B. D. Singh

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field.

Agrobacterium-mediated transformation of cauliflower: Optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.

Micropropagation of Centella asiatica (L.), a valuable medicinal herb

A protocol is described for rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Centella asiatica (L.) by enhanced axillary bud proliferation in nodal segments isolated from mature plants. Although bud break was dependent on BA supply, the synergistic combination of 22.2 μM BA and 2.68 μM NAA induced the optimum frequency (91%) of shoot formation as well as shoot number (4 to 5 shoots per node). Subculturing of nodal segments harvested from the in vitro derived axenic shoots on the multiplication medium enabled continuous production of healthy shoots with similar frequency. MS medium supplemented with 6.7 μM BA and 2.88 μM IAA was found most suitable for shoot elongation. Rooting was highest (90%) on full-strength MS medium containing 2.46 μM IBA. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics. This micropropagation procedure could be useful for raising a stock of genetically homogenous plant material for field cultivation.

Promotion of adventitious bud regeneration by ABA in combination with BAP in epicotyl and hypocotyl explants of sweet orange (Citrus sinensis L.Osbeck)

Abstract Epicotyl and hypocotyl segments (1–1.5 cm) from 2-week-old sweet orange (Citrus sinensis L. Osbeck var. mousambi) seedlings germinated in vitro were placed horizontally on Murashige and Skoog (MS) basal medium. Adventitious shoot regeneration from the cut ends occurred only in epicotyl explants. Addition to the MS medium of 6-benzylaminopurine (BAP) up to 2 mg l−1 had a promotive effect, both in terms of the frequency of responding explants and the number of shoots per explant, while higher concentrations of BAP had a depressive effect. Gibberellic acid (GA3) at 0.04 and 0.4 mg l−1 with or without 2 mg l−1 BAP suppressed shoot bud regeneration in both types of explants. Abscisic acid (ABA) alone suppressed shoot regeneration when added to basal MS medium, but in combination with 2 mg l−1 BAP it had a strong promotive effect, particularly at 0.2 mg l−1. A remarkable feature of this ABA effect was that the bulk of the buds regenerated away from the cut ends directly from the epidermis without any apparent callus formation. These shoot buds did not develop further even 8–10 weeks after their transfer to the basal MS medium, but showed normal growth on medium containing 0.04 mg l−1 GA3. This system, therefore, may be useful for germplasm conservation.

Identification of RAPD markers linked to the rust (Uromyces fabae) resistance gene in pea (Pisum sativum)

SummaryTwo RAPD markers linked to gene for resistance (assayed as pustule number cm−2 leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC1F1 [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F1, F2, BC1F1 and BC1F2 generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC1F1 individuals. Two RAPD makers, viz., SC10-82360 (primer, GCCGTGAAGT), and SCRI-711000 (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.

Individual and combined effects of berberine and santonin on spore germination of some fungi.

Berberine and santonin were isolated from rhizomes ofBerberis aristata and unexpanded flower buds ofArtemisia maritima, respectively. Efficacy of these two chemicals individually as well as of their mixtures, was tested against spore germination of some saprophytic and obligate fungi. While berberine individually was effective against most of the fungi,Helminthosporium spp. were least affected even at the highest dose (1500 ppm). Santonin was equally effective against several fungi. Mixture of both alkaloids found to be more effective than individual ones. Keeping the dose of berberine constant and santonin at two different concentrations (viz. 250 and 500 ppm) the spore germination ofHelminthosporium, oryzae was stimulated. Increasing concentration of santonin inhibited the spore germination of all other fungi tested,Colletotrichum capsici being affected only by 20 and 5% (at berberine concentration of 250 and 500 ppm, respectively). On the other hand, santonin being constant and berberine at different concentrations, the mixture was effective against all the fungi.

The effect of arsenic on pigment composition and photosynthesis in Hydrilla verticillata

The present study evaluated the effects of 100 and 500 μM arsenate (Na2HAsO4) on pigment composition and photosynthesis in Hydrilla verticillata (L.f.) Royle. Arsenic accumulation increased in concentration and duration dependent manner. The maximum accumulation [568 μg(As) g−1(d.m.)] was observed at 500 μM concentration and 96-h exposure. This concentration led to a significant decline in chlorophyll a content and PS II efficiency during the whole experiment, and in chlorophyll b and carotenoids after 96 h, but no significant changes in photosynthetic pigments were noticed at 100 μM arsenate. Net photosynthetic rate, electron transport rate, and water use efficiency declined whereas transpiration rate increased, and stomatal conductance and photochemical quenching did not show any effect or increased. The content of reactive oxygen species increased and content of reduced ascorbate declined at 500 μM arsenate in comparison to the control.

Identification and characterization of slow rusting components in pea (Pisum sativum L.)

Three hundred and forty five accessions of pea of diverse origin, height, leaf types and disease reaction were screened for rust disease severity and area under disease progress curve (AUDPC). The frequency of slow rusting types in the tall, dwarf, early and late groups appeared comparable. Of the 345 accessions, forty-four genotypes were evaluated for disease intensity, which was converted into AUDPC, number of pustules/leaf and pustule size. Wide range of variation was found for these traits. The slow rusting attribute of 16 genotypes was further confirmed by testing these under unprotected (inoculated) and protected (fungicidal spray) conditions for two successive years for disease intensity by assessing the AUDPC, seed yield/plot, and 100-seed weight. The fast rusting genotypes exhibited lower AUDPC, accompanied with increased seed yield and seed weight when grown under the protected condition, as compared to those raised under the unprotected condition. The genotypes Pant P 11, FC 1, HUDP 16, JPBB 3 and HUP 14 appeared as slow rusting genotypes.

Marker-Assisted Plant Breeding: Principles and Practices

Marker-assisted plant breeding involves the application of molecular marker techniques and statistical and bioinformatics tools to achieve plant breeding objectives in a cost-effective and time-efficient manner. This book is intended for beginners in the field who have little or no prior exposure to molecular markers and their applications, but who do have a basic knowledge of genetics and plant breeding, and some exposure to molecular biology. An attempt has been made to provide sufficient basic information in an easy-to-follow format, and also to discuss current issues and developments so as to offer comprehensive coverage of the subject matter. The book will also be useful for breeders and research workers, as it offers a broad range of up-to-the-year information, including aspects like the development of different molecular markers and their various applications. In the first chapter, the field of marker-assisted plant breeding is introduced and placed in the proper perspective in relation to plant breeding. The next three chapters describe the various molecular marker systems, while mapping populations and mapping procedures including high-throughput genotyping are discussed in the subsequent five chapters. Four chapters are devoted to various applications of markers, e.g. marker-assisted selection, genomic selection, diversity analysis, finger printing and positional cloning. In closing, the last two chapters provide information on relevant bioinformatics tools and the rapidly evolving field of phenomics

Wheat somaclonal variants showing earliness, improved spot blotch resistance and higher yield

Somaclones (R2, R3 and R4generations) were regenerated from immature embryos of two spring wheat varieties,HUW-206 and HUW-234. Many somaclones displayed improved earliness, enhanced resistance to spot blotch disease and increased yield over the parent. The superiority of variants for yield traits and disease resistance was established in R4 generation, confirming the possibility of wheat improvement through somaclonal variation.