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 (0.5–10μM) 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 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

The North-Eastern region of India is significant for biodiversity conservation because of its floristic richness and high levels of endemism. Deforestation levels are high in the region due to anthropogenic pressures. We accessed various literature sources to create a database for Meghalaya state containing information on plant species, habit, altitudinal distribution, endemism, and endangered status. Information on the existing protected area network (type, extent, and altitudinal representation) was added to the database. The database was used to assess the effectiveness of the existing protected area network in conserving the floristic biodiversity of the state. Of a total of 3331 plant species, 1236 (37.11%) are endemic of Meghalaya and 133 (4%) are confined to 'sacred forests'. However, 'sacred forests' are not legally protected areas. Only 32 220 ha (1.43% of the state's geographical area) is protected under the category of National Park or Sanctuary. Although 212 species (17.15% of the state's endemic species) occur only in Meghalaya at altitudes above 1500 m, none of the forests at these altitudes are protected as National Parks or Sanctuaries. We conclude that the existing protected area network does not effectively conserve the state's unique biodiversity and suggest measures by which its effectiveness might be increased.

Effectiveness of the Protected Area Network in Biodiversity Conservation: A Case-study of Meghalaya State

Genetic stability and uniformity of Foeniculum vulgare Mill. regenerated plants through organogenesis and somatic embryogenesis

SummaryA protocol has been developed for plant regeneration from encapsulated nodal segments of Tylophora indica. Nodal segments collected from a 2-year-old plant were encapsulated in calcium alginate beads. The best gel complex was achieved using 3% (w/v) sodium alginate and 100 mM CaCl2.2H2O. The maximum frequency (91%) of conversion of encapsulated beads into plantlets was achieved on Murashige and Skoog (MS) medium containing 2.5 µM 6-benzyladenine (BA) and 0.5 µM αnaphthalene acetic acid (NAA) after 6 weeks of culture. Encapsulated nodal segments stored at 4°C for 1 – 8 weeks also showed successful conversion, followed by development into complete plantlets when returned to regeneration medium. Conversion of encapsulated nodal segment into plantlets also occurred when the calcium alginate beads were sown directly into SoilriteTM moistened with ½2MS salts. Plants regenerated from encapsulated nodal segments could be hardened, acclimatised and established in soil, with a success rate of 90%. Encapsulat...

Regeneration of plants from alginate-encapsulated shoots of Tylophora indica (Burm. f.) Merrill, an endangered medicinal plant

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (TL-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l−1.

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l−1; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l−1; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l−1 (9.04 μM) 2.4-D and 0.01 mg l−1 (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l−1; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l−1; 2.46–14.76 μM) along with 0.01 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l−1 (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in tylophora indica (burm. f. merrill.)

Micropropagation of two species of rauvolfia (apocynaceae)

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.

Direct somatic embryogenesis and plantlet regeneration in oil palm

Biology and conservation of endangered plants : the need to study breedingsystems

Stevia rebaudiana (Bertoni), commonly called candy leaf or sweet leaf, endemic to South America, is an important medicinal plant. As a source of low calorie natural sweetener 'stevoside', it is used in obesity, diabetes, treatment of heartburn and tooth decay, and also serves as a food supplement. Large scale commercial propagation of S. rebaudiana demands a suitable protocol. Here, we propose an improved protocol for in vitro multiplication of S. rebaudiana from nodal explants. In this protocol, the effect of laboratory grade urea on multiple shoot induction from nodal explants was studied. The nodal explants were initially cultured on Murashige and Skoog (MS) basal media for 2 weeks which facilitated the axillary bud break. Further, culturing of these explants on MS medium fortified with 6 benzyl amninopurine (BAP) (2 mg/L) and Naphthalene acetic acid (NAA) (1 mg/L) with and .without urea (5 mg/L) for a period of 40 days revealed maximum shoot production of 44.56 from a single nodal explant in media supplemented with urea as compared to 22.44 without urea. The differences in the number of shoots produced were significant and these shoots readily rooted in MS media with NAA (4 mg/L). Primary and secondary hardening was successful in these plants. There were no visible morphological abnormalities observed in the micropropagated plantlets. Genetic analysis from random samples also revealed that these plants are genetically uniform. The advantage of the present protocol is that the complete process of multiple shoot induction, rooting and hardening could be completed within a period of 6 months as compared to the existing protocols.

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M. Jayanthi

Papers

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in tylophora indica (burm. f. merrill.)

Micropropagation of two species of rauvolfia (apocynaceae)

Direct somatic embryogenesis and plantlet regeneration in oil palm

Biology and conservation of endangered plants : the need to study breedingsystems

A rapid and efficient protocol for in vitro multiplication of genetically uniform Stevia rebaudiana (Bertoni).