In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro.

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans' AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22-25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.

Purification and characterization of a Ca(2+)-dependent protein kinase from sandalwood (Santalum album L.): evidence for Ca(2+)-induced conformational changes.

An early development-specific soluble 55 kDa Ca(2+)-dependent protein kinase has been purified to homogeneity from sandalwood somatic embryos and biochemically characterized. The purified enzyme, swCDPK, resolved into a single band on 10% polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK activity was strictly dependent on Ca(2+), K(0.5) (apparent binding constant) for Ca(2+)-activation of substrate phosphorylation activity being 0.7 microM and for autophosphorylation activity approximately 50 nM. Ca(2+)-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC(50) for W7=6 microM, for W5=46 microM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a K(m) value of 1.3 mg/ml for histone III-S and a V(max) value of 0.1 nmol min(-1) mg(-1). The enzyme exhibited high specificity for ATP with a K(m) value of 10 nM. Titration with calcium resulted in the enhancement of intrinsic emission fluorescence of swCDPK and a shift in the lambda(max) emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca(2+)-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca(2+) levels required for autophosphorylation and substrate phosphorylation help understand mode of Ca(2+) activation of this enzyme.

Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease.

Fertile transgenic plants of peanut (Arachis hypogaea L.) cv. TMV-2 expressing tobacco chitinase and neomycin phosphotransferase (npt II) genes were generated using an Agrobacterium tumefaciens (LBA4404/pBI121-pBTex)-mediated transformation system. A tissue culture-independent method wherein embryo in the mature seed is inoculated and reared into single plant transformant was used for transformation. Southern blot analysis of genomic DNA isolated from T(0) transformants and progeny plants (T(1)) demonstrated that the transgenes are stably integrated in the genome of transgenic peanut plants and inherited by the offspring. The expression of the heterologous chitinase gene driven by CaMV 35S promoter led to a high level of activity in some of the transgenic plants. Small-scale field tests indicated increased ability of these plants to resist the fungal pathogen Cercospora arachidicola (the causal organism of the leaf spot or Tikka disease of peanut), which is an important peanut pathogen. These results suggest that a heterologous chitinase gene was functional in peanut and expressed in healthy plants. The study also shows that peanut plants containing transgenically increased activity of chitinase were resistant to attack by the fungal pathogen C. arachidicola to different degrees. The strategy employed may be useful for the control of other fungal diseases of the crop.

Cryptosin - a new cardenolide in tissue culture and intact plants of Cryptolepis buchanani Roem. & Schult.

A steroidal glycoside of the digoxin type was isolated from fresh leaves and cultured cells of Cryptolepis buchanani Roem & Schult (Asclepiadaceae). Cultures were raised from leaf and shoot. Explants were cultured on MS medium containing 2,4-D, NAA, BA and coconut milk. Metabolically active calluses were proliferated and maintained on Gamborg's B5 medium with 2,4-D and Kn. The new compound, named cryptosin, was characterised by infrared nmr and mass spectroscopy. Cryptosin was also screened for its pharmacological properties and its cardiotonic action was established.

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.).

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.