Half-strength Murashige And Skoog Liquid Medium Research Articles

Micropropagation of Piper crassinervium: an improved protocol for faster growth and augmented production of phenolic compounds

The medicinal plant Piper crassinervium is a source of bioactive compounds with potential use in agrochemical and pharmaceutical industries. However, its propagation is slow and influenced by the composition of the culture medium and growth conditions. In the genus Piper, tissue oxidation limits the biomass and recovery of bioactive compounds such as phenols. We evaluated the effect of medium formulations, sucrose, growth regulators, medium consistency, explant type, gas exchange and irradiance levels on growth and phenols and total flavonoids content in P. crassinervium in vitro plants. Murashige and Skoog (MS) and DKW/Juglans (DKW/J) media induced the highest production of chlorophyll and phenolic compounds; however, the regeneration rate of the explants in the DKW/J medium was the lowest (~75%) in comparison with the other media (90%). The maximum biomass production was achieved when half-strength MS (½MS) liquid medium was used; while the optimum sucrose level depended on the medium salt concentration used. Notably, the irradiance altered the accumulation of biomass and phenolic compounds. The presence of one remaining leaf in the nodal segments, used as explants, produced taller plants in ½MS liquid medium, combined or not with 2-iP. The optimal growth conditions for biomass and phenolic compound production in P. crassinervium in vitro plants established were: nodal explant with a preexisting leaf cultured on ½MS stationary liquid medium, 15 g L−1 sucrose, using lids with one PTFE 0.45-µm-pore size membrane and an irradiance of 100 µmol m−2 s−1. Medium formulation, sucrose concentration, irradiance levels, gas exchange and explants with preexisting leaf improved the in vitro propagation and phenolic compounds production in Piper crassinervium.

English

Javanese ginseng (Talinum paniculatum) is an herb and one of the traditional medicinal plants in Indonesia which accumulate saponin in the root organ. However, slow root growth hampers the accumulation of the compound in this plant. Therefore, in vitro culture of adventitious roots offers an alternative way to increase the production of saponin. Furthermore, this study was performed to investigate the effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the growth and saponin content in adventitious root of Javanese ginseng. Adventitious root culture was induced on solid Murashige and Skoog (MS) medium supplemented by 10 µM IBA (indole-3-butyric acid). Subsequently, adventitious roots were subcultured into a half strength MS liquid medium with the addition of different concentrations of MeJA or SA and incubated for 5, 10 and 15 days. Despite inhibition on the root growth, saponin production increased by 1.5 and 1.3 fold upon elicitation with 0.2 mM MeJA and SA for 15 days, respectively. Thus, MeJA and SA elicitations regulate saponin biosynthesis in adventitious root culture of Javanese ginseng as a time and dose-dependent manner. Key words: Adventitious root, elicitation, Javanese ginseng, saponin, Talinum paniculatum.

Shoot Tips Derived-Somatic Embryogenesis in Mass Propagation of Dendrobium Indonesia Raya and #8216;Ina and #8217;

A mass propagation protocol of Dendrobium Indonesia Raya ‘Ina’ via somatic embryogenesis pathway in producing high qualified-seedlings was successfully established. Three different explant sources harvested from plantlets viz. shoot tips, lateral shoots and young leaves; half strength Murashige and Skoog (MS) and Vacin and Went (VW) basalt media; thidiazuron (TDZ) of 1.0, 1.5, and 2.0 mg/L; N6-benzylaminopurine (BAP) of 0.5 and 1.0 mg/L; kinetin (KIN) of 0.5 and 1.0 mg/L were tested for embryogenic callus (EC) initiation. TDZ (0, 0.1, 0.3 and 0.5 mg/L) and BAP (0, 0.05 and 0.1 mg/L) combination and callus densities of 1, 2, 3 and 4 g/25 ml liquid media were studied for EC proliferation. Three, five, ten and fourteen days period and 5 ± 2 °C, 18 ± 2 °C, and 24 ± 2 °C of desiccation were investigated for conversion of EC to somatic embryo (SE) and germination of SE. The VW, haft-strength MS, Growmore® (32N:10P:10K), Rosasol®, and Hyponex® containing 150 ml/L coconut water (CW) were used for plantlet preparation. Results of the studies revealed that shoot tips derived from in vitro plantlets cultured on half-strength MS semi-solid medium supplemented with 1.5 mg/L TDZ and 0.5 mg/L BAP resulted in the shortest of EC initiation time of 9.3 days after culture and 100% explant regeneration with 0.41 g EC fresh weight, and 4.27 mm3 volume of EC. Rapid growth and proliferation of EC was highly possible by culturing the 3 g EC per 25 ml in the half strength MS liquid medium fortified by 0.50 mg/L TDZ, 0.10 mg/L BAP, and 150 ml/L CW with 11.91 g fresh weight of EC, 9.91 g EC added, and 3.97 multiplication rate. The optimal conversion of EC into SEs as high as 92.7% with 35.9 SEs per clump and 86.9% SE germination with 31.2 germinated-SEs per clump were easily determined by culturing 7 days EC desiccation at 18 ± 2 °C on the half strength MS medium containing 0.05 mg/L BAP with conversion time of 12.7 days for EC into SEs and 19.3 days for germination. The best performance of plantlet growth with 7.07 cm height of plantlets, 5.15 cm leaf length, 8.67 number of roots, 5.30 cm root length, 0.71 g fresh weight of plantlets, and 0.076 g dry weight of plantlets was significantly noticed on 2 g/L Hyponex® medium containing 150 ml/L CW. The plantlets were successfully transferred to ex vitro condition on Cycas rumphii bulk with 91,6% survival rate. The established protocol is high possibly applied for other Dendrobium hybrids.

English

An efficient protocol had been developed for the root culture of Luffa acutangula (L.) Roxb. The root and leaf explants from 21 day old in vitro raised seedlings were cultured on half strength Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of Indole-3-acetic acid(IAA), Indole-3-butyric acid (IBA) and Napthalene acetic acid (NAA). IBA at 1.0 mgl-1 combined with NAA (1.0 mgl-1) induced maximum percentage of rooting from leaf explants, under total dark condition. After 3 weeks, well established roots were separated. Fresh root tissue from the leaf explants and root tips from in vitro seedlings were subcultured in half strength MS liquid medium supplemented with different concentrations and combinations of IAA, IBA and NAA, under continuous agitation at 80 rpm under total dark condition. The biomass of root culture (derived from leaf explants) in the MS medium fortified with IBA(1.0 mgl-1), NAA(1.0mgl-1) and L-glutamine (20 mgl-1) was increased to 5.38 g FW and the biomass of root culture (derived from root tip) in the MS medium supplemented with IBA (0.5 mgl-1), NAA (1.0 mgl-1) and L- glutamine (20 mgl-1) was increased to 5.82 g FW after 7 weeks of culture.

Enhanced production of withaferin-A in shoot cultures of Withania somnifera (L) Dunal

Withania somnifera (L) Dunal, commonly known as ashwagandha or Indian ginseng, is the source of large number of pharmacologically active withanolides. Withaferin-A (WS-3), a major withanolide of W. somnifera, has been proven to be an effective anti-cancer molecule. In this study, a liquid culture system for shoot proliferation, biomass accumulation and withaferin-A production of an elite accession (AGB002) of W. somnifera was investigated. The nodal explants cultured on Murashige and Skoog (MS) semi-solid medium supplemented with various concentrations of 6-benzyl adenine (BA) and Kinetin (Kn) elicited varied responses. The highest number of regenerated shoots per ex-plant (35 ± 3.25) and the maximum average shoot length (5.0 ± 0.25 cm) were recorded on MS medium supplemented with BA (5.0 μM). The shoots were further proliferated in half and full strength MS liquid medium supplemented with the same concentration BA. It was interesting to note that shoots cultured on MS half strength liquid medium fortified with 4 gL-1 FW (fresh weight) shoot inoculum mass derived from 5 week old nodal explants of W. somnifera showed highest accumulation of biomass and withaferin A content in 5 weeks. Withaferin A was produced in relatively high amounts (1.30 % and 1.10 % DW) in shoots cultured in half and full strength MS liquid media respectively as compared to natural field grown plants (0.85 % DW). A considerable amount of the withaferin A was also excreted in the culture medium. Successful proliferation of shoots in liquid medium and the synthesis of withaferin A in vitro opens new avenues for bioreactor scale-up and the large-scale production of the compound.

An efficient regeneration system via somatic embryogenesis in mango ginger (Curcuma amada Roxb.)

A reproducible protocol for somatic embryogenesis was established for mango ginger (Curcuma amada Roxb.)—an important horticultural aromatic rhizomatous plant. Embryogenic callus induction was obtained from leaf sheath explants of in vitro raised plants on Murashige and Skoog (MS) agar medium containing 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L 6-benzyladenine (BA). Embryogenic callus proliferation, somatic embryo (SE) formation and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose and BA on SE formation were also evaluated. Half strength MS liquid medium necessary for SE formation and optimal sucrose concentration was found to be 3.0 %. BA at 0.3 mg/L produced the highest number (84.71 %) of SEs from leaf sheath explants. Secondary somatic embryos originated from primary somatic embryos on the same medium supplemented with 0.4–0.6 mg/L BA. Stereo microscopic and scanning electron microscopic observation revealed that the globular and torpedo shaped somatic embryos resulted in suspension culture during development. Mature somatic embryos germinated readily and developed into normal plantlets after 3 weeks on half strength MS basal agar medium under dark condition. Well rooted plantlets were successfully acclimatized at the survival rate of 70 %.

Transformation through agroinfection on decapitated shoot apex of field-growing Phyllanthus amarus

A simple and easy transformation strategy was accomplished on field growing plants of Phyllanthus amarus, an anti-hepatitis B drug plant. Infection of Agrobacterium rhizogenes strains A4M70GUS and ATCC 15834 on decapitated shoots of field growing P. amarus induced hairy roots and crown gall, respectively. Infection with A4M70GUS yielded a mean of 23.2 roots from 40% plants in 40-day period. The crown gall induced on 30% plants after infection with ATCC 15834 grew to 5–10 mm in diameter. The roots and crown galls established in vitro on Murashige and Skoog (MS) basal medium grew well. The hairy roots yielded fivefold (6.91 g) biomass in half-strength MS liquid medium to that of the adventitious roots derived from internode explants in MS medium with 8.0 μM α-naphthaleneacetic acid (1.39 g). Histochemical assay and PCR analysis using the primers of uidA coding region confirmed the hairy roots induced by A4M70GUS. The crown galls induced by ATCC 15834 were confirmed by PCR analysis using rolB gene primers. The protocol enables an easy and early accomplishment of hairy roots.

In vitro propagation of Bambusa nutans Wall. ex Munro through axillary shoot proliferation

This communication describes for the first time an efficient and reproducible protocol for large-scale multiplication of Bambusa nutans. Nodal segments collected from field-grown clumps and cultured on Murashige and Skoog (MS) medium supplemented with 4.4 μM benzylaminopurine (BA) and 2.32 μM kinetin (Kin) gelled with 0.2% gelrite yielded 80% aseptic cultures with 100% bud-break. The in vitro-formed shoots obtained after bud-break were successfully multiplied in MS liquid medium supplemented with 13.2 μM BA, 2.32 μM Kin, and 0.98 μM indole-3-butyric acid (IBA). Sub-culturing of shoots every 3 weeks on fresh multiplication medium yielded a consistent proliferation rate of 3.5-fold. Shoot clusters containing three to five shoots were successfully rooted with 100% success on half-strength MS liquid medium supplemented with 9.8 μM IBA, 2.85 μM indole-3-acetic acid (IAA), 2.68 μM naphthaleneacetic acid (NAA), and 3% sucrose. Plantlets grown in vitro were acclimatized and subsequently transferred to the field. Inter-simple sequence repeat analysis has confirmed the genetic uniformity of the tissue-cultured plants up to 27 passages.

Production of withanolide-A from adventitious root cultures of Withania somnifera

Withanolides are biologically active secondary metabolites present in roots and leaves of Withania somnifera. In the present study, we have induced adventitious roots from leaf explants of W. somnifera for the production of withanolide-A, which is having pharmacological activities. Adventitious roots were induced directly from leaf segments of W. somnifera on half strength Murashige and Skoog (MS) semisolid medium (0.8% agar) with 0.5 mg l−1 indole-3-butyric acid (IBA) and 30 g l−1 sucrose. Adventitious roots cultured in flasks using half strength MS liquid medium with 0.5 mg l−1 IBA and 30 g l−1 showed higher accumulation of biomass (108.48 g l−1FW and 10.76 g l−1 DW) and withanolide-A content (8.8 ± 0.20 mg g−1 DW) within five weeks. Nearly 11-fold increment of fresh biomass was evident in suspension cultures and adventitious root biomass produced in suspension cultures possessed 21-fold higher withanolide-A content when compared with the leaves of natural plants. An inoculum size of 10 g l−1 FW favoured the biomass accumulation and withanolide-A production in the tested range of 2.5, 5.0, 10.0 and 20.0 g l−1 FW. Among different media tested [Murashige and Skoog (MS), Gamborg’s (B5), Nitsch and Nitsch (NN) and Chu’s (N6)], MS medium favoured both biomass accumulation and withanolide-A production. Half strength MS medium favoured the biomass accumulation and withanolide-A production among the different strength MS medium tested (0.25, 0.5, 0.75, 1.0, 1.5 and 2.0). The current results showed great potentiality of adventitious roots cultures for the production of withanolide-A.

Shoot regeneration from cotyledonary leaf explants of Jatropha curcas: 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 mu M kinetin (Kn), 4.5 mu M BAP, and 5.5 mu M a-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 mu M BAP and 8.5 mu M 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 mu M IBA, 5.7 mu M IAA, and 11 mu M 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.

Use of protocorm-like bodies for studying alkaloid metabolism in Pinellia ternata

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.

In vitro plant regeneration from embryogenic cell suspension culture of Astragalus chrysochlorus (Leguminoseae)

In vitro plant regeneration was achieved from embryogenic cell suspension culture of Astragalus chrysochlorus. When 30-day-old aseptically grown seedlings were cultured on Murashige and Skoog (MS) medium containing 0.1 mg/l -naphthaleneacetic acid (NAA) plus 1.0 mg/l 6-benzyladenine (BA), friable callus was formed within two weeks from the mesocotyl of the seedling. After three weeks, proliferated actively growing calli were transferred to MS liquid medium containing 2,4- dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) or NAA and subcultured at two week intervals. After two weeks, induction of somatic embryos up to the torpedo stage occured at all tested concentrations of 2,4-D, IAA or NAA. Somatic embryos developed only in MS medium containing 0.5 mg/l IAA within two weeks and 2% of globular embryos were developed into the cotyledonary stage embryos. Eighty one percent of somatic embryos cultured in MS medium supplemented with 0.5 mg/l IAA were found to be diploid by flow cytometric analysis. Plantlet propagation was achieved on half strength MS liquid medium supplemented with 3% (w/v) sucrose after four weeks of culture. After a month on half strength MS medium [1.5% (w/v) sucrose and 0.8% (w/v) agar] 29 of 71 shoots developed into rooted plantlets.

In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants

Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.

EFFECTIVENESS OF AUXIN INDUCED IN VITRO ROOT CULTURE IN CHICORY

An efficient protocol has been developed for the root culture of (Cichorium intybus L. cv. Focus), the leaf and hypocotyl explants from 25 days old in vitro raised seedlings were cultured on half-strength Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of Indole-3-acetic acid (IAA), Indole-3-butyric acid (IBA), α-Napthalenacetic acid (NAA). 0.5 mg/l NAA and 0.1 mg/l IBA induced highest percentage of rooting from matured leaf explants, under total dark condition. After three weeks well established roots were separated. Fresh root tissue, in amount of 0.5 was subcultured in half-strength MS liquid medium supplemented with 0.2 mg/l NAA and 0.5 mg/l IBA, under continuous agitation at 110 rpm and total dark condition. The biomass of root culture was increased to 5.820 g after 6 weeks of culture. The root culture was maintained up to the 8 weeks.

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

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.

Plant regeneration protocol of medicinally important Andrographis paniculata (Burm. F.) Wallich ex Nees via somatic embryogenesis

In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN6-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l−1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l−1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l−1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l−1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l−1 IBA supplemented medium. The shoots formed with 1.0 mg l−1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.

Micropropagation of Pittosporum napaulensis (DC.) Rehder & Wilson - a rare, endemic Himalayan medicinal tree.

An in vitro propagation protocol has been developed from mature trees of Pittosporum napaulensis. The best bud proliferation (83.1%), shoot number (21 axillary shoots/ explant) and shoot length (5.5 cm) was achieved in Murashige and Skoog (MS) medium supplemented with 5.0 μM N−6 benzyladenine and 0.1 μM α- naphthalene acetic acid. Of the three cytokinins tested (N−6 benzyladenine, kinetin and thidiazuron), N−6 benzyladenine proved to be the best for shoot induction. Shoot regeneration potential varied among genotypes. Regenerated shoots rooted after 48 hours treatment on half-strength MS liquid medium supplemented with 20 μM indole-3-butyric acid. Rooted shoots transferred to 120 g (w/v) soilrite + sand + soil (1:1:1) mixture showed 70% survival. Twenty-one plantlets are growing well in green house conditions.

In vitro regeneration of a mature leguminous liana ( Bauhinia vahlii Wight & Arnott)

An in vitro propagation protocol has been developed from mature lianas of Bauhinia valii. Browning was the major obstacle in the establishment of cultures. Explants collected during the growing season (April–June) showed maximum browning; however, browning was minimal during the dormant phase. This problem was circumvented by soaking the sterilized explants in a solution of antioxidant (50 mg l–1 ascorbic acid+75 mg l–1 citric acid). The explants were thereafter transferred to culture room conditions after an initial incubation in the dark at 4 °C for 48 h. Shoot proliferation (58%), shoot number (4.5) and shoot length (35 mm) was best in Murashige and Skoog (MS) medium supplemented with 2.5 μM kinetin + 100 mg l–1 adenine sulfate. Seasonal fluctuations significantly affected the proliferation potential of the explants. March– April was found to be the best season for shoot initiation. Microshoots were rooted on a half-strength, growth regulator-free, agar-gelled Murashige and Skoog medium after a dip in half-strength MS liquid medium containing 1-naph-thaleneacetic acid + indole-3-butyric acid (10 μM). Rooted plantlets were potted and acclimatized under culture room conditions for 4 weeks before transfer to a polyhouse.

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.).

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1-10 μM and α-naphthaleneacetic acid (0-0.5 μM showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 μM benzyl adenine and 0.25 μM α-naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 μM indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.