Vinod Kumar

AgNO3: a potential regulator of ethylene activity and plant growth modulator

The aim of this review is to critically analyze the role of silver nitrate (AgNO 3 ) in modulating plant growth and development. In recent years, basic studies on ethylene regulation opened new vistas for applied research in the area of micro-propagation, somatic embryogenesis, in vitro flowering, growth promotion, fruit ripening, and sex expression. Silver nitrate has proved to be a very potent inhibitor of ethylene action and is widely used in plant tissue culture. Few properties of silver nitrate such as easy availability, solubility in water, specificity and stability make it very useful for various applications in exploiting plant growth regulation and morphogenesis in vivo and in vitro . Silver ion mediated responses seem to be involved in polyamines, ethylene- and calcium- mediated pathways, and play a crucial role in regulating physiological process including morphogenesis. The molecular basis for regulation of morphogenesis under the influence of silver nitrate is completely lacking. This review compiles published reports of silver nitrate-mediated in vitro and in vivo studies and focuses on fundamental and applied aspects of plant growth modulation under the influence of silver nitrate.

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.

Peroxidase production from hairy root cultures of red beet (Beta vulgaris)

The genetically transformed roots of red beet have been shown, for the first time, to produce very high levels of peroxidase (POD; EC 1.11.1.7) accounting for 1.21 x 10 6 Units L -1 . Of the ten clones established using different strains of Agrobacterium rhizogenes , one was that from the strain LMG-150, three each from A 2/83, A 20/83 and A4. All the clones showed true integration of T-DNA when tested by PCR and Southern hybridization methods. Each clone differed significantly from the others in growth, hormone dependency and POD production where LMG-150 produced highest biomass (140 g FW L -1 ) as well as POD (ranging from 8000-9000 U g -1 FW and 1.18 x 10 6 U L -1 with a specific activity of 600 U mg -1 protein) on hormone-free medium, both in shake-flask as well as in bioreactor with a further enhancement to 1.21 x 10 6 U L -1 upon the addition of extra calcium chloride (5 mM). PAGE with active staining showed 4 distinct bands of R m 0.06, 0.16, 0.25, 0.38 and 0.46 in the biomass and bands at R m 0.06, 0.16, 0.25 and one extra band of R m 0.575 in the spent medium where isozymes of R m 0.38 and 0.46 were totally absent. The pH optima and other properties were grossly comparable with the standard horse-radish POD (HRP) with better thermal stability than HRP and therefore, the present source appears to offer a cheaper and additional alternative for the commercial production of POD.

Influence of different ethylene inhibitors on somatic embryogenesis and secondary embryogenesis from Coffea canephora P ex Fr.

The effect of cobalt chloride, salicylic acid, and silver nitrate for embryogenesis was studied in in vitro cultures of Coffea canephora. Murashige and Skoog (in Physiol. Plant. 15:473–497, 1962) medium containing 20 and 40xa0μM either of cobalt chloride, silver nitrate, or salicylic acid supplemented with 1.1xa0μM N6 benzyladenine and 2.85xa0μM indole-3-acetic acid was used for the study. At 20 and 40xa0μM silver nitrate treatment, 35–48% explants responded for embryogenesis, and 38u2009±u20097 and 153u2009±u200927 embryos were produced from each callus mass, respectively, whereas only 5% control explants responded on medium devoid of silver nitrate, cobalt chloride, or salicylic acid. Secondary embryogenesis was observed in 70–90% of the explants, and around 100–150 embryos were produced from each explant cultured on a medium containing silver nitrate, and only a 3% response was noticed in control embryo explants. Yellow friable embryogenic calluses were obtained from the cut edges of most of the tissues grown in a medium supplemented with cobalt chloride. The results clearly demonstrated that, among the tested ethylene inhibitors, silver nitrate is very effective in reprogramming the cellular machinery toward embryogenesis.

Induction of in vitro flowering in Capsicum frutescens under the influence of silver nitrate and cobalt chloride and pollen transformation

The influence of silver nitrate (AgNO 3 ) and cobalt chloride (CoCl 2 ) on shoot multiplication and in vitro flowering in Capsicum frutescens Mill. was investigated. Exogenous administration of AgNO 3 and CoCl 2 at a concentration of 30 µM resulted in the maximum tissue response in terms of shoot length and number of shoots after 45 days culturing on MS medium. Both silver nitrate (40 µM) and cobalt chloride (30 µM) influenced in vitro flowering after 25 and 45 days respectively. This is the first report on in vitro flowering in C. frutescens. The study also demonstrated successful transformation of pollen obtained from the in vitro flowers. Since capsicum is highly recalcitrant to in vitro plant regeneration, the results of the study may be highly useful in transformation of capsicum using germ free in vitro flowers.

Somatic embryogenesis, organogenesis, and regeneration from leaf callus culture of Decalepis hamiltonii Wight & Arn., an endangered shrub

SummaryA novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N6-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA3) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.

Direct shoot bud induction and plant regeneration in Capsicum frutescens Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28xa0μM indole-3-acetic acid, 10xa0μM silver nitrate and either of 13.31–89.77xa0μM benzyl adenine (BA), 9.29–23.23xa0μM kinetin, 0.91–9.12xa0μM zeatin, 2.46–9.84xa0μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63xa0μM) as a cytokinin source and 19.4xa0±xa04.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24xa0h photoperiod. These buds were elongated in MS medium containing 2.8xa0μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.

Developments in coffee biotechnology—in vitro plant propagation and crop improvement

Coffee is an important plantation crop grown in about 80 countries across the globe. In recent years, coffee attained lot of attention in the biotechnology research area. Since last three decades, there has been a steady flow of information on coffee biotechnology and now it is entering into the genomic era. Major milestones in coffee biotech research are successful in vitro manipulation and multiplication of coffee, development of gene transfer protocols and generation of transgenic coffee plants with specific traits. The isolation of genes involved in caffeine biosynthetic pathway has opened up new avenues for generating caffeine free transgenic coffee. With the initiation of international coffee genomics initiatives, the genomic research in coffee is expected to reach new dimensions. The IPR issues may play crucial role in sharing of benefits during international collaborations in near future. This review focuses on the basic and applied aspects of coffee biotechnology for newer potentials.

Effect of the Carotenoid-Producing Alga, Dunaliella bardawil, on CCl4-Induced Toxicity in Rats

Dunaliella bardawil is a carotenoid-producing alga that is being considered for use in nutraceuticals. To evaluate potential protective effects of consumption of this alga, rats were treated with two different doses of D. bardawil (2.5 and 5.0 g kg–1 body weight [bw]) as a biomass suspension daily for 14 days. Animals were tested against Carbon tetrachloride (CCl4; 2 ml kg–1)–induced liver toxicity as measured by various biochemical marker enzymes in liver and blood. All measurements were taken 6 h following the single dose of CCl4. The results of this study show that there was a slight, but statistically significant mean serum enzyme values, with D. bardawil treatment, compared to higher mean values in animals receiving CCl4 alone. Lipid peroxidation is measured by thiobarbituric acid–reactive substance (TBARS) activity was likewise slightly less elevated with algae treatment. The results also demonstrated protection against DNA strand breaks in hepatocytes, as measured by single cell gel electrophoresis. Liver histopathology was less severe with D. bardawil treatment, supporting the apparent protective action of 14-day treatment on hepatic oxidative injury.

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.