Debasis Pattanayak

Production and characterization of somatic hybrids between Solanum tuberosum L. and S. pinnatisectum Dun.

Interspecific somatic hybrids between the dihaploid Solanum tuberosum and the wild species S. pinnatisectum Dun. were produced via protoplast fusion. Protoplast isolation, electrofusion, culture of post-fusion products and regeneration of calli/shoots were undertaken following optimized protocols. Regenerants were characterized for hybridity, ploidy and resistance to Phytophthora infestans (Mont.) de Bery, causal fungal pathogen of late blight disease. From a total of 126 regenerated macrocalli, 12 somatic hybrids were confirmed by possessing species-specific diagnostic bands of their corresponding parents as revealed by RAPD, SSRs and cytoplasmic-DNA analyses. Tetraploid status of the 12 hybrids was determined using flow cytometry analysis. Intermediate phenotypes for leaf, flower, and tuber characteristics and high male fertility were observed in field-grown hybrid plants. Hybrids were highly resistant to foliage late blight based on field assessment for two seasons. In contrast, moderate level of resistance to foliage blight was observed in hybrids based on the detached leaf assay under laboratory conditions. Overall, somatic hybrids with moderate levels of resistance to foliage blight were identified, and these will be useful for in situ hybridization in potato breeding efforts.

Plant RNA Interference Pathways: Diversity in Function, Similarity in Action

Small non-coding RNA-mediated gene-silencing pathways, collectively called RNA interference (RNAi), are involved in regulation of endogenous gene expression and plant defence. It is manifested through two broad classes of small non-coding regulatory RNAs, small interfering RNA (siRNA) and microRNA (miRNA). siRNAs, generated from cleavage of long hairpin RNA by RNase III-class endonuclease, Dicer-like, mediate transcriptional or post-transcriptional gene silencing. At transcriptional level, 24-nucleotide (nt)-long-siRNAs guide an effector complex for DNA methylation, which leads to heterochromatinisation of target loci and consequently transcriptional silencing. At post-transcriptional level, a different size class of 21-nt-long siRNAs guides a silencing complex, called RNA-induced silencing complex, for cleavage of target mRNA. cis-acting siRNAs are involved in plant defence against viruses and transposons, and trans-acting siRNAs regulate endogenous genes involved in plant growth. miRNAs are generated from processing of imperfect stem-loop RNA precursors by Dicer-like. They regulate plant growth and adaptive stress responses by either degradation or translational repression of target mRNAs.

Genetic diversity of late blight resistant and susceptible Indian potato cultivars revealed by RAPD markers

Twenty-four tetraploid Indian potato cultivars were characterized by using RAPD markers to assess diversity within and between late blight resistant and susceptible cultivars. Sixty-four random decamer primers generated802 fragments, ranging in size from 60–3200 bp, with 96.4% fragment polymorphism. Shannons index of diversity was used to quantify the degree of variability present within and between the variety types. Most of the diversity was detected within variety types, with 88% of variation being within and 12% being between the resistant and susceptible cultivars. No clear groupings based on late blight resistance and susceptibility or kinship was reflected on the dendogram. The late blight resistant cultivars exhibited higher variability compared to susceptible cultivars and they were more dispersed on the PCO plot.

AFLP fingerprinting and genotypic characterization of some serovars of Bacillus thuringiensis.

Amplified fragment length polymorphism (AFLP)-based fingerprinting of 24 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 13 EcoR1 (+2) and Mse1 (+3) primer combinations for genotypic characterization. A high degree of polymorphism was established among the Bt serovars. A total of 1107 fragments ranging from 30–850 bp were generated out of which 1106 were polymorphic. Discrimination rates of different primer combinations at various band levels (1–5) among different Bt serovars were more than 90%. Cluster analysis revealed very low similarity values, ranging from 7–50%, among the Bt serovars indicating their remarkable genetic diversity. AFLP analysis establishes the molecular relatedness between the serovars and serotypes.

Host-mediated gene silencing of a single effector gene from the potato pathogen Phytophthora infestans imparts partial resistance to late blight disease.

RNA interference (RNAi) has proved a powerful genetic tool for silencing genes in plants. Host-induced gene silencing of pathogen genes has provided a gene knockout strategy for a wide range of biotechnological applications. The RXLR effector Avr3a gene is largely responsible for virulence of oomycete plant pathogen Phytophthora infestans. In this study, we attempted to silence the Avr3a gene of P. infestans through RNAi technology. The P. infestans inoculation resulted in lower disease progression and a reduction in pathogen load, as demonstrated by disease scoring and quantification of pathogen biomass in terms of Pi08 repetitive elements, respectively. Transgenic plants induced moderate silencing of Avr3a, and the presence and/or expression of small interfering RNAs, as determined through Northern hybridization, indicated siRNA targeted against Avr3a conferred moderate resistance to P. infestans. The single effector gene did not provide complete resistance against P. infestans. Although the Avr3a effector gene could confer moderate resistance, for complete resistance, the cumulative effect of effector genes in addition to Avr3a needs to be considered. In this study, we demonstrated that host-induced RNAi is an effective strategy for functional genomics in oomycetes.

Genetic Engineering for Tolerance to Climate Change-Related Traits

Climate change is expected to introduce new challenges for sustainable crop production worldwide. High temperature, less water availability, and emergence of new pests and pathogens calls for changing strategies and using biotechnological interventions to meet these challenges to sustaining food supply. Engineering biotic and abiotic stress tolerance will require concerted and combined efforts by plant breeders and biotechnologists alike. Several genes have been identified to have potential in mitigating climate change effects. These can be broadly classified as single-action genes and multiple action genes. Single action genes include osmoprotectants, detoxifying, LEA, HSP, ANPs, and ion transporters which have incremental roles in providing abiotic stress tolerance. Multiaction regulatory genes provide an attractive strategy to improve crop plants as these genes activate a cascade of genes which act together to enhance stress tolerance. CBF/DREB, SNAC, MYB, HSF, and AREB are some candidate genes of this category. Signal transduction genes such as osmosensors, AHK1, SNF1-related kinases are potential candidate genes for engineering stress tolerance in the near future. For insect resistance cry genes will remain the ideal choice however, engineering biotic resistance will involve new technologies such as RNAi and micro RNAs for combating insects and pests. Regulatory genes and genes involved in signal transduction will assume great importance in developing cultivars adapted to the threats of climate change. Here we review the target traits and potential genes for engineering stress tolerance in crop plants to meet climate change challenges for food production.

Light-mediated Regulation of Nitrate Reductase in Higher plants

Regulation of nitrate reductase (NR) by light is a complex process and is manifested through gene expression, both at the level of transcription and translation, covalent modification of the enzyme and supply of reductant. Light induces nia gene transcription by some still unknown mechanism. Light induction of nia mRNA is also mediated via phytochrome. By using protein synthesis inhibitors it has been observed that light enhances translation of NR mRNA. Light induction of NR synthesis follows the circadian rhythmicity. Cytokinin increases NR activity by regulating NR expression at the transcriptional level. In response to light-dark transition, NR is quickly inactivated/activated by phosphorylation/dephosphorylation. Inactivation of NR involves phosphorylation of the serine-543, located at the hinge 1 region connecting MoCo domain and cyt b domain of NR, by a Mg2+ dependent protein kinase and subsequent binding of an inhibitor protein. A type 2A protein phosphatase dephosphorylates/activates NR in response to light signal. Light also regulates supply of reductant via photosynthesis for activity of NR. Whether light is absolutely necessary for NR activity is also discussed.

Comparative Proteomic and Nutritional Composition Analysis of Independent Transgenic Pigeon Pea Seeds Harboring cry1AcF and cry2Aa Genes and Their Nontransgenic Counterparts

Safety assessment of genetically modified plants is an important aspect prior to deregulation. Demonstration of substantial equivalence of the transgenics compared to their nontransgenic counterparts can be performed using different techniques at various molecular levels. The present study is a first-ever comprehensive evaluation of pigeon pea transgenics harboring two independent cry genes, cry2Aa and cry1AcF. The absence of unintended effects in the transgenic seed components was demonstrated by proteome and nutritional composition profiling. Analysis revealed that no significant differences were found in the various nutritional compositional analyses performed. Additionally, 2-DGE-based proteome analysis of the transgenic and nontransgenic seed protein revealed that there were no major changes in the protein profile, although a minor fold change in the expression of a few proteins was observed. Furthermore, the study also demonstrated that neither the integration of T-DNA nor the expression of the cry genes resulted in the production of unintended effects in the form of new toxins or allergens.

Silencing of HaAce1 gene by host-delivered artificial microRNA disrupts growth and development of Helicoverpa armigera

The polyphagous insect-pest, Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.

Bt Insecticidal Crystal Proteins: Role in Insect Management and Crop Improvement

Insect pests are one of the major constraints that crops face during their growth period. Management of these insect pests has been prioritised since time immemorial. The insecticidal crystal proteins produced by Bacillus thuringiensis offer tangible options for insect management. They have not only been effective as biopesticides but also proved to be successful in plant biotechnology. A plethora of cry genes have been identified and used for the development of various transgenic crops of economic importance against insect pest. The success of Bt cotton globally demonstrates the utility of these genes in crop improvement programmes. This chapter describes the enormous information available on the various types and classes of cry genes and their mode of action on insects for the development of pest-resistant plants.