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2019 - 20203
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Author: Manjinder Singh × Start date: 2017 × Type: Thesis ×
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    Agrobacterium mediated genetic transformation of pigeon pea (Cajanus cajan L. Millsp.) for resistance to spotted pod borer Maruca vitrata
    (Punjab Agricultural University, Ludhiana, 2020) Manjinder Singh; Ajinder Kaur
    The present investigation dealing with introduction of cry1Ab gene for resistance to Maruca vitrata into pigeonpea (Cajanus cajan L. Millsp.) through Agrobacterium-mediated in planta transformation was carried out using three pigeonpea genotypes i.e. AL 15, AL 201 and PAU 881. Two methods of in planta transformation were used for generating transgenic plants, including inoculation of pricked embryo axes and floral dip transformation. Out of seven experiments on in planta transformation method involving inoculation of pricked embryo axes, only two (OD600 of Agrobacterium broth = 0.6 - 0.7 containing 100 mM acetosyringone, 1 h dipping of 2-day old pricked seedlings) gave positive results. A total of 5,022 half seeds were treated that gave rise to 4059 plants, out of which 15 (representing AL 15 and AL 201 genotypes) were PCR-positive with an overall transformation frequency of 0.36 %. Out of 15 primary transformants, only 11 plants showed transcript accumulation by semi-quantitative RT-PCR. The primary transformants were also analyzed for chimerism by ELISA-based protein accumulation in selected branches. Out of a total of 182 branches of 10 RT – PCR positive plants analyzed, 57, 43 and 49 branches had Cry1Ab protein content equal to or more than 1.08 µg/g [Positive calibrator, (PC) 5.0 ppb], 0.58 µg/g (PC, 2.5 ppb) and 0.08 µg/g (PC, 0.5 ppb), respectively. Besides, there were 33 such branches which did not show accumulation of Cry protein indicating the presence of chimerism in primary transformants. The putative transgenics were advanced to T1 generation, out of 904 plants analyzed, 97 were PCR-positive, thus exhibiting transformation efficiency of 11.70 % (with genotype AL 201) and 10.24 % (with genotype AL 15). Twenty three T1 plants were randomly taken for determination of Cry1Ab protein content, out of which 12 showed high protein content of more than 0.72 µg/g. These plants showed positive results in PCR and RT-PCR, and further used for determining the efficacy of cry1Ab gene against second instar larva of Maruca vitrata using insect bioassay. Both flowers and pods of these 12 T1 pigeonpea plants were used for bioassay experiments. All 12 T1 plants showed restricted increase in larval weight as compared to nontransgenic (control) plant. No insect mortality was observed, but larvae fed on two transgenic plants i.e. 201-344 and 15-537 showed no adult emergence as compared to other transgenic and control plants where adult emergence was normally observed. Both of these two plants (201-344 and 15-537) had high Cry protein content (0.88 µg/g) as compared to other transgenic plants, which established clearcut positive correlation between amount of protein accumulated and inhibition of M. vitrata larval growth. Further, T1 plants were advanced to T2 generation that were maintained in transgenic glasshouse. In case of floral dip transformation, a total of 139 flower buds were treated and 454 seeds were obtained. Ten out of 454 putative T1 plants were observed to be PCR-positive with a transformation frequency of 2.2 %.
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    Construction of New Ratio Type Estimator of Population Parameter
    (Punjab Agricultural University, Ludhiana, 2019) Manjinder Singh; Javed, Mohammed
    In the present project research, an improved ratio type estimator of population mean for the study variable (Y) has been proposed, using the information on the auxiliary variable (X). The sample was selected from the population using simple random sampling without replacement (SRSWOR). The expressions for large sample properties such as bias and mean squared error (MSE) of proposed ratio type estimator were obtained, up to the first order of approximation. To judge the performance of the proposed ratio type estimator of population mean, an empirical study was conducted on the basis of MSE and percent relative efficiency (PRE). To achieve the merits of the proposed ratio type estimator, a comparison study was carried out with respect to the existing ratio estimators present in the literature. The optimum value of the characterizing scalar (α) has been obtained and the minimum value of the mean square error of the proposed ratio type estimator for this optimum value has also been obtained. The theoretical results are validated numerically using four population data sets considered earlier by Murthy (1967) and Mukhopadhyay (2009). Also, the graphical illustrations shows that the proposed ratio-type estimator performs better than the existing estimators of population mean under certain conditions.
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    ThesisItemOpen Access
    Studies on morphological variability and DUS characterization in tuberose (Polianthes tuberosa L.)
    (Punjab Agricultural University, Ludhiana, 2019) Manjinder Singh; Madhu Bala
    The present investigations „Studies on morphological variability and DUS characterization in tuberose (Polianthes tuberosa L.)‟ were undertaken in the Department of Floriculture and Landscaping, Punjab Agricultural University, Ludhiana (India) during the year 2017-2018. The experimental material comprised of thirteen genotypes of tuberose viz. Local Single, Bidhan Rajani H-1, Bidhan Rajani H-2, Bidhan Rajani H-3, Phule Rajani, Prajwal, GKT-C4 and Arka Nirantara of single type and Pearl Double, Suvasini, Pune Double, Hyderabad Double and Vaibhav of double type. The experiment was replicated thrice using randomized block design (RBD). The observations on various growth and flowering parameters were recorded and data was subjected to statistical analysis. The results of the study revealed that for single type genotypes, plant height (53.66 cm), flowering duration (26.66 days), weight of floret per spike (52.32 g), diameter of bulbs (3.67 cm) and bulblets (1.87 cm), weight of bulblet (3.34 g) and vase life (6.95 days) was observed maximum in cv. „Prajwal‟, while cv. „Bidhan Rajani H-2‟ exhibited minimum days to spike emergence (65.8 days) and the maximum number of bulbs per clump (3.89). The spike length was the maximum (86.46 cm) in cv. „Bidhan Rajani H-1‟. In double type genotypes, the maximum plant height (43.92 cm), spike length (70.26 cm), number of florets per spike (56.86), diameter of floret (3.86 cm), weight of floret per spike (2.62 g) and vase life (7.37 days) was recorded in „Suvasini‟ while „Vaibhav‟ was earliest to spike emergence (76.73 days) and had maximum number of leaves per plant (52.14), flowering duration (26.40 days), number of bulbs per clump (2.06), number of bulblets per clump (22.8), diameter of bulbs (3.57 cm), diameter of bulblets (2.17 cm) and weight of bulblets (5.41 g). The morphological characterization of these genotypes showed differences for colour, variegation, length and breadth of leaf, length of bud and length, type and diameter of flower. The colour of bud and flower recorded no variation and were white in all genotypes. The shape of flower, rows of tepals, inflorescence length, number of flowers per inflorescence, days taken for flowering also exhibited variations among different genotypes when compared as per DUS 80 days. Among all the genotypes „Prajwal‟ and „Suvasini‟ performed best in all the growth, flowering and yield parameters under Punjab conditions for single and double types, respectively. All the varieties under study showed morphological variation with respect to different parameters as per DUS guidelines.