Plant Cell Reports
Plant Cell Reports (1986) 5:266-268
© Springer-Verlag 1986
Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.) G. Lakshmi Sita 1, S. Chattopadhyay 1, and D.H. Tejavathi 2 1 Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560 012, India 2 Botany Department, Bangalore University Jnanabbarathi, Bangalore 560 056, India Received July 16, 1985 / Revised version received June 10, 1986 - Communicated by O. L. Gamborg
ABSTRACT In vitro propagation of t r e e s using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation ol Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old t r e e s . Bud r e g e n e r a t i o n was obtained on MS media s u p p l e m e n t e d with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3-5 cm can be excised and rooted in White's medium supplemented with t-2 mg/L IAA. Rooted plants were successfully established in soil. Abbreviations; BA, Benzyladenine; CM, Coconut milk; 2,#-D, 2,# dichlorophenoxyacetic acid; IAA, Indoleacetic acid; 1BA, Indolebutyric acid; K, Kinetin; NAA, Naphthal e n e a c e t i c acid; PVP-360, Polyvinyl pyrrolidone INTRODUCTION Organogenesis has been r e p o r t e d eartier in a few forest trees. (Brown and Sommer 1982; Bajaj 1986). However routine shoot and root r e g e n e r a t i o n from callus is not common as in herbaceous species. R e g e n e r a t i o n from leguminous t r e e s has been difficult until r e c e n t l y . Only a few have been cultured in vitro like Acacia koa (Skolmen 1986), Albezzia lebbek, Sesbania sesban (Gharyal and Maheswari 1980) and Dalbergia sissoo (Mukhopadhyay and Mohan Ram 1981; D a t t a et al 1982). Several species of Dalbergia are the main source ol rosewood which is in g r e a t demand commercially. D__. latifolia Roxb. is one of the i m p o r t a n t indian species which makes up the bulk of the t o t a l production. Natural growing stock of this species is dwindling due to indiscrim i n a t e cutting. Also conventional propagation of rosewood is not s a t i s f a c t o r y due to low seed viability. Micropropagation by tissue culture methods can be used to speed up the production of planting stocks. In this paper we describe callus induction and plant r e g e n e r a t i o n by adventitious shoot buds as a method for rapid propagation. MATERIALS AND METHODS Young shoots, 10-15 cm long were collected from 5 year old potted plants oI Dalbergia latifotia Roxb. and washed thoroughly with d e t e r g e n t solution for two minutes prior to surface sterilization. The shoots were sterilized in 0.1% m e r c u r i c chloride solution for 15 minutes and then rinsed 3 # t i m e s with sterile
Offprint requests to: G. L. Sita
distilled water. They were cut into explants of 5 8 mm and were inoculated aseptically. The medium consisted of Murashige and Skoog's (1962) mineral salts and vitamins and was supplemented with 2% Sucrose (w/v). Medium was solidified with 0.g% (w/v) agar. G a m b o r g ' s B5 (1968) basal medium was used occassionally in some e x p e r i m e n t s . The growth subst a n c e s added most commonly were 2,#-D, NAA and BA. Coconut milk was used occassionally. Soluble PVP (PVP-360)at 1 gm/L was added to reduce the phenolics in the medium. The pH was adjusted to 5.6 t o 5 . g g w i t h 1N KOH solution before autoclaving at 151b/in" for 15 to 20 minutes. The callus was t r a n s f e r r e d at # week intervals. Cultures were illumin a t e d with white f l u o r e s c e n t light at 1000 - 1200' Lux for 16 hours at 25°C. C o m b i n a t i o n s of auxins and cytokinins were used to induce d i l f e r e n t i a t i o n . The t e m p e r a t u r e and light conditions were the same as those employed for callus induction. Each medium was t e s t e d with 30 samples and all the media have been t e s t e d several t i m e s over a period of 11/2 years. For microscopy the callus pieces with emerging shoot buds were fixed in f o r m a l i n - a c e t i c - a l c o h o l (FAA) overnight at room t e m p e r a t u r e prior to t r a n s f e r to 70% alcohol. Standard histological techniques were followed to achieve dehydration and embedded in paraffin. Sections were cut a t g - 10 um with a rotary m i c r o t o m e . The sections were stained with Heidenhain's ironalum haematoxylin and c o u n t e r s t a i n e d with erythrosin and mounted in canada balsam or DPX. RESULTS AND DISCUSSION Both nodal and internodal pieces produced callus on MS media containing 2,#-D or NAA at 1-5 mg/L within two weeks. Large amounts of friable callus was obtained on media s u p p l e m e n t e d with auxins alone in about 5-6 weeks. However, friable callus thus obtained was found to be unsuitable for shoot induction. Hence the media were subsequently supplem e n t e d with BA and CM. MS s u p p l e m e n t e d with 5 mg/L NAA, 1 mg]L 2,#-D and 1 mg]L BA along with I0% CM was found to be suitable for both callus induction and m a i n t e n a n c e . Callus on this medium is c o m p a c t and green in colour. Pieces of t h e s e calli were s e p a r a t e d and cultured on media s u p p l e m e n t e d with only BA and NAA. In t h e preliminary e x p e r i m e n t s (Lakshmi Sita and Sankara Rao 198#) with MS medium supplemented with 3 mg/L NAA and 1 mg/L BA,
267
Fig. 1. Differentiation of shoot buds from compace callus (early stage).
Fig. 2. Elongated shoots before rooting.
Fig. 3. Longitudinal section oI an emerging shoot bud.
Fig. 4. 6 month old potted plant.
268 brown shoot like structures emerged. Callus pieces with these shoot bud like structures were subcultured on media containing 3 mg/L BA and 1 mg/LNAA. Within four weeks more of these shoot like structures developed. Further reduction of NAA to 0.5 mg/L increased the number of buds. In one tube a maximum of twelve identifiable shoots were obtained. Some of these buds have elongated to 12-15 cm in length. Based on these preliminary experiments several combination of BA (1-5 rag/L) and NAA (0.I-1 rag/L) were tried. Table 1 shows the e f f e c t of BA and NAA on bud differentiation. Of these the MS medium supplemented with 3 mg/L BA and 0.I-1 mg/L NAA gave best differentiation. Table 1 : Effect of BA and NAA on bud diflerentiation
BA mg/L
NAA mg/L 0.1
0.5
differentiation was observed directly on root explants with or without callusing from seedlings (Mukhopadhyay and Mohan Ram 1981) and multiple shoots from nodal segments of mature trees (Datta et al 1982), Regeneration from forest trees in general and legumes in particular has been a difficult problem. In most of the cases regeneration is from seedling parts such as cotyledons and hypocotyls. As is well documented (Bonga 1977) from the tree improvement point of view regeneration from sections of embryos and young seedlings has the distinct disadvantage that the plantlets have a genotype different from that of the parent tree, and the superior characters of the parent are not necessarily retained by the propagules. Regeneration from adult material will be useful since the phenotype of the selected plant and its desirabilities are known. Regeneration from callus cultures of adult trees is so far limited to a few species (Bajaj 1986). In the present work, although the material was juvenile the procedures developed for obtaining plants from callus could be used for isolating faster growing and straight boled rosewood trees. Work is now in progress with mature materials.
1.0 ACKNOWLEDGEMENT
1
2 3 # 5
Nil 3-5 5-10 3-5 1-3
Nil Nil 5-10 3-5 1-3
Nil Nil 5-10 3-5 1-3
This work was supported by the Hindustan Lever Research Foundation grant to the senior author. REFERENCES Bajaj YPS (1986) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry,Vol.l; Trees I. Springer, Berlin Heidelberg New York, Tokyo, pp 1-23
The number of explants showing differentiation in all the concentrations of NAA was nearly the same. The number of shootbuds per explant varied Irom 5-10. For routine shoot bud induction, MS supplemented with 3 mg/L BA and 0.1 or 0.5 mg/L NAA was used. Approximately 1000 callus pieces were cultured and of these about 300 have formed shoots. Figs. 1 & 2 show differentiating shoots from the callus. Sections were cut to determine the origin of the buds. Fig. 3 shows a longitudinal section of an emerging shoot bud. Serial sections showed that m e r e s t e m a t i c regions are Iormed in the parench ymatous callus. The buds arise from these regions and from the axils of the leaf primordia. Shoot producing calli, as well as the medium turned dark brown during extended culture period. Shoots can be rooted by transferring them into white's medium, either solid or liquid, supl~lemented with 1-2 mg/L of IAA. Subculture onto MS medium with 1/2 strength MS salts and IBA (2-5 rag/L) also was found to be suitable. Rooting was always accompanied by leaf drop in the culture vessle. However after establishment in soil new leaves developed. Fig. # shows a 6 month old plant. The foregoing observations clearly indicate that the diflerentiation of shoot buds in callus was not from any preexisting shoot primordia. In D. sissoo
Bonga 3M (1977) In: Reinert 3, Bajaj YPS (eds) Applied and fundamental aspects of plant cell, tissue and organ culture, Springer, Berlin Heidelberg New York, pp 93-108 Brown CL, Sommer HE (1982) In: Bonga 3M, Durzan D3 (eds) Tissue culture in Forestry, Martinus Nijhoff/ Dr. W. 3unk, pp 109-1#8 Datta SK, Datta K, Pramanik tissue organ culture 2:15
T (1982)
Plant
cell
Gamborg OL, Miller RA, Ojima K (1968) Expt. cell Res 50:151-158 Gharyal PK, Maheswari SC (1980) Naturwissenschaften 8:379 Lakshmi Sita G, Sankara Rao K (198t~) t~lst Conference in the Easter School Series in Agricultural Sciences. Univ. of Nottingham Poster Presentation, pp 70 Mukhopadhyay A, Mohan Ram Expt. Biol. 19:1113-1115
HY
(1981)
Indian
3
Murashige T, Skoog F (1962) Physiol Plant. 15:#73-#97 Skoleman RG, Mapes MO (1976) 3. Hered. 6 7 : 1 1 4 - 1 1 5
Plant Cell Reports (1986) 5:266-268
© Springer-Verlag 1986
Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.) G. Lakshmi Sita 1, S. Chattopadhyay 1, and D.H. Tejavathi 2 1 Microbiology and Cell Biology Laboratory, Indian Institute of Science, Bangalore 560 012, India 2 Botany Department, Bangalore University Jnanabbarathi, Bangalore 560 056, India Received July 16, 1985 / Revised version received June 10, 1986 - Communicated by O. L. Gamborg
ABSTRACT In vitro propagation of t r e e s using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation ol Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old t r e e s . Bud r e g e n e r a t i o n was obtained on MS media s u p p l e m e n t e d with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3-5 cm can be excised and rooted in White's medium supplemented with t-2 mg/L IAA. Rooted plants were successfully established in soil. Abbreviations; BA, Benzyladenine; CM, Coconut milk; 2,#-D, 2,# dichlorophenoxyacetic acid; IAA, Indoleacetic acid; 1BA, Indolebutyric acid; K, Kinetin; NAA, Naphthal e n e a c e t i c acid; PVP-360, Polyvinyl pyrrolidone INTRODUCTION Organogenesis has been r e p o r t e d eartier in a few forest trees. (Brown and Sommer 1982; Bajaj 1986). However routine shoot and root r e g e n e r a t i o n from callus is not common as in herbaceous species. R e g e n e r a t i o n from leguminous t r e e s has been difficult until r e c e n t l y . Only a few have been cultured in vitro like Acacia koa (Skolmen 1986), Albezzia lebbek, Sesbania sesban (Gharyal and Maheswari 1980) and Dalbergia sissoo (Mukhopadhyay and Mohan Ram 1981; D a t t a et al 1982). Several species of Dalbergia are the main source ol rosewood which is in g r e a t demand commercially. D__. latifolia Roxb. is one of the i m p o r t a n t indian species which makes up the bulk of the t o t a l production. Natural growing stock of this species is dwindling due to indiscrim i n a t e cutting. Also conventional propagation of rosewood is not s a t i s f a c t o r y due to low seed viability. Micropropagation by tissue culture methods can be used to speed up the production of planting stocks. In this paper we describe callus induction and plant r e g e n e r a t i o n by adventitious shoot buds as a method for rapid propagation. MATERIALS AND METHODS Young shoots, 10-15 cm long were collected from 5 year old potted plants oI Dalbergia latifotia Roxb. and washed thoroughly with d e t e r g e n t solution for two minutes prior to surface sterilization. The shoots were sterilized in 0.1% m e r c u r i c chloride solution for 15 minutes and then rinsed 3 # t i m e s with sterile
Offprint requests to: G. L. Sita
distilled water. They were cut into explants of 5 8 mm and were inoculated aseptically. The medium consisted of Murashige and Skoog's (1962) mineral salts and vitamins and was supplemented with 2% Sucrose (w/v). Medium was solidified with 0.g% (w/v) agar. G a m b o r g ' s B5 (1968) basal medium was used occassionally in some e x p e r i m e n t s . The growth subst a n c e s added most commonly were 2,#-D, NAA and BA. Coconut milk was used occassionally. Soluble PVP (PVP-360)at 1 gm/L was added to reduce the phenolics in the medium. The pH was adjusted to 5.6 t o 5 . g g w i t h 1N KOH solution before autoclaving at 151b/in" for 15 to 20 minutes. The callus was t r a n s f e r r e d at # week intervals. Cultures were illumin a t e d with white f l u o r e s c e n t light at 1000 - 1200' Lux for 16 hours at 25°C. C o m b i n a t i o n s of auxins and cytokinins were used to induce d i l f e r e n t i a t i o n . The t e m p e r a t u r e and light conditions were the same as those employed for callus induction. Each medium was t e s t e d with 30 samples and all the media have been t e s t e d several t i m e s over a period of 11/2 years. For microscopy the callus pieces with emerging shoot buds were fixed in f o r m a l i n - a c e t i c - a l c o h o l (FAA) overnight at room t e m p e r a t u r e prior to t r a n s f e r to 70% alcohol. Standard histological techniques were followed to achieve dehydration and embedded in paraffin. Sections were cut a t g - 10 um with a rotary m i c r o t o m e . The sections were stained with Heidenhain's ironalum haematoxylin and c o u n t e r s t a i n e d with erythrosin and mounted in canada balsam or DPX. RESULTS AND DISCUSSION Both nodal and internodal pieces produced callus on MS media containing 2,#-D or NAA at 1-5 mg/L within two weeks. Large amounts of friable callus was obtained on media s u p p l e m e n t e d with auxins alone in about 5-6 weeks. However, friable callus thus obtained was found to be unsuitable for shoot induction. Hence the media were subsequently supplem e n t e d with BA and CM. MS s u p p l e m e n t e d with 5 mg/L NAA, 1 mg]L 2,#-D and 1 mg]L BA along with I0% CM was found to be suitable for both callus induction and m a i n t e n a n c e . Callus on this medium is c o m p a c t and green in colour. Pieces of t h e s e calli were s e p a r a t e d and cultured on media s u p p l e m e n t e d with only BA and NAA. In t h e preliminary e x p e r i m e n t s (Lakshmi Sita and Sankara Rao 198#) with MS medium supplemented with 3 mg/L NAA and 1 mg/L BA,
267
Fig. 1. Differentiation of shoot buds from compace callus (early stage).
Fig. 2. Elongated shoots before rooting.
Fig. 3. Longitudinal section oI an emerging shoot bud.
Fig. 4. 6 month old potted plant.
268 brown shoot like structures emerged. Callus pieces with these shoot bud like structures were subcultured on media containing 3 mg/L BA and 1 mg/LNAA. Within four weeks more of these shoot like structures developed. Further reduction of NAA to 0.5 mg/L increased the number of buds. In one tube a maximum of twelve identifiable shoots were obtained. Some of these buds have elongated to 12-15 cm in length. Based on these preliminary experiments several combination of BA (1-5 rag/L) and NAA (0.I-1 rag/L) were tried. Table 1 shows the e f f e c t of BA and NAA on bud differentiation. Of these the MS medium supplemented with 3 mg/L BA and 0.I-1 mg/L NAA gave best differentiation. Table 1 : Effect of BA and NAA on bud diflerentiation
BA mg/L
NAA mg/L 0.1
0.5
differentiation was observed directly on root explants with or without callusing from seedlings (Mukhopadhyay and Mohan Ram 1981) and multiple shoots from nodal segments of mature trees (Datta et al 1982), Regeneration from forest trees in general and legumes in particular has been a difficult problem. In most of the cases regeneration is from seedling parts such as cotyledons and hypocotyls. As is well documented (Bonga 1977) from the tree improvement point of view regeneration from sections of embryos and young seedlings has the distinct disadvantage that the plantlets have a genotype different from that of the parent tree, and the superior characters of the parent are not necessarily retained by the propagules. Regeneration from adult material will be useful since the phenotype of the selected plant and its desirabilities are known. Regeneration from callus cultures of adult trees is so far limited to a few species (Bajaj 1986). In the present work, although the material was juvenile the procedures developed for obtaining plants from callus could be used for isolating faster growing and straight boled rosewood trees. Work is now in progress with mature materials.
1.0 ACKNOWLEDGEMENT
1
2 3 # 5
Nil 3-5 5-10 3-5 1-3
Nil Nil 5-10 3-5 1-3
Nil Nil 5-10 3-5 1-3
This work was supported by the Hindustan Lever Research Foundation grant to the senior author. REFERENCES Bajaj YPS (1986) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry,Vol.l; Trees I. Springer, Berlin Heidelberg New York, Tokyo, pp 1-23
The number of explants showing differentiation in all the concentrations of NAA was nearly the same. The number of shootbuds per explant varied Irom 5-10. For routine shoot bud induction, MS supplemented with 3 mg/L BA and 0.1 or 0.5 mg/L NAA was used. Approximately 1000 callus pieces were cultured and of these about 300 have formed shoots. Figs. 1 & 2 show differentiating shoots from the callus. Sections were cut to determine the origin of the buds. Fig. 3 shows a longitudinal section of an emerging shoot bud. Serial sections showed that m e r e s t e m a t i c regions are Iormed in the parench ymatous callus. The buds arise from these regions and from the axils of the leaf primordia. Shoot producing calli, as well as the medium turned dark brown during extended culture period. Shoots can be rooted by transferring them into white's medium, either solid or liquid, supl~lemented with 1-2 mg/L of IAA. Subculture onto MS medium with 1/2 strength MS salts and IBA (2-5 rag/L) also was found to be suitable. Rooting was always accompanied by leaf drop in the culture vessle. However after establishment in soil new leaves developed. Fig. # shows a 6 month old plant. The foregoing observations clearly indicate that the diflerentiation of shoot buds in callus was not from any preexisting shoot primordia. In D. sissoo
Bonga 3M (1977) In: Reinert 3, Bajaj YPS (eds) Applied and fundamental aspects of plant cell, tissue and organ culture, Springer, Berlin Heidelberg New York, pp 93-108 Brown CL, Sommer HE (1982) In: Bonga 3M, Durzan D3 (eds) Tissue culture in Forestry, Martinus Nijhoff/ Dr. W. 3unk, pp 109-1#8 Datta SK, Datta K, Pramanik tissue organ culture 2:15
T (1982)
Plant
cell
Gamborg OL, Miller RA, Ojima K (1968) Expt. cell Res 50:151-158 Gharyal PK, Maheswari SC (1980) Naturwissenschaften 8:379 Lakshmi Sita G, Sankara Rao K (198t~) t~lst Conference in the Easter School Series in Agricultural Sciences. Univ. of Nottingham Poster Presentation, pp 70 Mukhopadhyay A, Mohan Ram Expt. Biol. 19:1113-1115
HY
(1981)
Indian
3
Murashige T, Skoog F (1962) Physiol Plant. 15:#73-#97 Skoleman RG, Mapes MO (1976) 3. Hered. 6 7 : 1 1 4 - 1 1 5
