D. Sudhakar

An improvedAgrobacterium-mediated transformation protocol for recalcitrant elite indica rice cultivars

We report here a high-efficiency transformation protocol for recalcitrant indica rice cultivars IR64 and IR72 with the selectable marker genehph and thegusA reporter gene. Factors that favor high-efficiency transformation were found to be use of 2-month-old mature seed-derived embryogenic calli, maltose as a source of carbon, a higher concentration of 2,4-dichlorophenoxyacetic acid, and both phytagel and agar as gelling agents. The putative transgenic (T0) plants were analyzed for integration of the transgene through polymerase chain reaction and Southern blotting analyses. Various factors thought to be responsible for increased transformation efficiency are discussed.

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.

An Efficient Rice Transformation System Utilizing Mature Seed-derived Explants and a Portable, Inexpensive Particle Bombardment Device

We developed a practical and efficient gene transfer system for indica rice utilizing mature-seed derived explants and a simple bombardment device which uses compressed helium for accelerating DNA-coated metal particles. Unlike instruments which have been described in the literature previously, this new bombardment device, which is an improvement of the particle inflow concept, does not require vacuum. This attribute simplifies the transformation procedure significantly and it makes rice transformation technology accessible to laboratories which may not have the resources to invest in more expensive particle bombardment instruments. We determined experimentally that we could recover transgenic rice plants utilizing three different particle bombardment instruments at comparable frequencies.

Transgenic rice as a system to study the stability of transgene expression: Multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds

Abstract The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11 diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding lines grown in different parts of the world.

A high throughput functional expression assay system for a defence gene conferring transgenic resistance on rice against the sheath blight pathogen, Rhizoctonia solani

Should fruits of transgenic research on engineering resistance against crop diseases reach needy farmers, such resistance needs to be characterised thoroughly. Assessment of transgenic resistance conferred by over-expression of defence genes under field conditions or in greenhouse is a time-consuming process. It becomes often necessary, though laborious, to screen a huge population of putative transgenic lines for their resistance against a target disease. Most standard evaluation systems (SES) available for individual plant � /pathogen interactions are applicable only to field conditions, and there has been a constant need for evolving a rapid laboratory method for assessment of efficacy of defence genes deployed against plant diseases. In this paper, we describe a high throughput, yet simple method for functional assessment of a rice chitinase gene chi11 known to confer resistance against Rhizoctonia solani , the rice sheath blight (ShB) pathogen. Accuracy of this method as compared with a SES already available for assessing field performance of test-varieties against ShB is discussed. # 2003 Elsevier Ireland Ltd. All rights reserved.

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice chi11 gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T2 homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T0) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T4 generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T4 generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.

Transgenic resistance by N gene of a Peanut bud necrosis virus isolate of characteristic phylogeny.

The nucleocapsid protein (N) gene of a Tospovirus devastating tomato crop in the south Indian state of Tamil Nadu was cloned and characterized. The high identity of the cloned sequence to a Peanut bud necrosis virus (PBNV) tomato isolate (97.8/99.6% nucleotide/amino acid) and a PBNV peanut isolate (94.4/96.3% nucleotide/amino acid) identified the Tospovirus as an isolate of PBNV, designated PBNV Coimbatore tomato (PBNV CT) isolate. Phylogenetic analysis of PBNV CT N gene provided useful insights into the movement and evolution of PBNV within Indian Territory. The characteristic phylogeny of PBNV CT N gene implied its potential to be an efficient transgene to confer effective PBNV resistance on crop plants. The efficacy of PBNV CT N gene in conferring PBNV resistance was studied by generating tobacco (Nicotiana tabacum L. cv Wisconsin) lines transgenic to the sense or antisense version of the gene. Several transgenic lines showed transgenic mRNA and/or protein accumulation, ranging from very high to undetectable levels, accompanied by different degrees of PBNV resistance. The undetectable or very low levels of transgene transcripts in certain PBNV-resistant sense or antisense N gene transgenic lines suggested RNA-mediated resistance by post-transcriptional gene silencing (PTGS) mechanism. However, PBNV resistance of certain transgenic lines with high levels of N gene transcripts was suggestive of possible operation of RNA-mediated non-PTGS mechanism(s) of resistance in those lines. Moreover, the high levels of N protein in certain PBNV-resistant sense N gene transgenic lines suggested protein-mediated resistance. The results predict the potential of PBNV CT N gene to confer effective PBNV resistance on tomato and other economically important crops.

Engineering Disease Resistance in Rice

Rice diseases cause substantial yield loss in rice. Through conventional breeding, resistance genes (R-gene) were transferred into elite rice genotypes particularly against the fungal blast and bacterial blight diseases. Main drawback of this approach is that, in the long term, breakdown of resistance occurs due to evolution of new virulent pathogen strains. In the current scenario, developing rice with durable broad-spectrum resistance through genetic transformation is gaining importance. In this direction, genetic transformation of rice was being carried out for the past two decades via expressing pathogenesis-related (PR) proteins, antimicrobial peptide, and genes governing signaling pathways as well as elicitor proteins. In spite of several reports, the expression of PR proteins and antimicrobial peptides did not yield desirable disease control in rice. Better understanding of disease resistance mechanism in plants helped in identifying critical transcription factors (TFs) involved in disease resistance. Overexpression of NPR1 encoding non-expressor of pathogenesis-related protein 1 and OsWRKY45 transcription factors in rice showed strong disease resistance to multiple pathogens and at the same time resulted in fitness cost. Recently, transgenic rice with high level of resistance to important rice diseases was achieved by expressing NPR1 and WRKY45 under tissue-specific/pathogen-responsive promoter; thereby agronomic traits are not altered. Rice transformants expressing the pathogen-derived elicitor proteins particularly from rice blast pathogen, Magnaporthe oryzae is a promising approach for imparting broad-spectrum disease resistance without yield penalty. Host-delivered RNAi technology is the latest of the approaches toward enhancing disease resistance against sheath blight and viral disease of rice. Recently, genome-editing tools are being deployed in rice to enhance resistance against diseases of rice.