Jagdeep Singh Sandhu

High throughput in vitro micropropagation of sugarcane (Saccharum officinarum L.) from spindle leaf roll segments: Cost analysis for agri-business industry

High throughput micropropagation (HTM) protocol in sugarcane through direct shoot regeneration comprising five stages, was developed for agri-business industry. The distinction of the protocol lies in direct adventitious shoot regeneration without an intervening callus phase and liberty over the number of subculture passages leading to high rates of synchronous plant production. Stage 0 dealt with selection and maintenance of field-grown stock plants; Stage I marked the initiation of in vitro propagation on culture initiation medium using spindle leaf roll segments from apical tops; Stage II involved five passages (of 14xa0days each) on shoot multiplication medium followed by one additional passage (of 10xa0days) on shoot multiplication-cum-elongation medium. The shoot multiplication rates ranged from 4 to 25-fold in CoPb 91 and CoJ 83 cultivars, respectively; Stage III involved in vitro rooting on root induction medium and hardening for 14xa0days at each step; Stage IV comprised separation of clumps into single plantlets and transfer to soil. The complete plants were produced in 157xa0days with 97xa0percent survival rate. The fidelity of the protocol for agri-business industry was tested by producing approximately 0.1 million saleable HTM sugarcane plants in a small scale (150xa0m2) tissue culture unit. The recurring (energy, labour, media, culture container, transplanting material) and non-recurring (laboratory infrastructure, glasshouse, equipment) parameters accounted for 75.56 and 24.44xa0percent production costs, respectively. The low-cost options for media, culture containers and transplanting material were incorporated and cost per plant was calculated to be US

Genetics of ascochyta blight resistance in chickpea

0.13. The HTM protocol was adopted by two sugar mills, and the HTM plants produced in our tissue culture unit were sold for seed multiplication to eight sugar mills and 430 farmers throughout North India.

Inheritance of a plant type mutant and its utilization in chickpea

Genetics of resistance to ascochyta blight was studied using different generations of fifteen crosses of chickpea (Cicer arietinum L.). Six parents comprising two susceptible varieties GL 769, C 214 and four resistant lines GG 1267, GL 90168, GL 96010 and GL 98010 were used to develop one Sxa0×xa0S, eight Sxa0×xa0R and six Rxa0×xa0R crosses and some of the back crosses and F3 generations were developed. Field screening technique was used to evaluate the different generations for disease reaction using mixture of ten prevalent isolates (ab1–ab10) of ascochyta blight (Ascochyta rabiei). Inheritance study showed digenic recessive control of resistance in the cross GL 769xa0×xa0C 214, whereas monogenic recessive control of resistance was found in the crosses GL 769xa0×xa0GL 98010 and C 214xa0×xa0GL 98010. Digenic dominant and recessive control of resistance was found in the crosses GL 769xa0×xa0GG 1267 and C 214xa0×xa0GG 1267 while the crosses GL 769xa0×xa0GL 90168 and C 214xa0×xa0GL 96010 showed the monogenic dominant control of resistance. Trigenic dominant and recessive control of resistance was observed in the crosses GL 769xa0×xa0GL 96010 and C 214xa0×xa0GL 90168. Allelic relationship studies showed that three resistant parents viz., GG 1267, GL 96010 and GL 90168 possessed allelic single dominant gene for resistance. Besides, GG 1267 possessed two minor recessive genes for resistance, one of them was allelic to the minor recessive gene possessed by GL 90168 and other with GL 96010. The resistant parents GL 90168 and GL 96010 possessed non-allelic minor gene for resistance. The resistant parent GL 98010 possessed two minor recessive genes for resistance which were allelic to respective single recessive gene for resistance possessed by the susceptible parents GL 769 and C 214. The susceptible parents GL 769 and C 214 also possessed single independent inhibitory dominant susceptibility gene. The inhibitory gene was epistatic to the corresponding recessive gene for resistance.

Single step direct transgenic plant regeneration from adventive embryos of agro-infected sugarcane ( Saccharum spp.) spindle leaf roll segments with assured genetic fidelity

SummaryA macro-mutant, E 100Y(M) in chickpea (Cicer arietinum L.) was found to affect several plant and seed characters. For plant type monogenic inheritance was observed. A single pair of recessive genes pt/pt was ascribed to this mutant. The mutant locus seemed to be exerting pleiotropic action. The utilization of this mutant for chickpea improvement has been discussed.

Pod borer resistant transgenic pigeon pea (Cajanus cajan L.) expressing cry1Ac transgene generated through simplified Agrobacterium transformation of pricked embryo axes

AbstractnSingle step direct transgenic plant regeneration from agro-infected sugarcane (Saccharum spp.) spindle leaf roll segments with assured genetic fidelity is reported. The pre-cultured (96xa0h) leaf segments of cultivar CoJ 83 were incubated (10xa0min) with intermittent shaking in Agrobacterium suspension (OD 1.0–1.2) carrying antifungal gene encoding β-1,3-glucanase under the control of CaMV 35S promoter and NOS terminator, in presence of acetosyringone. Thereafter, the segments were co-cultivated (72xa0h) and transferred for shoot regeneration. The scanning electron micrographs of sectioned leaf segments undergoing in vitro shoot regeneration revealed occurrence of globular structures in clusters near cut leaf edges within 10xa0days of incubation. In each cluster, two to ten structures of varying sizes occurred concurrently. The sectioning of globular structures confirmed that these were arising directly from pre-embryogenic determined palisade cells, had unicellular origin and were referred to as adventive embryos. The adventive embryos successively expressed their potential for direct shoot regeneration without callus interphase in 3xa0weeks and transgenic plant formation within a short period of 8xa0weeks. The integration of β-1,3-glucanase transgene and its expression in T0 plants were determined by reverse transcription-PCR and quantitative real time-PCR, respectively. Simple sequence repeat DNA markers did not detect any polymorphism in the transgenic plants, pointing towards assured genetic fidelity of the plants.

Red rot resistant transgenic sugarcane developed through expression of β-1,3-glucanase gene

Pod borer resistant transgenic pigeon pea (Cajanus cajan L.) plants expressing cry1Ac transgene were generated through Agrobacterium transformation of differentiated and meristematic cells in pricked embryo axes. The uptake of transgene by such cells was expected to produce chimeric T0 plants that were grown to maturity without characterization. The putative transformants were identified from thousands of T1 and T2 seedlings following high throughput paromomycin screening. The presence of transgene in putative T1 and T2 plants was established by polymerase chain reaction and its expression was confirmed in six T2 plants by reverse transcription-PCR and quantitative real time-PCR. The T2 plant PAU 881-21 showed upto 10-fold higher cry1Ac transgene expression as compared to non-transgenic plant. The in vitro bioassay of pods and leaves from PAU 881-21 using third instar Helicoverpa armigera larvae demonstrated 97.78u2009% larval mortality just after 48xa0h, pointing towards direct correlation between transgene expression and larval mortality. The pod borer resistant T2 plant PAU 881-21 was phenotypically normal, self fertile and the transgene segregated in 3:1 Mendelian pattern in its T3 progeny.

Control of Fungal Diseases in Agricultural Crops by Chitinase and Glucanase Transgenes

Sugarcane (Saccharum spp.) is a commercially important crop, vulnerable to fungal disease red rot caused by Colletotrichum falcatum Went. The pathogen attacks sucrose accumulating parenchyma cells of cane stalk leading to severe losses in cane yield and sugar recovery. We report development of red rot resistant transgenic sugarcane through expression of β-1,3-glucanase gene from Trichoderma spp. The transgene integration and its expression were confirmed by quantitative reverse transcription-PCR in first clonal generation raised from T0 plants revealing up to 4.4-fold higher expression, in comparison to non-transgenic sugarcane. Bioassay of transgenic plants with two virulent C. falcatum pathotypes, Cf 08 and Cf 09 causing red rot disease demonstrated that some plants were resistant to Cf 08 and moderately resistant to Cf 09. The electron micrographs of sucrose storing stalk parenchyma cells from these plants displayed characteristic sucrose-filled cells inhibiting Cf 08 hyphae and lysis of Cf 09 hyphae; in contrast, the cells of susceptible plants were sucrose depleted and prone to both the pathotypes. The transgene expression was up-regulated (up to 2.0-fold in leaves and 5.0-fold in roots) after infection, as compared to before infection in resistant plants. The transgene was successfully transmitted to second clonal generation raised from resistant transgenic plants. β-1,3-glucanase protein structural model revealed that active sites Glutamate 628 and Aspartate 569 of the catalytic domain acted as proton donor and nucleophile having role in cleaving β-1,3-glycosidic bonds and pathogen hyphal lysis.

Generation of interspecific hybrids between Trifolium vesiculosum and T. alexandrinum using embryo rescue

In agriculture, pathogens cause fungal diseases leading to loss in crop yield and quality. These diseases have been controlled by chemical fungicides. But their effects are often non-specific, targeting beneficial organisms as well as pathogens. The actual crop cultivars have disease responses and in-built genetic resistance against diseases for their defense against fungal pathogens. For instance, chitinases and β-glucanases have been proposed to have a role in fungal cell wall lysis by targeting key macromolecular components of the cell walls, i.e. chitin and β-glucan microfibrils. Chitin does not occur in plants, whereas in many plant-pathogenic fungi chitin comprises of 22–44 % cell wall material and maintains the structural integrity of hyphae. The glucans are structural polymers for maintaining rigidity and conferring protection.

Combining Ability for Yield and its Components in Kabuli Chickpea

Interspecific hybrids were developed between Trifolium alexandrinum cultivar Wardanxa0×xa0Trifolium vesiculosum and T. alexandrinum cultivar BL1xa0×xa0T. vesiculosum through embryo rescue, as the crosses failed to set seed under natural conditions. Trifolium vesiculosum was used as a donor/male parent in this study as it is reported to possess tolerance to stem rot and high forage yield. Fertilization in crossed florets of the crosses was manifested from the recovery of swollen ovaries (<xa07.80%) and confirmed from the presence of one degenerated ovule in most (>xa093.00%) of the swollen ovaries. The hybrid embryos at various developmental stages (heart, torpedo and cotyledonary) were rescued at a frequency of 2.56% from Wardanxa0×xa0T. vesiculosum and 6.12% from BL1xa0×xa0T. vesiculosum. Differentiation occurred only in the cotyledonary stage embryos, resulting in 17 putative interspecific hybrid plantlets. The assessment of plantlet hybridity through SSR markers (for the alleles inherited from the donor parent), micromorphological leaf traits (leaf texture and stomata) and morphological characters (plant height, leaflet length and width) confirmed production of two interspecific hybrids designated as AV1 and BV3 representing both the crosses. AV1 displayed moderate resistance and BV3 was resistant to stem rot.