Bir Pal Singh

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.

Improvement for Bacterial Wilt Resistance in Potato By Conventional and Biotechnological Approaches

Bacterial wilt (BW) of potato caused by the bacterium Ralstonia solanacearum (Rs) is considered a serious problem particularly in tropical, subtropical and warm temperate regions. Chemical-, cultural- and biological control of BW has limited success. Thus, the control of BW through resistance breeding and biotechnology is considered to be very important and necessary. Rs is considered a ‘species complex’ and has significant variation at physiological, serological and genetic levels. The bacterium has an unusually wide host range with over 400 hosts belonging to more than 50 botanical families. A large number of Solanum species have been screened for resistance to this bacterium, but so far no Solanum species has been found to have complete immunity. A high degree of resistance to Rs was found only in S. phureja, a diploid relative of cultivated tetraploid potatoes. The resistance has been transferred from S. phureja to cultivated potatoes through introgression breeding as well as somatic hybridization. Although moderate to highly resistant potato varieties have been released, high frequency of latent infection in tubers is still a major problem. Further, the resistant cultivars are not adapted to different agro-climatic zones and are not effective against all the strains of the pathogen. Biotechnological approaches involving the use of antimicrobial peptides, plant defence genes and plant resistance genes are being tried. This paper reviews the global situation with regard to screening of genetic resources and their utilization in resistance breeding for BW in potato and also the status and the opportunities that biotechnology offers to combat this disease.

Integrated Management of Whitefly, Bemisia tabaci (Gennadius) and Potato Apical Leaf Curl Virus in India

This paper reviews the importance of Potato apical leaf curl virus (PALCV) in India, the biology of its major vector Bemisia tabaci and possible control measures of PALCV and B. tabaci. PALCV has appeared as a new problem in potato growing areas of northwestern plains of India. The first report of PALCV was made from Hissar around 2000. PALCV is a whitefly (B. tabaci) transmitted viral disease and the symptoms are manifested primarily in the form of upward or downward curling of leaves, with 40–75% infection being recorded in cultivars grown in the Indo-Gangetic Plains of India. B. tabaci is a polyphagous pest that feeds on numerous fibre (particularly cotton), food, vegetable and ornamental plants. B. tabaci produces many generations in a year and reaches high population densities. To check the disease incidence, whitefly suppression using imidacloprid either as seed treatment or as foliar application early in the season is recommended. To minimize within-field spread of viruses, removal of virus-infected plants is suggested. Application of mineral and vegetable oils has been found to inhibit virus transmission and possibly can help to avoid difficulties with insecticide resistance in whiteflies.

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.

A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato, in infested soil

A fast and simple polymerase chain reaction method has been developed for detection of Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR amplification without a precipitation step. This amplification leads to detection of pathogen in infested soils before planting of crop. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. infestans detection to confirm positive inoculum level in potato seeds and elsewhere. With increasing amounts of standard DNA templates, the respective threshold cycle (Ct) values were determined and a linear relationship was established between these Ct values and the logarithm of initial template amounts. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of P. infestans oospores on a large-scale basis.

Impact of Climate Change on Potato

Potato, a native of temperate region was introduced in India and adapted to tropical short-day conditions where it covers >80% of total potato area. The crop is very sensitive to climatic variability, and therefore, the climate change and global warming will have a profound effect on potato growth in India. Even moderately high temperature drastically reduces tuber yield without much affecting the photosynthesis and total biomass production. Besides, high temperature affects tuber quality by causing heat sprouting and internal necrosis. The effect of elevated CO2 concentration suggests positive effect on growth and yield with only few negative influences. Study on the impact assessment of climate change on potato production using INFOCROP-POTATO model showed that the potato production will increase by 11.12% at elevated CO2 of 550 ppm and 1°C rise in temperature but further increase in CO2 to 550 ppm with a likely rise in temperature to 3°C will result in decline in potato production by 13.72% in the year 2050. The effect of elevated temperature on late blight at global level revealed that with rise in global temperature of 2°C, there will be lower risk of late blight in warmer areas ( 13°C) with early onset of the epidemics. Global warming will have a serious repercussion on viral diseases through the altered biology of insect vectors. The increase in temperature will enhance vector population thereby increasing the number of insecticide sprays for keeping the vector population in check. The effects of temperature and CO2 on potato growth and development, productivity, diseases and insect pests, and quality have been discussed in the present communication. Besides, regional vulnerability to climate change and adaptation measures for climate change and global warming are also discussed.

Allele Mining in Solanum Germplasm: Cloning and Characterization of RB-Homologous Gene Fragments from Late Blight Resistant Wild Potato Species

The late blight disease can be managed by introduction of resistance (R) genes from the wild Solanum species into the cultivated potato. The R genes are mostly comprised of the nucleotide binding site-leucine rich repeat (NBS-LRR) domains and share nucleotide sequence homology in the crop species. In this study, we used potato R gene-specific primers to amplify homologous genes from wild species. A total of 39 wild species were tested for late blight resistance by challenge inoculation of Phytophthora infestans under controlled conditions. Of these, only 15 species were highly resistant (HR) and these were PCR (polymerase chain reaction) amplified by 53 primers representing 21 R genes of potato. Further, only single, distinct, and reproducible gene fragments were cloned and sequenced. Following sequence processing and analysis, 17 non-redundant sequences of RB-homologous genes were identified with uninterrupted open reading frames (ORFs) and nucleotide sequence homologies to known late blight R genes. Finally, 17 RB-homologous gene fragments amplified by the primers of the RB gene were isolated from 11 wild species. The isolation and characterization of 17 RB-homologous gene fragments from wild potato species may serve as an important genomic resource for the novel gene discovery in late blight resistance breeding programs.

Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato

BackgroundLate blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance.ResultsPopulations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order.ConclusionThis study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype.

Translation initiation in plants: roles and implications beyond protein synthesis

Protein synthesis is a ubiquitous and essential process in all organisms, including plants. It is primarily regulated at translation initiation stage which is mediated through a number of translation initiation factors (eIFs). It is now becoming more apparent that in addition to synthesis of proteins, eIFs also regulate various aspects of plant development and their interaction with environment. Translation initiation factors, such as eIF3, eIF4A, eIF4E, eIF4G, and eIF5A affect different processes during vegetative and reproductive growth like embryogenesis, xylogenesis, flowering, sporogenesis, pollen germination, etc. On the contrary, eIF1A, eIF2, eIF4, and eIF5A are associated with interaction of plants with different abiotic stresses, such as high temperature, salinity, oxidative stress, etc. Similarly, eIF4E and eIF4G have roles in interaction with many viruses. Therefore, the translation initiation factors are important candidates for improving plant performance and adaptation. A large number of genes encoding eIFs can functionally be validated and utilized through genetic engineering approaches for better adaptability and performance of plants by inhibiting/minimizing or increasing expression of desired eIF(s).

Uniplex and duplex PCR detection of geminivirus associated with potato apical leaf curl disease in India

Apical leaf curl disease has emerged as a new disease in potato during the last decade in India due to a change in planting date and an increased whitefly population. Its incidence is on the rise threatening the cultivation of potato across the country. Hence, a PCR assay was developed for the detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV-Potato) which is the causal agent of apical leaf curl disease in potato. Primers specific to the coat protein (AV1) and replicase (AC1) gene regions were designed and used for standardization of the PCR. Some of the primers (LCVCPF1/LCVCPR1, LCVREPF2/LCVREPR2, LCrep1F/LCrep2R) could detect the virus in 2.4-0.24pg of total DNA of infected plant. A duplex PCR assay was optimized with the selected coat protein gene specific primers and primers specific to potato urease gene, a housekeeping gene served as an internal check. The suitability of these primers was examined for detection of the virus in 80 potato apical leaf curl disease samples from 11 different potato growing states of India and also from micro-plants grown in tissue culture. The selected coat protein primer pair (LCVCPF1/LCVCPR1) was found to be conserved in all 80 isolates except for a few isolates, which had a single nucleotide substitution in the forward primer sequence. These substitutions did not interfere with amplification of the coat protein gene. The primers could detect the virus using a print-capture PCR assay both in the presence and absence of an internal control. These results indicate the robustness of the PCR assay for virus indexing of mother stocks in the seed production system.