PiantCell Reports
Plant Cell Reports (1992) 11:609-613
9 Springer-Verlag1992
Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L. E Javier Vieitez, Antonio Ballester, and Ana M s Vieitez Instituto de Investigaciones Agrobioltgicas de Galicia. CSIC., Apartado 122, 15080 Santiago de Compostela, Spain Received June 5, 1992/Revised version received August 2, 1992 - Communicated by I. Potrykus
SUMMARY. Embryogenic cell suspension cultures and somatic embryos of five genotypes of b e e c h , w e r e o b t a i n e d f r o m aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2,4-dichlorophenoxyacetic acid and N6-benzyladenine. T h e o r i g i n of somatic embryos was traced from single cells. E m b r y o s r e m a i n e d a r r e s t e d at the g l o b u l a r s t a g e o n l i q u i d media, f u r t h e r development was achieved after plating embryogenic a g g r e g a t e s on M u r a s h i g e and Skoog's medium with half strength major salts supplemented with g l u t a m i n e and l o w l e v e l s of g r o w t h r e g u l a t o r s . C u l t u r e s of d i f f e r e n t g e n o t y p e s s h o w e d s i g n i f i c a n t differences in maturation frequency w h i c h w a s not a f f e c t e d b y the h o r m o n e treatments assayed. The frequency of c o n v e r s i o n of e m b r y o s into p l a n t l e t s w a s low. T h i s f r e q u e n c y i n c r e a s e d a f t e r c o l d s t o r a g e of e m b r y o s for u p t o 7 months. K e y W o r d s . Beech, Fagus sylvatica, C e l l suspension, Somatic Embryogenesis, Plant regeneration BA, N6-benzyladenine; 2,4-D, 2,4dichlorophenoxyacet~c acid; EtOH, ethanol; GA3, giberrellic acid; IAA, indole-3-aceticacid; IBA, indole3-butyric acid; MS, Murashige and Skoog (1962); NAA, naphthalene acetic acid; WPM, woody plant medium (Lloyd and McCown, 1980); Z, zeatin. Abbreviations:
INTRODUCTION
Like
other
Fagaceae,
the European
beech,
Fagus sylvatica L. is v e r y d i f f i c u l t to propagate vegetatively by conventional methods and has also proved to be difficult to propagate in v i t r o (Ahuja 1984; C h a l u p a 1985). F o r f o r e s t r y , b e e c h t r e e s are c h i e f l y produced by seedling propagation. However, mature trees do not p r o d u c e fertile seeds until approximately 35-40 y e a r s of age, g o o d h a r v e s t s o c c u r o n l y e v e r y 4 to 6 y e a r s and b e e c h n u t s are not e a s y to s t o r e n o r are t h e y l o n g - l i v e d in Correspondence to: F. Javier Vieitez
storage. Thus, recovery of plants t h r o u g h s o m a t i c e m b r y o g e n e s i s m a y be an a l t e r n a t i v e for t h e p r o d u c t i o n of b e e c h p r o p a g u l e s for r e f o r e s t a t i o n . So far, p r o d u c t i o n of b e e c h s o m a t i c embryos has only met with limited success. J 6 r g e n s e n (1988) r e p o r t e d the obtention of o n l y o n e s o m a t i c e m b r y o f r o m an a n t h e r - d e r i v e d callus, and later (J~rgensen 1990) induced somatic embryogenesis in cultured zygotic embryos, although plantlet regeneration w a s not r e p o r t e d . In t h i s study, w e r e p o r t t h e o b t e n t i o n of m a t u r e s o m a t i c e m b r y o s of b e e c h from embryogenic cell suspension cultures, and t h e i r c o n v e r s i o n i n t o p l a n t l e t s .
MATERIAL
AND
METHODS
Immature beechnuts were collected from local trees at the end of July 1989. Once isolated, seeds were surfacesterilized by successive immersions in 70% (v/v) EtOH for 30 sec and 15% (v/v) commercial bleach (40 g/l chlorine) for 15 min, followed by three rinses in sterile deionized water. Immature embryos were then dissected out, and the embryo axis and cotyledon fragments were singly transferred to test tubes containing 25 ml of solid primary medium, which consisted of the salts and vitamins of WPM, 100 mg/l minositol, 1000 mg/l l-gtutamine, 50 mg/l glycine, 50 mg/[ arginine, 3% sucrose, (0.45, 2.26 or 4.52 #M) 2,4D, 2.22 #M BA and 0.8% Difco Agar. Cultures were maintained either in _~2f'k~ess or under a 1 6 h photoperiod at 30 ~mol m ~ ' - , with 25=C days and 20~ nights. After 4-5 weeks, all cultures were transferred to solid WPM medium supplemented with 1000 mg/l lglutamine, (0.44-0.89 #M) BA and (0.27-0.54 #M) NAA (secondary medium) and maintained under the same light conditions as above. Celt Suspension Cultures. Samples (ca. I g fresh weight) of soft, friable, mucilaginous callus formed on cultures left for 4-5 months in the original secondary medium were used to initiate suspension cultures {n 250 m[ Erlenmeyer flasks containing 60 ml of liquid medium LMI consisting of the nitrates (I/3 strength), other major salts (I/2 strength), m-inositol and microelements of MS medium, 100 #M Fe-Na-EDTA, B-5 vitamins (Gamborg et al.1968), 10 mg/l tocopherol, 10 mg/t ascorbic acid,2
610 mM t-gtutamine, (0.22-2.26 #M) 2,4-D, 0.45 #M 8A and 2% sucrose. Cultures were kept in a shaker at 90 rpm in the dark or under low irradiance (6 #mot m'2s'1). Initially, cultures were maintained by removing 40-50 m[ of medium every week and adding the same volume of fresh LMI medium. Thereafter, suspensions were subcultured every 8-10 days by either decantation or filtration, followed by dilution. After 25-30 min setting, 15-20 m[ samples of suspension c o n t a i n i n g o n l y f r e e c e l l s and the smaller c e l l clumps were t r a n s f e r r e d to 40-50 mt of LM1 medium, the s e t t l e d c u l t u r e d was used f o r f u r t h e r experiments on embryo development.
Development of Somatic Embryos. Samples (5-10 ml) of settled celt mass containing the larger cell aggregates were transferred to 40-50 ml of LM2 medium, which possessed the same composition as LMI except for having no tocopherol or ascorbic acid and having Z and IBA instead of BA and 2,4-D. After two 10-12 d passages, the suspensions were filtered through a 40mesh sieve (380 #m pore size), the filtrate was discarded and samples of the retained cultures were used to initiate experiments on late embryo development. To determine embryo production, eight 250 #l samples were taken at random at the end of the second passage and the number of embryos was counted under a stereo microscope. The percentage of embryogenic cell aggregates was calculated with respect to the total number of aggregates present in each sample. Overall embryo production was determined using 8 replicates of 8 samples each. The fresh weight was determined by collecting cell aggregates from each sample on a pre-weighed slide, draining with filter paper and weighing again. Maturation and Conversion to P t a n t t e t s . Samples (3-5 mm in diameter) of c u l t u r e s r e t a i n e d on 40 mesh sieves were p l a t e d on 10 mm dishes containing 18 mt of s o l i d 1/2 MS
(half strength major salts) plus 3mM t-gtutamine, and combinations of (0.91-2.28 #M) Z or (0.89-2.22 #M) BA with (0.24-0.49 #M) IBA or (0.27-0.54 #M) NAA, and 0.7% agar. There were at least 3 dishes (12-16 samples each) per genotype and hormone treatment assayed. Data were statiscalty analysed by the 2-way ANOVA test. Embryos at the cotyledonary stage (2-5 mm in length) were transferred to jars containing 50 ml of solid MS or WPM medium, with no growth regulators or supplemented with various combinations of BA, GA3 and IBA. Embryos showing b o t h radicle and shoot emergence were transferred to solid MS medium supplemented with 0.46 #M Z. Cult~re~ were kept under a 16 h photoperiod at 30 #mo[ m- s" .
The pH was always adjusted to 5.8 prior to autoclaving at 121~ for 20 min. Glutamine, tocopherol, ascorbic acid and GA3, were filter-sterilized. Microscopic Examination. For r o u t i n e observations, samples of suspensions were t r a n s f e r r e d to small dishes and observed under an i n v e r t e d microscope. The embryogenic nature of c e l t s was determined by s t a i n i n g with 2% (w/v) acetocarmine (Gupta and burzan 1987).
RESULTS
a n d BA. W i t h i n a b o u t 3 w e e k s a f t e r transfer to secondary medium, all c u l t u r e s s t o p p e d g r o w i n g a n d s t a r t e d to turn brown. A few somatic embryos developed from one cotyledon culture to t h e c o t y l e d o n a r y s t a g e (Fig. l), but d i d not r e g e n e r a t e p l a n t l e t s . After 4-5 months in the original s e c o n d a r y m e d i u m ( w i t h o u t t r a n s f e r ) , i015% of aged (browned) cultures had f o r m e d a r e a s of s o f t t e x t u r e d callus. Two types of callus could be d i s t i n g u i s h e d . T y p e I w a s slimy, w h i t i s h and non-embryogenic, and grew quickly d u r i n g t h e f i r s t m o n t h a f t e r t r a n s f e r to solid WPM or MS supplemented with various combinations of growth regulators, thereafter,it stopped growing and slowly became o p a q u e and degenerated, more rapidly when cultured in l i q u i d m e d i a . Callus type II was yellowish, translucent and mucilaginous. This callus proliferated slowly after subculture in WPM or MS media supplemented with both auxins and c y t o k i n i n s . Later, t h i s c a l l u s p r o v e d to be e m b r y o g e n i c .
Cell Suspension Cultures Embryogenic cell suspensions of 5 genotypes were successfully established in LMI containing 2,4-D. Media with higher salt concentrations proved unsuitable for the maintenance of h e a l t h y s u s p e n s i o n s . H i g h l e v e l s of 2,4D (2.26 ~M) y i e l d e d fine suspensions composed of n u m e r o u s free cells and discrete cell aggregates, w h i l e lower concentrations of 2 , 4 - D ( 0 . 2 2 - 0 . 4 5 pM) yielded suspensions with relatively l a r g e and m o r e c o m p a c t c e l l a g g r e g a t e s . The m a j o r i t y of c e l l s f r o m b o t h t y p e s of medium exhibited cytological characteristics associated with embryogenic cells and strong a~finity for a c e t o c a r m i n e . T h e s e c e l l s w e r e r o u n d or o v a l - s h a p e d and were intermingled w i t h larger, v a c u o l a t e d n o n - e m b r y o g e n i c c e l l s (Fig.2) . C e l l s u s p e n s i o n s of g e n o t y p e s FIP3, F 0 A a n d F3D w e r e m a i n t a i n e d b y r e g u l a r subculture for over 2 years without completely losing their embryogenic p o t e n t i a l , but t h e i r a b i l i t y t o p r o d u c e somatic embryos decreased. When s u b c u l t u r e w a s d e l a y e d l o n g e r t h a n 14 days, t h e s u s p e n s i o n s b e c a m e brown, as t h e a c i d i t y of t h e m e d i u m i n c r e a s e d to a b o u t p H 3.5. Embryogenic suspensions of line FIS6 w e r e e s t a b l i s h e d in LMI, as d e s c r i b e d in M a t e r i a l and M e t h o d s , f r o m c a l l u s t y p e II d e v e l o p e d f r o m a g e r m i n a t i n g s o m a t i c e m b r y o of g e n o t y p e FIP3.
Development of Somatic Embryos After one month, a slight callus p r o l i f e r a t i o n h a d b e g u n on m o s t e x p l a n t s cultured on media containing both 2,4-D
The earliest stage identified as e m b r y o g e n i c c o n s i s t e d of s i n g l e cells,
611 u n d e r g o i n g a first asymmetric division leading to the form tion of two daughter cells of u n e q u a l s~ze (Backs-H~ssemann and R e i n e r t 1970; Nomura and Komamine 1986), a small cell with dense cytoplasm and strong a f f i n i t y for acetocarmine and a larger cell w i t h only slight affinity for a c e t o c a r m i n e (Fig.3). Subsequently, either the smaller cells underwent further p o l a r i z e d divisions to form proembryos (Fig. 4, 5), or both daughter cells u n d e r w e n t a series of anarchical divisions leading to the formation of cell a g g r e g a t e s or p r o e m b r y o g e n i c masses, from w h i c h m u l t i p l e embryos developed by cleavage polyembryony (Halperin, 1966). A f t e r two p a s s a g e s on LM2, containing 0.92 ~ M Z and 0.25 ~M IBA, numerous clumps of g l o b u l a r or slightly elongated e m b r y o s d e v e l o p e d (Fig.6). Suspensions of d i f f e r e n t g e n o t y p e s e x h i b i t e d different d e v e l o p m e n t a l capacities. The percentage of p r o e m b r y o g e n i c masses or microcallus b e a r i n g g l o b u l a r embryos varied from 20 to 90% d e p e n d i n g on genotype. Table 1 lists embryo p r o d u c t i o n data for embryogenic suspensions of the h i g h e s t y i e l d i n g genotype, FIP3, at the end of the second 12 day passage through LM2. On average, 18 globular embryos were formed per mg f.w. of callus or 307• e m b r y o s per ml of suspension. I. Embryo production in suspension cultures of genotype FIP3 after two passages in LM2. Each replicate means eight 250 gl samles. Table
Replicate
I 2 3 4 5 6 7 8
F.W mg
43.4 33.9 34.1 34.4 35.4 29.7 32.3 32.7
AGGREGATES No. EMBRYOS No. EmbryoTotal genic(%) Total per mg
107 75 63 108 86 75 97 91
89.3 88.6 86.0 78.7 89.5 86.6 81.4 90.1
803 634 530 610 667 547 550 577
18.59 18.88 15.91 18.53 19.00 18.41 17.02 17.64
Although embryos beyond the globular stage were occasionally observed in suspension cultures, most embryos r e m a i n e d a r r e s t e d at the globular stage, s h o w i n g the t e n d e n c y to shed outer cell layers. F u r t h e r growth and development into m a t u r e somatic embryos required that the cell a g g r e g a t e s be plated on solid 1/2 MS medium supplemented with 3mM g l u t a m i n e plus low levels of both auxins and cytokinins. Four h o r m o n e t r e a t m e n t s were assayed (in pM): i) 0.92 Z, 0.25 IBA; 2) 0.89 BA, 0.25 IBA; 3) 2.22 BA, 0.49 IBA; 4) 2.22 BA, 0.54 NAA. W i t h i n about 2 weeks after plating, microscopic examination of cultures showed a great p r o l i f e r a t i o n of small, globular embryos (Fig.7). After one month, embryos at all stages of
development were observed, t o g e t h e r with abnormal structures in the same cultures. There were no d i f f e r e n c e s among the t r e a t m e n t s assayed w i t h respect to the culture maturation frequency (MF), defined as the p e r c e n t a g e of cultures that developed embryos beyond the globular stage. However, there were significant differences in m a t u r a t i o n frequency (pS0.001) among genotypes (Table 2). The secondary embryogenic line FIS6 had the highest m a t u r a t i o n frequency, although the p e r c e n t a g e of FIS6 cultures w i t h c o t y l e d o n a r y embryos (C) was lower than in the case of its parent genotype FIP3. 2. Embryo development from plated suspension cultures. The maturation frecuencies (MF) and the percentage of cultures with cotyledonary embryos (C) were recorded after 4 weeks culture. Table
Genotype Hormone treatment
FIP3 MF
I) 2) 3) 4)
C
73.2 44.4 58.9 22.5 51.8 21.8 39.4 1.9
FOA MF
C
43.1 48.7 59.0 30.4
4.1 7.0 3.2 0.0
F3D MF
17.3 22.6 10.4 26.8
C
0.0 0.0 0.4 0.4
FIS6 MF
C
87.8 21.7 77.9 22.1 72.6 15.0 63.6 6.0
F-test (genotype) F-test (treatment) LSD I% 15.18 (*) 1.205 (ns) 21.89% * significant at p ~0.001 ; ns = not significant Embryo m a t u r a t i o n was s t i m u l a t e d by isolating cotyledonary embryos from clumps and t r a n s f e r r i n g t h e m to solid MS or WPM m e d i u m supplemented with low levels of either Z or BA plus IBA. When isolated cotyledonary embryos were cultured on h o r m o n e - f r e e medium, roots developed, but not shoots. W i t h i n about 2 weeks after transfer to MS m e d i u m s u p p l e m e n t e d w i t h (2.2-4.4 HM) BA, 0.29 HM GA 3 and 0.49 ~M IBA, about 40% of embryos had e l o n g a t e d radicles, after which their c o t y l e d o n s expanded and became green; shoot p r i m o r d i a were observed in a small proportion of embryos 3-4 weeks after transfer. However, spending longer in this medium, resulted in callused embryos and the appearance of secondary embryos. C o t y l e d o n a r y embryos of different size and m o r p h o l o g i e s were capable of giving rise to p l a n t l e t s (Fig. 8). Different approaches were taken to enhance the conversion of somatic embryos into plantlets. Best results were achieved when isolated cotyledonary embryos were p r e t r e a t e d by storing at 4~ for 7 months and then cultured on MS m e d i u m s u p p l e m e n t e d with 4.4 @M BA and 0.49 ~M IBA for 2-3 weeks, with subsequent t r a n s f e r to MS plus 0.46 @M Z. After 4 weeks, 10% of embryos (63 plantlets from 638 embryos) had developed roots, shoots and well-formed leaves (Fig.9).
612
Somatic embryogenesis in Fagus sylvatica L. Fig I. Development of somatic embryos from a browned cotyledon-derived culture, after 5 weeks on solid secondary medium (bar = Imm). Fig 2. Embryogenic cell suspension in LMI medium supplemented with 0.45 #M 2,4-D (x 300). Fig 3. Asymetric divisions of embryogenic cells. 3a Differential interference contrast optics (x 285); 3b Acetocarmine staining (x 285). Fig 4. Pluricellular proembryo-like structure suspended in LMI medium (x 380). Fig 5. Growth of an individual globular embryo with a narrow suspensor-like structure in LM2 medium (x 37.5). Fig 6. Proembryogenic masses and clusters of globular embryos, after two 12-day passages in LM2 medium (x 4). Fig 7. Proliferating proembryogenic masses, several days after plating. Note the numerous small proembryos developed from globular embryos through secondary embryogenesis (x 6). Fig 8. Cotyledonary embryos after 4 months of cold storage at 4~ (bar = I cm). Fig 9. Regenerated beech plantlet from a somatic embryo (bar = I cm).
613 DISCUSSION
B r o w n i n g of t i s s u e s is n o r m a l l y a p r o b l e m in m i c r o p r o p a g a t i o n . H o w e v e r , t h i s and other previous studies ( Sondhal and S h a r p 1977; T u l e c k e and M c G r a n a h a n 1985; Neuenschwander and Baumann 1992) had s h o w n t h a t b r o w n i n g of t i s s u e s did not impair the competence for somatic embryogenesis. The cell suspension culture results s u g g e s t t h a t 2 , 4 - D w a s e s s e n t i a l for the induction and maintenance of the e m b r y o g e n i c c o m p e t e n c e of t h e c u l t u r e s at the earliest embryonic stages, since somatic embryogenesis and embryo development were only achieved after c u l t u r i n g s a m p l e s of t h e t y p e II c a l l u s o n l i q u i d m e d i u m c o n t a i n i n g 2,4-D. W h e n t h i s t y p e of c a l l u s w a s c u l t u r e d on m e d i a supplemented with other growth regulators o n l y c a l l u s p r o l i f e r a t i o n w a s achieved, e v e n w h e n c u l t u r e d o n 1/2 MS s u p p l e m e n t e d w i t h 3 m M g l u t a m i n e , 0.92 g M Z and 0.25 g M IBA, w h i c h w a s f o u n d t o b e a p p r o p r i a t e for e m b r y o d e v e l o p m e n t . A c c o r d i n g t o S h a r p et a i . ( 1 9 8 0 ) , 2 , 4 - D m a y act e i t h e r as a p r i m a r y agent of d e t e r m i n a t i o n of e m b r y o g e n i c c e l l s or as an a c t i v a t o r of t h e d e v e l o p m e n t of soc a l l e d p r e - d e t e r m i n e d e m b r y o g e n i c cells. Apparently, our embryogenic cells were determined as such after several d i v i s i o n s in t h e p r e s e n c e of 2,4-D, w h i c h m a y h a v e c a u s e d d e d i f f e r e n t i a t i o n and the t e r m i n a t i o n of p r e v i o u s l y a c t i v e p a t h w a y s (Carman 1990) and further epigenetic r e d e t e r m i n a t i o n to t h e e m b r y o g e n i c s t a t e ( S h a r p et al. 1980). B a c k s - H ~ s e m a n n a n d R e i n e r t (1970), and N o m u r a a n d K o m a m i n e (1986) r e p o r t e d t h a t t h e f i r s t s t e p in t h e d i f f e r e n t i a t i o n of e m b r y o s is t h e o c c u r r e n c e of u n e q u a l cell division in s i n g l e cells. In our cell s u s p e n s i o n c u l t u r e s , f i r s t a s y m e t r i c or unequal divisions were observed in f l o a t i n g f r e e cells, as w e l l as in s i n g l e cells at the peryphery of cell a g g r e g a t e s . A p p a r e n t l y , t h e r e can be two patterns of beech somatic embryo initiation. One, direct from single cells; the other, indirect f r o m cell a g g r e g a t e s or p r o e m b r y o g e n i c m a s s e s w h i c h had been formed by successive divisions of an embryogenic cell and of its d a u g h t e r cells. T h o u g h w e d i d not f o l l o w all steps sequentially from the first asymmetric division to the globular stage, b e e c h s o m a t i c e m b r y o s s e e m t o be of u n i c e l l u l a r o r i g i n a n d t o b e a b l e to f o l l o w v a r i o u s courses of development to the cotyledonary stage rather than a single
rigid pattern of divisions. It t h u s a p p e a r s t h a t t h e in v i t r o e m b r y o g e n e s i s of b e e c h is d e v e l o p m e n t a l l y p l a s t i c , in l i n e w i t h t h e t h e o r i e s of V o n A d e r k a s and B o n g a (1988) a n d C a r m a n (1990). The differences between the embryogenic c a p a c i t i e s a n d m a t u r a t i o n f r e q u e n c i e s of c u l t u r e s of d i f f e r e n t g e n o t y p e s s u g g e s t s t h a t s o m a t i c e m b r y o g e n e s i s is u n d e r genetic control. These variables were not a f f e c t e d b y t h e h o r m o n e t r e a t m e n t s tried. Thus, it s e e m s t h a t g e n o t y p e m a y be a more significant factor than culture medium in affecting the b e h a v i o u r of b e e c h e m b r y o g e n i c cultures. Acknowledgements. This study was partially supported by the EEC ECLAIR Programme, contract AGRE-CT91-0067. REFERENCES
Ahuja MR (1984) Silvae Genetica 33 (6): 241-242 Backs-HQssemann D, Reinert J (1970) Protoplasma 70: 496O Carman JG (1990) In Vitro Cell. Dev. Biol. 26 : 746-753 Chatupa W (1985) Comm. Inst. For. Cechosloveniae 14: 6990 Gamborg DL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:
151-158
Gupta PK, Durzan DJ (1987) Biotechnology 5:147-151 Halperin W (1966) Am. J. Bot. 53 (5): 443-453 JSrgensen J (1988) J. Plant Physiol. 132: 638-640 J6rgensen J (1990) Abst. VII Int. Cong. Ptant Cell Tissue
Culture.Amsterdam, pp. 108
Ltoyd G, McCown BH (1980) Proc. Int. Plant Prop. Soc. 30:
421-428
Murashige S, Skoog F (1962) Physiol. Plant. 15:473-497 Neuenschwander B, Baumann TW (1992) Plant Cell Rep 10: 608-612 Nomura K, Komamine A (1986) New Phytol. 104:25-32 Sharp WR, Sondhal MR, Caldas LS, Marafa SB (1980) Hort. Rew. 2:268-310 Sondhal MR, Sharp WR (1977) Z. Pflanzenphysiol 81: 395408 Tulecke W, McGranahan GH (1985) Plant Sci. 40:53-67 Von Aderkas P, Bonga JM (1988) Am. J. Bot. 75 (5) : 690- 700
Plant Cell Reports (1992) 11:609-613
9 Springer-Verlag1992
Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L. E Javier Vieitez, Antonio Ballester, and Ana M s Vieitez Instituto de Investigaciones Agrobioltgicas de Galicia. CSIC., Apartado 122, 15080 Santiago de Compostela, Spain Received June 5, 1992/Revised version received August 2, 1992 - Communicated by I. Potrykus
SUMMARY. Embryogenic cell suspension cultures and somatic embryos of five genotypes of b e e c h , w e r e o b t a i n e d f r o m aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2,4-dichlorophenoxyacetic acid and N6-benzyladenine. T h e o r i g i n of somatic embryos was traced from single cells. E m b r y o s r e m a i n e d a r r e s t e d at the g l o b u l a r s t a g e o n l i q u i d media, f u r t h e r development was achieved after plating embryogenic a g g r e g a t e s on M u r a s h i g e and Skoog's medium with half strength major salts supplemented with g l u t a m i n e and l o w l e v e l s of g r o w t h r e g u l a t o r s . C u l t u r e s of d i f f e r e n t g e n o t y p e s s h o w e d s i g n i f i c a n t differences in maturation frequency w h i c h w a s not a f f e c t e d b y the h o r m o n e treatments assayed. The frequency of c o n v e r s i o n of e m b r y o s into p l a n t l e t s w a s low. T h i s f r e q u e n c y i n c r e a s e d a f t e r c o l d s t o r a g e of e m b r y o s for u p t o 7 months. K e y W o r d s . Beech, Fagus sylvatica, C e l l suspension, Somatic Embryogenesis, Plant regeneration BA, N6-benzyladenine; 2,4-D, 2,4dichlorophenoxyacet~c acid; EtOH, ethanol; GA3, giberrellic acid; IAA, indole-3-aceticacid; IBA, indole3-butyric acid; MS, Murashige and Skoog (1962); NAA, naphthalene acetic acid; WPM, woody plant medium (Lloyd and McCown, 1980); Z, zeatin. Abbreviations:
INTRODUCTION
Like
other
Fagaceae,
the European
beech,
Fagus sylvatica L. is v e r y d i f f i c u l t to propagate vegetatively by conventional methods and has also proved to be difficult to propagate in v i t r o (Ahuja 1984; C h a l u p a 1985). F o r f o r e s t r y , b e e c h t r e e s are c h i e f l y produced by seedling propagation. However, mature trees do not p r o d u c e fertile seeds until approximately 35-40 y e a r s of age, g o o d h a r v e s t s o c c u r o n l y e v e r y 4 to 6 y e a r s and b e e c h n u t s are not e a s y to s t o r e n o r are t h e y l o n g - l i v e d in Correspondence to: F. Javier Vieitez
storage. Thus, recovery of plants t h r o u g h s o m a t i c e m b r y o g e n e s i s m a y be an a l t e r n a t i v e for t h e p r o d u c t i o n of b e e c h p r o p a g u l e s for r e f o r e s t a t i o n . So far, p r o d u c t i o n of b e e c h s o m a t i c embryos has only met with limited success. J 6 r g e n s e n (1988) r e p o r t e d the obtention of o n l y o n e s o m a t i c e m b r y o f r o m an a n t h e r - d e r i v e d callus, and later (J~rgensen 1990) induced somatic embryogenesis in cultured zygotic embryos, although plantlet regeneration w a s not r e p o r t e d . In t h i s study, w e r e p o r t t h e o b t e n t i o n of m a t u r e s o m a t i c e m b r y o s of b e e c h from embryogenic cell suspension cultures, and t h e i r c o n v e r s i o n i n t o p l a n t l e t s .
MATERIAL
AND
METHODS
Immature beechnuts were collected from local trees at the end of July 1989. Once isolated, seeds were surfacesterilized by successive immersions in 70% (v/v) EtOH for 30 sec and 15% (v/v) commercial bleach (40 g/l chlorine) for 15 min, followed by three rinses in sterile deionized water. Immature embryos were then dissected out, and the embryo axis and cotyledon fragments were singly transferred to test tubes containing 25 ml of solid primary medium, which consisted of the salts and vitamins of WPM, 100 mg/l minositol, 1000 mg/l l-gtutamine, 50 mg/l glycine, 50 mg/[ arginine, 3% sucrose, (0.45, 2.26 or 4.52 #M) 2,4D, 2.22 #M BA and 0.8% Difco Agar. Cultures were maintained either in _~2f'k~ess or under a 1 6 h photoperiod at 30 ~mol m ~ ' - , with 25=C days and 20~ nights. After 4-5 weeks, all cultures were transferred to solid WPM medium supplemented with 1000 mg/l lglutamine, (0.44-0.89 #M) BA and (0.27-0.54 #M) NAA (secondary medium) and maintained under the same light conditions as above. Celt Suspension Cultures. Samples (ca. I g fresh weight) of soft, friable, mucilaginous callus formed on cultures left for 4-5 months in the original secondary medium were used to initiate suspension cultures {n 250 m[ Erlenmeyer flasks containing 60 ml of liquid medium LMI consisting of the nitrates (I/3 strength), other major salts (I/2 strength), m-inositol and microelements of MS medium, 100 #M Fe-Na-EDTA, B-5 vitamins (Gamborg et al.1968), 10 mg/l tocopherol, 10 mg/t ascorbic acid,2
610 mM t-gtutamine, (0.22-2.26 #M) 2,4-D, 0.45 #M 8A and 2% sucrose. Cultures were kept in a shaker at 90 rpm in the dark or under low irradiance (6 #mot m'2s'1). Initially, cultures were maintained by removing 40-50 m[ of medium every week and adding the same volume of fresh LMI medium. Thereafter, suspensions were subcultured every 8-10 days by either decantation or filtration, followed by dilution. After 25-30 min setting, 15-20 m[ samples of suspension c o n t a i n i n g o n l y f r e e c e l l s and the smaller c e l l clumps were t r a n s f e r r e d to 40-50 mt of LM1 medium, the s e t t l e d c u l t u r e d was used f o r f u r t h e r experiments on embryo development.
Development of Somatic Embryos. Samples (5-10 ml) of settled celt mass containing the larger cell aggregates were transferred to 40-50 ml of LM2 medium, which possessed the same composition as LMI except for having no tocopherol or ascorbic acid and having Z and IBA instead of BA and 2,4-D. After two 10-12 d passages, the suspensions were filtered through a 40mesh sieve (380 #m pore size), the filtrate was discarded and samples of the retained cultures were used to initiate experiments on late embryo development. To determine embryo production, eight 250 #l samples were taken at random at the end of the second passage and the number of embryos was counted under a stereo microscope. The percentage of embryogenic cell aggregates was calculated with respect to the total number of aggregates present in each sample. Overall embryo production was determined using 8 replicates of 8 samples each. The fresh weight was determined by collecting cell aggregates from each sample on a pre-weighed slide, draining with filter paper and weighing again. Maturation and Conversion to P t a n t t e t s . Samples (3-5 mm in diameter) of c u l t u r e s r e t a i n e d on 40 mesh sieves were p l a t e d on 10 mm dishes containing 18 mt of s o l i d 1/2 MS
(half strength major salts) plus 3mM t-gtutamine, and combinations of (0.91-2.28 #M) Z or (0.89-2.22 #M) BA with (0.24-0.49 #M) IBA or (0.27-0.54 #M) NAA, and 0.7% agar. There were at least 3 dishes (12-16 samples each) per genotype and hormone treatment assayed. Data were statiscalty analysed by the 2-way ANOVA test. Embryos at the cotyledonary stage (2-5 mm in length) were transferred to jars containing 50 ml of solid MS or WPM medium, with no growth regulators or supplemented with various combinations of BA, GA3 and IBA. Embryos showing b o t h radicle and shoot emergence were transferred to solid MS medium supplemented with 0.46 #M Z. Cult~re~ were kept under a 16 h photoperiod at 30 #mo[ m- s" .
The pH was always adjusted to 5.8 prior to autoclaving at 121~ for 20 min. Glutamine, tocopherol, ascorbic acid and GA3, were filter-sterilized. Microscopic Examination. For r o u t i n e observations, samples of suspensions were t r a n s f e r r e d to small dishes and observed under an i n v e r t e d microscope. The embryogenic nature of c e l t s was determined by s t a i n i n g with 2% (w/v) acetocarmine (Gupta and burzan 1987).
RESULTS
a n d BA. W i t h i n a b o u t 3 w e e k s a f t e r transfer to secondary medium, all c u l t u r e s s t o p p e d g r o w i n g a n d s t a r t e d to turn brown. A few somatic embryos developed from one cotyledon culture to t h e c o t y l e d o n a r y s t a g e (Fig. l), but d i d not r e g e n e r a t e p l a n t l e t s . After 4-5 months in the original s e c o n d a r y m e d i u m ( w i t h o u t t r a n s f e r ) , i015% of aged (browned) cultures had f o r m e d a r e a s of s o f t t e x t u r e d callus. Two types of callus could be d i s t i n g u i s h e d . T y p e I w a s slimy, w h i t i s h and non-embryogenic, and grew quickly d u r i n g t h e f i r s t m o n t h a f t e r t r a n s f e r to solid WPM or MS supplemented with various combinations of growth regulators, thereafter,it stopped growing and slowly became o p a q u e and degenerated, more rapidly when cultured in l i q u i d m e d i a . Callus type II was yellowish, translucent and mucilaginous. This callus proliferated slowly after subculture in WPM or MS media supplemented with both auxins and c y t o k i n i n s . Later, t h i s c a l l u s p r o v e d to be e m b r y o g e n i c .
Cell Suspension Cultures Embryogenic cell suspensions of 5 genotypes were successfully established in LMI containing 2,4-D. Media with higher salt concentrations proved unsuitable for the maintenance of h e a l t h y s u s p e n s i o n s . H i g h l e v e l s of 2,4D (2.26 ~M) y i e l d e d fine suspensions composed of n u m e r o u s free cells and discrete cell aggregates, w h i l e lower concentrations of 2 , 4 - D ( 0 . 2 2 - 0 . 4 5 pM) yielded suspensions with relatively l a r g e and m o r e c o m p a c t c e l l a g g r e g a t e s . The m a j o r i t y of c e l l s f r o m b o t h t y p e s of medium exhibited cytological characteristics associated with embryogenic cells and strong a~finity for a c e t o c a r m i n e . T h e s e c e l l s w e r e r o u n d or o v a l - s h a p e d and were intermingled w i t h larger, v a c u o l a t e d n o n - e m b r y o g e n i c c e l l s (Fig.2) . C e l l s u s p e n s i o n s of g e n o t y p e s FIP3, F 0 A a n d F3D w e r e m a i n t a i n e d b y r e g u l a r subculture for over 2 years without completely losing their embryogenic p o t e n t i a l , but t h e i r a b i l i t y t o p r o d u c e somatic embryos decreased. When s u b c u l t u r e w a s d e l a y e d l o n g e r t h a n 14 days, t h e s u s p e n s i o n s b e c a m e brown, as t h e a c i d i t y of t h e m e d i u m i n c r e a s e d to a b o u t p H 3.5. Embryogenic suspensions of line FIS6 w e r e e s t a b l i s h e d in LMI, as d e s c r i b e d in M a t e r i a l and M e t h o d s , f r o m c a l l u s t y p e II d e v e l o p e d f r o m a g e r m i n a t i n g s o m a t i c e m b r y o of g e n o t y p e FIP3.
Development of Somatic Embryos After one month, a slight callus p r o l i f e r a t i o n h a d b e g u n on m o s t e x p l a n t s cultured on media containing both 2,4-D
The earliest stage identified as e m b r y o g e n i c c o n s i s t e d of s i n g l e cells,
611 u n d e r g o i n g a first asymmetric division leading to the form tion of two daughter cells of u n e q u a l s~ze (Backs-H~ssemann and R e i n e r t 1970; Nomura and Komamine 1986), a small cell with dense cytoplasm and strong a f f i n i t y for acetocarmine and a larger cell w i t h only slight affinity for a c e t o c a r m i n e (Fig.3). Subsequently, either the smaller cells underwent further p o l a r i z e d divisions to form proembryos (Fig. 4, 5), or both daughter cells u n d e r w e n t a series of anarchical divisions leading to the formation of cell a g g r e g a t e s or p r o e m b r y o g e n i c masses, from w h i c h m u l t i p l e embryos developed by cleavage polyembryony (Halperin, 1966). A f t e r two p a s s a g e s on LM2, containing 0.92 ~ M Z and 0.25 ~M IBA, numerous clumps of g l o b u l a r or slightly elongated e m b r y o s d e v e l o p e d (Fig.6). Suspensions of d i f f e r e n t g e n o t y p e s e x h i b i t e d different d e v e l o p m e n t a l capacities. The percentage of p r o e m b r y o g e n i c masses or microcallus b e a r i n g g l o b u l a r embryos varied from 20 to 90% d e p e n d i n g on genotype. Table 1 lists embryo p r o d u c t i o n data for embryogenic suspensions of the h i g h e s t y i e l d i n g genotype, FIP3, at the end of the second 12 day passage through LM2. On average, 18 globular embryos were formed per mg f.w. of callus or 307• e m b r y o s per ml of suspension. I. Embryo production in suspension cultures of genotype FIP3 after two passages in LM2. Each replicate means eight 250 gl samles. Table
Replicate
I 2 3 4 5 6 7 8
F.W mg
43.4 33.9 34.1 34.4 35.4 29.7 32.3 32.7
AGGREGATES No. EMBRYOS No. EmbryoTotal genic(%) Total per mg
107 75 63 108 86 75 97 91
89.3 88.6 86.0 78.7 89.5 86.6 81.4 90.1
803 634 530 610 667 547 550 577
18.59 18.88 15.91 18.53 19.00 18.41 17.02 17.64
Although embryos beyond the globular stage were occasionally observed in suspension cultures, most embryos r e m a i n e d a r r e s t e d at the globular stage, s h o w i n g the t e n d e n c y to shed outer cell layers. F u r t h e r growth and development into m a t u r e somatic embryos required that the cell a g g r e g a t e s be plated on solid 1/2 MS medium supplemented with 3mM g l u t a m i n e plus low levels of both auxins and cytokinins. Four h o r m o n e t r e a t m e n t s were assayed (in pM): i) 0.92 Z, 0.25 IBA; 2) 0.89 BA, 0.25 IBA; 3) 2.22 BA, 0.49 IBA; 4) 2.22 BA, 0.54 NAA. W i t h i n about 2 weeks after plating, microscopic examination of cultures showed a great p r o l i f e r a t i o n of small, globular embryos (Fig.7). After one month, embryos at all stages of
development were observed, t o g e t h e r with abnormal structures in the same cultures. There were no d i f f e r e n c e s among the t r e a t m e n t s assayed w i t h respect to the culture maturation frequency (MF), defined as the p e r c e n t a g e of cultures that developed embryos beyond the globular stage. However, there were significant differences in m a t u r a t i o n frequency (pS0.001) among genotypes (Table 2). The secondary embryogenic line FIS6 had the highest m a t u r a t i o n frequency, although the p e r c e n t a g e of FIS6 cultures w i t h c o t y l e d o n a r y embryos (C) was lower than in the case of its parent genotype FIP3. 2. Embryo development from plated suspension cultures. The maturation frecuencies (MF) and the percentage of cultures with cotyledonary embryos (C) were recorded after 4 weeks culture. Table
Genotype Hormone treatment
FIP3 MF
I) 2) 3) 4)
C
73.2 44.4 58.9 22.5 51.8 21.8 39.4 1.9
FOA MF
C
43.1 48.7 59.0 30.4
4.1 7.0 3.2 0.0
F3D MF
17.3 22.6 10.4 26.8
C
0.0 0.0 0.4 0.4
FIS6 MF
C
87.8 21.7 77.9 22.1 72.6 15.0 63.6 6.0
F-test (genotype) F-test (treatment) LSD I% 15.18 (*) 1.205 (ns) 21.89% * significant at p ~0.001 ; ns = not significant Embryo m a t u r a t i o n was s t i m u l a t e d by isolating cotyledonary embryos from clumps and t r a n s f e r r i n g t h e m to solid MS or WPM m e d i u m supplemented with low levels of either Z or BA plus IBA. When isolated cotyledonary embryos were cultured on h o r m o n e - f r e e medium, roots developed, but not shoots. W i t h i n about 2 weeks after transfer to MS m e d i u m s u p p l e m e n t e d w i t h (2.2-4.4 HM) BA, 0.29 HM GA 3 and 0.49 ~M IBA, about 40% of embryos had e l o n g a t e d radicles, after which their c o t y l e d o n s expanded and became green; shoot p r i m o r d i a were observed in a small proportion of embryos 3-4 weeks after transfer. However, spending longer in this medium, resulted in callused embryos and the appearance of secondary embryos. C o t y l e d o n a r y embryos of different size and m o r p h o l o g i e s were capable of giving rise to p l a n t l e t s (Fig. 8). Different approaches were taken to enhance the conversion of somatic embryos into plantlets. Best results were achieved when isolated cotyledonary embryos were p r e t r e a t e d by storing at 4~ for 7 months and then cultured on MS m e d i u m s u p p l e m e n t e d with 4.4 @M BA and 0.49 ~M IBA for 2-3 weeks, with subsequent t r a n s f e r to MS plus 0.46 @M Z. After 4 weeks, 10% of embryos (63 plantlets from 638 embryos) had developed roots, shoots and well-formed leaves (Fig.9).
612
Somatic embryogenesis in Fagus sylvatica L. Fig I. Development of somatic embryos from a browned cotyledon-derived culture, after 5 weeks on solid secondary medium (bar = Imm). Fig 2. Embryogenic cell suspension in LMI medium supplemented with 0.45 #M 2,4-D (x 300). Fig 3. Asymetric divisions of embryogenic cells. 3a Differential interference contrast optics (x 285); 3b Acetocarmine staining (x 285). Fig 4. Pluricellular proembryo-like structure suspended in LMI medium (x 380). Fig 5. Growth of an individual globular embryo with a narrow suspensor-like structure in LM2 medium (x 37.5). Fig 6. Proembryogenic masses and clusters of globular embryos, after two 12-day passages in LM2 medium (x 4). Fig 7. Proliferating proembryogenic masses, several days after plating. Note the numerous small proembryos developed from globular embryos through secondary embryogenesis (x 6). Fig 8. Cotyledonary embryos after 4 months of cold storage at 4~ (bar = I cm). Fig 9. Regenerated beech plantlet from a somatic embryo (bar = I cm).
613 DISCUSSION
B r o w n i n g of t i s s u e s is n o r m a l l y a p r o b l e m in m i c r o p r o p a g a t i o n . H o w e v e r , t h i s and other previous studies ( Sondhal and S h a r p 1977; T u l e c k e and M c G r a n a h a n 1985; Neuenschwander and Baumann 1992) had s h o w n t h a t b r o w n i n g of t i s s u e s did not impair the competence for somatic embryogenesis. The cell suspension culture results s u g g e s t t h a t 2 , 4 - D w a s e s s e n t i a l for the induction and maintenance of the e m b r y o g e n i c c o m p e t e n c e of t h e c u l t u r e s at the earliest embryonic stages, since somatic embryogenesis and embryo development were only achieved after c u l t u r i n g s a m p l e s of t h e t y p e II c a l l u s o n l i q u i d m e d i u m c o n t a i n i n g 2,4-D. W h e n t h i s t y p e of c a l l u s w a s c u l t u r e d on m e d i a supplemented with other growth regulators o n l y c a l l u s p r o l i f e r a t i o n w a s achieved, e v e n w h e n c u l t u r e d o n 1/2 MS s u p p l e m e n t e d w i t h 3 m M g l u t a m i n e , 0.92 g M Z and 0.25 g M IBA, w h i c h w a s f o u n d t o b e a p p r o p r i a t e for e m b r y o d e v e l o p m e n t . A c c o r d i n g t o S h a r p et a i . ( 1 9 8 0 ) , 2 , 4 - D m a y act e i t h e r as a p r i m a r y agent of d e t e r m i n a t i o n of e m b r y o g e n i c c e l l s or as an a c t i v a t o r of t h e d e v e l o p m e n t of soc a l l e d p r e - d e t e r m i n e d e m b r y o g e n i c cells. Apparently, our embryogenic cells were determined as such after several d i v i s i o n s in t h e p r e s e n c e of 2,4-D, w h i c h m a y h a v e c a u s e d d e d i f f e r e n t i a t i o n and the t e r m i n a t i o n of p r e v i o u s l y a c t i v e p a t h w a y s (Carman 1990) and further epigenetic r e d e t e r m i n a t i o n to t h e e m b r y o g e n i c s t a t e ( S h a r p et al. 1980). B a c k s - H ~ s e m a n n a n d R e i n e r t (1970), and N o m u r a a n d K o m a m i n e (1986) r e p o r t e d t h a t t h e f i r s t s t e p in t h e d i f f e r e n t i a t i o n of e m b r y o s is t h e o c c u r r e n c e of u n e q u a l cell division in s i n g l e cells. In our cell s u s p e n s i o n c u l t u r e s , f i r s t a s y m e t r i c or unequal divisions were observed in f l o a t i n g f r e e cells, as w e l l as in s i n g l e cells at the peryphery of cell a g g r e g a t e s . A p p a r e n t l y , t h e r e can be two patterns of beech somatic embryo initiation. One, direct from single cells; the other, indirect f r o m cell a g g r e g a t e s or p r o e m b r y o g e n i c m a s s e s w h i c h had been formed by successive divisions of an embryogenic cell and of its d a u g h t e r cells. T h o u g h w e d i d not f o l l o w all steps sequentially from the first asymmetric division to the globular stage, b e e c h s o m a t i c e m b r y o s s e e m t o be of u n i c e l l u l a r o r i g i n a n d t o b e a b l e to f o l l o w v a r i o u s courses of development to the cotyledonary stage rather than a single
rigid pattern of divisions. It t h u s a p p e a r s t h a t t h e in v i t r o e m b r y o g e n e s i s of b e e c h is d e v e l o p m e n t a l l y p l a s t i c , in l i n e w i t h t h e t h e o r i e s of V o n A d e r k a s and B o n g a (1988) a n d C a r m a n (1990). The differences between the embryogenic c a p a c i t i e s a n d m a t u r a t i o n f r e q u e n c i e s of c u l t u r e s of d i f f e r e n t g e n o t y p e s s u g g e s t s t h a t s o m a t i c e m b r y o g e n e s i s is u n d e r genetic control. These variables were not a f f e c t e d b y t h e h o r m o n e t r e a t m e n t s tried. Thus, it s e e m s t h a t g e n o t y p e m a y be a more significant factor than culture medium in affecting the b e h a v i o u r of b e e c h e m b r y o g e n i c cultures. Acknowledgements. This study was partially supported by the EEC ECLAIR Programme, contract AGRE-CT91-0067. REFERENCES
Ahuja MR (1984) Silvae Genetica 33 (6): 241-242 Backs-HQssemann D, Reinert J (1970) Protoplasma 70: 496O Carman JG (1990) In Vitro Cell. Dev. Biol. 26 : 746-753 Chatupa W (1985) Comm. Inst. For. Cechosloveniae 14: 6990 Gamborg DL, Miller RA, Ojima K (1968) Exp. Cell Res. 50:
151-158
Gupta PK, Durzan DJ (1987) Biotechnology 5:147-151 Halperin W (1966) Am. J. Bot. 53 (5): 443-453 JSrgensen J (1988) J. Plant Physiol. 132: 638-640 J6rgensen J (1990) Abst. VII Int. Cong. Ptant Cell Tissue
Culture.Amsterdam, pp. 108
Ltoyd G, McCown BH (1980) Proc. Int. Plant Prop. Soc. 30:
421-428
Murashige S, Skoog F (1962) Physiol. Plant. 15:473-497 Neuenschwander B, Baumann TW (1992) Plant Cell Rep 10: 608-612 Nomura K, Komamine A (1986) New Phytol. 104:25-32 Sharp WR, Sondhal MR, Caldas LS, Marafa SB (1980) Hort. Rew. 2:268-310 Sondhal MR, Sharp WR (1977) Z. Pflanzenphysiol 81: 395408 Tulecke W, McGranahan GH (1985) Plant Sci. 40:53-67 Von Aderkas P, Bonga JM (1988) Am. J. Bot. 75 (5) : 690- 700
