Plant Cell Reports
Plant Cell Reports (1988) 7:550-552
© Springer-Verlag1988
Somatic embryogenesis and plant regeneration in embryo cultures of Euterpe edMis mart. (pal ae) Miguel Pedro Guerra 1 and Walter Handro Plant Tissue Culture Laboratory, Department of Botany, Institute of Biosciences, University of Silo Paulo, 05499 S~o Paulo, Brazil Received September 7, 1988/Revised version received October 12, 1988 - Communicated by I.K. Vasil
ABSTRACT The i n d u c t i o n of s o m a t i c e m b r y o g e n e sis in e m b r y o c u l t u r e s of E u t e r p e e d u l i s is d e s c r i b e d . The basal m e d i u m was c o m p o s e d of LS salts and Morel & W e t m o r e v i t a m i n s . Acti vated charcoal was a d d e d to p r e v e n t e x p l a n t o x i d a t i o n . 2,4-O higher than 50 mg/l was n e c e s s a r y for inducing e m b r y o g e n e s i s which occurs 4 5 - 1 8 0 days after the start of cultu res. E m b r y o s a r i s e d i r e c t l y from surface p r o l i f e r a t i n g t i s s u e s on the m a t r i x s t r u c t u re, w i t h o u t callus f o r m a t i o n . The transfer of t i s s u e s with e m b r y o c l u s t e r s to medium with NAA plus 2iP, or w i t h o u t g r o w t h regula tars, induces e m b r y o d e v e l o p m e n t into plantlets. Key words: LS: L i n s m a i e r & Skoog; 2,4-D: 2,4-dichlorophenoxiacetic acid; 2iP: 2-isop e n t e n y l a d e n i n e ; NAA: n a p h t h a l e n e a c e t i c acid. INTRODUCTION" The use of in v i t r o t e c h n i q u e s for m o r p h o g e n e t i c s t u d i e s and m i c r o p r o p a g a t i o n in n e o t r o p i c palms has i n c r e a s e d s i g n i f i c a n tly, s p e c i a l l y in s p e c i e s with high e c o n o m T c interest such as P h o e n i x d a c t y l i f e r a , Elaeis g u i n e e n s i s and Cocos n u c i f e r a (see R e y n o l d s 1982, T i s s e r a t ~ Pannetier & BuffardMorel 1986). The palm E u t e r p e e d u l i s is fndi genous to a n a r r o w area of moist f o r e s t s inS o u t h e r n and S o u t h e a s t e r n Brazil; its main product, the heart of palm, has an increa sing w o r l d c o n s u m p t i o n . This rising demand is t h r e a t e n i n g its e x t i n c t i o n , b e c a u s e to get the heart, the tree must be killed. The p r o p a g a t i o n of this s p e c i e s is t h r o u g h seeds. This makes the i m p r o v e m e n t , s e l e c t i o n and p r o p a g a t i o n of elite individuals from the r e m a i n i n g natural p o p u l a t i o n s d i f f i c u l t . The d e v e l o p m e n t of a t e c h n i q u e for in v i t r o plant r e g e n e r a t i o n of this species is d e s i r a b l e not only for rapid c l o n i n g of s e l e c t e d i n d i v i d u a l s , but also for imorovement using non c o n v e n t i o n a l m e t h o d s . Moreover, the e s t a b l i s h m e n t of tissue and cell c u l t u r e s of this s p e c i e s could p r o v i d e an useful s y s t e m to study p h y s i o l o g i c a l , genetic and m o r p h o g e n e t i c a l p r o b l e m s in the palm
fami]y. Here we d e s c r i b e the establishment of embryo cu]tures o f E. e d u l i s and p ] a n t r e g e neration, MATERIAL
AND
METHODS
E m b r y o s were isolated from m a t u r e and i m m a t u r e fruits from olants o c c u r r i n g natu rally in f o r e s t s in the State of Sao Pau]o . Before e m b r y o isolation f r u i t s were steri ]ized for 12 h in a s o l u t i o n having 4% Sodium h y p o c h ] o r i t e , 0,15% s t r e D t o m y c i n and 0.0]% m e r t h i o l a t e , on a r e c i p r o c a l shaker . Fruits were then immersed in 70% ethanol for 5 min, and f i n a l l y rinsed in s t e r i l e water. The basal m e d i u m was c o m p o s e d of LS salts ( L i n s m a i e r & Skoog 1965), v i t a m i n s (Mo re] & W e t m o r e ]951), 0.15% a c t i v a t e d charcoal, and 3% sucrose~ G r o w t h regulators (2,4-0, NAA, 2iP) were used at several conc e n t r a t i o n s d e o e n d i n g on the stage of the c u l t u r e s . The DH was a d i u s t e d to 5.5 and the m e d i u m q e l l e d with 0.45% M e r c k aqar before autoclavinf~.- The c u l t u r e s w e r e i n c u b a t e d at 26+_I°C in 16 h/day of liqht ( G r o - L u x lamps,
5
w.m-2).
RESULTS
AND
DISCUSSION
The s u c c e s s in the m a i n t e n a n c e of via ble e m b r y o c u l t u r e s d e p e n d e d e n t e r e l y on rapid e x c i s i o n and i n o c u l a t i o n , and the presen ce of a c t i v a t e d charcoal in the media. Consp i c u o u s and r e p e t i t i v e m o r p h o g e n e t i c responses were o b s e r v e d when 2,4-D was added to the basal m e d i u m at d i f f e r e n t c o n c e n t r a t i o n s ; until 50 mg/l e m b r y o s d e v e l o p e d s i m i l a r l y as in normal g e r m i n a t i o n , f o r m i n g the c o t y l e d o nary p e t i o l e and the h a u s t o r i u m . In c u l t u r e s with 10O mg/l 2,4-D, a g r a n u l a r tissue develops s h o w i n g red (from the c o t y ] e d o n a r y petiole) and w h i t e s e c t o r s (from the h a u s t o rium). A f t e r p e r i o d s r a n g i n g from 45 to 180 days in c u l t u r e , g l o b u l a r - t r a n s l u s c e n t stru~ tures were seen on the c o t y l e d o n a r y 0 e t i o ] e t i s s u e (Fig. l). T h e s e s t r u c t u r e s , t y p i c a l l y e m b r y o g e n i c , i n c r e a s e d r a p i d l y in size and n u m b e r and up to 50 of them could be seen in several stages of d e v e l o p m e n t (Fig. 2) Later, p e a r l y - s t a g e e m b r y o i d s a t t a c h e d to the m a t r i x t h r o U g h n a r r o w s u s p e n s o r s were
1 Permanent address." Department of Phytotechny, UFSC. C.P. 476, 88049 Florianopo!is, Brazil Offprint requests to." W. Handro
551
Fig. I - C u l t u r e d z y g o t i c e m b r y o 120 days a f t e r i n o c u l a t i o n sho wing f o r m a t i o n of e m b r y o g e n i c sectors. Fig. 2 - C l u s t e r s of glo bular s o m a t i c e m b r y o s 160 days a f t e r i n o c u l a t i o n of the zygotic e m b r y o . Fig. 3 - C l u s t e r s of p e a r l y - s t a g e s o m a t i c e m b r y o s 180 days a f t e r -inoculation. Fig. 4 - P l a n t l e t o r i g i n a t e d from s o m a t i c e m b r y o , 330 days a f t e r the start of c u l t u r e .
formed (Fig. 3). The i s o l a t i o n of this mass of e m b r y o s and t r a n s f e r to m e d i u m d e v o i d of 2,4-D (solid or liquid, with or w i t h o u t char coal) c a u s e d the d e v e l o p m e n t of the e m b r y o s into p l a n t l e t s (Fig. 4), but b e t t e r results were o b t a i n e d when the c u l t u r e s having emb r y o g e n i c mass were t r a n s f e r r e d to medium c o n t a i n i n g 2iP (5 mg/l) plus NAA (0.5 mg/1) f o l l o w e d by t r a n s f e r to m e d i u m d e v o i d of g r o w t h r e g u l a t o r s . The s o m a t i c e m b r y o masses are a l w a y s f o r m e d on c o m p a c t regions of the t i s s u e s from the c o t y l e d o n a r y p e t i o l e , never from h a u s t o r i u m tissues. M o r e o v e r , em bryos e x c i s e d from i m m a t u r e fruits were more s u i t a b l e for s o m a t i c e m b r y o g e n e s i s . In m o n o c o t y l e d o n s , o r g a n o g e n e s i s and e m b r y o g e n e s i s are two p o s s i b l e m o r p h o g e n e tic routes for o b t a i n i n g p l a n t s in v i t r o . E m b r y o g e n e s i s seems to be the most i m p o r t a n t p a t h w a y in P a l m a e (Blake 1983) and G r a m i n e a e (Vasil & Vasil 1982). Our r e s u l t s show c l e a r ly the o c c u r r e n c e of s o m a t i c e m b r y o g e n e s i s in E u t e r p e e d u l i s e m b r y o c u l t u r e s with liberation of b i p o l a r e m b r y o s from the m a t r i x s t r u c t u r e . F u r t h e r d e v e l o p m e n t of the somatic e m b r y o s into p l a n t l e t s is s i m i l a r to the d e v e l o p m e n t of z y g o t i c e m b r y o s and subsequent g e r m i n a t i o n as d e s c r i b e d by Bell inD e p o u x and Q u e i r o s (1971).
In most plant s p e c i e s and s p e c i a l l y in m o n o c o t y l e d o n s , 2 ~ 4 - D is r e s p o n s a b l e for c o n f e r i n g e m b r y o g e n i c c o m p e t e n c e to t i s s u e s c u l t u r e d in v i t r o ( A m m i r a t o 1983). As re ported by many p r e v i o u s w o r k e r s , the stage of d e v e l o p m e n t of the e x p l a n t s is p a r t i c u larly i m p o r t a n t in the e m b r y o g e n i c p r o c e s s . S o m a t i c e m b r y o g e n e s i s may be a c o n s e q u e n c e of a subtle i n t e r a c t i o n b e t w e e n the d e v e l o ~ mental stage and 2 , 4 - D c o n c e n t r a t i o n . If a d e q u a t e this i n t e r a c t i o n will result in the s u p r e s s i o n of z y g o t i c e m b r y o g r o w t h and i n d u c t i o n of s o m a t i c e m b r y o g e n e s i s with the new e m b r y o s a r i s i n g d i r e c t l y on the c o t y l e d o n a r y p e t i o l e surface, s h o w i n g a clear diff e r e n c e with o t h e r palms, w h e r e e m b r y o s or ! g i n a t e from c a l l u s ( T i s s e r a t 1984). In E. edu] is true c a l l u s e s w e r e not f o r m e d and p r o b a b l y a model of d i r e c t e m b r y o g e n e s i s o c c u r s s u g g e s t i n g an o r i g i n from single sup e r f i c i a l cells w h e r e e m b r y o s are linked to the m a t r i x t h r o u g h s u s p e n s o r s (see W i l l i a m s & M a h e s w a r a n 1986). This seems to be in a c c o r d a n c e with o b s e r v a t i o n s of S h a r p et al. I
( 1 9 8 0 ) and Evans e t a l . ( 1 9 8 1 ) t h a t di-r-~ct-embryogenesis occurs from cells determined previously b e f o r e in v i t r o culture, the only necessity being growth regulators and other special conditions for cell division
552 and
e x p r e s s i o n of e m b r y o g e n e s i s . In spite of the current idea that so matic e m b r y o g e n e s i s in tissues of m o n o c o t y ledons is a response to a sequence of trans fers from media with high auxin to media with low auxin or even devoid of growth regulators (Sharp et al. 1980), our results showed that primary cultures in high 2,4-D (50-100 mg/l) are able to produce somatic embryos and only one s u b s e q u e n t transfer to medium w i t h o u t growth r e g u l a t o r s or with 2iP and NAA allows g e r m i n a t i o n and s e e d l i n g development. For o b t a i n i n g a better u n d e r s t a n ding of the in vitro e m b r y o g e n i c process , structural studies are being carried out. Efforts are now u n d e r w a y to induce m o r p h o g ~ nesis in other types of tissues, such as leaves and i n f l o r e s c e n c e s . REFERENCES A m m i r a t o PV (]983) In: Evans DE Sharp WR A m m i r a t o PV Yamada Y (eds) H a n d b o o k of Plant Cell Culture v. I. M c M i l l a n , New York, pp 82-123. Belin-Depoux M Queiroz Bot. 78: 339-371.
MH
(1971)
Rev.
Linsmaier EM Skoog 18: I00-127.
F
Morel G W e t m o r e 138-140.
(1951)
RH
(1965)
Physiol.
Am.
J.
Plant.
Bot.
38:
Pannetier C Buffard-Morel J (1986) In: Bajaj YPS (ed) B i o t e c h n o l o g y in A g r i c u l t u re and F o r e s t r y l Trees I, S p r i n g e r - V e r lag, Berlin, pp 431-450. Reynolds (eds) tinus
JF (1982) In: Bonga JM Durzan DJ Tissue Culture in Forestry, MarNijhoff, The Hague, pp 182-207.
Sharp WR S~ndahl MR SB (1980) Hortic.
Caldas Rev. 2:
LS Maraffa 268-310.
Tisserat B Esan BB Murashige tic. Rev. I: 1-78.
T
(1979)
Hor
Tisserat B (1984) In: Sharp WR Evans DE A m m i r a t o PV Yamada Y (eds) H a n d b o o k of P|ant T i s s u e Culture, v. 2, McMillan, New York, pp 505-545. Vasil IK (1985) In: Henke RR Hughes KW C o n s t a n t i n MJ H o l l a e n d e r A (eds) Tis sue C u l t u r e in F o r e s t r y and Agriculture, Plenum Press, New York, pp 31-47.
Gen.
Blake I (1983) In: Dodos GH (ed) Tissue cul ture of trees. The Ari P u b l i s h i n g Co, W e s t p o i n t , pp 29-50. Evans DA Sharp WR Flick CE (1981) In: T h o r p e TA (ed) Plant Tissue Culture: Methods and A p p l i c a t i o n s in A g r i c u l t u r e , A c a d e m i c Press, pp 45-113.
Vasil V Vasil 1441-1449.
IK
(1982)
Williams EG Maheswaran 57: 443-462.
Amer. G
J.
(1986)
Bot. Ann.
69: Bot.
Plant Cell Reports (1988) 7:550-552
© Springer-Verlag1988
Somatic embryogenesis and plant regeneration in embryo cultures of Euterpe edMis mart. (pal ae) Miguel Pedro Guerra 1 and Walter Handro Plant Tissue Culture Laboratory, Department of Botany, Institute of Biosciences, University of Silo Paulo, 05499 S~o Paulo, Brazil Received September 7, 1988/Revised version received October 12, 1988 - Communicated by I.K. Vasil
ABSTRACT The i n d u c t i o n of s o m a t i c e m b r y o g e n e sis in e m b r y o c u l t u r e s of E u t e r p e e d u l i s is d e s c r i b e d . The basal m e d i u m was c o m p o s e d of LS salts and Morel & W e t m o r e v i t a m i n s . Acti vated charcoal was a d d e d to p r e v e n t e x p l a n t o x i d a t i o n . 2,4-O higher than 50 mg/l was n e c e s s a r y for inducing e m b r y o g e n e s i s which occurs 4 5 - 1 8 0 days after the start of cultu res. E m b r y o s a r i s e d i r e c t l y from surface p r o l i f e r a t i n g t i s s u e s on the m a t r i x s t r u c t u re, w i t h o u t callus f o r m a t i o n . The transfer of t i s s u e s with e m b r y o c l u s t e r s to medium with NAA plus 2iP, or w i t h o u t g r o w t h regula tars, induces e m b r y o d e v e l o p m e n t into plantlets. Key words: LS: L i n s m a i e r & Skoog; 2,4-D: 2,4-dichlorophenoxiacetic acid; 2iP: 2-isop e n t e n y l a d e n i n e ; NAA: n a p h t h a l e n e a c e t i c acid. INTRODUCTION" The use of in v i t r o t e c h n i q u e s for m o r p h o g e n e t i c s t u d i e s and m i c r o p r o p a g a t i o n in n e o t r o p i c palms has i n c r e a s e d s i g n i f i c a n tly, s p e c i a l l y in s p e c i e s with high e c o n o m T c interest such as P h o e n i x d a c t y l i f e r a , Elaeis g u i n e e n s i s and Cocos n u c i f e r a (see R e y n o l d s 1982, T i s s e r a t ~ Pannetier & BuffardMorel 1986). The palm E u t e r p e e d u l i s is fndi genous to a n a r r o w area of moist f o r e s t s inS o u t h e r n and S o u t h e a s t e r n Brazil; its main product, the heart of palm, has an increa sing w o r l d c o n s u m p t i o n . This rising demand is t h r e a t e n i n g its e x t i n c t i o n , b e c a u s e to get the heart, the tree must be killed. The p r o p a g a t i o n of this s p e c i e s is t h r o u g h seeds. This makes the i m p r o v e m e n t , s e l e c t i o n and p r o p a g a t i o n of elite individuals from the r e m a i n i n g natural p o p u l a t i o n s d i f f i c u l t . The d e v e l o p m e n t of a t e c h n i q u e for in v i t r o plant r e g e n e r a t i o n of this species is d e s i r a b l e not only for rapid c l o n i n g of s e l e c t e d i n d i v i d u a l s , but also for imorovement using non c o n v e n t i o n a l m e t h o d s . Moreover, the e s t a b l i s h m e n t of tissue and cell c u l t u r e s of this s p e c i e s could p r o v i d e an useful s y s t e m to study p h y s i o l o g i c a l , genetic and m o r p h o g e n e t i c a l p r o b l e m s in the palm
fami]y. Here we d e s c r i b e the establishment of embryo cu]tures o f E. e d u l i s and p ] a n t r e g e neration, MATERIAL
AND
METHODS
E m b r y o s were isolated from m a t u r e and i m m a t u r e fruits from olants o c c u r r i n g natu rally in f o r e s t s in the State of Sao Pau]o . Before e m b r y o isolation f r u i t s were steri ]ized for 12 h in a s o l u t i o n having 4% Sodium h y p o c h ] o r i t e , 0,15% s t r e D t o m y c i n and 0.0]% m e r t h i o l a t e , on a r e c i p r o c a l shaker . Fruits were then immersed in 70% ethanol for 5 min, and f i n a l l y rinsed in s t e r i l e water. The basal m e d i u m was c o m p o s e d of LS salts ( L i n s m a i e r & Skoog 1965), v i t a m i n s (Mo re] & W e t m o r e ]951), 0.15% a c t i v a t e d charcoal, and 3% sucrose~ G r o w t h regulators (2,4-0, NAA, 2iP) were used at several conc e n t r a t i o n s d e o e n d i n g on the stage of the c u l t u r e s . The DH was a d i u s t e d to 5.5 and the m e d i u m q e l l e d with 0.45% M e r c k aqar before autoclavinf~.- The c u l t u r e s w e r e i n c u b a t e d at 26+_I°C in 16 h/day of liqht ( G r o - L u x lamps,
5
w.m-2).
RESULTS
AND
DISCUSSION
The s u c c e s s in the m a i n t e n a n c e of via ble e m b r y o c u l t u r e s d e p e n d e d e n t e r e l y on rapid e x c i s i o n and i n o c u l a t i o n , and the presen ce of a c t i v a t e d charcoal in the media. Consp i c u o u s and r e p e t i t i v e m o r p h o g e n e t i c responses were o b s e r v e d when 2,4-D was added to the basal m e d i u m at d i f f e r e n t c o n c e n t r a t i o n s ; until 50 mg/l e m b r y o s d e v e l o p e d s i m i l a r l y as in normal g e r m i n a t i o n , f o r m i n g the c o t y l e d o nary p e t i o l e and the h a u s t o r i u m . In c u l t u r e s with 10O mg/l 2,4-D, a g r a n u l a r tissue develops s h o w i n g red (from the c o t y ] e d o n a r y petiole) and w h i t e s e c t o r s (from the h a u s t o rium). A f t e r p e r i o d s r a n g i n g from 45 to 180 days in c u l t u r e , g l o b u l a r - t r a n s l u s c e n t stru~ tures were seen on the c o t y l e d o n a r y 0 e t i o ] e t i s s u e (Fig. l). T h e s e s t r u c t u r e s , t y p i c a l l y e m b r y o g e n i c , i n c r e a s e d r a p i d l y in size and n u m b e r and up to 50 of them could be seen in several stages of d e v e l o p m e n t (Fig. 2) Later, p e a r l y - s t a g e e m b r y o i d s a t t a c h e d to the m a t r i x t h r o U g h n a r r o w s u s p e n s o r s were
1 Permanent address." Department of Phytotechny, UFSC. C.P. 476, 88049 Florianopo!is, Brazil Offprint requests to." W. Handro
551
Fig. I - C u l t u r e d z y g o t i c e m b r y o 120 days a f t e r i n o c u l a t i o n sho wing f o r m a t i o n of e m b r y o g e n i c sectors. Fig. 2 - C l u s t e r s of glo bular s o m a t i c e m b r y o s 160 days a f t e r i n o c u l a t i o n of the zygotic e m b r y o . Fig. 3 - C l u s t e r s of p e a r l y - s t a g e s o m a t i c e m b r y o s 180 days a f t e r -inoculation. Fig. 4 - P l a n t l e t o r i g i n a t e d from s o m a t i c e m b r y o , 330 days a f t e r the start of c u l t u r e .
formed (Fig. 3). The i s o l a t i o n of this mass of e m b r y o s and t r a n s f e r to m e d i u m d e v o i d of 2,4-D (solid or liquid, with or w i t h o u t char coal) c a u s e d the d e v e l o p m e n t of the e m b r y o s into p l a n t l e t s (Fig. 4), but b e t t e r results were o b t a i n e d when the c u l t u r e s having emb r y o g e n i c mass were t r a n s f e r r e d to medium c o n t a i n i n g 2iP (5 mg/l) plus NAA (0.5 mg/1) f o l l o w e d by t r a n s f e r to m e d i u m d e v o i d of g r o w t h r e g u l a t o r s . The s o m a t i c e m b r y o masses are a l w a y s f o r m e d on c o m p a c t regions of the t i s s u e s from the c o t y l e d o n a r y p e t i o l e , never from h a u s t o r i u m tissues. M o r e o v e r , em bryos e x c i s e d from i m m a t u r e fruits were more s u i t a b l e for s o m a t i c e m b r y o g e n e s i s . In m o n o c o t y l e d o n s , o r g a n o g e n e s i s and e m b r y o g e n e s i s are two p o s s i b l e m o r p h o g e n e tic routes for o b t a i n i n g p l a n t s in v i t r o . E m b r y o g e n e s i s seems to be the most i m p o r t a n t p a t h w a y in P a l m a e (Blake 1983) and G r a m i n e a e (Vasil & Vasil 1982). Our r e s u l t s show c l e a r ly the o c c u r r e n c e of s o m a t i c e m b r y o g e n e s i s in E u t e r p e e d u l i s e m b r y o c u l t u r e s with liberation of b i p o l a r e m b r y o s from the m a t r i x s t r u c t u r e . F u r t h e r d e v e l o p m e n t of the somatic e m b r y o s into p l a n t l e t s is s i m i l a r to the d e v e l o p m e n t of z y g o t i c e m b r y o s and subsequent g e r m i n a t i o n as d e s c r i b e d by Bell inD e p o u x and Q u e i r o s (1971).
In most plant s p e c i e s and s p e c i a l l y in m o n o c o t y l e d o n s , 2 ~ 4 - D is r e s p o n s a b l e for c o n f e r i n g e m b r y o g e n i c c o m p e t e n c e to t i s s u e s c u l t u r e d in v i t r o ( A m m i r a t o 1983). As re ported by many p r e v i o u s w o r k e r s , the stage of d e v e l o p m e n t of the e x p l a n t s is p a r t i c u larly i m p o r t a n t in the e m b r y o g e n i c p r o c e s s . S o m a t i c e m b r y o g e n e s i s may be a c o n s e q u e n c e of a subtle i n t e r a c t i o n b e t w e e n the d e v e l o ~ mental stage and 2 , 4 - D c o n c e n t r a t i o n . If a d e q u a t e this i n t e r a c t i o n will result in the s u p r e s s i o n of z y g o t i c e m b r y o g r o w t h and i n d u c t i o n of s o m a t i c e m b r y o g e n e s i s with the new e m b r y o s a r i s i n g d i r e c t l y on the c o t y l e d o n a r y p e t i o l e surface, s h o w i n g a clear diff e r e n c e with o t h e r palms, w h e r e e m b r y o s or ! g i n a t e from c a l l u s ( T i s s e r a t 1984). In E. edu] is true c a l l u s e s w e r e not f o r m e d and p r o b a b l y a model of d i r e c t e m b r y o g e n e s i s o c c u r s s u g g e s t i n g an o r i g i n from single sup e r f i c i a l cells w h e r e e m b r y o s are linked to the m a t r i x t h r o u g h s u s p e n s o r s (see W i l l i a m s & M a h e s w a r a n 1986). This seems to be in a c c o r d a n c e with o b s e r v a t i o n s of S h a r p et al. I
( 1 9 8 0 ) and Evans e t a l . ( 1 9 8 1 ) t h a t di-r-~ct-embryogenesis occurs from cells determined previously b e f o r e in v i t r o culture, the only necessity being growth regulators and other special conditions for cell division
552 and
e x p r e s s i o n of e m b r y o g e n e s i s . In spite of the current idea that so matic e m b r y o g e n e s i s in tissues of m o n o c o t y ledons is a response to a sequence of trans fers from media with high auxin to media with low auxin or even devoid of growth regulators (Sharp et al. 1980), our results showed that primary cultures in high 2,4-D (50-100 mg/l) are able to produce somatic embryos and only one s u b s e q u e n t transfer to medium w i t h o u t growth r e g u l a t o r s or with 2iP and NAA allows g e r m i n a t i o n and s e e d l i n g development. For o b t a i n i n g a better u n d e r s t a n ding of the in vitro e m b r y o g e n i c process , structural studies are being carried out. Efforts are now u n d e r w a y to induce m o r p h o g ~ nesis in other types of tissues, such as leaves and i n f l o r e s c e n c e s . REFERENCES A m m i r a t o PV (]983) In: Evans DE Sharp WR A m m i r a t o PV Yamada Y (eds) H a n d b o o k of Plant Cell Culture v. I. M c M i l l a n , New York, pp 82-123. Belin-Depoux M Queiroz Bot. 78: 339-371.
MH
(1971)
Rev.
Linsmaier EM Skoog 18: I00-127.
F
Morel G W e t m o r e 138-140.
(1951)
RH
(1965)
Physiol.
Am.
J.
Plant.
Bot.
38:
Pannetier C Buffard-Morel J (1986) In: Bajaj YPS (ed) B i o t e c h n o l o g y in A g r i c u l t u re and F o r e s t r y l Trees I, S p r i n g e r - V e r lag, Berlin, pp 431-450. Reynolds (eds) tinus
JF (1982) In: Bonga JM Durzan DJ Tissue Culture in Forestry, MarNijhoff, The Hague, pp 182-207.
Sharp WR S~ndahl MR SB (1980) Hortic.
Caldas Rev. 2:
LS Maraffa 268-310.
Tisserat B Esan BB Murashige tic. Rev. I: 1-78.
T
(1979)
Hor
Tisserat B (1984) In: Sharp WR Evans DE A m m i r a t o PV Yamada Y (eds) H a n d b o o k of P|ant T i s s u e Culture, v. 2, McMillan, New York, pp 505-545. Vasil IK (1985) In: Henke RR Hughes KW C o n s t a n t i n MJ H o l l a e n d e r A (eds) Tis sue C u l t u r e in F o r e s t r y and Agriculture, Plenum Press, New York, pp 31-47.
Gen.
Blake I (1983) In: Dodos GH (ed) Tissue cul ture of trees. The Ari P u b l i s h i n g Co, W e s t p o i n t , pp 29-50. Evans DA Sharp WR Flick CE (1981) In: T h o r p e TA (ed) Plant Tissue Culture: Methods and A p p l i c a t i o n s in A g r i c u l t u r e , A c a d e m i c Press, pp 45-113.
Vasil V Vasil 1441-1449.
IK
(1982)
Williams EG Maheswaran 57: 443-462.
Amer. G
J.
(1986)
Bot. Ann.
69: Bot.
