, ZEA - Zeatin. Introduction D i f f e r e n t species of P h y s a l i s (Solanaceae) c o n t a i n steroids of m e d i c a l importance, some of w h i c h h a v e b e e n isolated and c h a r a c t e r i z e d (Glotter et al. 1978, A l l u r i et al. 1976). In the genus P h y s a l i s in v i t r o g r o w t h and o r g a n o g e n e s i s has b e e n s u c c e s s f u l l y achieved in the species Ph. m i n i m a Linn. (Bapat and Rao 1977). In further i n v e s t i g a t i o n s S i p a h i m a l a n i et al. (1981) d e m o n s t r a t e d the b i o s y n t h e t i c p o t e n t i a l i t y of d i p l o i d and t r i p l o i d callus tissues and r e g e n e r a n t s of Ph. minima. Hence, it m a y be feasible to improve the steroid c o n t e n t of P h y s a l i s specfes b y i n c o r p o r a t i o n of a h a p l o i d selection step. Such a t e c h n i q u e is a v a l u a b l e tool for i n c r e a s i n g the e f f i c i e n c y in the b r e e d i n g p r o g r a m s of n u t r i t i o n a l and m e d i c a l p l a n t s (Nitzsche and Wenzel 1977). The p r e s e n t c o m m u n i c a t i o n r e p o r t s the p r o d u c t i o n of h a p l o i d p l a n t l e t s v i a microspore c u l t u r e w i t h i n the anther sac of Ph. ixocarpa. Such h a p l o i d s or h o m o z y g o u s d o u b l e d h a p l o i d s respectively m a y be useful also for studies aiming at the e l u c i d a t i o n of genetic control for s e c o n d a r y b i o s y n thesis and as a s t a r t i n g m a t e r i a l for the isolation, r e g e n e r a t i o n and m a n i p u l a t i o n of a p r o t o p l a s t system. Material
and M e t h o d s
Flower buds of 2-10 m m were c o l l e c t e d from Ph. ixocarpa plants (2n=24) grown in g r e e n h o u s e s (day temperature 22 J 4°C). In 2-3 m m long buds, a h i g h propor ± tion of the m i c r o s p o r e s were in the late u n i n u c l e a t e stage. These buds were h a r v e s t e d and p r e c u l t u r e d in d r y test tubes at 3°C for two d in the dark. After surface s t e r i l i z a t i o n of the buds w i t h O.1% HgCl for 20 m i n and subsequent w a s h i n g w i t h the a u t o c l a v e d water, the anthers were d i s s e c t e d o u t and p l a t e d on
the culture m e d i u m of NN (1969) or on MS (1962) basal m e d i u m s u p p l e m e n t e d w i t h p l a n t hormones. Simultaneously anthers w e r e also c u l t u r e d in the liquid media as float cultures as d e s c r i b e d by S u n d e r l a n d and Roberts (1979). For e x p e r i m e n t s on the isolated p o l l e n culture, anthers w e r e crushed and m i c r o s p o r e s w e r e s e p a r a t e d first by filtration and later by sucrose gradients as d e s c r i b e d b y Wenzel et al. (1975). After w a s h i n g w i t h the w a s h i n g m e d i u m the m i c r o s p o r e s w e r e c u l t u r e d in the liquid m e d i a containing d i f f e r e n t comb i n a t i o n s and c o n c e n t r a t i o n s of auxins and cytokinins. D i f f e r e n t i a t e d embryoids were t r a n s f e r r e d to the r e g e n e r a t i o n medium, a m o d i f i e d MS medium, for plantlet development. P l o i d y level of the p l a n t l e t s was d e t e r m i n e d by c o u n t i n g c h r o m o s o m e s from y o u n g leaves. For each experiment, solid and liquid medium, 15 anthers w e r e c u l t u r e d per 6 cm petri d i s h e s and each t r e a t m e n t c o n s i s t e d of 5 to 8 replicates. Half the cultures w e r e k e p t in the d a r k and half in the 12 h light regime of 2,000 lx at t e m p e r a t u r e of 26 ~ l°C. Results Of the two basal m e d i a tested (NN and MS), embryoid f o r m a t i o n from the m i e r o s p o r e s was a c h i e v e d only on NN medium, c o n t a i n i n g 5 mgl- 1 IAA and 0.2 mgl- i Kn. Fig. la d e m o n s t r a t e s the first d i v i s i o n of m i c r o s p o r e s on this medium. In c o n t r a s t to several other r e p o r t s (Maheshwari et al. 1980), a c t i v a t e d charcoal at 0.5% did not enhance the e m b r y o i d formation. I l l u m i n a t i o n was also not e s s e n t i a l and there was even a t e n d e n c y that from anthers kept in the dark about 10% more macroscopic structures developed. After iO d of anther culture p r o e m b r y o i d s could be isolated, w h i c h were just b u r s t i n g t h r o u g h the exine (Fig. ib). On average 50 - 60% of the p l a t e d 2,O00 anthers showed p r o e m b r y old formation. These structures d e v e l o p e d r a p i d l y and r e a c h e d the typical e m b r y o i d structure w i t h i n 15 d (Fig. ic). Normally, the embryoids d e v e l o p i n g from the m i c r o s p o r e s broke through the anther wall 6 weeks after plating, b u t sometimes they d r o p p e d o u t m u c h earlier and d e v e l o p e d on the m e d i u m in isolation from the anther. This o b s e r v a t i o n together w i t h the fact that u s u a l l y only one m a c r o s c o p i c structure per anther grew into a plantlet, a l t h o u g h m a n y more m i c r o s p o r e s started showing androgenesis, m a d e it feasible to try isolated mic r o s p o r e culture. However, so far no m i c r o s p o r e s showe d dii~£sions of further d e v e l o p m e n t after early separation from the anther tissue. A l s o anthers c u l t u r e d in the liquid NN m e d i u m as float cultures showed no
* Present address: Bhabha Atomic Research Centre, Bio-Organic Division, Bombay, India ** Present and correspondence address:Biologische Bundesanstalt fiir Land- und Forstwirtschaft, Institut fiir Resistenzgenetik,
D-8059 Griinbach, FRG
0721-7714/82/0001/0154/$ 01.00
155
Fig.
i: D e v e l o p m e n t
of P h y s a l i s
ixocarpa microspores
into h a p l o i d
plantlets.
a) I n i t i a t i o n of d i v i s i o n s on a microspore; b) d e v e l o p m e n t of an e m b r y o i d from a m i c r o s p o r e ; c) six celled embryoid; d) e m b r y o i d s g r o w i n g on MS r e g e n e r a t i o n medium; e) squash p r e p a r a t i o n of a young leaf s h o w i n g h a p l o i d number of c h r o m o s o m e s (n=12) ; f) p l a n t l e t s d e v e l o p i n g on G a m b o r g et al. B5 medium. e m b r y o i d formation. S h e d d i n g of the m i c r o s p o r e s was a slow process. C o m b i n a t i o n s of growth h o r m o n e s or the a d d i t i o n of O.O1% of c h a r c o a l failed to p r o v o k e any p o s i t i v e r e s p o n s e from such f l o a t i n g anthers. T h e y rem a i n e d green for m o r e than 6 weeks, p r o b a b l y d e m o n s t r a ting that the shift from the d e v e l o p m e n t a l route of p o l l e n grains to the s p o r o p h y t i c route was not induced successfully. The e m b r y o i d s o b t a i n e d from the m i c r o s p o r e s cultured w i t h i n an anther on the agar m e d i a w e r e isolated after 6 weeks f r o m the anther tissue and t r a n s f e r r e d to the r e g e n e r a t i o n medium, c o n t a i n i n g m a c r o - and micr o - n u t r i e n t s of MS medium, t o g e t h e r w i t h 10% CM and 0.3 mgl -I ZEA. On this m e d i u m w i t h i n 3 weeks m u l t i p l e bud f o r m a t i o n started (Fig. i d). F r o m these roots and normal p l a n t s w e r e d e v e l o p e d (Fig. i f] u p o n t r a n s f e r to the B5 m e d i u m of G a m b o r g et al. (1968) w h i c h g r e w to full m a t u r i t y in the greenhouse. S q u a s h p r e p a r a t ions f r o m y o u n g leaves showed the h a p l o i d n u m b e r of c h r o m o s o m e s (Fig. I e). Discussion The p r e s e n t i n v e s t i g a t i o n d e m o n s t r a t e s that the devel o p m e n t of e m b r y o i d s and h a p l o i d p l a n t l e t s (n=12) f r o m m i c r o s p o r e s of Ph. i x o c a r p a (2n=24) is possible; in c o n t r a s t to the a n d r o g e n e s i s of Ph. m i n i m a (2n-48) (George and Rao ~979), w h e r e all p l a n t s showed the t r i p l o i d n u m b e r of c h r o m o s o m e s . P r o b a b l y the .formation of 3n plants is m o r e f r e q u e n t f r o m p o l y p l o i d s as in Ph. m i n i m a than f r o m a true d i p l o i d species such as Ph. ixocarpa. For the r e g e n e r a t i o n e x p e r i m e n t s NN m e d i u m was s u p e r i o r o v e r the MS medium, but as in other species, c o c o n u t m i l k was e s s e n t i a l for further e m b r y o i d development. C o m p a r i s o n of d i f f e r e n t m i c r o spore c u l t u r e t e c h n i q u e s d e m o n s t r a t e d that the m o s t f r e q u e n t r e g e n e r a t i o n of h a p l o i d s was o b t a i n e d f r o m m i c r o s p o r e c u l t u r e w i t h i n the anther. E v e n b y o m i t t i n g
the agar, and c u l t u r i n g the anthers on identical med i u m in float c u l t u r e r n o r e s p o n s e was observed. Surp r i s i n g was the clear e m b r y o g e n i c c a p a c i t y of Ph. ixocarpa microspores. We did not o b s e r v e callus formation from the m i c r o s p o r e or as a s e c o n d a r y effect from an o r i g i n a l l y formed embryoid. This m i g h t be due to the low levels of sucrose in the medium. The r e l a t i v e h i g h c o n c e n t r a t i o n of the h e a t instable IAA was actually in the m e d i u m after a u t o c l a v i n g so low that it did n o t induce callus formation. The d i r e c t e m b r y o i d f o r m a t i o n h a s t e n e d p l a n t l e t f o r m a t i o n and, in addition, a v o i d e d the p o s s i b i l i t y for c h r o m o s o m e variations, w h i c h are f r e q u e n t d u r i n g longer callus stages. There are no data a v a i l a b l e yet in the steroid content of the h a p l o i d regenerants. A m o n g s t the d i f f e r e n t regenerants, a w i d e v a r i a b i l i t y is expected, as the donor m a t e r i a l will not be homozygous. It should be p o s s i b l e to increase v i a p o l y p l o i d i z a t i o n the secondary p r o d u c t formation, after simple s e l e c t i o n at the h a p l o i d level. In Ph. m i n i m a for example, the callus f r o m t r i p l o i d plants c o n % a i n e d a b o u t twice the steroid c o n c e n t r a t i o n c o m p a r e d to callus from d i p l o i d p l a n t s (Sipahimalani et al. 1981). A m o s t favorable a p p r o a c h for h a p l o i d genome c o m b i n a t i o n is via p r o t o p l a s t fusion. One p r e r e q u i s i t e for such e x p e r i m e n t s is the reg e n e r a t i o n of p r o t o p l a s t s from d i p l o i d s t a r t i n g material of Ph. i x o c a r p a which has b e e n a c c o m p l i s h e d (Bap a t and S c h i e d e r ~981). Acknowledgement We thank Mrs. A. T r u c k e n b r o d t and Mr. W. L e h m a n n for the t e c h n i c a l a s s i s t a n c e and Mr. D. B o c k for the photography. VAB wishes to thank B M F T / K F A J ~ l i c h for the f e l l o w s h i p and BARC, DEA, Bombay, India, for the deputation.
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