1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Somatic embryogenesis in rice (Oryza sativa L.)(Department of Plant Breeding and Genetics, College of Agriculture,Vellanikkara, 2023-05-17) Ardra, K S.; Sindhumole, PRice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.Item Somatic embryogenesis in rice (Oryza sativa L.)(Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2023) Ardra, K S; Sindhumole, PRice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.Item Locating high yielding lodging resistant lines from the segregating populations in rice (Oryza sativa L.)(Department of plant breeding and genetics, college of agriculture , Vellanikkara, 2023-07-25) Abhijith K Antony; Biju, SThe success of green revolution led to the introduction of high yielding and semidwarf genotypes in rice to a great extent. However, lodging remains a problem in many of the improved rice cultivars causing a significant reduction in grain yield and quality. With this aspect in mind, Keerthiraj et al. (2019) conducted a study to identify the lodging resistant genotypes among twenty-one different rice genotypes at Department of Plant Breeding and Genetics (PBGN). Three lodging resistant genotypes and four lodging prone genotypes were identified and they were crossed in Line × Tester design for the development of hybrids. Lodging resistance (LR) evaluation was carried out among the hybrids developed to identify the best lodging resistant hybrids. The evaluation of segregating generations would provide more insight into the lodging resistant character and higher yield of the hybrids developed. The present study was therefore conducted as two experiments to identify the best segregants for LR in F2 and F3 generations of two hybrids H8 (Vaisakh × Gouri) and H5 (Prathyasha × Gouri) having intermediate LR at Agricultural Research Station (ARS), Mannuthy. Observations were recorded for qualitative, quantitative and biochemical characters. The qualitative characters included flag leaf angle, culm angle, culm strength, panicle type and panicle axis. Quantitative observations consisted of plant height (cm), flag leaf length (cm), flag leaf width (cm), internodal length (cm), number of tillers, number of productive tillers, culm diameter (mm), culm thickness (mm), panicle length (cm), panicle weight (g), number of seeds per panicle, 1000 seed weight (gm), seed yield (g) and lodging percentage. Biochemical characters included silicon and potassium content. Correlation and path analysis were carried out to identify the effect of the characters towards grain yield. The F2 population of both the crosses showed high mean performance, range and variance over the parents, indicating higher variability among the segregating population. Correlation study revealed that grain yield per plant exhibited a significant positive association with number of tillers, number of productive tillers, panicle weight, number of seeds per panicle, culm diameter and culm thickness. Path analysis revealed that number of productive tillers and 1000 seed weight had the highest positive direct effect towards grain yield. Based on the observation recorded for quantitative characters individual plants were ranked for each character effecting lodging resistance. A selection criterion was fixed based on quantitative characters for selecting best lines to be forwarded to F3 generation. Six lines from F2’ s of H8 family and four lines from F2’s of H5 family were identified to be the best and forwarded to next generation. Silicon and potassium content were analyzed among the selected plants and parents, it was found to be high in selected plants indicating strong culms for the plants which to a great extent signifies lodging resistance of the selected plants. In experiment 2, the selected F2 plants were grouped into families consisting of 50 plants each and were evaluated for lodging resistance in F3 generation to identify the best segregants. Similar as in experiment 1 the observations recorded include qualitative, quantitative and biochemical characters. The mean performance and variability in F3 generation were found to be lower than F2 generation. Correlation studies revealed that grain yield per plant exhibited a significant positive association with number of tillers, number of productive tillers, panicle weight, number of seeds per panicle, culm diameter and culm thickness. Path analysis revealed that number of productive tillers and panicle weight had the highest positive direct effect towards grain yield in F3 generation. The selection criteria were fixed based on the quantitative observations recorded for lodging related traits in F3 generation. Based on this criterion and ranking of individual plants seven lines from F3’s of H8 family and three lines from F3’s of H5 family were identified as the best lodging resistant segregants from the evaluation and shall be forwarded to next generation for evaluation.Item Stability analysis of hybrids and pedigree selection in brinjal (Solanum melongena) with resistance to shoot and fruit borer(Department of plant breeding and genetics, college of agriculture , vellanikkara, 2023-07-15) Arya Sidharth, M; Jiji JosephBrinjal, also known as ‘eggplant’ or ‘Guinea squash,’ is one of the most popular and commercial crop grown in India and other parts of the world. It is often referred as vegetable of masses and believed to have originated in Indo-Burma region. The main factor limiting the production of eggplant in the Indian sub-continent is the infestation of the Brinjal Fruit and Shoot Borer (BSFB) or Eggplant Fruit and Shoot Borer (EFSB) (Leucinodes orbonalis Guen.). Farmers mostly rely on chemical insecticides to control EFSB, which lead to massive pesticide overuse. The environment friendly method to minimize pest-related damage is Host Plant Resistance (HPR). However, to get a satisfactory yield in addition to being resistant to fruit and shoot borer, the varieties should be stable in performance over a range of environments. The present study entitled “Stability analysis of hybrids and pedigree selection in brinjal (Solanum melongena) with resistance to shoot and fruit borer” comprised of two experiments. First experiment was an attempt to identify suitable high yielding hybrids resistant to shoot and fruit borer for general cultivation across three agroecological units of Pattambi, Vellanikkara and Vadakara. Materials used for the study comprised of four F1 hybrids (Ponni x Vengeri, Ponni x Arka Kusumakar, Haritha x Vengeri and Ponni x IC 618044) developed at Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, along with their parents and standard check for yield, Neelima. Field experiments were laid out in Randomized Block Design (RBD) with three replications. The second experiment was conducted with the objective of evaluation of F2 populations of two crosses ( Ponni x Vengeri and Haritha x IC 624241) and pedigree selection for resistance to shoot and fruit borer. The experimental materials included two crosses (200 plants each), their parents and standard check, Neelima. Observations were recorded on eleven characters: number of flowers per plant, number of fruits per plant, number of infested fruits per plant, number of shoots per plant, number of infested shoots per plant, number of primary branches per plant, fruit weight (g), fruit yield per plant (g), marketable yield per plant (g), per cent fruit infestation and percent shoot infestation. The recorded observations were subjected to individual location wise analysis of variance (ANOVA) followed by pooled analysis of variance (pooled ANOVA) over three locations. From the pooled ANOVA, the characters that exhibited significant genotype x environment (G x E) interaction were further assessed for stability using two models, Additive Main effects and Multiplicative Interaction effects (AMMI) model and the Genotype main effects plus Genotype-by Environment interaction effect (GGE) biplot. ANOVA revealed significant difference between genotypes for all the eleven characters considered. However, the error variances were homogeneous only for the characters number of fruits per plant, number of infested fruits per plant, number of shoots per plant, number of infested shoots per plant, fruit weight (g), marketable yield per plant (g) and per cent fruit infestation in Bartlett’s test. These seven characters were subjected to pooled ANOVA across three environments. Significant G x E interaction was observed in all characters considered and were hence forwarded for analysing with the stability. The AMMI model with its additive as well as multiplicative formulations could interpret the complex G x E patterns effectively through the AMMI biplots. Genotypes were scored and then ranked, according to their Stability Index (SI) value computed on the basis of rank of AMMI Stability Value (rASV) and the rank of performance for each character (rY). It was observed that Ponni x Vengeri and Neelima with lowest score, ranked as the best and stable genotypes followed by Vengeri. The GGE biplot model enabled effective interpretation of genotype x environment interaction by providing visual understanding of genotype and test environment evaluation through mean versus stability graph, ranking of genotypes, ranking of environments and which-won-where analysis. On an overall basis, it was identified from GGE biplot method that Neelima, Vengeri and Ponni x Vengeri were the most stable and highly performing genotypes. Comparison of results from the two models of stability confirmed Neelima, Vengeri and Ponni x Vengeri as the most stable and promising genotypes that these genotypes can be evaluated in large fields so as to confirm with the results and for checking the suitability of genotype Ponni x Vengeri to be released as a variety. In second experiment, F2 population of the cross Ponni x Vengeri exhibited wide range of segregation for all the characters evaluated. Correlation study revealed that marketable yield per plant (g) exhibited a significant positive association with number of flowers, number of fruits, number of shoots, fruit weight (g) and total yield (g), and a negative correlation with per cent fruit damage in the cross Ponni × Vengeri and the yield could be boosted through selection based on these characters. Ten best performing plants having a marketable yield of more than 600 g were selected and can be forwarded to the further generations. But in case of the cross Haritha × IC 624241 none of the plants performed well. So this cross need not be forwarded for further improvement.Item Breeding of ToLCV resistant high yielding tomato (Solanum lycopersicum L.) genotypes(Department of Plant Breeding and Genetics, College of Agriculture, Vellanikkara, 2023-05-15) Athira Sebastian; Deepthy Antony, PTomato (Solanum lycopersicum L.) is a warm season vegetable crop of solanaceae family, which is widely grown in India for use as vegetable as well as fruit. But, its production is hampered by disease incidence leading to severe crop loss. Among the diseases affecting tomato, whitefly transmitted tomato leaf curl virus disease (ToLCD) is very severe, and it can cause upto 100 per cent yield loss depending on stage of infection. As vector management using insecticides leads to environmental pollution and creates vulnerability, exploitation of host plant resistance is the best sustainable strategy to manage ToLCD. Hence, identified resistance sources can be utilised for the development of high yielding ToLCV resistant genotypes through hybridization programmes. In this context, the present study entitled “Breeding of ToLCV resistant high yielding tomato (Solanum lycopersicum L.) genotypes” was carried out during 2021 2023. Ten previously identified genotypes showing field resistance or carrying Ty genes (AVTO 0301, AVTO 0922, AVTO 1314, AVTO 1702, AVTO 1706, AVTO 1707, AVTO 1725, AVTO 1726, EC 519806 and Local Collection (Idukki)) were selected as source of resistance and were crossed with three agronomically superior released varieties viz., Akshaya, Manuprabha and Vellayani Vijay in Line x Tester mating design. Seventeen successful crosses along with parents and check variety, Anagha, were screened for ToLCV resistance in RBD design with two replications during March- June 2022. The disease incidence was assessed at 15, 30, 60 and 90 days after transplanting as per the score given by Banerjee and Kalloo (1987). Among the hybrids screened, Vellayani Vijay × EC 519806, Akshaya × AVTO 1726 and Akshaya × AVTO 1707 showed least Coefficient of Infection (CI) and were highly resistant under field condition. In the present study, the morphological characters affecting growth and yield such as growth habit, plant height, spread of the plant, number of primary branches per plant, days to flowering, fruit colour, fruit size, number of fruits per plant, number of locules per fruit, lycopene content, fruit weight and fruit yield per plant differed significantly among the hybrids. The crosses of EC 519806 followed by Akshaya × AVTO 1726 recorded the highest yield per plant. Moreover, the whitefly population assessment in the field also showed less whitefly infestation on Akshaya × AVTO 1726. Hybrids with significant positive Relative heterosis (RH), Heterobeltiosis (HB) and Standard heterosis (SH) for all growth and yield characters were obtained in the present study. Most of the hybrids exhibited significant SH for all the characters under study. And all the traits exhibited high heritability coupled with high genetic advance. Line x Tester analysis of nine F1’s, developed from three lines (Akshaya, Manuprabha and Vellayani Vijay) and three testers (AVTO 1707, EC 519806 and Local Collection (Idukki)), revealed significant sca effect for hybrids and significant gca effect for parents for days to flowering (Vellayani Vijay x EC 519806, Manuprabha x AVTO 1707); equatorial diameter and fruit size (Manuprabha × AVTO 1707); polar diameter (Vellayani Vijay × AVTO 1707); fruit weight (Akshaya × AVTO 1707); spread of the plant (Akshaya × Local Collection (Idukki), Manuprabha × EC 519806); number of fruits per plant and yield per plant (Vellayani Vijay × EC 519806). Significant sca effect was recorded for plant height (Manuprabha × AVTO 1707); equatorial diameter (Akshaya × EC 519806, Vellayani Vijay × Local Collection (Idukki)); polar diameter and fruit size (Akshaya × EC 519806, Akshaya × Local Collection (Idukki), Manuprabha × AVTO 1707); number of fruits per plant (Akshaya x Local Collection (Idukki), Manuprabha x AVTO 1707); number of primary branches per plant (Akshaya x EC 519806); spread of the plant (Manuprabha × AVTO 1707, Vellayani Vijay × Local Collection (Idukki)); yield per plant (Akshaya × AVTO 1707, Akshaya × Local Collection (Idukki), Manuprabha × AVTO 1707, Manuprabha × Local Collection (Idukki)). Although, significant gca for number of primary branches per plant (Manuprabha; Local Collection (Idukki)); number of fruits per plant (Vellayani Vijay; Local Collection (Idukki)) and spread of the plant (Akshaya; EC 519806, Manuprabha; Local Collection (Idukki)) was observed, their crosses did not have significant sca. Therefore, selection in segregating generations will be effective for these characters. Glandular and non glandular trichome density on both abaxial and adaxial leaf surfaces demonstrated that glandular abaxial trichome density is more effective for ToLCV resistance than adaxial trichomes. Among the hybrids, Akshaya × AVTO 1726 and Akshaya × AVTO 1707 had above average glandular and below average non glandular trichome density. To confirm ToLCV resistance, whitefly mediated artificial inoculation was done on the selected hybrids and observed that Akshaya × AVTO 1726 was highly resistant under artificial screening as well. All crosses of EC 519806 (except Akshaya × EC 519806) and Local Collection (Idukki) were susceptible under artificial screening. Screening for six reported Ty genes using the primers 562 JB/830 JB, SCAR2, P6-25, TY-1/3_K, C2_AT5g51110, AVRDC-TM273 and SLM 10-46 linked to ToLCV resistance genes Ty 1, Ty 2, Ty 3, Ty 1/3, Ty 4, ty 5 and Ty 6 respectively, revealed presence of Ty 1, Ty 2, Ty 3 and Ty 1/3 in all the crosses of AVTO 1726 and AVTO 1707. Whereas, AVTO 0922, AVTO 0301 and AVTO 1314 contributed Ty 1 and Ty 2; Ty 1, Ty 2 and Ty 3; Ty 1 and Ty 3 genes respectively in their crosses. None of the markers showed resistant amplicon in EC 519806. In the present study, none of the genotypes exhibited presence of resistant amplicon for Ty 4. AVRDC-TM273 linked to ty 5 failed to detect polymorphism in the genotypes. Whereas, resistant allele of the marker linked to Ty 6 was amplified only in Local Collection (Idukki) and its crosses. The present study revealed that Akshaya × AVTO 1726 is a very promising hybrid with high yield, ToLCV resistance large sized fruits. Akshaya × EC 519806, Akshaya × AVTO 1707 and Manuprabha × AVTO 1707 are high to medium yielding hybrids with moderate resistance to ToLCV. Vellayani Vijay × EC 519806 was the highest yielding genotype. Therefore, these crosses can be forwarded to further generations to identify superior genotypes from segregating generationsItem Seed characterization and standardization of in vitro seed germination protocol in Rhynchostylis retusa (L.) Blume.(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2023-03-16) Akhila Rajan; Seeja ,GThe present study entitled “Seed characterization and standardization of in vitro seed germination protocol in Rhynchostylis retusa (L.) Blume.” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during the year 2020-22. The experiment was aimed at seed characterization and standardization of asymbiotic seed germination protocol in Rhynchostylis retusa (L.) Blume. Germplasm repository maintained at Saraswathy Thangavelu Extension Centre of JNTBGRI, Puthenthope, Thiruvananthapuram was utilized to observe the flower characters and the capsules for the characterization and in vitro seed germination study. The research work was divided into four experiments. Experiment I - evaluation of floral characters, experiment II -morphological and morphometric characterization of seeds, experiment III- preparation of in vitro seed germination media, and experiment IV -in vitro seed germination in culture media and evaluation. All the experiments were carried out in five replicates and mean was calculated. In the first experiment, various floral, pollen and capsule characters were noted and qualitative characters were expressed descriptively after observing the plants behaviour closely and quantitative characters were assessed and mean was worked out. Rhynchostylis retusa was seasonal (June-August) in flowering behaviour. Two types of plants were observed, one with white flowers having purple spots or tessellation and other with blush of purple on white background. Anthesis was between late evening and early morning. The stigma was receptive in young flowers until five days after anthesis and after which the stigma become dried out. Morning hours until 10.30 am showed maximum receptivity than other times of the day. Under natural conditions capsule setting was noticed within seven days of flowering. Morphology of pollinia was observed visually. They were crushed and stained using acetocarmine to understand the individual pollen structure, number, arrangement, viability etc. The pollen load per pollinium estimated by haemocytometer averaged to 2,06,000. Furthermore, almost all the pollen was found to be viable and 73.64% of them germinated in sucrose solution of 10 per cent strength. The fruit or capsule at maturity was 2.18 cm long and 0.86 cm wide. Undehisced mature capsules of 145-180 days of pollination was found to be best for in vitro culture. 66 In experiment II, seeds of R. retusa was characterized morphologically under compound light microscope and morphometric data analysed using microscopic software package Leica LAZ 500. The seeds were fusiform shaped, elongated, with prolate spheroid embryo of violet colour. The testa cells were yellowish white and reticulated. At maturity 78.74 percent of the seed volume was filled with air space. The viability of seeds used for seed culture was worked out to be 78.87 percentage. Out of the millions of seeds in a capsule 41.87 percentage of seeds were found to be well filled with large embryo of regular shape. In experiment III- for the in vitro seed germination studies four different media viz., MS, half-strength MS, Knudson C orchid media and WPM including liquid and solid with and without growth hormones were prepared and used. The seeds from mature disinfected capsules were scooped out and inoculated under sterile conditions. Five replicates of each media was inoculated with seeds. Observations were recorded on the number of days for showing seed greening and chlorophyll embryo, for protocorm development, for number of protocorms produced and number of protocorms with shoot apex and multiple shoots, number of days for root and leaf initiation. Half MS medium (liquid) was observed to be the best among the different media used for asymbiotic seed germination of Rhynchostylis retusa. Half MS was best media for greening and protocorm development. If we compare the overall response for different stages of development half MS with 0.5 mgL-1 BAP (solid) was found best for callusing and shoot proliferation. Half MS with 0.5 mgL-1 BAP and 1 mgL-1 NAA (solid) for multiple shoots, small leaves and root development. Initiation of germination was seen within two months of culture inoculation. The grain shaped protocorm needed to be subcultured at proper time and calloid like structures with multiple shoots could be divided to separate units and each unit developed to separate plantlets. Seed characterization provides an insight into understanding biology, ecology and evolutionary background. Mass multiplication by asymbiotic seed culture can be a viable option for restoring this threatened species and can be utilized for further genetic improvement programme. 67Item Performance analysis of Sub 1 pyramided F5 population in rice(Department of Plant Breeding and Genetics, College of Agriculture , Vellayani, 2023-04-10) Aravind Krishnan ,P S.; Veena VighneswaranRice (Oryza sativa L.) is the most important primary food crop that provides food to about half of the population. The cultivated species of rice are semi aquatic plants of normal height ranging from 0.90m – 1.8m, but some can grow up to 5 metres in deep water. In lowlands and deep-water rice field, over 16 million hectares of the world’s rice lands are unfavourably impacted by floods due to total submergence and one of the major constraints in rice cultivation worldwide is submergence stress. This mainly affects flood prone areas like kuttanadu, pokkali as well as low lying paddy fields in Kerala. Even though hundreds of high yielding varieties were developed in the nation, they are not suitable for cultivation in the problematic areas affected by various abiotic stresses. The easiest way is to incorporate the abiotic stress tolerant (Quantitative Trait Loci) QTLs into the already bred high yielding varieties without altering any of the characters through molecular breeding. Sub1 is a major quantitative trait locus (QTL) representing a cluster of three ethylene responsive factor (ERF) genes: Sub1 A, Sub1B and Sub1C that confers submergence tolerance to rice genotype. Submergence tolerant rice variety is credited with maintaining energy and alcoholic fermentation which require high levels of carbohydrates. Sub1 locus contain Sub1A, Sub1B and Sub1C, all of which encode ethylene responsive factor and are up regulated under submergence, but only Sub1A is responsible for flash flood tolerance. The near isogenic lines generated by the introgression of the Sub1 from FR13A into to susceptible cultivar shows restricted shoot elongation similar to FR13A. The present study was undertaken for the performance assessment of Sub 1 QTL in the BC3F5 progenies of submergence susceptible rice varieties Jaya, Jyothi and Uma. The research was conducted in the Department of Plant Breeding and Genetics, Rice Research Station, Vyttila during the period 2021-2022. The experimental material used for the study included 50 accessions of BC3F5 progenies of Jaya, Jyothi and Uma obtained from Rice Research Station, Vyttila. 47 SSR markers distributed across the rice genome were used for polymorphism survey between recurrent parents Jaya, Jyothi and Uma and Swarna Sub-1 96 Progenies of BC3F5 was evaluated in the field to assess the submergence tolerance of Sub-1 introgressed lines. In-vitro screening of BC3F5 progenies were performed by pot culture experiment. 14 days old seedlings were subjected to complete submergence and the pots were desubmerged after 14 days. The survival percentage and elongation percentage were scored based on the observation. Most of the selected lines have shown good recovery percentage resembling its Sub-1 donor Swarna Sub-1. Rice varieties with the Sub1 gene were shown higher survival rate with better yield even after 2 weeks of submergence stress. The advancement of marker assisted selection (MAS) have striking impacts on developing improved Sub1 varieties that can meet the increasing demands of consumers along with the population hike. In this present study, 31 rice genotypes were observed with the Sub1 QTL using SSR markers through molecular characterisation in order to find out the best submergence tolerant lines. More over this finding lays the foundation for further researches with Sub1 QTL for crop improvement. 2 SSR markers ART-5 and Sub1BC2 were used as foreground markers to identify the Sub-1 QTL. Background markers are used to identify the recurrent parent marker allele. A total of 45 polymorphic background markers were used for background screening. 9Item Somatic embryogenesis in rice (Oryza sativa L.)(Department of Plant Breeding and Genetics, College of Agriculture , Vellanikkara, 2023-05-17) Ardra, K S.; Sindhumole,PRice is the staple food for more than half of the world’s population, but over time the yields have plateaued and there are higher incidences of pest and diseases. This problem can be tackled by combining biotechnological tools with crop improvement methods. Genetic modifications can be used to improve yield, tolerance to pest and disease and even incorporate climate resilience in the crop, which is the need of the hour for the rapidly growing population. For the application of genetic transformation techniques, standardised in vitro regeneration protocols are necessary and among the many techniques, somatic embryogenesis is a promising technique as it produces large number of plantlets. Somatic embryogenesis is a type of asexual reproduction whereby somatic or gametic cells are induced to form somatic embryos under favorable in vitro conditions by exploiting the cellular totipotency of plant cells to form entire plantlets. Thus, this study was conducted to standardise the protocol for somatic embryogenesis and regeneration in two rice varieties, Jyothi (PTB 39) and Nagina 22 (N22). Jyothi is a very popular, red and long bold grained rice variety cultivated predominantly in the Kole and Kuttanad regions of Kerala. N22 is a deep rooted aus type Indian rice variety, well known for its high tolerance to drought and heat. The mature seeds and leaves of these varieties were used as explants in this research programme. Experiment 1 involved studies to standardise the medium for callus induction and somatic embryogenesis. In the preliminary study, the effect of medium (MS and N6), carbon source (sucrose and maltose) and 2,4-D at different levels (0, 1, 2, and 3 µM) were observed, and the treatment combination MS + 2,4-D (3 µM) with maltose performed the best among the sixteen treatments in both varieties. The effect of medium with 2,4-D and BAP on callus induction from leaf explants was studied and none of the varieties responded to the treatments. When the effect of 2,4-D and kinetin on somatic embryogenesis was investigated, the somatic embryogenesis per cent was higher in the treatment combination MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) in Jyothi (80%) and the treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Moreover, both varieties exhibited some rhizogenesis from the callus, which reduced the capacity of the calli for somatic embryogenesis as well as regeneration. During this study, the seeds germinated regardless of treatment and the germination per cent varied from 75 to 97.22 per cent in Jyothi while it ranged from 58.33 to 100 per cent in N22. Regarding the callus induction frequency (CIF), 2,4-D (3 µM) in MS medium containing maltose exhibited the highest CIF in both the varieties (N22 with 100 per cent CIF and Jyothi with 69.44 per cent CIF). The callus was developed from the scutellar region of the seeds. Callus induction required six to seven days in both the varieties. It was observed that three week old calli started forming somatic embryos when sub cultured on MS medium supplemented with 2,4-D (0 and 0.4 µM) and Kinetin (0, 0.4, 1, 1.5 and 2 µM), and incubated in dark. Embryogeneic calli per cent was highest in treatment MS + 2,4-D (0.4 µM) + Kinetin (0.4 µM) in Jyothi (87.50%) and in treatments MS + 2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) in N22 (85.71%). Non-embryogenic calli formation was the lowest in the treatment MS +2,4-D (0.4 µM) + Kinetin (0.4 µM) for Jyothi (12.50%), while MS +2,4-D (0.4 µM) + Kinetin (1.5 µM) and MS + 2,4-D (0.4 µM) + Kinetin (2 µM) for N22 (14.29 %). For the development of somatic embryos into plantlets, the embryogenic calli were sub cultured on medium with NAA and BAP and incubated in 16 hours light and 8 hours dark conditions. Embryoid formation was observed in both varieties within two weeks, but not all the embryoids developed into plantlets due to degeneration. In Experiment 2 for regeneration studies, N22 formed two plantlets from somatic embryos in the treatment combinations MS + NAA (0.2 µM) + BAP (0.8 µM) and MS + NAA (1 µM) + BAP (3.5 µM) whereas, Jyothi required the addition of maltose (20 and 30 g/l) and sorbitol (25 and 30 g/l) in the medium for embryoid development. Seventeen embryoids formed in Jyothi, but only three developed into plantlets in the treatment MS + NAA (1 µM) + BAP (3.5 µM) with 30 g/l each of maltose and sorbitol. Jyothi plantlets needed an average of 15.67 days for shoot initiation and 29.67 days for root initiation. In N22, the average number of days for shoot initiation and root initiation were 8.50 and 18.50 respectively. All the plants obtained from this study were green and healthy, with the characteristics of normal rice plants formed from seeds. However, these plantlets required additional time for proper root development in basal MS medium or MS medium with IBA and NAA, prior to planting out into pots. Experiment 3 entailed hardening and pot culture. The plantlets of Jyothi and N22 were first transferred to distilled water for a few days to let the leaves to unfurl and then planted in sterilised potting mixture. The plants were then kept under 16 hour photoperiod for 10 days. These plants were further hardened in a polytunnel and later grown in a rain shelter using standard agronomic practices. N22 and Jyothi plants grew with healthy leaves and formed panicles within two months but N22 exhibited poor tillering.Item Introgression of TY-2 gene for resistance to tomato leaf curl virus(ToLCV) into tomato variety anagha through marker assisted backcross(Department of plant breeding and genetics, college of agriculture, Vellayani, 2023-09-16) Bhaskar Reddy, S; Beena ThomasThe present research work entitled ‘Introgression of Ty-2 gene for resistance to Tomato leaf curl virus (ToLCV) into tomato variety Anagha through marker assisted backcross’ was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during the year 2019-2023, with an objective to develop high yielding and ToLCV resistant backcross progenies by introgression of Ty2 gene into tomato variety Anagha through marker assisted backcross and also to understand the nature and magnitude of gene effects of different yield related characters. The 17 BC2F1 lines containing Ty2 gene developed in the study can be carried forward for the development of a ToLCV resistant essentially derived variety (EDV). Pyramiding of resistance genes Ty-1, Ty-3, Ty-4, ty-5 or Ty-6 along with Ty2 may enhance the spectrum and durability of resistance. In generation mean analysis, additive and additive x additive gene effects are significant for attributes investigated. Non-additive gene effects were observed to be overpower additive gene effects. Hence, breeding procedure which could exploit non-additive gene action would be suitable for improvement. These methods are used for improvement followed by conventional selection.Item Introgression of TY-2 gene for resistance to tomato leaf curl virus(ToLCV) into tomato variety anagha through marker assisted backcross(Department of plant breeding and genetics, college of agriculture, Vellayani, 2023-09-16) Bhaskar Reddy, S.; Beena ThomasThe present research work entitled ‘Introgression of Ty-2 gene for resistance to Tomato leaf curl virus (ToLCV) into tomato variety Anagha through marker assisted backcross’ was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during the year 2019-2023, with an objective to develop high yielding and ToLCV resistant backcross progenies by introgression of Ty2 gene into tomato variety Anagha through marker assisted backcross and also to understand the nature and magnitude of gene effects of different yield related characters. The 17 BC2F1 lines containing Ty2 gene developed in the study can be carried forward for the development of a ToLCV resistant essentially derived variety (EDV). Pyramiding of resistance genes Ty-1, Ty-3, Ty-4, ty-5 or Ty-6 along with Ty2 may enhance the spectrum and durability of resistance. In generation mean analysis, additive and additive x additive gene effects are significant for attributes investigated. Non-additive gene effects were observed to be overpower additive gene effects. Hence, breeding procedure which could exploit non-additive gene action would be suitable for improvement. These methods are used for improvement followed by conventional selection.