Huge yield losses and deterioration of quality of cultivated plants have been realized due to continuous exposure of plants to pathogens and insect-pests. The plants and their obligate pathogens and pests have evolved for co-existence. This natural balance has been disturbed by deployment of genes for race-specific vertical resistance and excessive use of pesticides leading to a vicious cycle of emergence of new virulences and search for new genes and potent pesticides. The genetic base for resistance to diseases and insect pests is extremely narrow or does not exist for same plant-pathogen or plant-insect combinations. With the recent advances of cellular and molecular biology and understanding of molecular mechanism of plant-parasite interactions and disease resistance it has been possible to clone, modify and mobilize hitherto inaccesible genes from diverse sources for engineering disease and insect-pest resistant plants. A number of plant species have been successfully transformed for resistance to bacteria, viral, fungal pathogens, nematodes and insects. The transgenic plants have been extensively field tested meeting the stringent biosafety guidelines and released for commercial cultivation since 1990 occupying more than 12 million hectares. Perspectives and strategies for overcoming crucial constraints and concern for engineering high levels of resistance and its exploitation are discussed.
Many angiosperm plants express self-incompatibility (SI), through which they can recognize self-pollen and restrict fertilization to non-self-pollen. In species of Brassica, SI is sporophytically expressed, regulated by a single locus, S, with multiple alleles. Two stigma-specific genes, SLG and SRK, both of which locate at the S locus, are believed to play a role in the recognition reaction on the stigma side. Reviewed here are findings about SLG and SRK genes, the molecular characterization of S-multigene family, the genomic structure of S locus, and some aspects on signal transfer by the proteins encoded by these genes.
An in-vitro procedure is described for simple and rapid regeneration of shoots and roots from small calli of tobacco (Nicotiana tabacum L. cv. Xanthi). The calli induced from stem pith tissue were subcultured for 2 to 3 times at 2-week intervals. Then, the calli were crushed on a stainless-steel mesh with a stainless-steel spoon and passed through the mesh. Micro-calli of uniform size (0.5 or 1.0mm in diameter) were obtained by this procedure. For shoot regeneration, micro-calli of 0.5mm in diameter were suspended in a liquid MS medium supplemented with 0.5mgl-1 IBA and 0.05mgl-1 BA and rotation-cultured. After 7 days, the culture bottles were transferred to static culture under continuous light of 5400 lux. After 11 days of static culture, an adventitious shoot was formed in about 50% of the calli. The diameter of shoot-forming calli was 1 to 2mm and the number of shoots per callus was usually one. For root regeneration, the micro-calli (1.0mm in diameter) were rotation-cultured in the dark for 5 days in a liquid MS medium supplemented with 1.0mgl-1 NAA and 0.1mgl-1 kinetin, and then put separately on a 0.06-ml Gelrite drop containing the same medium composition and cultured in darkness. A regeneration frequency of about 45% was obtained by day 30 of microculture. The diameter of calli at root initiation was 4 to 5mm and the number of roots per callus was usually one or two.
The effects of phyA and phyB mutations on shoot bud regeneration in Arabidopsis thaliana have been studied. The phyB mutant showed a significantly lower frequency of shoot bud regeneration than did the wild-type, indicating a role for phytochrome B in the regulation of in-vitro shoot bud regeneration. Interestingly, the shoot bud regeneration frequency of the phyA mutant was not significantly different from that of the wild-type in a light and dark, suggesting that phytochrome A may not be involved in the regulation of in-vitro shoot bud regeneration. The effects of gibberellin (GA) were also studied because previously we showed that a reduction in endogenous GA level results in a higher shoot bud regeneration frequency. Exogenous paclobutrazol and prohexadione (GA biosynthesis inhibitors) increases, and exogenous GA reduces, the shoot bud regeneration frequency in the phyB mutant. Thus shoot regeneration in the phyB mutant is still responsive to regulation via the GA signalling system.
To develop an efficient procedure for obtaining a desired mutation, we performed ion beam exposure on tobacco anthers and selected Potato Virus Y (PVY)-resistant mutants in the haploid generation. Anthers were exposed to C ion beams of 5 to 200Gy or He ion beams of 5 to 400Gy, and cultured on modified Nakata's medium. Anther culture response, the percentage of anthers producing plantlets, was markedly reduced by the exposure and, at higher doses, all plantlets were vitrified. RD50 based on the anther culture response was about 5Gy for C ion beams and about 10Gy for He ion beams. The frequency of mitotic cells with chromosome aberrations in root tips of plantlets derived from exposed anthers, ranged from 11.9% to 16.4%, compared to 3.1% in the control. All plants in the non-exposed regimen died within 21 days after inoculation of PVY. On the other hand, 15 of 472 plants in the exposed regimen were viable and continued to grow, with minor disease symptoms observed in some plants after 50 days. Accordingly, these plants were considered to be PVY-resistant. In particular, one plant showed no disease symptoms and grew vigorously.
Four hundred and six anther culture developed progeny plants of eight different families of Lolium perenne L. were evaluated for their ploidy level, genetic variation at isozyme loci and performance at field level. Seventy six percent of the total progeny plants were found to be diploids, (2n=14) but the rate of diploidization varied from family to family. The segregations of eight isozyme loci among the progeny families and their level of heterozygosity were assessed. The heterozygosity was found to be low for all, only one family segregated at four isozyme loci and the rest were even lower. The field performance of the artificial homozygous naturally out-breeding species was evaluated and none could survive to the extreme environmental conditions over the winter, however they grew under controlled conditions.
Electrofusion was conducted to use the embryogenic callus cells of satsuma mandarin (Citrus unshiu cv. ‘Juman’ unshiu) and mesophyll cells of yuzu (C. junos) and lemon (C.limon cv. ‘Eureka’ lemon). Two plants were regenerated from the combination of satsuma mandarin and yuzu, and one plant from that of satsuma mandarin and lemon. The plants had 18 chromosomes (2n=2×=18 in each parent), and showed the same nuclear rDNA fragment pattern as that of the mesophyll parent. The mitochondrial DNA analysis showed the presence of specific yuzu or lemon bands in addition to specific satsuma mandarin bands. From these results with morphological evidence, the regenerated plants were considered as cybrids with recombined mitochondrial DNA.
We investigated the differences between liquid- and air-heat shock (HS) treatments performed on transgenic Nicotiana plumbaginifolia leaves harboring Arabidopsis HSP18.2 promoter-GUS chimeric gene. The optimum temperatures for heat-induced GUS activity in leaves were 42°C for 2h in liquid-HS treatments, and 45°C for 2h in air-HS treatments. Thus, we found a great difference between the GUS activity in leaves exposed to HS in air and liquid.
The leaf disks of Nicotiana plumbaginifolia were irradiated by an 14N heavy-ion beam and then were cultured in shoot-inducing medium in order to produce mutants for elucidating the functions of the cell wall in plant morphogenesis. Non-organogenic and loosely attached callus, which had the same morphological features as non-embryogenic callus of carrot was formed on 11.8% of the haploid leaf disks irradiated by 14N heavy-ion beam (5 Gy) at high frequency. The cells stimulated by the heavy-ion beam loosely attached to each other resulting in a random morphology of cell clusters, whereas the non-irradiated cells formed a tight callus with multiple shoots.
Plant oxidosqualene cyclase (OSC) is classified into two types depeneding on its catalytic mechanisms and products: cycloartenol synthase and triterpene synthase. A cDNA probably encoding OSC was cloned from bulbs of Allium macrostemon by RT-PCR. The cDNA contains a 2289-by open reading frame, encoding 762 amino acids. A higher degree of homology (73%) was found for the gene product with the known cycloartenol synthases than with the triterpene synthases (51-55%), indicating its most likely function as cycloartenol synthase. This is the first cloning example of a putative cDNA encoding OSC from a monocotyledonous plant.
Differential display of immature anther cDNA was carried out between restorer and maintainer lines of [ms-bo] cytoplasmic male sterile rice. One clone (DD2-3) was identified to be specific to restorer line. However DD2-3 was mapped on the chromosome 11 and was not linked to fertility-restorer gene, Rf-1. Full length eDNA of DD2-3 was obtained and the predicted amino acid sequence contains leucine zipper structure, followed by nucleotide binding site (NBS) and leucine rich repeats (LRR). These structures are characteristics of the NBS-LRR class of plant disease-resistant genes. The transcripts of DD2-3 were detected in the anthers at the microspore stage and leaves.