PlantCell Reports
Plant Cell Reports (1986) 3:190-191
© Springer-Verlag 1986
Plant regeneration of Sapindus trifoliatus L. (soapnut) through somatic embryogenesis Harsha V. Desai, Prashant N. Bhatt, and Atul R. Mehta Department of Botany, The M.S. University, Baroda-390002, India Received November 26, 1985 / Revised version received March 31, t986 - Communicated by I. K. Vasil
ABSTRACT
Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from ]eaves of mature (over 60years old) Soapnut (Sapindus trifoliatus L,) tree. Callus was induced from leaf explants and grown on Murashige and Skoog's medium supplemented with 2,4-dichlorophenoxy acetic acid (2~4-D) and kinetin. Reduction of 2,4-D concentration during subsequent subcultures resulted in formation of embryoids. These embryoids developed further when transferreff to a medium containing benzylaminopurine and kinetin and then to a hormone-free medium. Unless 5-methyl tryptophan was added and the level of sucrose raised, the embryoids began to recallus and failed to form plantlets. INTRODUCTION Reports of somatic embryogenesis from explants of mature tree species are sparse in comparison to herbaceous plants. Embryo like structures have been grown to form plants, but with the exception of citrus, coffee and sandalwood, there has been hardly any work to make this a reliable avenue Ior vegetative propagation (Mott, 1984). Soapnut (Sapindus trifoliatus L.) is an economically important tree of the tropical region. In the present study we report the in vitro regeneration of plantlets through somatic embryogenesis from callus cultures derived from the leaves of a mature tree. MATERIALS AND METHODS Young leaves were collected lrom a Soapnut tree growing in the University Botanical Garden. Aiter surface sterilization with 0.1% HgCl2 (w/v) for 3 minutes and 4-5 rinses in sterile distilled water, 5 mm discs were excised and transferred to the medium. Only young leaves gave satisfactory callus. We have used MS (Murashige and Skoog, 1962) medium supplemented with 2% sucrose, 100 mg/l inositol and gelled with 0.85% agar as the basal medium. Callus was initiated and maintained on MS-medium supplemented with 2 mg/l 2,4-D and 0.5 mg/l kinetin. Embryoids were induced on a medium containing 0.5 m g / l , 2,4-D and 0.5 mg/1 kinetin. For Further growth of embryoids, they
Offprint requests to: A. R. Mehta
were transferred te a medium containing 2 mg/l benzylaminopurine and 0.5 mg/l kinetin and finally to a hormone free medium. Further growth of embryoids was achieved .in liquid MS medium supplemented with 0.1 mg/l 5-methyltryptophan followed by agar based basal medium with 4% sucrose. The culture media were adjusted to an initial PH of 5.8 prior to autoclaving. The culture vessels (with leaf discs, callus or embryoids) were incubated in a culture room at 25"C with 16 h photoperiod. Squash preparations were made from the embryogenic callus and stained with Coomassie blue. RESULTS AND DISCUSSION Callus induction and maintenance: Leaf discs lormed white friable Callus within 10 days of inoculation of MS medium containing 2 mg/l 2,4-D and 0.5 mg/l kinetin. The callus was subcultured on this medium after every 4 weeks. It remained essentially undifferentiated on this medium (Fig.l). .!nduction and growth ol somatic embryos: In order to induce somatic embryogenesis from soapnut callus, it was necessary to reduce the concentration of 2,t~-D gradually, while maintaining the original level of kinetin upon each subculture. Direct transfer of callus to a medium devoid of 2,4-D failed to induce somatic embryogenesis. Thus the level of 2,t~-D was reduced to 1 mg/l from 2 rag/1. No embryoids were visible even after 4 weeks of incubation. The level of 2,4-D was further reduced to 0.5 mg/l in the next subculture. Kinetin level was maintained at 0.5 mg/l during this period. The friable callus turned nodular and green on this medium within 10 days and numerous groups of embryogenic cells and globular and heart-shaped embryos with small suspensors became visible after t~ weeks (Figs. 2-4). The somatic embryos were loosely attached to the original callus. Growth of heart shaped embryos to mature embryos was not achieved on this medium as the residual 2,4-D was sufficient to induce callus from the heart-shaped embryos. Therefore, the entire callus bearing somatic embryos was first transferred to a medium containing 2 rag/1 benzylaminopurine 0.5 mg/l kinetin and totally devoid of 2,tc-D. After 2 weeks to a hormone-free
191 an auxin and a cytokinin, followed by transfer of callus to a medium with a reduced level of growth hormones or with a different combination of growth hormones (G]eddie et al 1983; Kamat and Rao 1978; Matsuoka and Hinata, 1979; Rao et a] 1973; Thomas and Street 1970).
Fig. 1
Undifferentiated Callus of Sapindus trifoliatus L. on MS mediumsupplemented with 2 m~-[ 2,#-D and 0.5 mg/l kinetin.
Fig.
Squash preparation of embryogenic
In the present case, somatic embryos never germinated on a medium containing cytokinins or on an auxin-free medium even after prolonged subculture; instead they began to recallus. We reasoned that by inhibiting the growth of this callus, it may be possible to trigger the germination of embryos. Several growth inhibitors were then tested in the concentration range of 0.i mg/] to 1.0 mg/l. T h u s antiauxins like B-naphthelene acetic acid, Phenylpropionic acid,triiodobenzoic acid and 2(O-chlorophenoxy) 2-methylpropionic acid as well as abscisic acid prevented the recaIlusing of embryoids. However, they could not induce germination of the embryoids. A tryptophan analogue, 5-methyl-tryphgophan was found effective not only in preventing recallusing but it also induced elongation of the embryonic radicle in about 40% of somatic embryos (Fig.6). The efficiency 5-methyltryptophan was further enhanced on transfer to the liquid medium, as almost all the embryoids germinated. Chandra et al (1978) have shown that in the presence of an antiauxin, 3,5-dichlorophenoxy acetic acid in media containing 2,g-D, carrot suspension cultures maintained active growth through three successive passages and continued to give a high' yield of globular embryoids. The small plantlets obtained on a medium containing 5-methyl-tryptophan, were transferred to agar based basal MS medium containing 4% sucrose. A complete plantlet that could be transferred to vermiculite was obtained in about 2 weeks (Fig.7). Lower sucrose concentration (below 4%) at this stage did not support continued growth. Further, mannitol in conjunction with 3% sucrose to give osmotic strength equivalent to 4% sucrose also failed to support growth. A total of 350 to g00 plantlets were obtained from a single 5 mm diameter leaf-disc in a period of about 19 weeks. ACKNOWLEDGEMENTS
Fig.
3
Callus stained with coomassie blue (x 125).
Fig.
g
Globular embryo bearing suspensor (x 125).
One of us (HVD) is thankful to the University Grants Commission, New Delhi, for the award of Research Associateship. We also thank Dr.D.K. Srivastava for his help in squash preparations.
2
Fig.
5
Somatic embryos on basal medium
REFERENCES
Fig.
6
Somatic embryos with radicle on MS medium containing o.1 mg/l, 5-methyl-tryptophan.
Fig.
7
Ammirato PV, (1983) In: Handbook of Plant Cell Culture; Vol. 1 (Evans DA, Sharp WR, Ammirato, PV, Yamada Y. (ed.) MacMillan Publ., London pp 82-123
Complete plarit from somatic embryo on basal medium with 4% sucrose.
medium growth of somatic embryos was continued as seen by massive enlargement of cotyledons (Fig.5). In some of the somatic embryos, the cotyledons were even larger than those of the zygotic embryos. Somatic embryogenesis has been reported from the callus derived from v e g e t a t i v e parts of several plants; a comprehensive list is provided by Ammirato (1983). In a number of plants, the basic protocol followed to obtain embryoids involves induction of callus growth in an auxin enriched medium and somatic embryogenesis upon transfer of callus to a medium free of the auxin. In some cases, as in the present one, requirements for somatic embryogenesis are far more complex, involving callus induction in a medium supplemented with
Chandra N, Lam TH, Street HE (1978) Z. Pflanzen Physiol, 61:55-60 Gleddie S, KellerW, Setterfield G (1983) Can. 3.Bot. 61:656-666. Kamat MG, Rao PS (1978) Plant. Sci. Lett. 13:57-85 Matsuokka H, Hinata K (1979) 3. Expt. Bot. 30:363-370 Mott RL (1984) In: Cloning Agricultural Plants via in vitro techniques. (Conger BY ed), CRC Press Inc.,217-256. Murashige T, Skoog F (1962) Physiol. Rao PS, Handdro W, Haradda Plant. 28:458/~63. Thomas
E, Street
34:657/669.
HE (1970)
Plant 15:g73-497
H (1973) Physiol. Ann. Bot. (London)
Plant Cell Reports (1986) 3:190-191
© Springer-Verlag 1986
Plant regeneration of Sapindus trifoliatus L. (soapnut) through somatic embryogenesis Harsha V. Desai, Prashant N. Bhatt, and Atul R. Mehta Department of Botany, The M.S. University, Baroda-390002, India Received November 26, 1985 / Revised version received March 31, t986 - Communicated by I. K. Vasil
ABSTRACT
Somatic embryogenesis and subsequent formation of plantlets was obtained from callus cultures derived from ]eaves of mature (over 60years old) Soapnut (Sapindus trifoliatus L,) tree. Callus was induced from leaf explants and grown on Murashige and Skoog's medium supplemented with 2,4-dichlorophenoxy acetic acid (2~4-D) and kinetin. Reduction of 2,4-D concentration during subsequent subcultures resulted in formation of embryoids. These embryoids developed further when transferreff to a medium containing benzylaminopurine and kinetin and then to a hormone-free medium. Unless 5-methyl tryptophan was added and the level of sucrose raised, the embryoids began to recallus and failed to form plantlets. INTRODUCTION Reports of somatic embryogenesis from explants of mature tree species are sparse in comparison to herbaceous plants. Embryo like structures have been grown to form plants, but with the exception of citrus, coffee and sandalwood, there has been hardly any work to make this a reliable avenue Ior vegetative propagation (Mott, 1984). Soapnut (Sapindus trifoliatus L.) is an economically important tree of the tropical region. In the present study we report the in vitro regeneration of plantlets through somatic embryogenesis from callus cultures derived from the leaves of a mature tree. MATERIALS AND METHODS Young leaves were collected lrom a Soapnut tree growing in the University Botanical Garden. Aiter surface sterilization with 0.1% HgCl2 (w/v) for 3 minutes and 4-5 rinses in sterile distilled water, 5 mm discs were excised and transferred to the medium. Only young leaves gave satisfactory callus. We have used MS (Murashige and Skoog, 1962) medium supplemented with 2% sucrose, 100 mg/l inositol and gelled with 0.85% agar as the basal medium. Callus was initiated and maintained on MS-medium supplemented with 2 mg/l 2,4-D and 0.5 mg/l kinetin. Embryoids were induced on a medium containing 0.5 m g / l , 2,4-D and 0.5 mg/1 kinetin. For Further growth of embryoids, they
Offprint requests to: A. R. Mehta
were transferred te a medium containing 2 mg/l benzylaminopurine and 0.5 mg/l kinetin and finally to a hormone free medium. Further growth of embryoids was achieved .in liquid MS medium supplemented with 0.1 mg/l 5-methyltryptophan followed by agar based basal medium with 4% sucrose. The culture media were adjusted to an initial PH of 5.8 prior to autoclaving. The culture vessels (with leaf discs, callus or embryoids) were incubated in a culture room at 25"C with 16 h photoperiod. Squash preparations were made from the embryogenic callus and stained with Coomassie blue. RESULTS AND DISCUSSION Callus induction and maintenance: Leaf discs lormed white friable Callus within 10 days of inoculation of MS medium containing 2 mg/l 2,4-D and 0.5 mg/l kinetin. The callus was subcultured on this medium after every 4 weeks. It remained essentially undifferentiated on this medium (Fig.l). .!nduction and growth ol somatic embryos: In order to induce somatic embryogenesis from soapnut callus, it was necessary to reduce the concentration of 2,t~-D gradually, while maintaining the original level of kinetin upon each subculture. Direct transfer of callus to a medium devoid of 2,4-D failed to induce somatic embryogenesis. Thus the level of 2,t~-D was reduced to 1 mg/l from 2 rag/1. No embryoids were visible even after 4 weeks of incubation. The level of 2,4-D was further reduced to 0.5 mg/l in the next subculture. Kinetin level was maintained at 0.5 mg/l during this period. The friable callus turned nodular and green on this medium within 10 days and numerous groups of embryogenic cells and globular and heart-shaped embryos with small suspensors became visible after t~ weeks (Figs. 2-4). The somatic embryos were loosely attached to the original callus. Growth of heart shaped embryos to mature embryos was not achieved on this medium as the residual 2,4-D was sufficient to induce callus from the heart-shaped embryos. Therefore, the entire callus bearing somatic embryos was first transferred to a medium containing 2 rag/1 benzylaminopurine 0.5 mg/l kinetin and totally devoid of 2,tc-D. After 2 weeks to a hormone-free
191 an auxin and a cytokinin, followed by transfer of callus to a medium with a reduced level of growth hormones or with a different combination of growth hormones (G]eddie et al 1983; Kamat and Rao 1978; Matsuoka and Hinata, 1979; Rao et a] 1973; Thomas and Street 1970).
Fig. 1
Undifferentiated Callus of Sapindus trifoliatus L. on MS mediumsupplemented with 2 m~-[ 2,#-D and 0.5 mg/l kinetin.
Fig.
Squash preparation of embryogenic
In the present case, somatic embryos never germinated on a medium containing cytokinins or on an auxin-free medium even after prolonged subculture; instead they began to recallus. We reasoned that by inhibiting the growth of this callus, it may be possible to trigger the germination of embryos. Several growth inhibitors were then tested in the concentration range of 0.i mg/] to 1.0 mg/l. T h u s antiauxins like B-naphthelene acetic acid, Phenylpropionic acid,triiodobenzoic acid and 2(O-chlorophenoxy) 2-methylpropionic acid as well as abscisic acid prevented the recaIlusing of embryoids. However, they could not induce germination of the embryoids. A tryptophan analogue, 5-methyl-tryphgophan was found effective not only in preventing recallusing but it also induced elongation of the embryonic radicle in about 40% of somatic embryos (Fig.6). The efficiency 5-methyltryptophan was further enhanced on transfer to the liquid medium, as almost all the embryoids germinated. Chandra et al (1978) have shown that in the presence of an antiauxin, 3,5-dichlorophenoxy acetic acid in media containing 2,g-D, carrot suspension cultures maintained active growth through three successive passages and continued to give a high' yield of globular embryoids. The small plantlets obtained on a medium containing 5-methyl-tryptophan, were transferred to agar based basal MS medium containing 4% sucrose. A complete plantlet that could be transferred to vermiculite was obtained in about 2 weeks (Fig.7). Lower sucrose concentration (below 4%) at this stage did not support continued growth. Further, mannitol in conjunction with 3% sucrose to give osmotic strength equivalent to 4% sucrose also failed to support growth. A total of 350 to g00 plantlets were obtained from a single 5 mm diameter leaf-disc in a period of about 19 weeks. ACKNOWLEDGEMENTS
Fig.
3
Callus stained with coomassie blue (x 125).
Fig.
g
Globular embryo bearing suspensor (x 125).
One of us (HVD) is thankful to the University Grants Commission, New Delhi, for the award of Research Associateship. We also thank Dr.D.K. Srivastava for his help in squash preparations.
2
Fig.
5
Somatic embryos on basal medium
REFERENCES
Fig.
6
Somatic embryos with radicle on MS medium containing o.1 mg/l, 5-methyl-tryptophan.
Fig.
7
Ammirato PV, (1983) In: Handbook of Plant Cell Culture; Vol. 1 (Evans DA, Sharp WR, Ammirato, PV, Yamada Y. (ed.) MacMillan Publ., London pp 82-123
Complete plarit from somatic embryo on basal medium with 4% sucrose.
medium growth of somatic embryos was continued as seen by massive enlargement of cotyledons (Fig.5). In some of the somatic embryos, the cotyledons were even larger than those of the zygotic embryos. Somatic embryogenesis has been reported from the callus derived from v e g e t a t i v e parts of several plants; a comprehensive list is provided by Ammirato (1983). In a number of plants, the basic protocol followed to obtain embryoids involves induction of callus growth in an auxin enriched medium and somatic embryogenesis upon transfer of callus to a medium free of the auxin. In some cases, as in the present one, requirements for somatic embryogenesis are far more complex, involving callus induction in a medium supplemented with
Chandra N, Lam TH, Street HE (1978) Z. Pflanzen Physiol, 61:55-60 Gleddie S, KellerW, Setterfield G (1983) Can. 3.Bot. 61:656-666. Kamat MG, Rao PS (1978) Plant. Sci. Lett. 13:57-85 Matsuokka H, Hinata K (1979) 3. Expt. Bot. 30:363-370 Mott RL (1984) In: Cloning Agricultural Plants via in vitro techniques. (Conger BY ed), CRC Press Inc.,217-256. Murashige T, Skoog F (1962) Physiol. Rao PS, Handdro W, Haradda Plant. 28:458/~63. Thomas
E, Street
34:657/669.
HE (1970)
Plant 15:g73-497
H (1973) Physiol. Ann. Bot. (London)
