Govind C. Sharma

Controlled-release fertilizers and horticultural applications

Abstract For optimum plant growth, it is essential that a fertilizer material provides sufficient nutrients for the initial start followed by a uniform supply that synchronizes well with the nutrient requirement of the crop. A wide array of controlled-release fertilizer (CRF) materials is available, varying in release duration from 3 to 24 months, for use in ornamental and nursery crops, floricultural and foliage crops, turf and forage usage, vegetable crops and tree crop production. Interest in these materials is primarily for one or more of the following reasons: Availability of nutrients during the entire growing-season; Reduced capital and labor outlay in horticultural crop production; Reduced nutrient loss via leaching and run-off; Reduced chemical and biological immobilization reactions in soil which cause plant-unavailable forms; Reduction of rapid nitrification and nitrogen loss through ammonia volatilization and denitrification; Reduced seed or seedling damage from high local concentrations of salts; Reduced leaf burn from heavy rates of surface-applied fertilizers; Better seasonal distribution of growth and better acclimatization in home or display environment; Improved storage and handling properties of fertilizer materials; Product differentiation resulting in improved market potential. The purpose of this review is to summarize the chemical and physical nature, methods of evaluation, release characteristics and soil transformations, utilization in various crop production systems, and the economic status of CRF materials in the U.S.A.

Picloram induced somatic embryogenesis in Gasteria and Haworthia

Various leaf sections of Gasteria verrucosa Haw. and Haworthia fasciata Haw. were cultured on media to examine the effect of picloram (4-amino 3, 5, 6-trichloropicolinic acid) and 2, 4-D (2, 4-dichlorophenoxy acetic acid) on somatic embryogenesis. Picloram (0.5, 1.0, 2.0, 3.0 mgl-1) outperformed 2, 4-D (0, 1.0, 2.0, 3.0 mgl-1) as the auxin source of both earliness of callus and embryo induction and final yield of embryos produced at both kinetin levels examined (0.25, 1.0 mgl-1). Embryos arose initially as a yellow, compact globular masses from the area just beneath the epidermis in linear pattern parallel with the main axis of the leaf and then developed a heartshaped appearance. Embryo formation was preceded by growth of callus almost crystalline in appearance on the cut surface. Subsequent shoot formation developed from green pigmented loci in crystalline callus derived from embryos. Shoot and root development in Gasteria was induced on a defined medium containing quarter strength MS or B5 salts with no hormonal supplementation.

SOMATIC EMBRYO INITIATION AND GERMINATION IN DIPLOID COTTON (GOSSYPIUM ARBOREUM L.)

SummaryThe diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A2-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M2 [0.04M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)] On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M1 (0.2M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M2 medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.

RNA Interference for Functional Genomics and Improvement of Cotton (Gossypium sp.)

RNA interference (RNAi), is a powerful new technology in the discovery of genetic sequence functions, and has become a valuable tool for functional genomics of cotton (Gossypium sp.). The rapid adoption of RNAi has replaced previous antisense technology. RNAi has aided in the discovery of function and biological roles of many key cotton genes involved in fiber development, fertility and somatic embryogenesis, resistance to important biotic and abiotic stresses, and oil and seed quality improvements as well as the key agronomic traits including yield and maturity. Here, we have comparatively reviewed seminal research efforts in previously used antisense approaches and currently applied breakthrough RNAi studies in cotton, analyzing developed RNAi methodologies, achievements, limitations, and future needs in functional characterizations of cotton genes. We also highlighted needed efforts in the development of RNAi-based cotton cultivars, and their safety and risk assessment, small and large-scale field trials, and commercialization.

Characterization of the two intra-individual sequence variants in the 18S rRNA gene in the plant parasitic nematode, Rotylenchulus reniformis.

The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1 (RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene.

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were ‘Corbit’ (first subculture); ‘GAF/Park’ and ‘88Ab3073’ (second subculture); and ‘GAF/Park’ and ‘87Ab5932’ (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. ‘GAF/Park’, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes ‘GAF/Park’, ‘88Ab3073’, and ‘Tibor’. The smaller clusters only regenerated plants for ‘GAF/Park’ and ‘88Ab3073’. From one gram of callus used to initiate suspensions of ‘GAF/Park’ and ‘88Ab3073’, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of ‘GAF/Park’, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.

Putrescine-enhanced somatic embryos and plant numbers from elite oat (Avena spp. L.) and reciprocal crosses

SummaryMature embryos from hulled, regenerable GP-1 (A. sativa L.), hull-less, recalcitrant Tibor (A. nuda L.) and reciprocal crosses were cultured in vitro on a putrescine- (Put) containing medium. Hormone-free Murashige and Skoog medium (MS-0) or shoot proliferation medium (SPM) [2.0 mgl−1 (9.0 μM) 2,4-dichlorophenoxyacetic acid (2,4-D)], with and without 0.5 mM Put or 1 mM Put, were tested for effects on somatic embryogenesis and plant regeneration. Put/SPM (0.5mM) was the best medium for both somatic embryos (SEs) and plant numbers per gram of callus, regardless of genotype. This effect was most evident in Tibor, which produced no somatic embryos or plants on SPM, a previously published regeneration medium, and in Tibor ×GP-1, which produced reduced numbers of SE and plants on the remaining media. The number of SEs per gram of callus for GP-1 and GP-1× Tibor showed little significant differences between the different media. Put treatments produced plants from the four genotypes but the regeneration efficiency on Put-containing medium was similar or even better than on SPM for explants containing maternal GP-1 germplasm. This suggests that Put-containing MS-0 medium can be used for testing regeneration of other oat lines. In addition, SPM containing 0.5 mM Put can be used to induce significant regeneration of plants from normally recalcitrant genotypes. This improvement greatly increases the number of potential germplasms for further transformation efforts.

A comparison of ward wheat suspension cultures containing clumps and single cells

Abstract Six wheat, two rye and three primary triticale lines were screened for adaptability to suspension culture and from these a durum wheat variety Ward was further studied. Ward cell numbers in suspension containing clumps doubled by the 6th day and packed cell volume (PCV) by the 9th day. Decreases in cell dry weight, mean cell volume and mean cell dry weight paralleled increases in cell number (CN) and cell volume. Clump dry weight increased as dry weight of free-floating single cells in the suspension decreased. An optimum initial cell population for suspensions containing clump was 6.3 × 10 4 cells/ml. Filtered cultures containing only single cells declined in CN and PCV irrespective of subculture interval and showed no cell division at any cell density examined. All parameters measured have underscored the necessity for the presence of clumps for continued cell division.

Development of New Candidate Gene and EST-Based Molecular Markers for Gossypium Species

New source of molecular markers accelerate the efforts in improving cotton fiber traits and aid in developing high-density integrated genetic maps. We developed new markers based on candidate genes and G. arboreum EST sequences that were used for polymorphism detection followed by genetic and physical mapping. Nineteen gene-based markers were surveyed for polymorphism detection in 26 Gossypium species. Cluster analysis generated a phylogenetic tree with four major sub-clusters for 23 species while three species branched out individually. CAP method enhanced the rate of polymorphism of candidate gene-based markers between G. hirsutum and G. barbadense. Two hundred A-genome based SSR markers were designed after datamining of G. arboreum EST sequences (Mississippi Gossypium arboreum   EST-SSR: MGAES). Over 70% of MGAES markers successfully produced amplicons while 65 of them demonstrated polymorphism between the parents of G. hirsutum and G. barbadense RIL population and formed 14 linkage groups. Chromosomal localization of both candidate gene-based and MGAES markers was assisted by euploid and hypoaneuploid CS-B analysis. Gene-based and MGAES markers were highly informative as they were designed from candidate genes and fiber transcriptome with a potential to be integrated into the existing cotton genetic and physical maps.

Characterization of the reniform nematode genome by shotgun sequencing.

The reniform nematode (RN), a major agricultural pest particularly on cotton in the United States, is among the major plant-parasitic nematodes for which limited genomic information exists. In this study, over 380 Mb of sequence data were generated from pooled DNA of four adult female RNs and assembled into 67,317 contigs, including 25,904 (38.5%) predicted coding contigs and 41,413 (61.5%) noncoding contigs. Most of the characterized repeats were of low complexity (88.9%), and 0.9% of the contigs matched with 53.2% of GenBank ESTs. The most frequent Gene Ontology (GO) terms for molecular function and biological process were protein binding (32%) and embryonic development (20%). Further analysis showed that 741 (1.1%), 94 (0.1%), and 169 (0.25%) RN genomic contigs matched with 1328 (13.9%), 1480 (5.4%), and 1330 (7.4%) supercontigs of Meloidogyne incognita, Brugia malayi, and Pristionchus pacificus, respectively. Chromosome 5 of Caenorhabditis elegans had the highest number of hits to the RN contigs. Seven putative detoxification genes and three carbohydrate-active enzymes (CAZymes) involved in cell wall degradation were studied in more detail. Additionally, kinases, G protein-coupled receptors, and neuropeptides functioning in physiological, developmental, and regulatory processes were identified in the RN genome.