Genetic analysis in haploids provides unconventional yet powerful advantages not available in diploid organisms. In Arabidopsis thaliana, haploids can be generated through seeds by crossing a wild-type strain to a transgenic strain with altered centromeres. Here we report the development of an improved haploid inducer (HI) strain, SeedGFP-HI, that aids selection of haploid seeds prior to germination. We also show that haploids can be used as a tool to accelerate a variety of genetic analyses, specifically pyramiding multiple mutant combinations, forward mutagenesis screens, scaling down a tetraploid to lower ploidy levels and swapping of nuclear and cytoplasmic genomes. Furthermore, the A. thaliana HI can be used to produce haploids from a related species A. suecica and generate homozygous mutant plants from strong maternal gametophyte lethal alleles, which is not possible via conventional diploid genetics. Taken together, our results demonstrate the utility and power of haploid genetics in A. thaliana.

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A haploid genetics toolbox for Arabidopsis thaliana.

Isolated microspore culture (IMC) represents a unique system of single cell reprogramming in plants wherein a haploid male gametophyte, the microspore, switches its default gametophytic developmental pathway toward embryogenesis by specific stress treatment. The application of a stress treatment(s) is necessary for efficient embryogenesis induction. Depending on species, microspores are often induced by cold and heat shock, osmotic stress, starvation, anti-microtubular agents, stress hormones, antibiotics, or polyamines. This technique (IMC) is likely to remain as a well-known method in plant breeding since it allows for the rapid production of completely homozygous lines while, in the context of developmental biology, it allows for in vitro embryogenesis to be explored in greater detail. Isolated microspores also represent ideal recipients for several gene transfer techniques including electroporation, microprojectile bombardment, and Agrobacterium-mediated transformation. IMC is also extensively used for genetic studies, i.e., studying inheritance of quantitative traits, quantitative trait loci (QTL) mapping, and genomics and gene identification, for mutation and selection and also used for producing reversible male-sterile lines. Male sterility avoids the labor costs of manual emasculation and serves as a molecular strategy for transgene containment by preventing pollen release to the environment. Combination of this technique with doubled haploid (DH) production leads to an innovative environmentally friendly breeding technology. In addition, the usefulness of DHs for reverse breeding program, an applied plant breeding technique introduced to directly produce parental lines for any hybrid plant, is also generally discussed.

Isolated Microspore Culture and Its Applications in Plant Breeding and Genetics

Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride, by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride. Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%-70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina, Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the mycoparasitism effect of the extracellular enzymes.

https://www.tandfonline.com/doi/pdf/10.1080/13102818.2014.978206

Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.

Trichoderma spp are free living filamentous fungi. They are cosmopolitan and versatile in nature. They have the potential to produce several enzymes that can degrade the cell wall materials. Also, they release a number of fungi toxic substances that can inhibit the growth of the fungal pathogens. Many mechanisms have been described on how Trichoderma exert beneficial effects on plants as a bio-control agent. But due to its versatile nature, its potential cannot be explored to its full extent. And it is a developing science in the field of bio-control with its new discoveries adding to the usefulness of the fungi as a bio-control agent. Its development as a bio-control agent passes through many phases and each phase adding novel ideas that will help in the development of an efficient bio-agent which in turn will help in the crop improvement and disease management. The studies on their various aspects responsible for bio-control will open a flood gate to the development of Trichoderma as an efficient and reliable bio-agent and provide a better scope for implementation in crop and disease management. The in vitro antagonistic activity of Trichoderma viride against phytopathogens (Sclerotium rolfsii, Fusarium oxysporum f.s.p. ciceri, Fusarium oxysporum f.s.p. udum) was studied and it was found to be potentially effective against F. oxysporum f.s.p. ciceri followed by F. oxysporum f.s.p. udum and Sclerotium rolfsii.

https://journals.ansfoundation.org/index.php/jans/article/download/927/886

Advent of Trichoderma as a bio-control agent- A review

Six Trichoderma strains (collected from IARI, New Delhi and MTCC, Chandigarh) were tested for their ability to inhibit soil-borne pathogen of groundnut mainly Sclerotium rolfsii (causing stem rot on groundnut). In vitro percent growth inhibition of S. rolfsii by various Trichoderma strains were recorded at 5th day after inoculation (DAI). Results obtained from the antagonism study indicated that Trichoderma viride (NBAII Tv 23) inhibited 61% growth of phytopathogenic fungi S. rolfsii followed by Trichoderma harzianum (NBAII Th1) (55% growth inhibition of pathogen). The specific activities of cell wall degrading enzymes chitinase, β-1,3 glucanase, protease and cellulase were tested during different incubation period (48, 72 and 96 h) when Trichoderma spp. grew in the presence of pathogen cell wall in synthetic media. The antagonist T. viride (NBAII Tv 23) induced higher chitinase and protease activity. The growth inhibition of pathogen during antagonism were positively correlated with coiling pattern of antagonists at 14th day after inoculation (DAI) as well as with the induction of chitinase, β-1,3 glucanase and total phenol content. However, the amount of cellulase and polygalacturonase recorded was least in these antagonists treatment. A significant positive correlation (p = 0.01) between percentage growth inhibition of test fungus and lytic enzymes (chitinase, β-1,3-glucanase and protease) in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these enzymes. Among all the tested Trichoderma strains, T. viride (NBAII Tv 23) was found to be the best strain to be used in biological control of plant pathogen S. rolfsii.

	Key words: Antagonism, soil-borne pathogens, lytic enzyme, Trichoderma, biocontrol agent.

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Production of lytic enzymes by Trichoderma strains during in vitro antagonism with Sclerotium rolfsii, the causal agent of stem rot of groundnut

Majority of higher plants are outbreeding, highly heterozygous and undergo a long developmental period before reaching their reproductive stage. Traditional breeding and cross- pollinating procedures are both unpredictable and time-consuming. Haploids are plants with a gametophytic chromosome number and doubled haploids are haploids that have undergone chromosome duplication, represent a particularly attractive biotechnological method to accelerate plant breeding. They can occur either spontaneously or can be induced by modified pollination methods in vivo, or by in vitro culture of immature male or female gametophytes. Biotechnologies provide powerful tools for plant breeding, and among these ones, tissue culture, particularly haploid and doubled haploid technology, can effectively help to select superior plants. In vitro haploid production is, thus, the most prolific and desirable approach of haploid production. This review describes the range of techniques available for the isolation or induction of haploids by in vivo or by in vitro, estimation of haploid lavel either through chromosome counting or flowcytometry and also discourse the value of haploids.

Haploid Production in Higher Plant

Antagonistic Activity of Trichoderma Spp, (A Bio-Control Agent) Against Isolated and Identified Plant Pathogens

l -asparaginase (EC 3.5.1.1) is an important antineoplastic agent, used in the acute lymphoblastic leukemia chemotherapy and well known as food processing aid for reducing the acrylamide formation in starch rich foods. In the present study, we have cloned, expressed and purified recombinant l -asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli. To study the best condition for performance of recombinant enzymes, pH and temperature of process conditions were optimized using response surface methodology. Optimal pH and temperature for enzyme were found to be 7.79 and 47.21 °C. Furthermore, the purified recombinant enzyme was subjected to thermal deactivation studies. pH and thermal stability of enzyme was studied at various combinations of pH and temperature. In addition, thermodynamic parameters of recombinant l -asparaginase II were evaluated. It was observed that recombinant l -asparaginase II enzyme is more stable at pH 8.5 at 35 °C relatively pH 6.5, 7.5 and 9.5. It was also observed that recombinant l -asparaginase II is more stable at pH 8.5 than pH 9.6 and 7.6 at higher temperature.

Optimization of process conditions and evaluation of pH & thermal stability of recombinant l-Asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli

Vijay Kumar Mishra

Papers

Haploid Production in Higher Plant

Antagonistic Activity of Trichoderma Spp, (A Bio-Control Agent) Against Isolated and Identified Plant Pathogens

Optimization of process conditions and evaluation of pH & thermal stability of recombinant l-Asparaginase II of Erwinia carotovora subsp. atroseptica SCRI 1043 in E. coli