Plant Cell Reports (1985) 4: 108-111
Plant Cell Reports © Springer-Verlag 1985
Somatic embryogenesis and plant regeneration in Cichorium intybus L. (Witloof, Compositae) Kristin M. G. Heirwegh 1, Nirmalya Banerjee 1, Karel van Nerum 2, and Edmond de Langhe 1 1 Laboratorium voor Teeltfysiologie en Tropische Fytotechnie K.U.Leuven, K. Mercierlaan, 92, B-3030 Leuven (Heverlee), Belgium 2 Studiecentrum voor Tuinbouwgronden K.U.Leuven, B-3030 Leuven, Belgium Received March 15, 1985 - Communicated by D. von Wettstein
Abstract Somatic e m b r y o g e n e s i s of C i c h o r i u m intybus L. var. 'Carolus' is induced using cubical pieces of mature tap roots with an i n t e r v e n i n g callus phase. A M u r a s h i g e and Skooq's (MS) semi solid basal m e d i u m s u p p l e m e n t e d with 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid 0 . 0 2 or 0.2 m g / O and b e n z y l a m i n o p u r i n e ~ . 2 6 m g / l ) a n d a liquid MS m e d i u m devoid of growth regulators are used r e s p e c t i v e l y for induction of callus and somatic e m b r y o i d s and for further d e v e l o p m e n t and germination. R e g e n e r a t i o n from the nodular p r o e m b r y o n a l stage to the full grown embryoids occurs following d i f f e r e n t m o r p h o l o g i c a l pathways d e p e n d i n g on the physical and chemical e n v i r o n m e n t of the culture. Further d e v e l o p m e n t of these embryos into p l a n t l e t s and the p o s s i b i l i t i e s of a p p l i c a t i o n of this technique in p l a n t b r e e d i n g have b e e n discussed. Abbreviations MS, M u r a s h i g e and Skoog medium; BAP, benzylaminopurine; 2,4-D, 2,4-dichlorop h e n o x y a c e t i c acid. Introduction C i c h o r i u m intybus L., a member of the Compositae is a popular vegetable crop in w e s t e r n e u r o p e a n countries. It is also c o n s i d e r e d as one of the most important export crop products of Belgium. However, the genetic i m p r o v e m e n t of the plant is still b e i n g h a n d i c a p p e d by a p r e v a l e n t self i n c o m p a t i b i l i t y (Eeninck, 1979) w h i c h creates extreme d i f f i c u l t y in o b t a i n i n g homozygous parent p o p u l a t i o n s needed for the p r o d u c t i o n of h y b r i d seeds. This p r o b l e m of s e l f - i n c o m p a t i b i l i t y could be c i r c u m v e n t e d by the a p p l i c a t i o n of somatic e m b r y o g e n e s i s techniques. Non-homozygous, not inbred parent plants could e v e n t u a l l y be r e p r o d u c e d rapidly on a large scale via somatic embryogenesis. F r o m the p l a n t l e t s of two i n d i v i d u a l parent p o p u l a t i o n s showing good c o m b i n a t i o n capacity, r e l a t i v e l y u n i f o r m hybrids with increased v i t a l i t y could be obtained.
Offprint requests to: K.M.G. Heirwegh
N o t w i t h s t a n d i n g the fact that the cases of e s t a b l i s h e d e m b r y o g e n e s i s in somatic cultures already cover a wide taxonomic spectrum, those in the members of C o m p o s i t a e a r e e x t r e m e l y meagre. Only in C i c h o r i u m endivia L. the p r o d u c t i o n of clonal plantlets through somatic e m b r y o g e n e s i s has been reported (Vasil et al. 1964, Vasil and H i l d e b r a n d t 1966). These structures however didn't survive t r a n s f e r to soil. In the present paper we describe the i n d u c t i o n of somatic embryos in a n o t h e r species of Compositae and the successive d e v e l o p m e n t a l stages of these embryos leading to p l a n t l e t r e g e n e r a t i o n and growth in the soil. M a t e r i a l s and }~thods Storage roots of C. intybus L. var. Carolus were used as the source of explants for the induction of callus. Well cleaned roots were surface s t e r i l i s e d by 90% ethanol and 1.5% c a l c i u m h y p o c h l o r i t e for 15 seconds and 15 minutes r e s p e c t i v e l y f o l l o w e d by repeated w a s h i n g in a u t o c l a v e d d i s t i l l e d water. Roots were peeled off and excised a s e p t i c a l l y into small cubical pieces of 5 ntm d i m e n s i o n and t r a n s f e r r e d i m m e d i a t e l y to culture tubes c o n t a i n i n g 35 ml of MS basal m e d i u m (Murashige and Skoog 1962) s u p p l e m e n t e d w i t h MS vitamins, iOO mg/l meso-inositol, 2,4-D (0.02 or 0.2 mg/l) and BAP (0.26 mg/l). The m e d i u m was solidified w i t h 6 g/l oxoid technical agar LI3 and the pH was a d j u s t e d to 5.7. This m e d i u m was used for the induction and m a i n t e n a n c e of the callus as well as for the induction of somatic embryoids. Cultures were incubated at 25 + 1 °C in n r e s e n c e of 16, hours of light of ~O00 lux f o l l o w e d by 8 hours of dark regime. For the d i f f e r e n t i a t i o n of somatic embryos from nodular stage to the full grown stage liquid or solid m e d i u m was used. For g e r m i n a t i o n of the full grown e m b r y o i d s d i f f e r e n t media were tested by v a r y i n g the c o m p o s i t i o n of the culture medium, for example lowering the nH of the m e d i u m from 5.7 to 5 and/or lowering the sucrose c o n c e n t r a t i o n to 20 and i0 g/l, lowering the strength of major
109 i n o r g a n i c salts, c h a n g i n g c o m p o s i t i o n as well as c o n c e n t r a t i o n of growth regulators. Well rooted p l a n t l e t s d e r i v e d t h r o u g h somatic e m b r y o g e n e s i s were t r a n s f e r r e d to Dots c o n t a i n i n g n o n - s t e r i l e b l a c k soil in the glass-house. Results
and D i s c u s s i o n
I N D U C T I O N OF CALLUS AND D E V E L O P M E N T EMBRYOS ON S E M I S O L I D M E D I U M
OF SOMATIC
White to pale green calli were visible w i t h i n 1 week after the i n o c u l a t i o n on the swollen surface Of the explanto(F±g, i). The m o r p h o g e n e t i c response of the p r i m a r y e x n l a n t tissue is given in table i. Table
i: M o r p h o g e n e t i c response in p r i m a r y cultures on s e m i s o l i d MS c o m p l e m e n t e d w i t h d i f f e r e n t growth regulators. (mg/l)
growth regulator
-
callus
shooting
rooting
somatic embryos
+
+
+
-
BAP
0.26
+
+
+
-
2,4-D
0.02
+
+
+
-
BAP 2,4-D
0.26 0.02
+
+
+
+
BAP 0.26 2,4-D 0.20
+
+
+
+
The p r i m a r y e x p l a n t s s p o n t a n e o u s l y formed callus f o l l o w e d by shoot and root o r g a n o g e n e s i s i r r e s p e c t i v e of the type and c o n c e n t r a t i o n of the growth r e g u l a t o r s added to the medium. Upon p r o l o n g e d (8 to 12 weeks) i n c u b a t i o n the cultures in p r e s e n c e of 2,4-D and BAP formed pale brown n a d u l a r callus in w h i c h the early stages of zygotic like e m b r y o g e n e s i s could be d i s t i n g u i s h e d . Full grown somatic embryos could be o b t a i n e d when this n o d u l a r callus was t r a n s f e r r e d to MS w i t h o u t any growth r e g u l a t o r s (Fig. 2,3). These somatic embryos are b i p o l a r s t r u c t u r e s w i t h a r a d i c u l a r end and two c o t y l e d o n s at the o p p o s i t e end (Fig. 2). M o r p h o l o g i c a l l y these structures r e s e m b l e d their zygotic c o u n t e r p a r t s a l t h o u g h the latter have r e l a t i v e l y larger and thicker cotyledons. A l t h o u g h the shoot d e v e l o p e d some new leaves (F£g. 4), none of these embryos p r o c e e d e d further to form a normal plantlet. This forms a c o n t r a s t w i t h the e m b r y o i d s w h i c h d e v e l o p e d c o m p l e t e l y in l i q u i d medium. A b n o r m a l e m b r y o i d s w i t h three c o t y l e d o n s w e r e also observed.
DEVELOPMENT
OF SOMATIC
EMBRYOS
IN LIQUID M E D I U M
The s u b c u l t u r e d callus when b r o u g h t into s u s p e n s i o n after 2 or 3 weeks (MS w i t h o u t growth regulators) d e v e l o p e d pale brown globules on the surface w i t h i n 4 weeks. A l t h o u g h one could observe free cells in the s u s p e n s i o n w h i c h d i v i d e d r a p i d l y and gave rise to small clumps of d e n s e l y c y t o p l a s m i c cells the e m b r y o i d s we o b t a i n e d had d e v e l o p e d from the small globules formed on the callus surface a n d not from the free cell groups. These g l o b u l a r structures, p r i n c i p a l l y c o n s i s t e d of h i g h l y m e r i s t e m a t i c small cells, could e a s i l y be s e p a r a t e d from the callus surface. When the e m b r y o g e n i c callus was i n c u b a t e d at 16 h p h o t o p e r i o d , the nodules formed first a central green spot and later on a small shoot (Fig. 5) or a shoot t o g e t h e r w i t h a root s i m u l t a n e o u s l y b r e a k i n g t h r o u g h the nodule (Fig. 7). In most cases the shoots c o n s i s t e d of two opposite leaves c o m p a r a b l e to the cotyledons. Similar structures in liquid m e d i u m had been o b t a i n e d in the cell cultures of C. e n d i v i a where the root had d e v e l o p e d first followed by the o u t g r o w t h of the shoot (Vasil & H i l d e b r a n d t 1966). To date, that is the only p u b l i s h e d report of somatic e m b r y o g e n e s i s in the family Compositae. In the genus T a r a x a c u m (Compositae), n o d u l a r structures were o b t a i n e d d u r i n g somatic e m b r y o g e n e s i s in liquid m e d i u m w h i c h f o l l o w e d the s u c c e s s i v e d e v e l o p m e n t a l p a t t e r n c o m p a r a b l e to our findings (Ammirato, p e r s o n a l communication). A l t h o u g h the d e v e l o p m e n t a l stages of somatic e m b r y o g e n e s i s in liquid m e d i u m in these 3 species ~iffer from the c o n v e n t i o n a l zygotic pathway, it m i g h t be c o n s i d e r e d as an a l t e r n a t i v e p a t t e r n typical for C~mpositae. In some cases more than One embryo d e v e l o p e d on the nodule in liquid medium. These embryos arose from the p e r i p h e r a l cells of the nodule and followed the zygotic m o r p h o g e n e t i c n a t t e r n c o m p a r a b l e to those o b t a i n e d on s e m i s o l i d m e d i u m (Fig. 6). GE~4INATION
OF THE SOMATIC
EMBRYOS
Of the d i f f e r e n t media tested to r e g e n e r a t e p l a n t l e t s from the s u s p e n s i o n d e r i v e d embryos, MS s e m i s o l i d m e d i u m of pH 5.8 c o n t a i n i n half c o n c e n t r a t i o n of major salts s u n o l e m e n t e d w i t h 2% sucrose and vitamins was found suitable. L o w e r i n g the nH and sucrose c o n c e n t r a t i o n did not have any p o s i t i v e effect on the g e r m i n a t i o n of the somatic embryos. R o o t e d p l a n t l e t s (Fig. 8) when t r a n s f e r r e d to soil showed normal growth (Fig. 9). To date somatic e m b r y o - d e r i v e d p l a n t l e t s (Fig. 8) show much p h e n o t y p i c r e s e m b l a n c e to the y o u n g seedlings (Fig. iO) . After seven weeks in the g r e e n h o u s e each plant had d e v e l o p e d one single central root of about 0.5 cm d i a m e t e r and seemed to have kent the original phenotype. The whole r e g e n e r a t i o n cycle b e g i n n i n g w i t h the i n d u c t i o n of callus p a s s i n g through the phases of i n d u c t i o n and m a t u r a t i o n of somatic e m b r y o s in liquid m e d i u m t e r m i n a t i n g at the h a r v e s t i n g of small p l a n t l e t s took
110
Fig. 1-9. P l a n t l e t r e g e n e r a t i o n through somatic e m b r y o g e n e s i s in C. intybus in vitro. i. Embryogenic callus, 2-4. Embryoids on semi-solid medium, 2,3. Zygotic-like embryoids, 4. Embryoid w i t h d e v e l o p i n g leaves, 5-7. Embryoids in liquid medium, 8. P l a n t l e t d e v e l o p e d from a somatic embryo in liquid medium, before transfer to soil, 9. D e v e l o p e d plant w i t h single taproot. Symbols: C = Cotyledon, r = root, S = Shoot.
11i about 3 months. To date, the genetic s t a b i l i t y seemed to be maintained, although this aspect has to be further i n v e s t i g a t e d and the output of plant p r o d u c t i o n by somatic e m b r y o g e n e s i s has to be b o o s t e d up before the method could be applied s u c c e s f u l l y in plant breeding. Acknowledgements We wish to thank Beatrice Longly, Lindsey Withers, P.V. Ammirato, H. B i n d i n g for their useful comments and the people of the lab for their help. References
Fig. I0. G e r m i n a t i o n and sequential d e v e l o p m e n t of C. intybus seeds.
Eeninck AH (1979) Z a a d b e l a n g e n 33:260-266 M u r a s h i g e T, Skoog F (1962) Physiol Plant 15:473-497 Vasil IK, H i l d e b r a n d t AC (1966) Am J Bot 53:860-869 Vasil IK, H i l d e b r a n d t AC, Riker AJ (1964) Science 146:76-77
Plant Cell Reports © Springer-Verlag 1985
Somatic embryogenesis and plant regeneration in Cichorium intybus L. (Witloof, Compositae) Kristin M. G. Heirwegh 1, Nirmalya Banerjee 1, Karel van Nerum 2, and Edmond de Langhe 1 1 Laboratorium voor Teeltfysiologie en Tropische Fytotechnie K.U.Leuven, K. Mercierlaan, 92, B-3030 Leuven (Heverlee), Belgium 2 Studiecentrum voor Tuinbouwgronden K.U.Leuven, B-3030 Leuven, Belgium Received March 15, 1985 - Communicated by D. von Wettstein
Abstract Somatic e m b r y o g e n e s i s of C i c h o r i u m intybus L. var. 'Carolus' is induced using cubical pieces of mature tap roots with an i n t e r v e n i n g callus phase. A M u r a s h i g e and Skooq's (MS) semi solid basal m e d i u m s u p p l e m e n t e d with 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid 0 . 0 2 or 0.2 m g / O and b e n z y l a m i n o p u r i n e ~ . 2 6 m g / l ) a n d a liquid MS m e d i u m devoid of growth regulators are used r e s p e c t i v e l y for induction of callus and somatic e m b r y o i d s and for further d e v e l o p m e n t and germination. R e g e n e r a t i o n from the nodular p r o e m b r y o n a l stage to the full grown embryoids occurs following d i f f e r e n t m o r p h o l o g i c a l pathways d e p e n d i n g on the physical and chemical e n v i r o n m e n t of the culture. Further d e v e l o p m e n t of these embryos into p l a n t l e t s and the p o s s i b i l i t i e s of a p p l i c a t i o n of this technique in p l a n t b r e e d i n g have b e e n discussed. Abbreviations MS, M u r a s h i g e and Skoog medium; BAP, benzylaminopurine; 2,4-D, 2,4-dichlorop h e n o x y a c e t i c acid. Introduction C i c h o r i u m intybus L., a member of the Compositae is a popular vegetable crop in w e s t e r n e u r o p e a n countries. It is also c o n s i d e r e d as one of the most important export crop products of Belgium. However, the genetic i m p r o v e m e n t of the plant is still b e i n g h a n d i c a p p e d by a p r e v a l e n t self i n c o m p a t i b i l i t y (Eeninck, 1979) w h i c h creates extreme d i f f i c u l t y in o b t a i n i n g homozygous parent p o p u l a t i o n s needed for the p r o d u c t i o n of h y b r i d seeds. This p r o b l e m of s e l f - i n c o m p a t i b i l i t y could be c i r c u m v e n t e d by the a p p l i c a t i o n of somatic e m b r y o g e n e s i s techniques. Non-homozygous, not inbred parent plants could e v e n t u a l l y be r e p r o d u c e d rapidly on a large scale via somatic embryogenesis. F r o m the p l a n t l e t s of two i n d i v i d u a l parent p o p u l a t i o n s showing good c o m b i n a t i o n capacity, r e l a t i v e l y u n i f o r m hybrids with increased v i t a l i t y could be obtained.
Offprint requests to: K.M.G. Heirwegh
N o t w i t h s t a n d i n g the fact that the cases of e s t a b l i s h e d e m b r y o g e n e s i s in somatic cultures already cover a wide taxonomic spectrum, those in the members of C o m p o s i t a e a r e e x t r e m e l y meagre. Only in C i c h o r i u m endivia L. the p r o d u c t i o n of clonal plantlets through somatic e m b r y o g e n e s i s has been reported (Vasil et al. 1964, Vasil and H i l d e b r a n d t 1966). These structures however didn't survive t r a n s f e r to soil. In the present paper we describe the i n d u c t i o n of somatic embryos in a n o t h e r species of Compositae and the successive d e v e l o p m e n t a l stages of these embryos leading to p l a n t l e t r e g e n e r a t i o n and growth in the soil. M a t e r i a l s and }~thods Storage roots of C. intybus L. var. Carolus were used as the source of explants for the induction of callus. Well cleaned roots were surface s t e r i l i s e d by 90% ethanol and 1.5% c a l c i u m h y p o c h l o r i t e for 15 seconds and 15 minutes r e s p e c t i v e l y f o l l o w e d by repeated w a s h i n g in a u t o c l a v e d d i s t i l l e d water. Roots were peeled off and excised a s e p t i c a l l y into small cubical pieces of 5 ntm d i m e n s i o n and t r a n s f e r r e d i m m e d i a t e l y to culture tubes c o n t a i n i n g 35 ml of MS basal m e d i u m (Murashige and Skoog 1962) s u p p l e m e n t e d w i t h MS vitamins, iOO mg/l meso-inositol, 2,4-D (0.02 or 0.2 mg/l) and BAP (0.26 mg/l). The m e d i u m was solidified w i t h 6 g/l oxoid technical agar LI3 and the pH was a d j u s t e d to 5.7. This m e d i u m was used for the induction and m a i n t e n a n c e of the callus as well as for the induction of somatic embryoids. Cultures were incubated at 25 + 1 °C in n r e s e n c e of 16, hours of light of ~O00 lux f o l l o w e d by 8 hours of dark regime. For the d i f f e r e n t i a t i o n of somatic embryos from nodular stage to the full grown stage liquid or solid m e d i u m was used. For g e r m i n a t i o n of the full grown e m b r y o i d s d i f f e r e n t media were tested by v a r y i n g the c o m p o s i t i o n of the culture medium, for example lowering the nH of the m e d i u m from 5.7 to 5 and/or lowering the sucrose c o n c e n t r a t i o n to 20 and i0 g/l, lowering the strength of major
109 i n o r g a n i c salts, c h a n g i n g c o m p o s i t i o n as well as c o n c e n t r a t i o n of growth regulators. Well rooted p l a n t l e t s d e r i v e d t h r o u g h somatic e m b r y o g e n e s i s were t r a n s f e r r e d to Dots c o n t a i n i n g n o n - s t e r i l e b l a c k soil in the glass-house. Results
and D i s c u s s i o n
I N D U C T I O N OF CALLUS AND D E V E L O P M E N T EMBRYOS ON S E M I S O L I D M E D I U M
OF SOMATIC
White to pale green calli were visible w i t h i n 1 week after the i n o c u l a t i o n on the swollen surface Of the explanto(F±g, i). The m o r p h o g e n e t i c response of the p r i m a r y e x n l a n t tissue is given in table i. Table
i: M o r p h o g e n e t i c response in p r i m a r y cultures on s e m i s o l i d MS c o m p l e m e n t e d w i t h d i f f e r e n t growth regulators. (mg/l)
growth regulator
-
callus
shooting
rooting
somatic embryos
+
+
+
-
BAP
0.26
+
+
+
-
2,4-D
0.02
+
+
+
-
BAP 2,4-D
0.26 0.02
+
+
+
+
BAP 0.26 2,4-D 0.20
+
+
+
+
The p r i m a r y e x p l a n t s s p o n t a n e o u s l y formed callus f o l l o w e d by shoot and root o r g a n o g e n e s i s i r r e s p e c t i v e of the type and c o n c e n t r a t i o n of the growth r e g u l a t o r s added to the medium. Upon p r o l o n g e d (8 to 12 weeks) i n c u b a t i o n the cultures in p r e s e n c e of 2,4-D and BAP formed pale brown n a d u l a r callus in w h i c h the early stages of zygotic like e m b r y o g e n e s i s could be d i s t i n g u i s h e d . Full grown somatic embryos could be o b t a i n e d when this n o d u l a r callus was t r a n s f e r r e d to MS w i t h o u t any growth r e g u l a t o r s (Fig. 2,3). These somatic embryos are b i p o l a r s t r u c t u r e s w i t h a r a d i c u l a r end and two c o t y l e d o n s at the o p p o s i t e end (Fig. 2). M o r p h o l o g i c a l l y these structures r e s e m b l e d their zygotic c o u n t e r p a r t s a l t h o u g h the latter have r e l a t i v e l y larger and thicker cotyledons. A l t h o u g h the shoot d e v e l o p e d some new leaves (F£g. 4), none of these embryos p r o c e e d e d further to form a normal plantlet. This forms a c o n t r a s t w i t h the e m b r y o i d s w h i c h d e v e l o p e d c o m p l e t e l y in l i q u i d medium. A b n o r m a l e m b r y o i d s w i t h three c o t y l e d o n s w e r e also observed.
DEVELOPMENT
OF SOMATIC
EMBRYOS
IN LIQUID M E D I U M
The s u b c u l t u r e d callus when b r o u g h t into s u s p e n s i o n after 2 or 3 weeks (MS w i t h o u t growth regulators) d e v e l o p e d pale brown globules on the surface w i t h i n 4 weeks. A l t h o u g h one could observe free cells in the s u s p e n s i o n w h i c h d i v i d e d r a p i d l y and gave rise to small clumps of d e n s e l y c y t o p l a s m i c cells the e m b r y o i d s we o b t a i n e d had d e v e l o p e d from the small globules formed on the callus surface a n d not from the free cell groups. These g l o b u l a r structures, p r i n c i p a l l y c o n s i s t e d of h i g h l y m e r i s t e m a t i c small cells, could e a s i l y be s e p a r a t e d from the callus surface. When the e m b r y o g e n i c callus was i n c u b a t e d at 16 h p h o t o p e r i o d , the nodules formed first a central green spot and later on a small shoot (Fig. 5) or a shoot t o g e t h e r w i t h a root s i m u l t a n e o u s l y b r e a k i n g t h r o u g h the nodule (Fig. 7). In most cases the shoots c o n s i s t e d of two opposite leaves c o m p a r a b l e to the cotyledons. Similar structures in liquid m e d i u m had been o b t a i n e d in the cell cultures of C. e n d i v i a where the root had d e v e l o p e d first followed by the o u t g r o w t h of the shoot (Vasil & H i l d e b r a n d t 1966). To date, that is the only p u b l i s h e d report of somatic e m b r y o g e n e s i s in the family Compositae. In the genus T a r a x a c u m (Compositae), n o d u l a r structures were o b t a i n e d d u r i n g somatic e m b r y o g e n e s i s in liquid m e d i u m w h i c h f o l l o w e d the s u c c e s s i v e d e v e l o p m e n t a l p a t t e r n c o m p a r a b l e to our findings (Ammirato, p e r s o n a l communication). A l t h o u g h the d e v e l o p m e n t a l stages of somatic e m b r y o g e n e s i s in liquid m e d i u m in these 3 species ~iffer from the c o n v e n t i o n a l zygotic pathway, it m i g h t be c o n s i d e r e d as an a l t e r n a t i v e p a t t e r n typical for C~mpositae. In some cases more than One embryo d e v e l o p e d on the nodule in liquid medium. These embryos arose from the p e r i p h e r a l cells of the nodule and followed the zygotic m o r p h o g e n e t i c n a t t e r n c o m p a r a b l e to those o b t a i n e d on s e m i s o l i d m e d i u m (Fig. 6). GE~4INATION
OF THE SOMATIC
EMBRYOS
Of the d i f f e r e n t media tested to r e g e n e r a t e p l a n t l e t s from the s u s p e n s i o n d e r i v e d embryos, MS s e m i s o l i d m e d i u m of pH 5.8 c o n t a i n i n half c o n c e n t r a t i o n of major salts s u n o l e m e n t e d w i t h 2% sucrose and vitamins was found suitable. L o w e r i n g the nH and sucrose c o n c e n t r a t i o n did not have any p o s i t i v e effect on the g e r m i n a t i o n of the somatic embryos. R o o t e d p l a n t l e t s (Fig. 8) when t r a n s f e r r e d to soil showed normal growth (Fig. 9). To date somatic e m b r y o - d e r i v e d p l a n t l e t s (Fig. 8) show much p h e n o t y p i c r e s e m b l a n c e to the y o u n g seedlings (Fig. iO) . After seven weeks in the g r e e n h o u s e each plant had d e v e l o p e d one single central root of about 0.5 cm d i a m e t e r and seemed to have kent the original phenotype. The whole r e g e n e r a t i o n cycle b e g i n n i n g w i t h the i n d u c t i o n of callus p a s s i n g through the phases of i n d u c t i o n and m a t u r a t i o n of somatic e m b r y o s in liquid m e d i u m t e r m i n a t i n g at the h a r v e s t i n g of small p l a n t l e t s took
110
Fig. 1-9. P l a n t l e t r e g e n e r a t i o n through somatic e m b r y o g e n e s i s in C. intybus in vitro. i. Embryogenic callus, 2-4. Embryoids on semi-solid medium, 2,3. Zygotic-like embryoids, 4. Embryoid w i t h d e v e l o p i n g leaves, 5-7. Embryoids in liquid medium, 8. P l a n t l e t d e v e l o p e d from a somatic embryo in liquid medium, before transfer to soil, 9. D e v e l o p e d plant w i t h single taproot. Symbols: C = Cotyledon, r = root, S = Shoot.
11i about 3 months. To date, the genetic s t a b i l i t y seemed to be maintained, although this aspect has to be further i n v e s t i g a t e d and the output of plant p r o d u c t i o n by somatic e m b r y o g e n e s i s has to be b o o s t e d up before the method could be applied s u c c e s f u l l y in plant breeding. Acknowledgements We wish to thank Beatrice Longly, Lindsey Withers, P.V. Ammirato, H. B i n d i n g for their useful comments and the people of the lab for their help. References
Fig. I0. G e r m i n a t i o n and sequential d e v e l o p m e n t of C. intybus seeds.
Eeninck AH (1979) Z a a d b e l a n g e n 33:260-266 M u r a s h i g e T, Skoog F (1962) Physiol Plant 15:473-497 Vasil IK, H i l d e b r a n d t AC (1966) Am J Bot 53:860-869 Vasil IK, H i l d e b r a n d t AC, Riker AJ (1964) Science 146:76-77
