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Regeneration of birch plants from catkin tissue cultures
Plant 8¢@nt~ Letters, 22 (1981) 879--386
879
© EimvierfNorth-Hollami 8eimmtifioPablbhem Ltd.
REGENERATION OF BIRCH PLANTS FROM CATKIN TISSUE CULTURES
P.8. 8RIVASTAVA and ~ 8TEINHAUER Lehretuhi ffir Fomtgenetik und Forstpflanzenziichtung, Bil~enweg 2, der Univemit~t G6ttin~n, 3400 C~ttingen (F.R.G.)
(Received December 12th, 1980) (Revblon received April 8th, 1981) (Accepted April 15th, 1981)
SUMMARY Catkins of Betula pendula cultured in vitro produced callus, and differentiated plantlets. In some explants rooting occurred within 4 weeks after very little callusing. Differentiation of roots and shoot buds occurred on defined White's medium (DWM) + 3-indoleacetic acid (IAA) + kinetin + casein hydrolysate (CH). However, much better growth of the shoots and formation of complete plantlets was possible only when a defined basal medium (DBM) without CH but with adenine was used. On this m e d i u m approx. 20 plantlets per culture could be harvested. No.loss in organogenesis has been observed although the callus is maintained since the last 10 months. Chromosome constitution has remained unchanged. In the present report early stages in embryogeny and induction of complete plantlets from catkin tissue cultures of birch is described.
INTRODUCTION During recent years much emphasis has been laid u p o n employing the technique of plant tissue culture in crop [1] and forest tree improvement [2--4]. It is now becoming possible to achieve clonal propagation of even older trees [5,6]. In vitro methods have rendered trees to a convenient size for experimentation [7] and manipulation for various genetic purposes. *l~mmmant addmm: Dep~mmm of Botany, ~G.T.B. Kiudsa Colkge, Uniwmity of Drift, Delhi 110007, India. AbbrevbstionJ: BAP, 6-bmu~laminopurJne, CI-I,casein hydml~mte;DBM, de~aml basal medium; DWM, defined Whitek modium; LA~ 3-tndoleseetic a~id; IBA~ind01o-8.butyrie add; NAA, a-Ml~]m~ucettc acid.
380 Although success has been achieved in raising plants from different explants of a number of plants, in vitro culture of tree explants has not been so successful [ 8--10]. In our studies, attempts are being made to raise tissue cultures of forest trees with a view to raising genetically
381
1~. 1. (s) 4-week~kl eatkka p b s on DWM + XAA + kinetin + CH. Note callusing of the explants and root (with root h a ~ ) diffenmti~ion; (b) prob_.. ~llu.inf and diffmntiation of shoots; (e) fuzthe~ growth of shoot upon trande~ to DBM + IAA + kine~in + adee~bm; (d) eomplete p ~ (medium as e); (e) ehromoeome eounts (2n = 28) in squash p ~ i o m of roots from the differentiated plantle~.
382
well~leveloped root hairs (Fig. la). However, further growth of roots was arrested. The callus became compact after 6 weeks in diffused daylight and differentiated shoots in 1 ~ cultures. A few poorly~ieveloped plantlets were also formed. If maintained under dark, the callus remained fluffy and light brown. Under continuous light the cultures became black and succumbed. Upon transfer to DBM supplemented with IAA (2 ppm) + kinetin (5 ppm) + adenine (30 ppm), 78% cultures produced more green callus, formed a number of shoot buds (Fig. lb), and differentiated plantlets in 69% cultures (Fig. lc,d). The remaining cultures exhibited small, shoot bud-like structures. These buds grew further and developed into normal shoots. In fact, many roots and shoots differentiated simultaneously from the callus. These formed a c o m m o n axis resulting into plantlets. In cultures where only shoots differentiated, the growth was n o t satisfactory. These shoots were excised and implanted on a fresh medium. Surprisingly, within 4 weeks, the shoots attained a size of 2.0 cm (original size 5.0 mm) and produced 4--5 pairs of normal leaves. These formed roots and ultimately complete plantlets could be achieved. Since the cultures showed distinct differences in response on DWM and DBM, in a set of experiment percentage of cultures showing plantlets (Fig. 2A), shoots (Fig. 2B), roots (Fig. 2C) and growth of callus (Fig. 2D) was compared on these two different media containing IAA + kinetin + adenine. Plantlet produced per callus was also scored (Fig. 2E). As is evident in Fig. 2, DBM proved more effective for organogenesis than DWM.
DWM L.)
70 ~£ 60 50
,]300 200
DBM
"•
300
470 ~"
4,°
200
l
~ 3O
~
I0 0
' t'°°,
E
(~ I00
DIc
B
A
"x.
13°
{ b
0 N
Fig. 2. Growth responses o f callus on two different media DWM, and DBM (both containing I A A + kinetin + adenine) after 6 weeks. The callus was originally derived on DWM + I A A + kinetin + CH. (A, B, C) per cent cultures showing organogenesis; (D) fresh wt. o f t h e callus (initial wt. o f callus was 70 rag); (E) number o f plantlets produced per calhts (average o f 50 callus pieees).
383
Therefore, in all subsequent experiments D B M + I A A + kinetin + adenine was used. In an attempt to increase the percentage of cultures differentiating plantlets, different concentrations of kinetin and adenine were tried. M a x i m u m number of cultures showing plantlets was achieved with I A A (2 ppm) + kinetin (6 ppm) + adenine (40 ppm) (Figs. 3 and 4). Early transfer (after 4 weeks) of callus (obtained on DWM + IAA + kinetin + CH) to DBM + IAA + kinetin + adenine, resulted in systematic division of cells showing early stages in embryogeny (Fig. 5a--e). A number of small plantlets (14--20) could be separated from the rest of the callus even by a gentle shaking of the culture vessels. These tiny plantlets when transferred to a fresh medium grew further. The plantlets started showing senescence effect on hormone-fetched medium. They were, therefore, transferred to hormone-free medium for 2 weeks before transplanting to soil. Attempts are being made to induce flowering in these plants. Histological studies of the callus revealed cells of different shape and size. Cell clusters simulating early stages in embryogeny were also noticed (Fig. 5a--e). Further development of these embryoids was quite irregular. In some cultures, small plantlets having two poorly~leveloped cotyledonlike structures with a long hypocotyl and a normal root were observed. These seem to arise from embryoids. The vasculature was fully differentiated. Embryogenesis in tissue cultures of tree species has been reported only in sandalwood [13--16]. The callus has been continuously growing on IAA (2 ppm) + kinetin (5 ppm) + CH (1000 ppm) m e d i u m since last 10 months but its organogenetic capacity is still maintained u p o n transfer to IAA + kinetin + adenine medium. We have also not yet found any change in chromosome number as well (Fig. le). 75
70 ;C ;C ;C
®
50
g 'C 'C
=~ 25
,,,,,,
0
0.5 10
20 ' 50 60 kinetin (pprn)
ZO
G
I0
20
30 ~0 adenine (pprn)
Fig. 3. R ~ p o m e o f callus to diffe~alt e o n e e ~ m t i o m of kJ0~etin. Medium: DBM + IAA (2 ppm) + kine£in + sdmine (30 ppm). Fig. 4. Callus response to varying eoncentr~ions o f adeuine. Medium: DBM + IAA (2 ppm) + klnetin (5 plan) + mlenlrune.
384
Fig. 5. Histological studies mhowing cell clustent m]zemlatlltge~rly ~ q e a in embryolleny (globular and dicotyledonous proembryos).
385 DISCUSSION
Tree breeders have been stymied by the long, complex life cycle and uncertainties of laborious genetic crosses with economically important forest trees. One of the main limitations of tree breeding is the relatively slow life cycle of trees and minimum of 15--20 years between generations. For the expression of a particular trait in trees one has to wait for generations. With the aid of in vitro regeneration however, the desired characters can be studied in the same generation. Also, in vitro regenerants offer a tool for the domestication of our wild forest resources. There are very few reports about the successful culture of tree explants [8,17,18]. While tissue culture has been obtained from other parts of the trees, catkins have never been tried earlier. The embryogenesis in tree tissue cultures is rare [13--16]. The meagre report of successful regeneration of plantlets in vitro of forest trees may be due to the failure of selection of proper media. By proper manipulation of culture media it is possible to obtain clonal multiplication of even 100-year-old teak [5], and 20-year-old Eucalyptus [6] trees. With modified media we have also succeeded in inducing growth in explants of other trees (Alnus, Fagus, Quercus) as well (to be reported later). The role of adenine in bud induction has been emphasized earlier [ 19]. In our studies, adenine played a significant role in shoot induction and ultimately in the differentiation of plantlets. We tried a number of media but none proved as effective. Our studies amply demonstrate that catkins of birch c/an be cultured in vitro, their morphogenic development can be reversed to vegetative phase and, can be induced to callusing, somatic embryogenesis and differentiation of plantlets. We had earlier observed only occasional cell clusters resembling proembryos [20]. In forest tree propagation, uniformity of types and phenotypicaily desirable individuals are an important consideration. However, clonai propagation of genetically-defined woody trees by vegetative means is not always simple. The process, frequently, is very slow and unsatisfactory. Plant tissue culture technique has demonstrated the feasibility of using this method in clonal propagation of many horticultural and some agricultural crops [1] and also of a few tree species [5,10,15]. Our investigations can be suitably used in vegetative propagation of geneticatly-defined clones of birch. Selection of 3--4-year-old vigorous plantlets, rapidly propagated by tissue culture method may possibly reduce time to obtain improved planting stock for regeneration. ACKNOWLEDGEMENT This work is supported by a DFG grant to Professor Dr. H.H. Hattemer,
386 D i r e c t o r o f t h e institute. We are t h a n k f u l t o Miss C. B e c k e r and Mr. S. K r a k u h n f o r e x c e l l e n t t e c h n i c a l assistance. REFERENCES 1 B.M. Johri, P.S. Srivastava and A.P. Raste, Indian J. Agric. Sci., 50 (1980) 108. 2 L. Winton and O. Huhtinen, Tissue culture of trees, in: J.P. Miksche (Ed.), Modem Methods in Forest Genetics, Springer-Verlag, Berlin, 1976, p. 243. 3 D.J. Durzan and R.A. Campbell, Can. J. For. Res., 4 (1974) 151. 4 T. Murashige, Annu. Rev. Plant Physiol., 25 (1974) 135. 5 P.K. Gupta, A.L. Nadgir, A.F. Masca~nhas and V. JagannathRn, Plant Sci. Lett., 17 (1980) 159. 6 P.K. Gupta, A.F. Mascaranhas and V. Jagannathan, Plant Sci. Lett., 20 (1981) 195. 7 V. Chalupa, Biol. Plant., 16 (1974) 316. 8 J.M. Bonga, Applications of tlssue culture in forestry, in: J. Reinert and Y.P.S. Bajaj (Eds.), Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, Springe~-Verlag, Berlin, 1977, p. 93. 9 B.M. Johri and P.S. Srivastava, Z. Pflanzenphysiol., 70 (1973) 285. 10 LK. Vaml and Vimla Vasil, Clonal propagation, in: I.K. Vasil (Ed.), Perspectives in Plant Cell and Tissue Cultures, Int. Rev. Cytology, Suppl. A, Academic Press, New York, 1980, p. 145. 11 B.M. Johri and P.S. Srivastava, In vitro growth responses of mature endosperm of Ricinus communis L., in: Adv. Pl. Morph., V. Puri Comm. Vol., Sarita Prakashan, India, 1973, p. 389. 12 P.S. Srivastava, A. Varga and J. Bruinsma, Z. Pflanzenphysiol., 98 (1980) 347. 13 P.S. Rao and N.S. Rangmmamy, Biol. Plant., 13 (1971) 200. 14 G. Lakshmi Sita, N.Y. Raghava Ram and C.S. Vaidyanathan, Plant Sci. Lett., 15 (1979) 265. 15 G. Lakahmi Sita, J. Shobha and C.S. VaidyRn~than, Curt. Sci., 49 (1980) 196. 16 G. Lakshmi Sita, N.V. Raghava Ram and C.S. Vaidyanathan, Plant Sci. Lett., 20 (1980) 63. 17 P.S. Srivutava, Z. Pfi~nT~nphydoL, 69 (1973) 270. 18 P.S. SrivMtava and B.M. Johri, Beitr. Biol. Pflanzanphysiol., 54 (1978) 881. 19 J.P. Nit~h, C. Nit~h, L.M.E. Rossini and B.D. Ha, Phytomorphology, 17 (1967) 446. 20 A. Steinhauer, H. (}lock and P.S. Srivutava, In vitro culture methods and its implications in genetics and forest tree breeding, 2nd Int. Congr. on Cell Biol., Ear. J. Cell. Biol., 22 (1980) 608.
879
© EimvierfNorth-Hollami 8eimmtifioPablbhem Ltd.
REGENERATION OF BIRCH PLANTS FROM CATKIN TISSUE CULTURES
P.8. 8RIVASTAVA and ~ 8TEINHAUER Lehretuhi ffir Fomtgenetik und Forstpflanzenziichtung, Bil~enweg 2, der Univemit~t G6ttin~n, 3400 C~ttingen (F.R.G.)
(Received December 12th, 1980) (Revblon received April 8th, 1981) (Accepted April 15th, 1981)
SUMMARY Catkins of Betula pendula cultured in vitro produced callus, and differentiated plantlets. In some explants rooting occurred within 4 weeks after very little callusing. Differentiation of roots and shoot buds occurred on defined White's medium (DWM) + 3-indoleacetic acid (IAA) + kinetin + casein hydrolysate (CH). However, much better growth of the shoots and formation of complete plantlets was possible only when a defined basal medium (DBM) without CH but with adenine was used. On this m e d i u m approx. 20 plantlets per culture could be harvested. No.loss in organogenesis has been observed although the callus is maintained since the last 10 months. Chromosome constitution has remained unchanged. In the present report early stages in embryogeny and induction of complete plantlets from catkin tissue cultures of birch is described.
INTRODUCTION During recent years much emphasis has been laid u p o n employing the technique of plant tissue culture in crop [1] and forest tree improvement [2--4]. It is now becoming possible to achieve clonal propagation of even older trees [5,6]. In vitro methods have rendered trees to a convenient size for experimentation [7] and manipulation for various genetic purposes. *l~mmmant addmm: Dep~mmm of Botany, ~G.T.B. Kiudsa Colkge, Uniwmity of Drift, Delhi 110007, India. AbbrevbstionJ: BAP, 6-bmu~laminopurJne, CI-I,casein hydml~mte;DBM, de~aml basal medium; DWM, defined Whitek modium; LA~ 3-tndoleseetic a~id; IBA~ind01o-8.butyrie add; NAA, a-Ml~]m~ucettc acid.
380 Although success has been achieved in raising plants from different explants of a number of plants, in vitro culture of tree explants has not been so successful [ 8--10]. In our studies, attempts are being made to raise tissue cultures of forest trees with a view to raising genetically
381
1~. 1. (s) 4-week~kl eatkka p b s on DWM + XAA + kinetin + CH. Note callusing of the explants and root (with root h a ~ ) diffenmti~ion; (b) prob_.. ~llu.inf and diffmntiation of shoots; (e) fuzthe~ growth of shoot upon trande~ to DBM + IAA + kine~in + adee~bm; (d) eomplete p ~ (medium as e); (e) ehromoeome eounts (2n = 28) in squash p ~ i o m of roots from the differentiated plantle~.
382
well~leveloped root hairs (Fig. la). However, further growth of roots was arrested. The callus became compact after 6 weeks in diffused daylight and differentiated shoots in 1 ~ cultures. A few poorly~ieveloped plantlets were also formed. If maintained under dark, the callus remained fluffy and light brown. Under continuous light the cultures became black and succumbed. Upon transfer to DBM supplemented with IAA (2 ppm) + kinetin (5 ppm) + adenine (30 ppm), 78% cultures produced more green callus, formed a number of shoot buds (Fig. lb), and differentiated plantlets in 69% cultures (Fig. lc,d). The remaining cultures exhibited small, shoot bud-like structures. These buds grew further and developed into normal shoots. In fact, many roots and shoots differentiated simultaneously from the callus. These formed a c o m m o n axis resulting into plantlets. In cultures where only shoots differentiated, the growth was n o t satisfactory. These shoots were excised and implanted on a fresh medium. Surprisingly, within 4 weeks, the shoots attained a size of 2.0 cm (original size 5.0 mm) and produced 4--5 pairs of normal leaves. These formed roots and ultimately complete plantlets could be achieved. Since the cultures showed distinct differences in response on DWM and DBM, in a set of experiment percentage of cultures showing plantlets (Fig. 2A), shoots (Fig. 2B), roots (Fig. 2C) and growth of callus (Fig. 2D) was compared on these two different media containing IAA + kinetin + adenine. Plantlet produced per callus was also scored (Fig. 2E). As is evident in Fig. 2, DBM proved more effective for organogenesis than DWM.
DWM L.)
70 ~£ 60 50
,]300 200
DBM
"•
300
470 ~"
4,°
200
l
~ 3O
~
I0 0
' t'°°,
E
(~ I00
DIc
B
A
"x.
13°
{ b
0 N
Fig. 2. Growth responses o f callus on two different media DWM, and DBM (both containing I A A + kinetin + adenine) after 6 weeks. The callus was originally derived on DWM + I A A + kinetin + CH. (A, B, C) per cent cultures showing organogenesis; (D) fresh wt. o f t h e callus (initial wt. o f callus was 70 rag); (E) number o f plantlets produced per calhts (average o f 50 callus pieees).
383
Therefore, in all subsequent experiments D B M + I A A + kinetin + adenine was used. In an attempt to increase the percentage of cultures differentiating plantlets, different concentrations of kinetin and adenine were tried. M a x i m u m number of cultures showing plantlets was achieved with I A A (2 ppm) + kinetin (6 ppm) + adenine (40 ppm) (Figs. 3 and 4). Early transfer (after 4 weeks) of callus (obtained on DWM + IAA + kinetin + CH) to DBM + IAA + kinetin + adenine, resulted in systematic division of cells showing early stages in embryogeny (Fig. 5a--e). A number of small plantlets (14--20) could be separated from the rest of the callus even by a gentle shaking of the culture vessels. These tiny plantlets when transferred to a fresh medium grew further. The plantlets started showing senescence effect on hormone-fetched medium. They were, therefore, transferred to hormone-free medium for 2 weeks before transplanting to soil. Attempts are being made to induce flowering in these plants. Histological studies of the callus revealed cells of different shape and size. Cell clusters simulating early stages in embryogeny were also noticed (Fig. 5a--e). Further development of these embryoids was quite irregular. In some cultures, small plantlets having two poorly~leveloped cotyledonlike structures with a long hypocotyl and a normal root were observed. These seem to arise from embryoids. The vasculature was fully differentiated. Embryogenesis in tissue cultures of tree species has been reported only in sandalwood [13--16]. The callus has been continuously growing on IAA (2 ppm) + kinetin (5 ppm) + CH (1000 ppm) m e d i u m since last 10 months but its organogenetic capacity is still maintained u p o n transfer to IAA + kinetin + adenine medium. We have also not yet found any change in chromosome number as well (Fig. le). 75
70 ;C ;C ;C
®
50
g 'C 'C
=~ 25
,,,,,,
0
0.5 10
20 ' 50 60 kinetin (pprn)
ZO
G
I0
20
30 ~0 adenine (pprn)
Fig. 3. R ~ p o m e o f callus to diffe~alt e o n e e ~ m t i o m of kJ0~etin. Medium: DBM + IAA (2 ppm) + kine£in + sdmine (30 ppm). Fig. 4. Callus response to varying eoncentr~ions o f adeuine. Medium: DBM + IAA (2 ppm) + klnetin (5 plan) + mlenlrune.
384
Fig. 5. Histological studies mhowing cell clustent m]zemlatlltge~rly ~ q e a in embryolleny (globular and dicotyledonous proembryos).
385 DISCUSSION
Tree breeders have been stymied by the long, complex life cycle and uncertainties of laborious genetic crosses with economically important forest trees. One of the main limitations of tree breeding is the relatively slow life cycle of trees and minimum of 15--20 years between generations. For the expression of a particular trait in trees one has to wait for generations. With the aid of in vitro regeneration however, the desired characters can be studied in the same generation. Also, in vitro regenerants offer a tool for the domestication of our wild forest resources. There are very few reports about the successful culture of tree explants [8,17,18]. While tissue culture has been obtained from other parts of the trees, catkins have never been tried earlier. The embryogenesis in tree tissue cultures is rare [13--16]. The meagre report of successful regeneration of plantlets in vitro of forest trees may be due to the failure of selection of proper media. By proper manipulation of culture media it is possible to obtain clonal multiplication of even 100-year-old teak [5], and 20-year-old Eucalyptus [6] trees. With modified media we have also succeeded in inducing growth in explants of other trees (Alnus, Fagus, Quercus) as well (to be reported later). The role of adenine in bud induction has been emphasized earlier [ 19]. In our studies, adenine played a significant role in shoot induction and ultimately in the differentiation of plantlets. We tried a number of media but none proved as effective. Our studies amply demonstrate that catkins of birch c/an be cultured in vitro, their morphogenic development can be reversed to vegetative phase and, can be induced to callusing, somatic embryogenesis and differentiation of plantlets. We had earlier observed only occasional cell clusters resembling proembryos [20]. In forest tree propagation, uniformity of types and phenotypicaily desirable individuals are an important consideration. However, clonai propagation of genetically-defined woody trees by vegetative means is not always simple. The process, frequently, is very slow and unsatisfactory. Plant tissue culture technique has demonstrated the feasibility of using this method in clonal propagation of many horticultural and some agricultural crops [1] and also of a few tree species [5,10,15]. Our investigations can be suitably used in vegetative propagation of geneticatly-defined clones of birch. Selection of 3--4-year-old vigorous plantlets, rapidly propagated by tissue culture method may possibly reduce time to obtain improved planting stock for regeneration. ACKNOWLEDGEMENT This work is supported by a DFG grant to Professor Dr. H.H. Hattemer,
386 D i r e c t o r o f t h e institute. We are t h a n k f u l t o Miss C. B e c k e r and Mr. S. K r a k u h n f o r e x c e l l e n t t e c h n i c a l assistance. REFERENCES 1 B.M. Johri, P.S. Srivastava and A.P. Raste, Indian J. Agric. Sci., 50 (1980) 108. 2 L. Winton and O. Huhtinen, Tissue culture of trees, in: J.P. Miksche (Ed.), Modem Methods in Forest Genetics, Springer-Verlag, Berlin, 1976, p. 243. 3 D.J. Durzan and R.A. Campbell, Can. J. For. Res., 4 (1974) 151. 4 T. Murashige, Annu. Rev. Plant Physiol., 25 (1974) 135. 5 P.K. Gupta, A.L. Nadgir, A.F. Masca~nhas and V. JagannathRn, Plant Sci. Lett., 17 (1980) 159. 6 P.K. Gupta, A.F. Mascaranhas and V. Jagannathan, Plant Sci. Lett., 20 (1981) 195. 7 V. Chalupa, Biol. Plant., 16 (1974) 316. 8 J.M. Bonga, Applications of tlssue culture in forestry, in: J. Reinert and Y.P.S. Bajaj (Eds.), Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture, Springe~-Verlag, Berlin, 1977, p. 93. 9 B.M. Johri and P.S. Srivastava, Z. Pflanzenphysiol., 70 (1973) 285. 10 LK. Vaml and Vimla Vasil, Clonal propagation, in: I.K. Vasil (Ed.), Perspectives in Plant Cell and Tissue Cultures, Int. Rev. Cytology, Suppl. A, Academic Press, New York, 1980, p. 145. 11 B.M. Johri and P.S. Srivastava, In vitro growth responses of mature endosperm of Ricinus communis L., in: Adv. Pl. Morph., V. Puri Comm. Vol., Sarita Prakashan, India, 1973, p. 389. 12 P.S. Srivastava, A. Varga and J. Bruinsma, Z. Pflanzenphysiol., 98 (1980) 347. 13 P.S. Rao and N.S. Rangmmamy, Biol. Plant., 13 (1971) 200. 14 G. Lakshmi Sita, N.Y. Raghava Ram and C.S. Vaidyanathan, Plant Sci. Lett., 15 (1979) 265. 15 G. Lakahmi Sita, J. Shobha and C.S. VaidyRn~than, Curt. Sci., 49 (1980) 196. 16 G. Lakshmi Sita, N.V. Raghava Ram and C.S. Vaidyanathan, Plant Sci. Lett., 20 (1980) 63. 17 P.S. Srivutava, Z. Pfi~nT~nphydoL, 69 (1973) 270. 18 P.S. SrivMtava and B.M. Johri, Beitr. Biol. Pflanzanphysiol., 54 (1978) 881. 19 J.P. Nit~h, C. Nit~h, L.M.E. Rossini and B.D. Ha, Phytomorphology, 17 (1967) 446. 20 A. Steinhauer, H. (}lock and P.S. Srivutava, In vitro culture methods and its implications in genetics and forest tree breeding, 2nd Int. Congr. on Cell Biol., Ear. J. Cell. Biol., 22 (1980) 608.