Pyramiding and evaluation of segregating lines containing lectin and protease inhibitor genes for aphid resistance in Brassica juncea-Supplementary Data

Aphids are one of the most devastating pests, affecting the potential yield and quality ofBrassica juncea. In the current study, we have attempted to pyramid two transgenic lines containing chickpea lectin (CHPL, P1) and urdbean protease inhibitor (UPI, P2) in each under the phloem specific rolC promoter, through conventional breeding approach. In the derived F2 population, both lectin and protease inhibitor genes were segregating in a 9:3:3:1 ratio (p-value: 0.81), indicative of a single copy of the transgenes in the parents. Furthermore, the parental, as well as pyramided progenies were evaluated for their potential resistance to aphids in terms of mortality and natality. The lines containing both the transgenes were found to be superior over single gene transgenics as a higher mortality rate (96%) was found in F2on the 9th day as compared to single gene transgenics (86% and 80% in P1 and P2 respectively). A significant decrease in the number of nymphs was observed in P1 and P2 but most in F2 plants as almost 43, 32.08, and 107.5 times decrease in the number of nymphs was found in P1, P2, and F2 individuals over control. Expression profiling was done to see if there was any impact of gene pyramiding on the expression pattern of both transgenes before and after aphid treatment, and no significant changes were observed, indicating constitutive expression of transgenes in pyramided lines also. In conclusion, pyramided lines were found to be promising and were superior for aphid resistance.

Pyramiding and evaluation of segregating lines containing lectin and protease inhibitor genes for aphid resistance in Brassica juncea

Aphids are one of the most devastating pests, affecting the potential yield and quality ofBrassica juncea. In the current study, we have attempted to pyramid two transgenic lines containing chickpea lectin (CHPL, P1) and urdbean protease inhibitor (UPI, P2) in each under the phloem specific rolC promoter, through conventional breeding approach. In the derived F2 population, both lectin and protease inhibitor genes were segregating in a 9:3:3:1 ratio (p-value: 0.81), indicative of a single copy of the transgenes in the parents. Furthermore, the parental, as well as pyramided progenies were evaluated for their potential resistance to aphids in terms of mortality and natality. The lines containing both the transgenes were found to be superior over single gene transgenics as a higher mortality rate (96%) was found in F2on the 9th day as compared to single gene transgenics (86% and 80% in P1 and P2 respectively). A significant decrease in the number of nymphs was observed in P1 and P2 but most in F2 plants as almost 43, 32.08, and 107.5 times decrease in the number of nymphs was found in P1, P2, and F2 individuals over control. Expression profiling was done to see if there was any impact of gene pyramiding on the expression pattern of both transgenes before and after aphid treatment, and no significant changes were observed, indicating constitutive expression of transgenes in pyramided lines also. In conclusion, pyramided lines were found to be promising and were superior for aphid resistance.

Purification of a protease inhibitor from Dolichos biflorus using immobilized metal affinity chromatography.

Plant protease inhibitors (PIs) are generally small proteins which play key roles in regulation of endogenous proteases and may exhibit antifeedant, antifungal, antitumor and cytokine inducing activities. Dolichos biflorus (horse gram) is an unexploited legume, which is rich in nutrients and also has therapeutic importance. It contains a double-headed PI, which is an anti-nutritional factor. As there is no report available on its simultaneous removal and purification in single step, in this study, a double-headed PI active against both trypsin and chymotrypsin was purified from Dolichos biflorus to ~14-fold with ~84% recovery using an immobilized metal affinity chromatography (IMAC) medium consisting of Zn-alginate beads. The method was single-step, fast, simple, reliable and economical. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 16 kDa and was stable over a pH range of 2.0-12.0 and up to a temperature of 100°C for 20 min. The optimum temperature for trypsin and chymotrypsin inhibitor was observed to be 50°C and 37°C, respectively and pH optimum was pH 7.0 and 8.0, respectively. Thus, IMAC using Zn-alginate beads was useful in simultaneous purification and removal of an anti-nutritional factor from horse gram flour in single step. This procedure may also be employed for purification of other plant PIs in one step.

Improved and convenient method of RNA isolation from polyphenols and polysaccharide rich plant tissues.

It has been difficult to extract a good quality total RNA from the plant parts (such as seeds) which contain high levels of phenolic compounds, carbohydrates and other compounds that bind and/or co-precipitate with RNA. A simple, rapid and efficient method for isolating total RNA from polyphenols and polysaccharide rich plant tissues has been developed. Seeds of leguminosae family were chosen for the study. The good quality and high yield of total RNA was achieved with A260/A280 ratio of 1.9. Seeds of three different crops (Cajanus cajan, Dolichos biflorus and Vigna mungo) at different developmental stages were evaluated for total RNA extraction using standardized protocol. Seeds at 21 days after flowering (DAF) gave the best results among others (7 DAF and dry seeds). Quality of isolated RNA from all the three crops was further checked by cDNA synthesis. The extracted RNA was found suitable for further molecular applications such as reverse transcription and cDNA library construction.