In this article we will discuss about:- 1. Introduction to Genes Transformation of Rice 2. Presence of Molecular-Marker in Genes Transformation of Rice 3. Genes for Rice Transformation.
Introduction to Genes Transformation of Rice:
A number of useful genes have been transferred from wild species to rice. Khush transferred the gene for grassy stunt virus resistance from O. nivara to rice through backcrossing. Grassy stunt virus resistance has been bred into several rice cultivars.
Recently, useful genes were transferred from other wild species. Jena and Khush transferred genes for resistance to three Philippine biotypes of BPH from O. officinalis into the elite breeding line IR31917-45-3-2. Some of the derived lines have also shown resistance to BPH populations in Bangladesh and India.
These lines are free from the undesirable features of wild species such as grain shattering and poor plant type, and are on par with the recurrent parent for grain yield. They are being used as parents in rice breeding at IRRI and in other national programmes. Four breeding lines resistant to BPH derived from O. officinalis have been released as varieties in Vietnam. Genes for blast, bacterial blight and BPH resistance have been transferred to rice from other wild species – O. minuta; O. latifolia; O. australiensis; and O. brachyantha.
Presence of Molecular-Marker in Genes Transformation of Rice:
Numerous genes of economic importance, such as those for disease and insect resistance, are repeatedly transferred from one varietal background to another by plant breeders. Most genes behave in a dominant and recessive manner and require time-consuming efforts to transfer.
Sometimes the screening procedures are cumbersome and expensive, and require large field space. If such genes can be tagged by tight linkage with DNA or iso-enzyme markers, time and money can be saved in transferring these genes from one varietal background to another.
The presence or absence of the associated molecular marker would indicate, at a very early stage, the presence or absence of the desired target gene. Co-dominance of the associated molecular marker allows all the possible genotypes to be identified in any breeding scheme, even if the gene for economic traits cannot be scored directly. A molecular marker very closely linked to the target gene can act as a ‘tag’ which can be used for indirect selection of the gene(s) in a breeding programme.
Genes for Rice Transformation:
Major efforts are under way to isolate genes for disease and insect resistance and abiotic stress tolerance, and to introduce them into rice in order to increase the level of host resistance. Several laboratories have produced transgenic plants both in japonica and indica rices. Recently, transgenic rice carrying agronomically important genes for resistance to stem borer, virus tolerance, resistance to fungal pathogens and herbicide tolerance have also been produced.
Genes for Insect Resistance:
As early as 1987, genes coding for toxins from Bacillus thuringiensis (Bt) were transferred to tomato, tobacco and potato where they provided protection against lepidopteran insects. A major target for Bt deployment in transgenic rice is the yellow stem borer, Scirpophaga incertulas (Walker). The pest is widespread in Asia and has the potential to cause substantial crop losses. Improved rice cultivars are either susceptible to the insect or have only moderate levels of resistance.
Thus, Bt transgenic rice has much appeal for controlling the yellow stem borer. Fujimoto introduced a truncated endotoxin gene, cry 1A (b), of B. thuringiensis. The coding sequence was modified on the basis of the codon usage of rice genes. Transgenic rice plants efficiently expressed the modified cry 1A (b) gene at both the mRNA and protein levels. Transgenic plants in the R2 generation expressing the cry 1A (b) protein had increased resistance to striped stem borer and leaf folder.
Wuhn introduced the cry 1A (b) gene into IR58 through particle bombardment. The transgenic plants in the R0, R1 and R2 generations showed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed up to 100% mortality for yellow stem borer and striped stem borer. Recently, Nayak et.al transformed IR64 through particle bombardment using the cry 1A(c) gene placed under the control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator.
Six independent transgenic lines showing high expression of insecticidal crystal protein were identified. The transferred synthetic cry 1A(c) gene was stably expressed in the T2 of these lines and the transgenic rice plants were highly toxic to yellow stem borer larvae, resulting in little feeding damage.
Another category of genes for transforming rice for insect resistance is the inhibitors of insect digestive enzymes. The storage tissues of most plants contain chemicals that limit predation by insects and other herbivores. Some are the protease inhibitors of insect digestive enzymes, and the genes coding for them are sources for insect resistance. Chen has purified and characterized five starch digestive enzymes (a-amylase isoenzymes) from the gut of three major storage pests (the rice weevil, the red flour beetle and the yellow mealworm).
Hilder have purified a putative inhibitor from cowpea, which is a typical serine protease inhibitor with broad spectrum effectiveness against insect pests. The cowpea gene (CpTi) was transferred to tobacco, where it conferred resistance to tobacco bud worm and other insect pests. This gene is now being tested for its effectiveness against BPH, a very serious pest of rice. Additional inhibitors from other plants such as the mung-bean trypsin inhibitor, potato proteinase inhibitors I and II, arrowhead proteinase inhibitors, and towel gourd trypsin inhibitors are being evaluated against the digestive enzymes of rice insects.
Irie transformed the japonica rice cultivar Nipponbare by introducing corn cystatin (CC) cDNA through electroporation of protoplasts. The transgenic plants showed strong inhibitory activity against rice proteinases that occur in the gut of the insect pest, Sitophilus zeamais.
We are attempting to transform rice with the soybean trypsin inhibitor (SBTi) gene. Hygromycin-resistant plants have been regenerated that were co-transformed with the SBTi. Rice plants transformed with the SBTi are likely to be toxic to yellow stem borer and they may delay the breakdown of Bt toxins within the larval mid-gut and thereby enhance the action of Bt toxins.
Other genes that might confer enhanced insect resistance are genes encoding a-amylase inhibitors, lectins and chitinases. a-amylase inhibitors might be toxic to insects through interference in the digestion of dietary carbohydrates. It is widely assumed that the proteinase inhibitors and oc-amylase inhibitors that accumulate in wounded tissue are produced as a normal defence against insects. Many lectins are toxic to insects, presumably through some deleterious interaction with intestinal glycoproteins. Chitinases might be toxic to insects if they are able to degrade the chitin layer of the peritropic membrane, which protects the insect’s gut epithelium.
Genes for Disease Resistance:
Several viral, fungal and bacterial diseases attack rice and cause serious yield losses. Sources of resistance to some diseases (blast and bacterial blight) have been identified within cultivated rice germplasm, and improved germplasm with resistance has been developed. However, sources of resistance to sheath blight are not available and only a few donors for resistance to tungro disease, caused by two virus particles, are known.
A highly successful strategy, termed coat-protein (CP)-mediated protection, has been employed against certain viral diseases such as tobacco mosaic virus in tobacco and tomato. When expressed in the transgenic crop, a chimeric gene made by combining a strong promoter with the virus gene encoding the capsid protein results in the accumulation of capsid protein in plant cells.
Such plants are resistant to infection by the virus from which the gene stems. While the mechanism of resistance is not understood, it appears that disassembly of the virus is inhibited and that this is due to the accumulated capsid protein and not to the mRNA.
CP genes for the two component viruses that cause tungro disease have been cloned and efforts are under way to express these genes in rice plants. A coat protein gene for rice stripe virus was introduced into two japonica varieties by electroporation of the protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of the total soluble proteins) and exhibited a significant level of resistance to virus infection, and this resistance was inherited.
In addition to CP cross-protection in RNA viruses, other mechanisms for transgenic virus resistance have been reported. Of particular relevance to transgenic tungro resistance is the report that the DNA virus (tomato golden mosaic virus) has been controlled in transgenic tobacco expressing the antisense product of the viral AL1 gene. The AL1 protein is involved in the replication of viral DNA. It may be possible to exploit a similar antisense approach in controlling rice tungro bacilliform virus, a DNA virus.
Sheath blight of rice is caused by, the pathogen Rhizoctonia solani, which has a wide host range. Transgenic tobacco and canola plants with enhanced resistance to R. solani have been obtained by introducing the bean chitinase gene under the control of the CaMV35S promoter. Similarly, Logemann transformed tobacco plants with a barley gene encoding a ribosome inactivating protein under the control of the wound inducible wun-2 promoter from potato. We are exploring the possibility of transforming rice with similar gene constructs to develop resistance to sheath blight.
Chitinases and glucanases degrade the major structural polysaccharides of the fungal cell wall. These enzymes have both a binding domain and a catalytic domain for their respective polysaccharides. Alone or in combination, they attack the growing hyphal tip and are potent inhibitors of fungal growth. About six chitinase genes have been identified in rice and are being manipulated to increase the level of resistance to fungal diseases.
Lin introduced a 1.1 kb rice genomic DNA fragment containing a chitinase gene through PEG-mediated transformation. The presence of this chimeric chitinase gene in T0 and T1 transgenic plants was detected by Southern blot analysis. Western blot analysis of transgenic plants and progeny revealed the presence of two proteins that reacted with the chitinase antibody.
Progeny from the chitinase positive plants were tested for resistance to sheath blight pathogen. The degree of resistance correlated with the level of chitinase expression.
Genes for Abiotic Stress Tolerance:
Transgenic mechanisms for resistance to abiotic stresses in rice may also be feasible. Tarczynski have reported the use of the mannitol-1-phosphate dehydrogenase gene, mtlD, from Escherichia coli to provide salt tolerance in transgenic tobacco. The mechanism of resistance involves the accumulation of mannitol in plant tissues. Given the importance of osmolytes in drought tolerance, such mechanisms of salt tolerance may also provide some protection against drought. Other abiotic stresses for which transgenic mechanisms of protection have been reported are heavy metal ions and oxidative stress.
Genes for Nuclear Male Sterility and Fertility Restoration:
Nuclear male sterility has been engineered in tobacco and oilseed rape by expression of a bacterial ribonuclease (barnase) in the tapetal cells that provide the nutrition to developing microspores. Tapetum specific expression of the ribonuclease was achieved through the use of a tapetum specific promoter from tobacco. Restoration of male fertility was achieved by crossing transgenic plants containing the barnase gene with transgenic plants expressing the bacterial gene encoding the barnase inhibitor barstar, again under the control of the tapetum specific promoter.
Sufficient barstar was produced in some crosses to block barnase activity. Such a restoration mechanism of nuclear male sterility would be valuable in rice and would permit diversification of limited sources of cytoplasmic male sterility for hybrid rice production.