Sanjeev Kumar

Effects of explant age, germination medium, pre-culture parameters, inoculation medium, pH, washing medium, and selection regime on Agrobacterium -mediated transformation of tomato

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato (Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog’s (MS) basal medium containing 8.9xa0μM benzyladenine (BA) produced the most suitable explant material. Sixxa0days of explant pre-culture and 5xa0min inoculation with Agrobacterium culture in MS medium, containing 8.9xa0μM BA, 9.3xa0μM kinetin, and 0.4xa0mgxa0l−1 thiamine at pHxa05.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20xa0mgxa0l−1 in the selection medium for the initial 10xa0d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3xa0%. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5xa0% of the independent transgenic lines from self-fertilized T1 progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars.

Detection of genetic components of variation for some biometrical traits in Linum usitatissimum L. in sub-mountain Himalayan region

SummaryGene action for seed yield and its component traits in linseed were studied using triple test cross analysis. The main objective was to determine the nature and magnitude of genic effects for different biometrical traits under variable environments that could support further improvements of linseed productivity using appropriate breeding methodology. Epistasis was observed for technical height in E1, 1,000-seed weight in both the environments, plant height, 1,000-seed weight and biological yield per plant in combined over environments. Both [i] and [jxa0+xa0l] type of epistatic interactions were significant for all these traits except technical height and 1,000-seed weight in E1, for which only [i] type of interaction was significant. Additive (D) gene action was significant for most of the traits whereas non-additive (H) gene action was significant for only seeds per capsule (E1, E2 & E3), 1,000-seed weight (E2 & E3), technical height (E2) and biological yield per plant (E3). Additive type of gene action was preponderant for plant height (E1, E2 & E3), technical height (E1, E2 & E3), capsules per plant (E3), seed yield per plant (E1 & E3), 1,000-seed weight (E1, E2 & E3), straw yield per plant (E1 & E3), and harvest index (E1, E2 & E3), whereas non-additive type of gene action was preponderant for seeds per capsule (E1, E2 & E3), and biological yield per plant (E3). The presence of additive gene action for most of the traits including seed yield per plant implies that early generation selection may be useful for the improvement of these traits. However, for traits showing both additive and dominance components of variance, heterosis breeding may be useful but chances of exploiting hybrid vigour through hybrid varieties in linseed due to its autogamous nature are bleak at present. The autogamous nature of crop and absence of genetic-cytoplasmic male sterility warrants diallel selective mating/biparental mating or recurrent selection followed by pedigree method of selection for its improvement.

Identification of putative candidate genes for red rot resistance in sugarcane ( Saccharum species hybrid) using LD-based association mapping

Red rot is a serious disease of sugarcane caused by the fungus Colletotrichum falcatum that has a colossal damage potential. The fungus, prevalent mainly in the Indian sub-continent, keeps on producing new pathogenic strains leading to breakdown of resistance in newly released varieties and hence the deployment of linked markers for marker-assisted selection for resistance to this disease can fine tune the breeding programme. This study based on a panel of 119 sugarcane genotypes fingerprinted for 944 SSR alleles was undertaken with an aim to identify marker-trait associations (MTAs) for resistance to red rot. Mixed linear model containing population structure and kinship as co-factor detected four MTAs that were able to explain 10–16xa0% of the trait variation, individually. Among the four MTAs, EST sequences diagnostic of three could be BLAST searched to the sorghum genome with significant sequence homology. Several genes encoding important plant defence related proteins, viz., cytochrome P450, Glycerol-3-phosphate transporter-1, MAP Kinase-4, Serine/threonine-protein kinase, Ring finger domain protein and others were localized to the vicinity of these MTAs. These positional candidate genes are worth of further investigation and possibly these could contribute directly to red rot resistance, and may find a potential application in marker-assisted sugarcane breeding.

Marker-trait association study for sucrose and yield contributing traits in sugarcane (Saccharum spp. hybrid)

Linkage disequilibrium (LD)-based marker-trait association (MTA) was used to identify markers for sucrose and yield contributing traits in a panel of 108 sugarcane genotypes from sub-tropical India. Population structure (Q), kinship (K), and MTA study exploited a set of 989 marker loci generated from 123 genomic- and expressed sequence tag-SSR primers. The mixed linear model (MLM) coupled with a modified algorithm for population structure (Q) analysis was able to control both type I and type II errors and provided a deeper understanding of the genetics, population stratification and its manifestations on LD in the sugarcane genome. Significant associations were identified for four markers with cane diameter, seven markers each with cane length and number of millable canes (NMCs), eleven markers with number of nodes, six with sucrose per cent, and five markers with average cane weight. A total of 15 markers stable for all the 3xa0years of study explained 57xa0% trait variation for NMCs, 34xa0% for cane width, 27xa0% for cane length, 20xa0% for sucrose content, and 19xa0% for number of nodes. The frequent deviation of structure-based profiles from pedigree-based grouping in this complex heterozygous system reinforced the importance of using genotypic data for selection and breeding. The results contribute to a deeper insight of the complex genome and the identified MTAs could be exploited to fine-tune marker-assisted breeding programmes in genetically complex sugarcane crop.

Analysis of Genetic Differentiation and Phylogenetic Relationships among Sugarcane Genotypes Differing in Response to Red Rot

The selection of parents is the most crucial step in any breeding programme. A better understanding of genetic diversity among the available genotypes would help the breeder to make better crosses. Advancements of DNA marker techniques in many crops have supplemented the morphological traits with molecular markers to identify the diverse genotypes. The genetic diversity of 30 sugarcane genotypes differing in response to red rot resistance was carried out using simple sequence repeat (SSR) markers. The polymorphism information content (PIC) ranged from 0.216 to 0.813 with an average of 0.525. The highest values (0.86) of proportion of polymorphic loci (P) and expected heterozygosity (He) were obtained in the susceptible population (S) and moderately resistant population (MR), respectively. FST values between populations ranged from −0.043 to 0.041. However, AMOVA did not show much variation among the groups. Cluster analysis clearly distinguished all the genotypes from each other. The resistant genotypes namely ISH150 and SES594 emerged out to be most distinct genotypes, whereas the rest of the genotypes could be grouped in two broad clusters separating the moderately resistant and susceptible sugarcane genotypes. The clustering pattern was fairly supported by Mantel’s test (rxa0=xa00.894) and high bootstrap value (75.0%). Thus, information given in the present study can be used in genetic resource management as well as in broadening the genetic base of cultivated sugarcane for red rot and selection of suitable parent for generating the mapping population for tagging the red rot resistance gene(s) in sugarcane.

Genetic and molecular characterisations of Tomato leaf curl virus resistance in tomato (Solanum lycopersicum L.)

SUMMARY Four different F2 populations were developed involving Solanum habrochaites accession ‘EC-520061’ that is highly resistant to Tomato leaf curl virus (ToLCV) disease and four ToLCV-susceptible cultivars of tomato (S. lycopersicum L.), ‘Punjab Chhuhara’ (PBC),‘H-24’, ‘H-86’, and ‘DVRT-2’.The segregation ratios of tomato leaf curl disease development in all four F2 populations were 13:3 (resistant:susceptible), indicating a dominant inhibitory gene for leaf curl disease resistance, and 3:1 (indeterminate:determinate) indicating a single dominant gene for plant growth habit. The patterns of inheritance were shown to be additive with dominant non-allelic gene interactions for the traits of ToLCV resistance and fruit yield. Using bulk segregant analysis (BSA) and one of the F2 populations (‘PBC’ X ‘EC-520061’) above, two SSR markers (SSR218170–145 and SSR304158–186) were identified for ToLCV resistance. The SSR marker SSR218170–145 was located at 15 cM on chromosome 10, and the marker SSR304158–186 was located at 35 cM on chromosome 7. This study will support the development of new ToLCV- resistant tomato varieties via breeding programmes.

Trichoderma-Mediated Suppression of Red Rot of Sugarcane Under Field Conditions in Subtropical India

AbstractThirty-eight isolates of Trichoderma spp. established from the rhizosphere of sugarcane were characterized using a multiplex PCR assay and further assessed for the production of hydrolytic enzymes chitinase and cellulase. The results of multiplex PCR assay successfully identified 29 isolates and revealed T. harzianum to be the predominant species (21 isolates) in sugarcane rhizosphere followed by T. longibrachiatum (8 isolates). In enzymatic assays, chitinase production was recorded in 18 isolates and cellulase production observed in 17 isolates. However, there was a considerable variability in both chitinase and cellulase production potential across the isolates. Three T. longibrachiatum isolates (STr-52, STr-83 and STr-108) exhibited high production of both chitinase and cellulase. Talc formulation of two promising isolates (STr-83 and STr-108) was prepared and evaluated in the field conditions for their potential to suppress red rot under three different delivery systems, viz. sett treatment, soil application and their combination. All Trichoderma treatments resulted in considerable reduction in red rot (29.5–56.3%) over untreated control. However, the level of reduction accorded was much higher when the Trichoderma isolates were applied as a combination of sett and soil treatment (>u200953% reduction) as compared to soil application (43–49% reduction) or sett treatment alone (<u200935% reduction). Delivery of Trichoderma isolates as a combination of sett and soil treatment also exhibited highest NMCs and yield over untreated control. The application methods of talc formulations of these two isolates offer a feasible and effective option for large-scale application of these isolates for the management of red rot disease in sugarcane growing regions.n

Characterization of leaf transcriptome, development and utilization of unigenes-derived microsatellite markers in sugarcane ( Saccharum sp. hybrid)

Sugarcane (Saccharum species hybrid) is the major source of sugar (>u200980% sugar) in the world and is cultivated in more than 115 countries. It has recently gained attention as a source of biofuel (ethanol). Due to genomic complexity, the development of new genomic resources is imperative in understanding the gene regulation and function, and to fine tune the genetic improvementxa0of sugarcane. In this study, a cDNA library was constructed from mature leaves so as to develop ESTs resources which were further compared with nucleotide and protein databases to explore the functional identity of sugarcane genes. The non-redundant ESTs (unigenes) were categorized into 18 metabolic functions. The major categories were bioenergetics and photosynthesis (4%), cell metabolism (5%), development related protein (3%), membrane-related, mobile genetic elements (5%), signal transduction (2%), DNA (1%), RNA (1%) and protein (2%) metabolism, other metabolic processes (3%), transcription factors (1%), transport (4%) and proteinsxa0related to stress/defense (4%). From 540 unique ESTs, 212 simple sequence repeats were identified, of which 206 were from 463 singlets and six were mined from 77 contig sequences. A total of 540 unique EST sequences were used for SSR search of which 97 (17.9%) contained specified SSR motifs, generating 212 unique SSRs. The genes characterized in this study and the EST-derived microsatellite markers identified from the cDNA library will enrich genomic resources for association- and linkage-mapping studies in sugarcane.

Identification of marker-trait associations for morphological descriptors and yield component traits in sugarcane

Ninety two sugarcane varieties from sub-tropical India were subjected to molecular profiling with 174 simple sequence repeat markers and characterized for 23 qualitative (morphological descriptors) and nine quantitative traits that directly or indirectly contribute to yield and juice quality. Using STRUCTURE-based population stratification study and a mixed linear model for marker-trait association (MTA) analysis, a total of 60 MTAs were identified for 22 qualitative traits that were able to explain a significantly higher (up to 40%) proportion of the phenotypic variations compared to all the previous reports of MTA studies in sugarcane. In addition, 21 MTAs stable over the three years of study were also identified for nine quantitative traits that explained 16–37% of the total trait variation. It could be concluded that the qualitative traits that are governed mostly by one or a few genes are more responsive to MTA studies and hence have a better potential to be adopted in marker-assisted breeding programmes in sugarcane. The MTAs identified in this study could also find significant applications in upcoming more stringent IP regime, which may necessitate tracking of specific alleles integrated in breeding programmes.