Plant Cell Reports
Plant Cell Reports (1990) 9:370-373
9 Springer Verlag 1990
Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis * M. M. Subbaiah and S. C. Minocha ** Department of Plant Biology, University of New Hampshire, Durham, NH 03824, USA Received June 11, 1990/Revised version received August 24, 1990 - Communicated by A. R. Gould
ABSTRACT
INTRODUCTION
Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/1 benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/1 BA, 500 mg/1 polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4-6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.
Regeneration of shoots from callus and suspension cultures is a critical requirement for application of in vitro selection, somaclonal variation, and genetic engineering techniques for genetic improvements in plants. In woody plants, although micropropagation is possible through shoot tip and axillary bud culture, organogenesis and embryogenesis from callus are limited to only a few species (Bonga and Durzan 1987 a,b). Various species of Eucalyptus have been studied extensively for adventitious shoot formation from cultures of seedling explants (Bennet and McComb 1982; Diallo and Duhoux 1984; Kitahara and Caladas 1985; Lakshmi Sita 1979; Oka et al. 1982; Gupta and Mascarenhas 1987). Explants from mature trees of E. marginata and E. sideroxylon have been used to regenerate shoots and whole plants (Bennet and McComb 1982; Burger 1987). Shoot formation from leaf explants of E. camaldulensis and E. leichow has also been reported (Boulay 1 9 8 6 ; Muralidharan and Mascarenhas 1987; Quyang et al. 1981). Somatic embryogenesis from callus has been obtained in E. citriodora (Muralidharan and Mascarenhas 1987; Muralidharan et al. 1989) and E. leichow (Quyang et al. 1981). This communication describes a procedure for high efficiency shoot regeneration from leaf and stem callus of E. tereticornis.
ABBREVIATIONS BA: Benzyladenine, 2,4-D : 2,4dichlorophenoxyacetic acid, IBA: Indolebutyric acid, NAA: Naphthaleneacetic acid, PVP: Potyvinylpyrrolidone, mWP: modified Woody Plant medium.
MATERIALS AND METHODS
* Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station. ** Corresponding author.
Seeds of E. tereticornis SM were obtained from the Forestry Department, University of Agricultural Sciences, Bangalore, India, and stored at 4~ Prior to use, the seeds were surfacesterilized with 0.2% mercuric chloride for 2 min, washed with sterile distilled water and germinated on hormone-free B5 medium (Gamborg et al. 1968) in 125 ml Erlenmeyer flasks containing 50
371 ml medium. For multiple shoot production, 1 cm long hypocotyl segments from 4 to 6 week old seedlings were subcultured onto B5 medium with 0.5 mg/1 BA. The plantlets were excised and rooted in vitro on mWP (Muralidharan and Mascarenhas 1987) medium with 0.5 mg/1 IBA. Leaf and stem segments (0.5-1.0 cm) were excised from rooted plantlets and placed on B5 medium containing different concentrations of NAA and BA in the dark for callus induction. Callus was then transferred to mWP with 0.5 mg/1 BA, 500 mg/1 PVP (MW 360,000, Sigma Chemical Co., St. Louis, MO) and 5 to 10% (v/v) coconut milk for shoot induction. These shoots were rooted and transplanted to soil and kept under intermittent mist for 2 to 4 weeks before normal growth in the greenhouse. For the initiation of suspension cultures, approximately 500 mg of callus clumps were forced through a 1 mm sieve and transferred to 50 ml liquid medium with different growth regulators in 125 ml Erlenmeyer flasks. The flasks were placed on a gyratory shaker at 150 rpm. Except for callus induction, all cultures were maintained under 16 h light (50 umol.m-2.s -1) and 8 h dark cycles at 25_+2~ All media were adjusted to pH 5.5 and autoclaved for 25 to 30 min at 15 psi. The constituents of B5 medium used in these studies were purchased from Sigma Chemical Co. in a pre-mixed powder form. The media were solidified with 1% agar (Sigma Chemical Co.) RESULTS Multiple shoots were produced from the excised ends of hypocotyl segments of 4 to 6 week old seedlings. As many as 30 to 50 shoots were produced from each segment within 30-40 days on B5 medium containing 0.5 mg/l BA. These adventitious shoots could be rooted with 100% efficiency in the presence of 0.5 mg/1 IBA in mWP medium. A small callus clump was formed first on the cut end from which the multiple shoots were produced. When stem and leaf explants taken from rooted plantlets growing in vitro were cultured on different media in order to induce callus formation, in most cases the explants turned brown. Large quantities of dark brown exudates (presumably phenolics) were secreted into the medium. The browning of explants could be prevented by the addition of 500 mg/1 PVP to the medium. Healthy callus was formed from leaf and stem explants in the dark within 10 to 12 days (Fig. IA). Explants grown in light generally turned brown and callus formation was poor. Of the different concentrations tested, 0.1 rag/1 BA plus 3.0 or 5.0 m g / l NAA yielded the best callus in terms of growth rate and appearance (Table 1).
Table I. The effect of different concentrations of BA and NAA on the induction of callus from leaf explants of E. tereticornis on B5 medium. The cultures were grown in the dark for 14 days. The results presented are from one of the three replicate experiments with similar results.
BA NAA (mg/l) (mg/l)
0.0 0.0 0.i 0.i 0.1 0.i
0.0 0.05 0.5 2.0 3.0 5.0
Total explants
50 42 41 43 41 41
Explants Relative growth forming callus of callus
0 40 40 39 40 40
+ ++ +++ +++++ ++++
The callus was pinkish green in the beginning and turned brown if allowed to grow on the same medium for more than 3-4 weeks. When either the leaf or the stem callus was excised and transferred to mWP medium with different concentrations of BA alone, no shoots were produced in any case. However, when 10% (v/v) coconut milk was added to the medium along with 0.5 mg/1 BA, shoot formation occurred in about 30% of the explants (Table 2, Fig. IB). The callus always turned pink prior to the appearance of shoot primordia. No shoots were produced in BA concentrations higher than 3 rag/1 or lower than 0.5 mg/1. The shoot primordia were visible within 30 days. Some of the shoots were also pink at first, but gradually they all turned green. The results were similar with both leaf and stem callus. These shoots were excised and rooted with 100% efficiency on mWP medium with 0.5 mg/l IBA. Normal roots were produced within two weeks (Fig. IC). Rooted plantlets were successfully transplanted to soil in the greenhouse. Prior to growth under normal greenhouse conditions, they had to be placed under intermittent mist for 2 to 3 weeks. Table 2. The effect of different concentrations of BA on shoot regeneration from leaf callus of E. tereticornis. Excised callus pieces were transferred to mWP medium containing 10Z coconut milk and 500 mg/l PVP. All cultures were grown for 45 days before counting the number of shoots. The data are from one of the three replicate experiments with similar results.
BA Total (m~/l) explants
0.0 0.05 0.5 3.0 5.0 10.0
20 25 35 25 20 25
Explants forming shoots
0 0 10 5 0 0
Shoots per explant
0 0 15 +_ 2 7 +2 0 0
372
Fig. 1. A. Formation of callus from leaf explants. B. Regeneration of shoots from callus. C. Rooted plantlets produced from regenerated shoots.
When callus produced on mWP medium containing 0.1 m g / l BA and 3.0 m g / l NAA was transferred either to liquid mWP or B5 medium containing the same hormones as used for callus induction, no growth was observed. The callus usually turned brown. A range of concentrations (0.1 to 1.0 mg/1) of 2,4-D was tested for the maintenance of suspension cultures; 0.5 mg/1 2,4D combined with subculturing at 15 days intervals showed the best results. The callus grew in suspension as spherical clumps (nodules) with smooth surface. Growth was similar in light or in the dark. When suspension cultures were treated with various combinations of auxins and cytokinins which had been found to be effective in causing shoot regeneration from callus on the solid medium (Table 2), the nodules turned pink but did not differentiate. Hand sections of nodules revealed no organization of shoot or root apex (results not presented). DISCUSSION Regeneration of multiple shoots from stem explants of a number of Eucalyptus species has been reported (Gupta and Muscarenhas 1987; Muralidharan and Mascarenhas 1987). With the exception of a few cases of callus formation and multiple shoot regeneration directly from stem segments of mature trees, juvenile seedling materials have been often used. Gupta et al. (1983) reported multiple shoot production from nodal explants of mature E. torelliana and E. camaldulensis trees on media containing 0.2 mg/1
kinetin and 0.5 mg/1 BA, respectively. Activated charcoal was used in the medium for rooting of the shoots following auxin treatment. Nodal explants of mature trees of E. grandis produced multiple shoots on media containing 0.5 to 2.0 m g / l BA (Lakshmi Sita and Shoba Rani 1985). Oka et al. (1982) reported production of multiple shoots from 6 day old seedling hypocotyl segments of E. globulus on medium containing 0.02 rag/1 NAA and 0.2 mg/1 BA. Burger (1987) obtained auxiliary shoots from nodal explants of mature E. sideroxylon on medium containing 0.4 to 0.8 mg/1 BA and 0.2 to 0.4 rag/1 NAA. In the present study, large number of multiple shoots were formed from excised hypocotyl segments of 4 to 6 week old seedlings on 0.5 mg/1 BA alone. These shoots rooted readily in media containing 0.5 mg/1 IBA. Callus from hypocotyl segments and cotyledons of young seedlings of E. citriodora was produced in the presence of l mg/1 zeatin plus 0.2 m g / l indoleacetic acid and 10% coconut milk under diffused light (Lakshmi Sita 1975). Only cotyledonary callus differentiated into shoots which could be rooted with low concentrations of auxins. Explants from mature trees produced callus in the presence of relatively higher concentrations (5 mg/1) of NAA or indoleacetic acid (Lakshmi Sita 1975). In our studies also a relatively high concentration of NAA was needed in combination with BA for callus induction. As reported by Muralidharan and Mascarenhas (1987), we also observed better callus production in the dark.
373 Shoot regeneration from callus has been reported for only a few species of Eucalyptus (Muralidharan et al. 1989). Diallo and Duhoux (1984) reported shoot initiation from cotyledonary callus of E. camaldulensis with 0.5 mg/1 BA. Muralidharan and Mascarenhas (1987) obtained shoots from dark-grown leaf callus of E. camaldulensis on mWP media containing 10% coconut milk and 0.5 mg/1 BA. Our results on the regeneration of shoots from callus in E. tereticornis (Table 2) are similar to those of Muralidharan and Mascarenhas (1987) and Gupta et al. (1983) for E. camaldulensis and E. mar ginata, respectively. Rooting of the regenerated shoots in Eucalyptus does not seem to be a problem. In few cases, the need for a brief liquid culture exposure with low auxin levels before rooting has been reported (Muralidharan and Mascarenhas 1987). In other cases, solid medium with 0.1-0.5 m g / l auxin seems sufficient (Diallo and Duhoux 1984). A common problem with Eucalyptus tissue culture, i.e., the production of phenolics, has been partially or fully overcome either by using activated charcoal (Gupta et al. 1983, Lakshmi Sita 1979) or PVP (Lakshmi Sita and Shoba Rani, 1985). In our studies, a combination of PVP with culturing in the dark produced the best callus growth. Muralidharan and Mascarenhas (1987) obtained similar results by modifying sucrose concentration, supplementing the medium with casein hydrolysate and growing the cultures in the dark. Suspension cultures of Eucalyptus have been reported only recently (Teulieres et al., 1989). Once again, the difficulty of maintaining liquid cultures is probably due to the production of phenolics in the medium. In our studies, suspension cultures could be maintained by subculture at two week intervals in light in the presence of 0.5 m g / l 2,4-D. The cell clumps never differentiated into shoots even in the Rpresence of growth regulators which were quite effective in causing shoot formation from callus on the solid medium. ACKNOWLEDGEMENTS We thank Mr. C. G. Kushalappa of the University of Agricultural Sciences, Bangalore, India, for supplying the seeds and Mr. Eun Noh for useful suggestions and discussions throughout the project. Nancy Jackson's help in word processing is highly appreciated.
REFERENCES Aneja S, Atal CK (1969) Curr. Sci. 30:69-71. Bennet I J, McComb JA (1982) Aust. For. Res. 12:121-127. Bonga JM, Durzan DJ (eds) (1987a) Cell and Tissue Culture in Forestry. Vol. 1, Martinus Nijhoff Publ, Boston. pp. 447. Bonga JM, Durzan DJ (eds) (1987b) Cell and Tissue Culture in Forestry. Vol. 2, Martinus Nijhoff Publ, Boston. pp. 422. Bonga JM, Durzan DJ (eds) (1987c) Cell and Tissue Culture in Forestry. Vol. 3, Martinus Nijhoff Publ, Boston. pp. 416. Boulay M (1986) In: Proc. 6 Intl. Cong. Plant Tissue and Cell Culture. Minneapolis, Minnesota. pp. 367-382. Burger DW (1987) HortScience 22:496-497. Clapham DH, Eckberg I (1986) Scand. J. For. Res. 1:435-437. Diallo N, Duhoux E (1984) J. Plant. Physiol. 115:177-182. Gamborg OL, Miller A, Ojima K (1968) Exp. Cell. Res. 50:151-158. Gupta PK, Mehta UJ, Mascarenhas AF (1983) Plant Cell. Reports 2:296-299. Gupta PK, Masearenhas AF (1987) In: Bonga JM and Durzan DJ (eds) Cell and Tissue Culture in Forestry. Vol. 3, Martinus Nijhoff Publ., Boston, pp. 385-399. Kitahara EH, Caladas LS (1975) For. Sci. 21:242243. Lakshmi Sita G (1979) Plant Sci. Lett. 14:63-65. Lakshmi Sita G, Shoba Rani B (1985) Plant Cell Reports 4:63.-65. Muralidharan EM, Mascarenhas AF (1987) Plant Cell. Reports 6:256-259. Muralidharan EM, Gupta PK, Mascarenhas AF (1989) Plant Cell Reports 8:41-43. Oka S, Young EC, Thorpe TA (1982) Newzealand. J. For. Sci. 12:501-509. Quyang Q, Peng HZ, Li QQ (1981) Sci. Silvae Sin. 17:1-7 Teulieres C, Feuillet C, Boudet AM (1989) Plant Cell Reports 8:407-410. Teulieres C, Alibert G, Boudet AM, Marien JN (1989) J. Plant. Physiol. 134:316-319.
Plant Cell Reports (1990) 9:370-373
9 Springer Verlag 1990
Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis * M. M. Subbaiah and S. C. Minocha ** Department of Plant Biology, University of New Hampshire, Durham, NH 03824, USA Received June 11, 1990/Revised version received August 24, 1990 - Communicated by A. R. Gould
ABSTRACT
INTRODUCTION
Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/1 benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/1 BA, 500 mg/1 polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4-6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.
Regeneration of shoots from callus and suspension cultures is a critical requirement for application of in vitro selection, somaclonal variation, and genetic engineering techniques for genetic improvements in plants. In woody plants, although micropropagation is possible through shoot tip and axillary bud culture, organogenesis and embryogenesis from callus are limited to only a few species (Bonga and Durzan 1987 a,b). Various species of Eucalyptus have been studied extensively for adventitious shoot formation from cultures of seedling explants (Bennet and McComb 1982; Diallo and Duhoux 1984; Kitahara and Caladas 1985; Lakshmi Sita 1979; Oka et al. 1982; Gupta and Mascarenhas 1987). Explants from mature trees of E. marginata and E. sideroxylon have been used to regenerate shoots and whole plants (Bennet and McComb 1982; Burger 1987). Shoot formation from leaf explants of E. camaldulensis and E. leichow has also been reported (Boulay 1 9 8 6 ; Muralidharan and Mascarenhas 1987; Quyang et al. 1981). Somatic embryogenesis from callus has been obtained in E. citriodora (Muralidharan and Mascarenhas 1987; Muralidharan et al. 1989) and E. leichow (Quyang et al. 1981). This communication describes a procedure for high efficiency shoot regeneration from leaf and stem callus of E. tereticornis.
ABBREVIATIONS BA: Benzyladenine, 2,4-D : 2,4dichlorophenoxyacetic acid, IBA: Indolebutyric acid, NAA: Naphthaleneacetic acid, PVP: Potyvinylpyrrolidone, mWP: modified Woody Plant medium.
MATERIALS AND METHODS
* Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station. ** Corresponding author.
Seeds of E. tereticornis SM were obtained from the Forestry Department, University of Agricultural Sciences, Bangalore, India, and stored at 4~ Prior to use, the seeds were surfacesterilized with 0.2% mercuric chloride for 2 min, washed with sterile distilled water and germinated on hormone-free B5 medium (Gamborg et al. 1968) in 125 ml Erlenmeyer flasks containing 50
371 ml medium. For multiple shoot production, 1 cm long hypocotyl segments from 4 to 6 week old seedlings were subcultured onto B5 medium with 0.5 mg/1 BA. The plantlets were excised and rooted in vitro on mWP (Muralidharan and Mascarenhas 1987) medium with 0.5 mg/1 IBA. Leaf and stem segments (0.5-1.0 cm) were excised from rooted plantlets and placed on B5 medium containing different concentrations of NAA and BA in the dark for callus induction. Callus was then transferred to mWP with 0.5 mg/1 BA, 500 mg/1 PVP (MW 360,000, Sigma Chemical Co., St. Louis, MO) and 5 to 10% (v/v) coconut milk for shoot induction. These shoots were rooted and transplanted to soil and kept under intermittent mist for 2 to 4 weeks before normal growth in the greenhouse. For the initiation of suspension cultures, approximately 500 mg of callus clumps were forced through a 1 mm sieve and transferred to 50 ml liquid medium with different growth regulators in 125 ml Erlenmeyer flasks. The flasks were placed on a gyratory shaker at 150 rpm. Except for callus induction, all cultures were maintained under 16 h light (50 umol.m-2.s -1) and 8 h dark cycles at 25_+2~ All media were adjusted to pH 5.5 and autoclaved for 25 to 30 min at 15 psi. The constituents of B5 medium used in these studies were purchased from Sigma Chemical Co. in a pre-mixed powder form. The media were solidified with 1% agar (Sigma Chemical Co.) RESULTS Multiple shoots were produced from the excised ends of hypocotyl segments of 4 to 6 week old seedlings. As many as 30 to 50 shoots were produced from each segment within 30-40 days on B5 medium containing 0.5 mg/l BA. These adventitious shoots could be rooted with 100% efficiency in the presence of 0.5 mg/1 IBA in mWP medium. A small callus clump was formed first on the cut end from which the multiple shoots were produced. When stem and leaf explants taken from rooted plantlets growing in vitro were cultured on different media in order to induce callus formation, in most cases the explants turned brown. Large quantities of dark brown exudates (presumably phenolics) were secreted into the medium. The browning of explants could be prevented by the addition of 500 mg/1 PVP to the medium. Healthy callus was formed from leaf and stem explants in the dark within 10 to 12 days (Fig. IA). Explants grown in light generally turned brown and callus formation was poor. Of the different concentrations tested, 0.1 rag/1 BA plus 3.0 or 5.0 m g / l NAA yielded the best callus in terms of growth rate and appearance (Table 1).
Table I. The effect of different concentrations of BA and NAA on the induction of callus from leaf explants of E. tereticornis on B5 medium. The cultures were grown in the dark for 14 days. The results presented are from one of the three replicate experiments with similar results.
BA NAA (mg/l) (mg/l)
0.0 0.0 0.i 0.i 0.1 0.i
0.0 0.05 0.5 2.0 3.0 5.0
Total explants
50 42 41 43 41 41
Explants Relative growth forming callus of callus
0 40 40 39 40 40
+ ++ +++ +++++ ++++
The callus was pinkish green in the beginning and turned brown if allowed to grow on the same medium for more than 3-4 weeks. When either the leaf or the stem callus was excised and transferred to mWP medium with different concentrations of BA alone, no shoots were produced in any case. However, when 10% (v/v) coconut milk was added to the medium along with 0.5 mg/1 BA, shoot formation occurred in about 30% of the explants (Table 2, Fig. IB). The callus always turned pink prior to the appearance of shoot primordia. No shoots were produced in BA concentrations higher than 3 rag/1 or lower than 0.5 mg/1. The shoot primordia were visible within 30 days. Some of the shoots were also pink at first, but gradually they all turned green. The results were similar with both leaf and stem callus. These shoots were excised and rooted with 100% efficiency on mWP medium with 0.5 mg/l IBA. Normal roots were produced within two weeks (Fig. IC). Rooted plantlets were successfully transplanted to soil in the greenhouse. Prior to growth under normal greenhouse conditions, they had to be placed under intermittent mist for 2 to 3 weeks. Table 2. The effect of different concentrations of BA on shoot regeneration from leaf callus of E. tereticornis. Excised callus pieces were transferred to mWP medium containing 10Z coconut milk and 500 mg/l PVP. All cultures were grown for 45 days before counting the number of shoots. The data are from one of the three replicate experiments with similar results.
BA Total (m~/l) explants
0.0 0.05 0.5 3.0 5.0 10.0
20 25 35 25 20 25
Explants forming shoots
0 0 10 5 0 0
Shoots per explant
0 0 15 +_ 2 7 +2 0 0
372
Fig. 1. A. Formation of callus from leaf explants. B. Regeneration of shoots from callus. C. Rooted plantlets produced from regenerated shoots.
When callus produced on mWP medium containing 0.1 m g / l BA and 3.0 m g / l NAA was transferred either to liquid mWP or B5 medium containing the same hormones as used for callus induction, no growth was observed. The callus usually turned brown. A range of concentrations (0.1 to 1.0 mg/1) of 2,4-D was tested for the maintenance of suspension cultures; 0.5 mg/1 2,4D combined with subculturing at 15 days intervals showed the best results. The callus grew in suspension as spherical clumps (nodules) with smooth surface. Growth was similar in light or in the dark. When suspension cultures were treated with various combinations of auxins and cytokinins which had been found to be effective in causing shoot regeneration from callus on the solid medium (Table 2), the nodules turned pink but did not differentiate. Hand sections of nodules revealed no organization of shoot or root apex (results not presented). DISCUSSION Regeneration of multiple shoots from stem explants of a number of Eucalyptus species has been reported (Gupta and Muscarenhas 1987; Muralidharan and Mascarenhas 1987). With the exception of a few cases of callus formation and multiple shoot regeneration directly from stem segments of mature trees, juvenile seedling materials have been often used. Gupta et al. (1983) reported multiple shoot production from nodal explants of mature E. torelliana and E. camaldulensis trees on media containing 0.2 mg/1
kinetin and 0.5 mg/1 BA, respectively. Activated charcoal was used in the medium for rooting of the shoots following auxin treatment. Nodal explants of mature trees of E. grandis produced multiple shoots on media containing 0.5 to 2.0 m g / l BA (Lakshmi Sita and Shoba Rani 1985). Oka et al. (1982) reported production of multiple shoots from 6 day old seedling hypocotyl segments of E. globulus on medium containing 0.02 rag/1 NAA and 0.2 mg/1 BA. Burger (1987) obtained auxiliary shoots from nodal explants of mature E. sideroxylon on medium containing 0.4 to 0.8 mg/1 BA and 0.2 to 0.4 rag/1 NAA. In the present study, large number of multiple shoots were formed from excised hypocotyl segments of 4 to 6 week old seedlings on 0.5 mg/1 BA alone. These shoots rooted readily in media containing 0.5 mg/1 IBA. Callus from hypocotyl segments and cotyledons of young seedlings of E. citriodora was produced in the presence of l mg/1 zeatin plus 0.2 m g / l indoleacetic acid and 10% coconut milk under diffused light (Lakshmi Sita 1975). Only cotyledonary callus differentiated into shoots which could be rooted with low concentrations of auxins. Explants from mature trees produced callus in the presence of relatively higher concentrations (5 mg/1) of NAA or indoleacetic acid (Lakshmi Sita 1975). In our studies also a relatively high concentration of NAA was needed in combination with BA for callus induction. As reported by Muralidharan and Mascarenhas (1987), we also observed better callus production in the dark.
373 Shoot regeneration from callus has been reported for only a few species of Eucalyptus (Muralidharan et al. 1989). Diallo and Duhoux (1984) reported shoot initiation from cotyledonary callus of E. camaldulensis with 0.5 mg/1 BA. Muralidharan and Mascarenhas (1987) obtained shoots from dark-grown leaf callus of E. camaldulensis on mWP media containing 10% coconut milk and 0.5 mg/1 BA. Our results on the regeneration of shoots from callus in E. tereticornis (Table 2) are similar to those of Muralidharan and Mascarenhas (1987) and Gupta et al. (1983) for E. camaldulensis and E. mar ginata, respectively. Rooting of the regenerated shoots in Eucalyptus does not seem to be a problem. In few cases, the need for a brief liquid culture exposure with low auxin levels before rooting has been reported (Muralidharan and Mascarenhas 1987). In other cases, solid medium with 0.1-0.5 m g / l auxin seems sufficient (Diallo and Duhoux 1984). A common problem with Eucalyptus tissue culture, i.e., the production of phenolics, has been partially or fully overcome either by using activated charcoal (Gupta et al. 1983, Lakshmi Sita 1979) or PVP (Lakshmi Sita and Shoba Rani, 1985). In our studies, a combination of PVP with culturing in the dark produced the best callus growth. Muralidharan and Mascarenhas (1987) obtained similar results by modifying sucrose concentration, supplementing the medium with casein hydrolysate and growing the cultures in the dark. Suspension cultures of Eucalyptus have been reported only recently (Teulieres et al., 1989). Once again, the difficulty of maintaining liquid cultures is probably due to the production of phenolics in the medium. In our studies, suspension cultures could be maintained by subculture at two week intervals in light in the presence of 0.5 m g / l 2,4-D. The cell clumps never differentiated into shoots even in the Rpresence of growth regulators which were quite effective in causing shoot formation from callus on the solid medium. ACKNOWLEDGEMENTS We thank Mr. C. G. Kushalappa of the University of Agricultural Sciences, Bangalore, India, for supplying the seeds and Mr. Eun Noh for useful suggestions and discussions throughout the project. Nancy Jackson's help in word processing is highly appreciated.
REFERENCES Aneja S, Atal CK (1969) Curr. Sci. 30:69-71. Bennet I J, McComb JA (1982) Aust. For. Res. 12:121-127. Bonga JM, Durzan DJ (eds) (1987a) Cell and Tissue Culture in Forestry. Vol. 1, Martinus Nijhoff Publ, Boston. pp. 447. Bonga JM, Durzan DJ (eds) (1987b) Cell and Tissue Culture in Forestry. Vol. 2, Martinus Nijhoff Publ, Boston. pp. 422. Bonga JM, Durzan DJ (eds) (1987c) Cell and Tissue Culture in Forestry. Vol. 3, Martinus Nijhoff Publ, Boston. pp. 416. Boulay M (1986) In: Proc. 6 Intl. Cong. Plant Tissue and Cell Culture. Minneapolis, Minnesota. pp. 367-382. Burger DW (1987) HortScience 22:496-497. Clapham DH, Eckberg I (1986) Scand. J. For. Res. 1:435-437. Diallo N, Duhoux E (1984) J. Plant. Physiol. 115:177-182. Gamborg OL, Miller A, Ojima K (1968) Exp. Cell. Res. 50:151-158. Gupta PK, Mehta UJ, Mascarenhas AF (1983) Plant Cell. Reports 2:296-299. Gupta PK, Masearenhas AF (1987) In: Bonga JM and Durzan DJ (eds) Cell and Tissue Culture in Forestry. Vol. 3, Martinus Nijhoff Publ., Boston, pp. 385-399. Kitahara EH, Caladas LS (1975) For. Sci. 21:242243. Lakshmi Sita G (1979) Plant Sci. Lett. 14:63-65. Lakshmi Sita G, Shoba Rani B (1985) Plant Cell Reports 4:63.-65. Muralidharan EM, Mascarenhas AF (1987) Plant Cell. Reports 6:256-259. Muralidharan EM, Gupta PK, Mascarenhas AF (1989) Plant Cell Reports 8:41-43. Oka S, Young EC, Thorpe TA (1982) Newzealand. J. For. Sci. 12:501-509. Quyang Q, Peng HZ, Li QQ (1981) Sci. Silvae Sin. 17:1-7 Teulieres C, Feuillet C, Boudet AM (1989) Plant Cell Reports 8:407-410. Teulieres C, Alibert G, Boudet AM, Marien JN (1989) J. Plant. Physiol. 134:316-319.
