A. Chandrashekar

Bacterial synthesis of poly(hydroxybutyrate‐ co‐hydroxyvalerate) using carbohydrate‐rich mahua (Madhuca sp.) flowers

Aims:  The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp‐256.

Stable transformation and direct regeneration in Coffea canephora P ex. Fr. by Agrobacterium rhizogenes mediated transformation without hairy-root phenotype

A system for genetic transformation of Coffea canephora by co-cultivation with Agrobacterium rhizogenes harbouring a binary vector has been developed. The objective of the present study was the genetic transformation and direct regeneration of transformants through secondary embryos bypassing an intervening hairy root stage. Transformants were obtained with a transformation efficiency up to 3% depending on the medium adjuvant used. A. rhizogenes strain A4 harbouring plasmid pCAMBIA 1301 with an intron uidA reporter and hygromycin phosphotransferase (hptII) marker gene was used for sonication-assisted transformation of Coffea canephora. The use of hygromycin in the secondary embryo induction medium allowed the selection of transgenic secondary embryos having Ri T-DNA along with the T-DNA from the pCAMBIA 1301 binary vector. In addition transgenic secondary embryos devoid of Ri-T-DNA but with stable integration of the T-DNA from the binary vector were obtained. The putative transformants were positive for the expression of the uidA gene. PCR and Southern blot analysis confirmed the independent, transgenic nature of the analysed plants and indicated single and multiple locus integrations. The study clearly demonstrates that A. rhizogenes can be used for delivering transgenes into tree species like Coffea using binary vectors with Agrobacterium tumefaciens T-DNA borders.

Eggplant polyphenol oxidase multigene family: Cloning, phylogeny, expression analyses and immunolocalization in response to wounding

Though polyphenol oxidase (PPO) genes from tomato and potato have been extensively studied, information about PPO genes in eggplant (Solanum melongena) is lacking. The main objective of this study is to understand the structural and functional aspects of eggplant PPO genes. Six eggplant PPO genes (SmePPO1-6) cloned by RACE and genome walking were found to be intronless and correspond to eight eggplant unigenes. Comprehensive sequence analyses indicated that the eggplant PPO genes exhibit considerable variation in the transit peptide regions, copper-binding domains and UTRs, and fall into two distinct structural classes. Further, PPO gene members appear to exist in clusters on eggplant chromosome 8 as seen in the case of tomato and potato PPOs. During normal growth and development, SmePPO1 and 2 are expressed in roots, whereas the transcript levels of all the eggplant PPO genes vary considerably in leaves, flowers and fruits. SmePPO1 was expressed in Escherichia coli as a GST fusion protein, and immunoblot using rabbit polyclonal antiserum to GST-SmePPO1 detected a major protein band (~70 kDa) and a minor band (~67 kDa) in eggplant fruit extract. Tissue printing indicated the predominant presence of PPO in the exocarp and the areas surrounding the seeds in the mesocarp of eggplant fruits. Immunolocalization of PPOs in eggplant infested with shoot-and-fruit borer revealed localization of the PPO at the site of infection in tender shoots and fruits, and further inside the mature tissues. The upregulation of eggplant PPO gene transcripts following mechanical injury shows that all the genes except SmePPO2 are induced in the fruit over 6h. On the contrary, the transcripts of SmePPO2 and PPO3 are not detectable in the stem, and expression seems to be prominent over a 2h period for SmePPO1 and SmePPO4-6. Our results show that eggplant PPO genes are structurally different, and are differentially expressed in various tissues of eggplant indicating their functional diversity.

Characterization of bacteriocinogenic strains of lactic acid bacteria in fowl and fish intestines and mushroom

Abstract Lactic acid‐producing bacteria (LAB) isolated from fowl, fish intestines and mushroom were identified and characterized with reference to the production of bacteriocins and its genetic markers. Among the isolated cultures, two isolate each of Lactobacillus casei ssp casei C‐40 and M‐50, Pediococcus pentosaceus C‐6 and M‐10, and Pediococcus acidilactici C‐20 and F‐58 and one isolate of Pediococcus sp. F‐7 were potent producers of bacteriocin active against related strains of LAB. With reference to the genetic characterization of bacteriocin production, the potent cultures appeared to posses either one or two plasmids of 4–8 Kb size and a megaplasmid. Plasmid curing of bacteriocinogenic strain of Lact. casei ssp. casei C‐40 resulted in the loss of bacteriocin production and an altered pattern of sugar utilization. Antibiogram of bacteriocinogenic cultures of LAB and bacteriocin indicators was carried out to evaluate as a markers in genetic characterization. The bacteriocinogenic cultures were resis...

A Novel Modified Endosperm Texture in a Mutant High-Protein Digestibility/High-Lysine Grain Sorghum (Sorghum bicolor (L.) Moench)

ABSTRACT Development of high-protein digestibility (HPD)/high-lysine (hl) sorghum mutant germplasm with good grain quality (i.e., hard endosperm texture) has been a major research objective at Purdue University. Progress toward achieving this objective, however, has been slow due to challenges posed by a combination of genetic and environmental factors. In this article, we report on the identification of a sorghum grain phenotype with a unique modified endosperm texture that has near-normal hardness and possesses superior nutritional quality traits of high digestibility and enhanced lysine content. These modified endosperm lines were identified among F6 families developed from crosses between hard endosperm, normal nutritional quality sorghum lines, and improved HPD/hl sorghum mutant P721Q-derived lines. A novel vitreous endosperm formation originated in the central portion of the kernel endosperm with opaque portions appearing both centrally and peripherally surrounding the vitreous portion. Kernels exhi...

Quantification and distribution of kafirins in the kernels of sorghum cultivars varying in endosperm hardness

Abstract Kafirins extracted from the endosperm of seven sorghum [ Sorghum bicolor (L.) Moench] cultivars were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). Kafirins extracted from the vitreous and floury endosperm portions within the kernel were also analysed by these techniques. The ELISA results indicated that the level of all the three kafirins was high in the hard endosperm kernels. The level of γ-kafirin was particularly higher in the vitreous endosperm portions of these kernels. The ratio of γ-kafirin to the α-kafirin was, however, higher for the floury portions of soft kernels. Tissue print immunoblotting revealed that the β- and γ-kafirins were concentrated in the central floury endosperm portions of soft kernels, whilst α-kafirin was distributed more uniformly throughout the endosperm. In contrast, all three kafirins were distributed uniformly throughout the endosperms of hard kernels. The data indicate that the content, as well as the distribution, of kafirins within the kernel is different in grains varying in endosperm hardness.

Effect of reducing agents on the in vitro protein and starch digestibilities of cooked sorghum

Abstract A low-tannin sorghum cultivar M-35-1 was used in this study. Investigation showed that the in vitro protein digestibility (IVPD) decreased considerably when sorghum flour was cooked in water, while it increased when cysteine, sodium metabisulphite, or ascorbic acid were added to the cooking medium. The increase in the IVPD was significantly higher with increasing concentrations of cysteine up to 0.25 M and it continued to increase to 0.5 M for sodium metabisulphite; with ascorbic acid it increased up to 0.1 M then decreased. The in vitro starch digestibility (IVSD) of the treated gruel initially increased in the presence of either cysteine, sodium metabisulphite or ascorbic acid. The increase was parallel to that shown by IVPD; however, at high levels of cysteine or sodium metabisulphite the IVSD was low. Removal of cysteine from the gruel by alcohol gave higher IVSD. Altered viscosity patterns for all the treatments led to increase in the gelatinization temperature, peak viscosity and breakdown. However, the setback decreased in all treatments. Cysteine and ascorbic acid gave a negative setback but when the pH was adjusted to 4.5 or 7.0 a normal setback was obtained. ©

Regulation of astaxanthin and its intermediates through cloning and genetic transformation of β-carotene ketolase in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, β-carotene ketolase (BKT) on β-carotene through intermediates echinenone and canthaxanthin. In this study, the β-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2-3-fold higher, while the intermediates like echinenone and canthaxanthin were found to be 8-10-fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and β-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.

Ascorbic acid-induced loss of a pediocin-encoding plasmid in Pediococcus acidilactici CFR K7.

A strain of Pediococcus acidilactici CFR K7 grown in presence of 1 mm ascorbate for 18 h resulted in 35% colony-forming units (CFU) irreversibly losing their ability to produce pediocin. Agarose gel electrophoresis of plasmid DNA and dot-blot hybridization showed concomitant loss of a 7.8 kb plasmid coding for pediocin from these colonies. Sensitivity of these colonies to nitrofurantoin was also observed after ascorbate treatment. Since ascorbic acid is a readily available and non-hazardous compound in contrast to other conventional plasmid curing agents, the possibility of its use in determining plasmid-encoded traits in food grade lactic acid bacteria is also proposed.

Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of Coffea canephora P. ex Fr.

The influence of polyamines, polyamine inhibitors and ethylene inhibitors were tested in Coffea canephora for in vitro morphogenetic response and caffeine biosynthesis. Coffea canephora produced non-embryogenic and embryogenic calli. Somatic embryos were produced only from the embryogenic callus. Endogenous polyamine pools were estimated in these tissues. Somatic embryos were subjected to secondary embryogenesis under the influence of putrescine, silver nitrate and specific inhibitors of polyamine biosynthesis. Estimation of endogenous total polyamines revealed that embryogenic callus contained 11-fold more spermine and 3.3-fold higher spermidine when compared to non-embryogenic callus. Incorporation of polyamines resulted in 58% explant response for embryogenesis when compared to control with 42% response. Incorporation of silver nitrate resulted in 65% response for embryogenesis. Incorporation of polyamine biosynthetic pathway inhibitors DFMO and DFMA resulted in 83% reduction in embryogenic response with concomitant increase in caffeine levels by two-fold as compared to control. These results have clearly demonstrated that polyamines play a crucial role in embryogenesis and caffeine biosynthesis.