K. Kumar

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.

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.

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.

Identification of nif genes of heterotrophic and endophytic diazotrophs associated with rice (Oryza sativa L.,) by targeted DNA finger printing.

Heterotrophic and endophytic diazotrophs were isolated from rhizosphere soil, rhizoplane, roots and stems of different rice varieties. A total of thirteen isolates obtained were subjected to acetylene reduction assay (ARA) and eight isolates recorded significant amount of nitrogenase activity in a range of 31.65 to 91.95 nmoles of ethylene mg-1 cells h-1 . Targeted PCR fingerprinting using nif H primers generated specific DNA band of approximately 750 bp, confirming the presence of nif genes in these isolates. Two isolates of heterotrophic diazotrophs, HDM 7 and HDT 1, generated multiple bands ranging from 500 – 1000 bp.

Combination of driselase and lysing enzyme in one molar potassium chloride is effective for the production of protoplasts from germinated conidia of Fusarium verticillioides

Various cell wall degrading enzymes and the protoplasting media were evaluated for the production of protoplast in Fusarium verticillioides. Among the various enzymes tested, driselase at 12.5 mg/ml in 1 M KCl protoplasting medium produced the maximum number of protoplast. Next to driselase, lysing enzyme at 10 mg/ml in 1.2 M MgSO4 protoplasting medium was found to be the second best enzyme for the production of protoplast. More interestingly, the combined use of driselase @ 12.5 mg/ml and lysing enzyme @ 10 mg/ml in 1 M KCl exhibited the additive effect on protoplast formation. Germinated conidia of F. verticillioides are the most susceptible fungal material for protoplast production. The use of sucrose at 1.2 M in the regeneration medium supported the maximum regeneration of protoplast. From the present study, we recommend driselase (12.5 mg/ml) and lysing enzyme (10 mg/ml) in 1 M KCl protoplasting medium and germinated conidia of F. verticillioides for the maximum production of protoplasts and 1.2 M sucrose is the best osmoticum for the regeneration of protoplasts.

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.