Somatic embryogenesis and shoot regeneration from leaf derived callus of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

Abstract

The development of in vitro regeneration systems for Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh, a medicinal plant used for treating neurological disorders, is described. For the first time in this variety, somatic embryogenesis and shoot regeneration were induced from leaf-derived callus. Well-formed plantlets were obtained through both shoot regeneration and somatic embryogenesis, with separate morphogenetic programmes. Proembryogenic masses were obtained in liquid MS and B5 media supplemented with 5.8 μM 2,4-D, 1.34 μM NAA, and 1.16 μM Kin; after being transferred onto hormone-free medium, they formed whitish and spherical structures that subsequently developed into the heart and torpedo stages.

On MS agarized medium containing thidiazuron (TDZ) at different concentrations (3, 6, 9, 12 μM) combined with 2 μM IBA, only shoot regeneration, and not somatic embryogenesis, was obtained. The mean number of shoots increased significantly when the concentration of TDZ was 3 μM.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (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 supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

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.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Efficient plant regeneration through callus in Zanthoxylum armatum DC: an endangered medicinal plant of the Indian Himalayan region

Abstract

An efficient in vitro propagation protocol for Zanthoxylum armatum DC has been developed via indirect organogenesis using aseptic leaf explants. The explants were soaked for different time duration (12, 24 or 36?h) in liquid woody plant medium (WPM) supplemented with various concentrations (15.0, 25.0 or 50.0?μM) of thidiazuron (TDZ). The pre-exposed explants transferred for callus induction onto WPM supplemented with different concentrations of TDZ (2.0, 4.0, 6.0, 8.0 and 10.0?μM) either alone or in combination with varied concentrations (0.5, 1 and 1.5?μM) of naphthaleneacetic acid (NAA). Of the tested concentrations and combinations, best response for pretreated (15?μM TDZ for 24?h) explants was achieved on WPM augmented with 6.0?μM TDZ and 0.5?μM NAA after 8?weeks of incubation. For shoot induction, the callus clumps were excised into small pieces (～0.5?g) and were transferred onto WPM fortified with different concentrations (2.0–9.0?μM) of benzylaminopurine (BA), indole-3-acetic acid (IAA, 1.0?μM) and gibberellic acid (GA_3, 0.5–3.0?μM). Maximum shoot number (10.4?±?0.74) and average shoot length (4.75?±?0.71?cm) were observed in WPM enriched with 2.0?μM BAP, 1.0?μM IAA and 1.5?μM GA₃ after 8?weeks of incubation. The developed shoots (4?cm) were excised, pulse-treated for 24?h in half-strength WPM containing indole-3-butyric acid (IBA, 50.0?μM) prior to their transfer on hormone-free MS medium, where 100% rooting was achieved. The regenerated plants were implanted in soil-filled poly bags, acclimatized properly and subsequently placed under sunlight with 80% survival rate after 60?days recorded. This is the first report for propagation of Z. armatum via callus phase with high rate of shoot proliferation and can be effectively utilized for generating sufficient planting material in promoting its re-cultivation and conservation programme.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together 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 embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated 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 2.25 μM BAP and 8.5 μM IAA. 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 four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

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.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Pretreatment of seeds with thidiazuron delimits its negative effects on explants and promotes regeneration in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Thidiazuron (TDZ) is an important cytokinin-like substance used in plant tissue culture. However, long exposure of plant tissues to TDZ results in abnormal regeneration. The present study reports an efficient regeneration protocol that avoids the TDZ overexposure to chickpea (Cicer arietinum L.) tissues. Two separate experiments were designed for seed pretreatment with TDZ, of which the first one involved short-term pretreatment with various concentrations of TDZ (15, 20 and 25 µM), whereas the second one lacked TDZ pretreatment. Axillary meristem explants prepared from TDZ-pretreated and TDZ-non-pretreated seeds were then analyzed in shoot induction medium (SIM) with or without 4 µM TDZ. Thus, four conditions were chosen to analyze the effect of TDZ: (i) TDZ pretreatment only without TDZ in SIM, (ii) TDZ pretreatment along with TDZ in SIM, (iii) no TDZ pretreatment but there was TDZ in SIM, and (iv) there was neither TDZ pretreatment nor TDZ supplied in SIM (control). The response in terms of percent regeneration (69%), number of shoots per explant (20.66?±?0.5), minimum number of days taken for multiple shoot induction (7.3?±?0.5) and effective rooting was the highest under the condition (i) with 20 μM TDZ pretreatment without subsequent TDZ supplementation into the SIM. In addition, shoot elongation medium (SEM-3) having 5 μM benzyladenine, 2 μM kinetin and 2 μM gibberellic acid showed the highest branching and maximum shoot length. In conclusion, we reported an easy and efficient regeneration method in chickpea using TDZ pretreatment only, which showed qualitative multiple shooting and effective rooting.     

Rapid in vitro propagation of Matteuccia struthiopteris (L.) Todaro – an edible fern

A rapid and efficient in vitro plant regeneration method was developed for Matteuccia struthiopteris (L.) Todaro (Ostrich fern). Side shoots, originating in meristems of sectioned rhizomes, were used as explant material. A very high rate of meristem multiplication was achieved by culturing the explants in half-strength MS liquid medium supplemented with 2.0 mg/l N-(4-Pyridyl)-N′-phenylurea (4-PU) and 0.5 mg/l thidiazuron (TDZ). Multiplication of the shoot primordia was faster in suspension culture than on solid medium. Rhizogenesis and growth of regenerants were best achieved on hormone-free one-quarter-strength MS solid medium amended with 0.4% agar and 1.0% activated charcoal. Regenerated plantlets continued to grow after transfer to soil in a phytotron. Received: 19 March 1998 / Revision received: 17 July 1998 / Accepted: 3 August 1998     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

An optimized method for in vitro propagation of African baobab (Adansonia digitata L.) using two-node segments

Adansonia digitata L. (African baobab), is an important multi-purpose tree, whose distribution is at present limited to wild or semi-domesticated individuals widespread in Africa. Its distribution is threatened by seedling clearance for other land use and potentially by overharvesting induced by growing commercial use of baobab fruit. Recently, efforts have been made to establish baobab domestication and conservation strategies, with mixed results due to the low germinability of baobab seeds, a factor that hinders the possibility of developing commercial A. digitata plantations. Here, micropropagation was tested as a method for clonal propagation of explants from in vivo-grown seedlings. In vitro shoot multiplication was achieved by enhanced axillary bud proliferation of sterilized two-node segments. Bud break was dependent on cytokinin supply, but the combination of 1.0 or 10.0 μM zeatin riboside and 10.0 μM indole-3-butyric acid (IBA) increased the formation of microshoots after 8 weeks of culture. Regenerated microshoots rooted successfully in in vitro nutrient medium containing 10.0 μM IBA and normally grew in a greenhouse after acclimatization.     

Factors influencing direct shoot regeneration from mature leaves of Jatropha curcas, an important biofuel plant

In order to establish a highly efficient and sustainable regeneration system, we systematically researched the key factors affecting direct shoot regeneration from Jatropha curcas leaves that were collected from Hainan (HN1-1), Lijiang (LJ3-1), and Yuxi (YX2-12) provinces in China. The L₉(3⁴) orthogonal test of thidiazuron (TDZ), kinetin (Kn), and gibberellic acid (GA₃) were studied, and the explant type, growth age, and cultivar of leaves were subsequently investigated. Simultaneously, the combinations of plant growth regulators (PGRs) promoting shoot bud proliferation, elongation, and root establishment were examined. The results showed that the best medium for shoot bud induction was Murashige and Skoog (MS) medium supplemented with 1.0 mg/L TDZ, 0.5 mg/L Kn, and 0.5 mg/L GA₃. TDZ was the key PGR, while Kn and GA₃ played an important role in shoot bud elongation and the number of shoots per leaf disk, respectively. The induced shoot buds proliferated and readily elongated in MS medium with 0.3 mg/L 6-benzylaminopurine and 0.01 mg/L indole-3-butyric acid (IBA) and established roots in half-strength MS medium supplemented with 2.0 mg/L IBA. Using the previously described methods, the third to fifth leaves were found to be the best explant source for shoot bud induction, with a high induction rate, large shoot numbers per disk, excellent proliferation, and consistent rooting. With the use of this regeneration system, the shoot bud induction rate increased from the reported rate of 53.5% to more than 90% using different explants and cultivars, and the shoot number per leaf disk (shoot length?≥?0.5 cm) increased from 1.6 to 3.5. Thus, this optimized regeneration system will effectively promote the propagation and genetic transformation of J. curcas.     

Zeatin and TDZ-induced Shoot Proliferation and Use of Bioreactor in Clonal Propagation of Medicinal Herb, Roseroot (Rhodiola rosea L)

A procedure for in vitro propagation of roseroots (Rhodiola rosea L), a medicinal plant, was developed using a RITA bioreactor system containing liquid medium, combined with a gelled medium. Wild roseroot clones: ‘RCi’, ‘RC2’ and ‘RC3’ were established on a basal medium (BM) from in vitro-germinated seedlings on half-strength Murashige and Skoog (MS) salts. TDZ at 2–4 μM supported shoot proliferation but inhibited shoot elongation of ‘RCi’ shoots on gelled medium. Clones differed significantly with respect to multiplication rate with ‘RCi’ producing the most shoots per explant on gelled BM with 2 μM zeatin. In a bioreactor system, TDZ supported rapid shoot proliferation at lower concentration (0.5 μM) but induced hyperhydricity at more than 0.5 μM. Bioreactor-multiplied hyperhydric shoots of all clones when transferred to gelled medium containing 1–2 μM zeatin produced normal shoots within 4 wk of culture. Shoots were rooted in vitro on BM void of growth regulators. Almost all (9U to 95%) in vitro plantlets survived when transferred to potting medium.     

Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.