RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes.

Our containment trials have established cold tolerance in Nicotiana tabacum osmotin (Nt Osm) transgenic tomato (Solanum lycopersicum L. cv. Pusa Ruby). Though, the stress tolerance mechanisms have been studied at physio-biochemical levels, molecular mechanisms underlying the tolerant response are still not well studied. Therefore, quantitative transcript expression of Osmotin and other stress responsive genes (CBF1, P5CS and APX) was studied in response to cold (4°C; 2 and 24 h) treatment in the transgenic and wild type tomato plants. The expression analysis revealed differential transcript regulation in the transgenic and wild type plants on the cold exposure. In general, the genes were either earlier induced or the extent of fold change in transcript expression over the respective untreated controls was higher in transgenic than in the wild type plants on cold exposure. The transcript expression data also supported the metabolite analysis on free Proline and ascorbate content. The results thus suggest that constitutive over expression of the Osmotin modulate transcript abundance and functional expression products of the other stress responsive genes thereby, imparting cold tolerance in the transgenic tomato plants.

Cold tolerance in thiourea primed capsicum seedlings is associated with transcript regulation of stress responsive genes.

Benefits of seed priming in seedling establishment and tolerance to subsequent stress exposure are well reported. However, the molecular mechanisms underlying the priming mediated benefits are not much discovered. Results of our earlier experiments established that thiourea (TU) seed priming imparts cold tolerance to capsicum seedlings. Therefore, to understand molecular mechanisms underlying priming mediated cold stress tolerance, quantitative transcript expression of stress responsive genes involved in transcript regulation (CaCBF1A, CaCBF1B, Zinc Finger protein, CaWRKY30), osmotic adjustment (PROX1, P5CS, Osmotin), antioxidant defence (CAT2, APX, GST, GR1, Cu/Zn SOD, Mn SOD, Fe SOD), signaling (Annexin), movement of solutes and water (CaPIP1), and metabolite biosynthesis through phenylpropanoid pathway (CAH) was studied in response to cold (4 °C; 4 and 24 h) stress in seedlings grown from the TU primed, hydroprimed and unsoaked seeds. The transcript expression of CaWRKY30, PROX1, Osmotin, Cu/Zn SOD and CAH genes was either higher or induced earlier on cold exposure in thiourea priming than that of hydroprimed and unsoaked over the respective unstressed controls. The results thus suggest that the TU priming modulate expression of these genes thereby imparting cold tolerance in capsicum seedlings.