The abscisic acid receptor OsPYL6 confers drought tolerance to indica rice through dehydration avoidance and tolerance mechanisms.

Abscisic acid (ABA) is a key regulator of plant development and stress tolerance. Here we report functional validation of the ABA receptor OsPYL6 by constitutive and stress-inducible overexpression and RNAi silencing, in an indica rice cultivar 'Pusa Sugandh 2'. Overexpression of OsPYL6 conferred ABA hypersensitivity during germination and promoted total root length. Overexpression and RNAi silencing of OsPYL6 resulted in enhanced accumulation of ABA in seedlings under non-stress conditions, at least, in part through up-regulation of different 9-cis epoxycarotenoid dioxygenase (NCED )genes. This suggests that PYL6 expression is crucial for ABA homeostasis. Analysis of drought tolerance of OsPYL6 transgenic and wild type plants showed that OsPYL6 overexpression enhanced the expression of stress-responsive genes and dehydration tolerance. Transgenic rice plants overexpressing OsPYL6 with AtRD29A (Arabidopsis thaliana Responsive to Dehydration 29A) promoter also exhibited about 25% less whole plant transpiration, compared with wild type plants under drought, confirming its role in activation of dehydration avoidance mechanisms. However, overexpression of PYL6 reduced grain yield under non-stress conditions due to reduction in height, biomass, panicle branching and spikelet fertility. RNAi silencing of OsPYL6 also reduced grain yield under drought. These results showed that rice OsPYL6 is a key regulator of plant development and drought tolerance, and fine-tuning of its expression is critical for improving yield and stress tolerance.

Overexpression of Arabidopsis ICE1 enhances yield and multiple abiotic stress tolerance in indica rice.

ICE1 (Inducer of CBF Expression 1), a MYC-type bHLH transcription factor, is a regulator of cold tolerance in Arabidopsis. Indica rice, which occupies the major rice cultivated area, is highly sensitive to cold stress. Hence in this study, Arabidopsis ICE1 (AtICE1) was overexpressed in indica rice to analyze its role in reproductive stage cold and other abiotic stress tolerance to indica rice. AtICE1 was overexpressed by using stress inducible AtRD29A promoter in mega rice cv. MTU1010. Under cold stress conditions, AtICE1 overexpression lines showed lower accumulation of MDA and H2O2, higher membrane stability, and thus higher seedling survival rate than the WT plants. Expression levels of OsDREB1A, OsMYB3R2, and OsTPP1 were significantly higher in transgenics as compared with WT under cold stress conditions. AtICE1 transgenic rice plants produced 44-60% higher grain yield as compared with WT plants under control conditions in three independent experiments. Of the three AtICE1 overexpression lines, two lines produced significantly higher grain yield as compared with WT plants after recovery from cold, salt and drought stresses. AtICE1 overexpression lines showed significantly higher stomatal density and conductance under non-stress conditions. qRT-PCR analysis showed that expression levels of stomatal pathway genes viz., OsSPCH1, OsSPCH2, OsSCR1, OsSCRM1, OsSCRM2 and OsMUTE were significantly higher in AtICE1 transgenics as compared with WT plants. The components of water use viz., stomatal conductance, photosynthesis, and instantaneous WUE were higher in transgenics as compared with WT plants. The results showed that AtICE1 confers multiple stress tolerance to indica rice, and the role of ICE1 in stress tolerance and stomatal development is conserved across species.

Overexpression of ABA Receptor PYL10 Gene Confers Drought and Cold Tolerance to Indica Rice.

Abscisic acid (ABA) plays versatile functions in regulating plant development and tolerance to various biotic and abiotic stresses. Towards elucidating the functions of one of the ABA receptors (ABARs) in rice, OsPYL10 was cloned from drought tolerant rice cv. Nagina 22 and was overexpressed under stress inducible RD29A promoter in a mega rice variety MTU1010 by using Agrobacterium mediated genetic transformation. Four single copy transgenic lines selected based on Southern blot analysis were used for physiological and molecular analysis. PYL10 receptor appears to regulate its ligand ABA accumulation as PYL10 overexpressing transgenics accumulated 2-3.3-fold higher levels of ABA than that of WT in flag leaf at anthesis under non-stress conditions. The enhanced accumulation of ABA was associated with enhanced expression of genes for ABA biosynthesis viz., ZEP1, NCED1, NCED2, NCED3, and NCED4 in transgenics than in WT plants. At seedling stage, PYL10 transgenics showed significantly higher survival rate under cold stress as compared with WT plants. qRT-PCR analysis showed that expression levels of cold responsive genes viz., DREB1F, MYB3R2, TPP1, COR410, DEHYDRIN, and LEA3 were significantly higher in PYL10 overexpressing transgenic lines as compared to WT plants under cold stress. PYL10 transgenic and WT plants grown in the same pot were subjected to -80 kPa drought stress and recovery treatments at vegetative and reproductive stages. At vegetative stage drought stress, three overexpressing lines showed significantly higher grain yield (40-58%) and at reproductive stage drought stress one of these overexpression lines showed two-fold higher grain yield than that of WT plants. Excised leaf water loss analysis showed that PYL10 transgenic lost about 20% less water than WT plants. At reproductive stage, OsPYL10 transgenic maintained higher RWC, membrane stability index, chlorophyll content, and accumulated lower amount of MDA and H2O2 as compared with WT plants. qRT-PCR analysis showed that expression levels of RAB16, Dehydrin, LEA3, and ABA45 were higher in PYL10 transgenics as compared with WT plants under drought stress. Thus, overall results showed that OsPYL10 overexpression has potential to improve both drought and cold stress tolerance of indica rice.

In vitro regeneration from protocorms in Dendrobium aqueum Lindley - An imperiled orchid.

An efficient in vitro plant regeneration protocol from protocorms of Dendrobium aqueum was developed. The uniformly developed protocorms (in vitro origin) having shoot initials were cultured on half macro strength MS medium (1/2 MS) supplemented with cytokinins (BA, 2iP, KIN and TDZ) at 1, 3, 5, 7, 10 mg l-1, natural additives (BP and CW) at 1%, 3%, 5%, 7%, 10% and auxins (IBA, NAA, 2,4-D) at 1, 3, 5, 7, 10 mg l-1 to study their efficacy on complete plant development. A maximum of 9.4 shoots per explant were generated on 3 mg l-1 of NAA followed by 3% of BP (7.0 shoots). Shoot elongation (1.52 cm) was achieved on 1/2 MS medium fortified with NAA 7 mg l-1 followed by TDZ 7 mg l-1 (1.37 cm). Shoots cultured on 1/2 MS medium supplemented with IBA 5 mg l-1 produced an average of 8.75 roots per shoot, however the lengthiest roots (1.48 cm) were noted in NAA 7 mg l-1. Healthy rooted plantlets successfully acclimatized in ex vitro condition. The role of complete plantlet production by natural additives could be useful for conservation and cost effective commercial production of orchids.

Organogenesis from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis -a vulnerable species of Western Ghats

An efficient system was developed for indirect plant regeneration from in vitro-derived leaf and internode explants of Hoya wightii ssp. palniensis. Maximum percentage of the organogenic callus was obtained on MS medium supplemented with NAA (1.0 mg/l) and 2,4-D (2.0 mg/l). The best shoot bud induction was observed on MS medium with BA (1.0 mg/l) +IBA (0.5 mg/l). The coconut water (15%) was better, resulting in a differentiation of the shoot initials in to well-developed shoots. The elongated shoots (› 3cm long) were rooted on a full strength MS basal medium, supplemented with 0.2 mg/l of IBA. Finally, the rooted plants were transferred to the soil with 80% success rate. This protocol was utilized for the in vitro propagation of this endangered plant species.

Effect of picloram, additives and plant growth regulators on somatic embryogenesis of Phyla nodiflora (L.) Greene

The present study describes the plant regeneration via somatic embryogenesis in suspension culture derived from the leaf and stem explants of Phyla nodiflora. The medium type, plant growth regulators, complex extract (coconut milk and malt extract) and anti-oxidant (activated charcoal, ascorbic acid, Polyvinylpyrrolidone and citric acid) markedly influenced the embryo regeneration of P. nodiflora. MS with 2,4-D and activated charcoal (10 mg/L) gave the highest stimulation of embryogenic callus growth. Optimized callus was transfered into suspension culture, which showed the globular, heart shaped embryos in MS with 2,4-D + BA + picloram (0.1 mg/L), coconut milk (10 ml/L), citric acid (100 mg/L) on 6th subcultures. Further development stages such as torpedo and cotyledonary stage embryos and fostered maturation of embryos were observed at 8th and 10th subculture. However, the high frequency embryo germination and plantlet (45 plants/20 mg cotyledonary stages embryos) formation was obtained in half-strength MS medium without growth regulators from cotyledonary embryos. All the plantlets established in the field exhibited morphological characters similar to those of the mother plant.

In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R. Br. ex roemer and schultes through callus culture under abiotic stress conditions.

Plant secondary metabolites have enormous potential for research and new drug development. Many secondary metabolites have a complex and unique structure and their production is often enhanced by biotic and abiotic stress conditions. Gymnemic acid (C(43)H(68)O(14)), a pentacyclic triterpenoid isolated from the leaves of Gymnema sylvestre, exhibits potent inhibitory effect on diabetes. The gymnemic acid content is determined by chromatographic methods: Camag HPTLC system equipped with a sample applicator Linomat IV and TLC scanner and integration software CAT 4.0. In HPLC C(18) (ODS) reverse phase column; water 486 UV detector; mobile phase, water/methanol (35:65, HPLC grade) + 0.1% acetic acid are used. Sample (20 microL) is applied with a flow rate of 1 mL/min and read at 230 nm with UV detector. The production of gymnemic acid is significantly higher in callus treated with 2,4-dichloro phenoxy acetic acid (2,4-D) and kinetin (KN). The blue light increases gymnemic acid accumulation upto 4.4-fold as compared with fluorescent light treatment and out of which 2.8 is found in leaves. Gymnemic acid is isolated from callus, grown under stress conditions followed by preparative TLC, simple and reproducible character based on HPTLC and high performance liquid chromatography.