<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

An improved plant regeneration system and ex vitro acclimatization of <Emphasis Type="Italic">Ocimum </Emphasis><Emphasis Type="Italic">basilicum</Emphasis> L.

An efficient, rapid and large scale propagation of a multipurpose herb, Ocimum basilicum through in vitro culture of nodal segments with axillary buds from mature plants has been accomplished. Among the cytokinins, 6-benzyladenine (BA), thidiazuron (TDZ), kinetin (Kin) and 2-isopentenyl adenine (2-iP) tested as supplements to Murashige and Skoog (MS) medium, 5.0 μM BA was optimum in inducing bud break. The highest rate of shoot multiplication was achieved on half-strength MS medium supplemented with 2.5 μM BA and 0.5 μM indole-3-acetic acid (IAA) combination. The shoots regenerated from TDZ supplemented medium when subcultured to hormone-free MS medium considerably increased the rate of shoot multiplication and shoot length by the end of third subculture. For rooting, MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) proved to be better than that supplemented with IAA or α-naphthalene acetic acid (NAA). The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. Chlorophyll a and b, carotenoids and net photosynthetic rate were measured in leaves during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Firstly these parameters showed a decreasing trend but subsequently increased after 7 days of acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions is more extended in time than generally accepted.     

Shoot regeneration from cotyledonary leaf explants of <Emphasis Type="Italic">Jatropha curcas</Emphasis>: a biodiesel plant

A simple, high frequency, and reproducible method for plant regeneration through direct organogenesis from cotyledonary leaf explants of Jatropha curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) or 6-benzyl aminopurine (BAP). Medium containing TDZ has greater influence on regeneration as compared to BAP. The induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP, and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA, and indole-3-butyric acid (IBA). MS medium with 2.25 μM BAP and 8.5 μM IAA was found to be the best combination for shoot elongation. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA, and NAA for 4 days, followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg l⁻¹ activated charcoal. Elongated shoot treated with 15 μM IBA, 5.7 μM IAA, and 11 μM NAA resulted in highest percent rooting. The rooted plants could be established in soil with more than 90% survival rate. The method developed may be useful in improvement of J. curcas through genetic modification.     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Metabriggsia ovalifolia</Emphasis>

An efficient propagation and regeneration system via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia, was established. High activity cytokinins [6-benzyladeneine (BA) and thidiazuron (TDZ)] and low activity auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA)] could directly induce adventitious shoots from leaf or petiole explants within 5 weeks. Cytokinins (TDZ or BA) combined with auxin (NAA) in the induction media induced more adventitious shoots than when auxins or cytokinins were used alone. Adventitious shoots could be induced and also mass-propagated on media containing 2.5–5.0 μM TDZ (or BA) and 0.25–0.5 μM NAA. Adventitious roots differentiated at the proximal end of shoots on rooting media containing half-strength MS salts and 0.5 μM IBA, 0.5 μM NAA, 0.1% activated charcoal or no plant growth regulators. Over 90% of plantlets survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite) in basins.     

An efficient micropropagation system for Tylophora indica: an endangered, medicinally important plant

An efficient protocol is described for the rapid in vitro multiplication of an endangered medicinal plant, Tylophora indica (Burm. f.) Merrill, via enhanced axillary bud proliferation from nodal explants collected from young shoots of a two-year-old plant. The physiological effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)], ascorbic acid (AA), different strengths of Murashige and Skoog (MS) medium and various pH levels on in vitro morphogenesis were investigated. The highest number (8.6 ± 0.71) of shoots and the maximum average shoot length (5.2 ± 0.31 cm) were recorded on MS medium supplemented with 2.5 μM BA, 0.5 μM NAA and 100 mg/l AA at pH 5.8. Rooting was best achieved on half-strength MS medium augmented with 0.5 μM IBA. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with a 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long-term in vitro conservation of T. indica.     

Direct adventitious shoot induction and plant regeneration of <Emphasis Type="Italic">Embelia ribes</Emphasis> Burm F.

An efficient micropropagation system via direct shoot organogenesis from hypocotyl segments of Embelia ribes Burm F. was developed. A high frequency (84%) of adventitious shoot induction was obtained on Murashige and Skoog (MS) medium supplemented with additives (283.85 μM ascorbic acid [AA], 118.96 μM citric acid [CA], 142.33 μM cysteine, and 684.22 μM glutamine) and 1.13 μM of thidiazuron (TDZ) after 4 weeks following culture. Further development of shoot primordia into well-grown shoots of 4–5 cm in length was achieved by sub-culturing explants along with shoot primordia on MS medium supplemented with 0.44 μM benzyl adenine (BA) and 0.49 μM indole butyric acid (IBA) for three sub-culture periods with an interval of 15 days between them. The highest shoot multiplication was obtained when explants were incubated on MS medium supplemented with 2.2 μM BA and 0.49 μM IBA in 4 weeks. All in vitro developed shoots, 3–4 cm in length, rooted when grown on half-strength MS basal medium along with 2.47 μM IBA within 4 weeks. Moreover, 100% of shoots developed roots when these were treated with 4.93 μM IBA for 20 min and then transferred to pots containing soilrite mix and grown in the greenhouse. In vitro and ex vitro rooted plants showed a survival of 85 and 95% respectively, during hardening in the greenhouse for a 6-week period.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of Indian Kino (<Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.) using Thidiazuron

An efficient protocol was developed for micropropagation of an economically important timber-yielding multipurpose tree, Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes (CNs) derived from 18-d-old axenic seedlings on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.1–10 μM). The highest shoot regeneration frequency (90%) and maximum number (15.2 ± 0.20) of shoots per explant was recorded on MS medium amended with 0.4 μM TDZ. Continuous presence of TDZ inhibited shoot elongation. In the primary medium, TDZ-initiated cultures were transferred to the secondary medium supplemented with another cytokinin, 6-benzyladenine (BA), for shoot growth and elongation. Maximum (90%) shoot elongation with an average shoot length of 5.4 ± 0.06 cm was observed at 5 μM BA. To further enhance the number of shoots per explant, mother tissue was repeatedly subcultured on fresh shoot induction medium after each harvest of newly formed shoots. Thus, by adopting this strategy, an average of 44 shoots per explant could be obtained. About 65% of in vitro regenerated shoots produced a maximum number (4.4 ± 0.2) of roots per shoot by a two-step culture procedure employing pulse treatment and subsequent transfer of treated shoots to a low concentration of 0.2 μM indole-3-butyric acid along with phloroglucinol (3.96 μM). The in vitro-raised plantlets were successfully acclimatized first under culture room conditions, then to greenhouse with 70% survival rate.     

Rapid clonal propagation of Nothapodytes foetida (Wight) sleumer—A threatened medicinal tree

Summary Nothapodytes foetida (Wight) is a small evergreen tree and the extract from this tree is used to make the antileukaemia and antitumoral compound camptothecin. Due to exploitation of this resource, efficient methods for rapid propagation of N. foetida are highly desirable. Multiple shoots were induced on hypocotyl segments of 20–25-d-old seedlings of N. foetida cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of cytokinins. The highest shoot multiplication was achieved on MS medium containing thidiazuron (TDZ) at the concentration of 2.2 μM. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to medium containing 2.2 μM benzylaminopurine which produced healthy shoots after three additional subcultures. The production of shoots was further promoted by repeated subculturing of original explants on fresh multiplication medium after each harvesting of the newly formed shoots. In vitro rooting was best induced (87%) in shoots excised from proliferated shoot cultures on one-fourth MS medium augmented with 5.7 μM indole-3-acetic acid and 2.4 μM indolebutryic acid (IBA). In vitro-developed shoots were also rooted ex vitro by dipping in 49 μM IBA for 10 min. In vitro- and ex vitro-rooted plants were successfully acclimatized and established in greenhouse conditions.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Regeneration of plants from <Emphasis Type="Italic">Fraxinus americana</Emphasis> hypocotyls and cotyledons

A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5, respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting (78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to the emerald ash borer.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of <Emphasis Type="BoldItalic">Embelia ribes</Emphasis> Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March–May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 μM and indole-3-butyric acid (IBA) at 0.49 μM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 μM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 μM) for the third subculture. Treating the explants with an antioxidant mixture of 568 μM ascorbic acid, 119 μM citric acid, and 307 μM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 μM) and IBA (0.49 μM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 μM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 μM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

Thidiazuron induced high frequency axillary shoot multiplication in Psoralea corylifolia

The effect of thidiazuron (TDZ) was studied on in vitro axillary shoot proliferation from nodal explant of Psoralea corylifolia - an endangered medicinal plant. Proliferation of shoots was achieved on Murashige and Skoog (MS) medium supplemented with 0.5, 1, 2, 3, 4 and 5 μM TDZ. The maximum number (13.6 ± 1.4) of shoots per explant were obtained from nodal segment cultured on 2 μM TDZ for 4 weeks and this increased to 29.7 ± 2.1 on hormone free MS medium after 8 weeks. The in vitro proliferated and elongated shoots were transferred individually on a root induction medium containing 0.5 μM indole-3-butyric acid (IBA) and within 4 weeks 4.5 ± 0.5 roots per shoot were produced. The regenerated plantlets were transferred to 1:1 soil and vermiculite mixture and acclimatized with 80 % survival rate. Fully acclimatized plants were grown in garden soil in greenhouse and their morphological and physiological parameters were comparable with seedlings.     

Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium

Leaf explants of Jatropha curcas cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ; 0.90 μM) in combination with indole-3-butyric acid (IBA; 0.98μM) produced adventitious shoot buds directly on the surface of the explants without formation of intervening callus while shoot bud formation was accompanied with callus formation on medium supplemented with 6-benzylaminopurine (BAP; 13.3 μM) and IBA (2.46 μM). TDZ treatment resulted in more than twice higher rate of shoot bud induction than BAP. Shoot buds were multiplied and elongated following repeated transfers to medium containing BAP (2.22 μM) and gibberellic acid (GA₃; 1.44 μM). The effect of copper sulphate on differentiation of shoot buds from leaf segments was also investigated. Both shoot induction and multiplication media were supplemented with different levels of CuSO₄ (0–5 μM). Significant improvement in shoot bud induction was observed when the concentration of CuSO₄ was increased to 10 times the normal MS level. Healthy elongated shoots were rooted on half strength MS medium supplemented with IBA (2.46 μM). Rooted plantlets were transferred to field and survived. Histological analysis revealed direct formation of shoot buds from leaf explants.     

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.     

Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.