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ThesisItem Open Access Intra and inter generic hybridization and molecular charatrization in monopodial orchids(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2008) Beena, Thomas; KAU; Lekha Rani, CA research programme entitled “Intra and inter generic hybridization and molecular characterization in monopodial orchids” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during 2005-2008 with the objective of undertaking intra and intergeneric hybridization, in vitro embryo culture and molecular characterization in monopodial orchids, as a preliminary step to develop novel hybrids. Fifteen monopodial orchid genotypes comprising of six genera viz., Aranda, Aranthera, Kagawara, Mokara, Renanthera and Vanda, with good cut flower qualities and high demand in the market were selected as parents after initial evaluation. They were evaluated adopting completely randomized design with five replications. Analysis of variance revealed significant differences for almost all the characters studied. Genotypic and phenotypic coefficients of variation were high for thickness of leaf, leaf area and number of aerial roots. High heritability (>70 %) combined with high genetic advance (>70 %) was observed for number of aerial roots, width of leaf, thickness of leaf, leaf area, number of spikes per shoot and number of flowers per inflorescence. Significant positive inter-correlation at genotypic and phenotypic levels was observed for length of flower and width of flower with number of spikes per shoot. The character number of spikes per shoot recorded significant positive correlation with leaf area. Number of flowers per inflorescence was positively correlated with number of leaves per shoot and length of inflorescence. Out of the six genera studied, four viz., Aranda, Aranthera, Kagawara and Mokara, exhibited free-flowering nature. Seasonal flowering was observed mainly from June to December in Vanda, whereas it was confined to two seasons, from February to March and August to October in Renanthera. Inflorescence axis was found to be arching in Aranthera and Renanthera while the rest of the genotypes produced erect inflorescence axis. The 15 parental genotypes were crossed in all possible combinations after preliminary studies on floral biology. A total of 225 cross combinations were attempted including 105 crosses, 105 reciprocals and 15 selfs. Incompatibility reactions were noticed at different stages ranging from flower abscission before the onset of any visible post pollination change to instances where seeds germinated but aborted in culture. Mature green capsules were harvested from 70 combinations at 70 to 90 per cent maturity. Among them 15 combinations did not yield any seeds in the capsule while the remaining 55 combinations were cultured axenically. Among the 55 combinations inoculated in vitro, no germination was obtained from seeds of 12 combinations. Out of the 43 combinations that germinated successfully, seven combinations showed arrested development. Thus out of the total 55 combinations inoculated in vitro 36 combinations developed successfully. These were subcultured three to four times. Seedlings having 2-3 leaves and 2-3 roots were deflasked and planted out. MS half strength was selected as the best basal medium. For improving the in vitro growth of hybrid monopodial orchid seedlings refinement of this medium by supplementing with IAA (8 mg l-1) and NAA (2 mg l-1) was beneficial. Significant differences among the combinations were observed with respect to number of days taken for germination initiation, number of days taken for development of protocorms, chlorophyll, first leaf, first shoot and first root primordia and for deflasking. Significant differences in seedling morphology were observed among the 36 hybrid combinations at deflasking. These were kept in humidity chamber for acclimatization for one month, transferred to net house for hardening and maintained there for further growth. In the present study, RAPD was employed for studying the genetic diversity and for the fingerprinting of 20 monopodial orchid hybrids, making use of arbitrary primers to amplify random DNA sequences in the genome. To identify the promising primers for RAPD analysis, 70 decamer primers of kit A, B, C and D were screened using the DNA of hybrid H-2. Based on the performance in DNA amplification, eight decamer primers were identified for RAPD analysis. Primers that produced highest number of polymorphic bands which were intense and reproducible were selected. They were OPB-07, OPB-15, OPB-l7, OPC-04, OPC-05, OPC-08, OPC-15 and OPD-02. a total of 57 scorable bands (average of 7.125 bands per primer) were generated by the selected eight primers of which six were monomorphic and the remaining 51 were polymorphic (89.47%). The estimation of Jaccard’s coefficients and construction of dendrogram by using UPGMA revealed the presence and extent of genetic similarities among the 20 monopodial orchid hybrids. The overall similarity coefficients ranged from 0.40 to 0.84. Cluster analysis revealed that at 0.69 similarity coefficient, the 20 monopodial orchid hybrids got divided into six groups. Among the 20 hybrids, H-18, H-7, H-13, H-17, H-19 and H-20 stood separately in clusters II, III A, III B, IV, V and cluster VI respectively. This substantiates the moderately broad distribution of genetic variability, which can be attributed to the broad genetic base in their ancestry. Cluster I A contained four hybrids viz., H-1, H-10, H-3 and H-9. This grouping is justified by the presence of a common parent i.e., Arachnis Maggie Oei Red Ribbon in their parentage. Moreover, two common species viz., Arachnis hookeriana and Arachnis flos-aeris are involved in their ancestry. All these support their belonging to the same cluster. The three hybrids such as H-5, H-6 and H-8 fell in cluster I B. RAPD technique is relatively simpler, quicker, less expensive and non-radioactive than other molecular characterization techniques. The results of present investigation proved that it can detect sufficient polymorphisms in genetic distance studies in monopodial orchids. A research programme entitled “Intra and inter generic hybridization and molecular characterization in monopodial orchids” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during 2005-2008 with the objective of undertaking intra and intergeneric hybridization, in vitro embryo culture and molecular characterization in monopodial orchids, as a preliminary step to develop novel hybrids. Fifteen monopodial orchid genotypes comprising of six genera viz., Aranda, Aranthera, Kagawara, Mokara, Renanthera and Vanda, with good cut flower qualities and high demand in the market were selected as parents after initial evaluation. They were evaluated adopting completely randomized design with five replications. Analysis of variance revealed significant differences for almost all the characters studied. Genotypic and phenotypic coefficients of variation were high for thickness of leaf, leaf area and number of aerial roots. High heritability (>70 %) combined with high genetic advance (>70 %) was observed for number of aerial roots, width of leaf, thickness of leaf, leaf area, number of spikes per shoot and number of flowers per inflorescence. Significant positive inter-correlation at genotypic and phenotypic levels was observed for length of flower and width of flower with number of spikes per shoot. The character number of spikes per shoot recorded significant positive correlation with leaf area. Number of flowers per inflorescence was positively correlated with number of leaves per shoot and length of inflorescence. Out of the six genera studied, four viz., Aranda, Aranthera, Kagawara and Mokara, exhibited free-flowering nature. Seasonal flowering was observed mainly from June to December in Vanda, whereas it was confined to two seasons, from February to March and August to October in Renanthera. Inflorescence axis was found to be arching in Aranthera and Renanthera while the rest of the genotypes produced erect inflorescence axis. The 15 parental genotypes were crossed in all possible combinations after preliminary studies on floral biology. A total of 225 cross combinations were attempted including 105 crosses, 105 reciprocals and 15 selfs. Incompatibility reactions were noticed at different stages ranging from flower abscission before the onset of any visible post pollination change to instances where seeds germinated but aborted in culture. Mature green capsules were harvested from 70 combinations at 70 to 90 per cent maturity. Among them 15 combinations did not yield any seeds in the capsule while the remaining 55 combinations were cultured axenically. Among the 55 combinations inoculated in vitro, no germination was obtained from seeds of 12 combinations. Out of the 43 combinations that germinated successfully, seven combinations showed arrested development. Thus out of the total 55 combinations inoculated in vitro 36 combinations developed successfully. These were subcultured three to four times. Seedlings having 2-3 leaves and 2-3 roots were deflasked and planted out. MS half strength was selected as the best basal medium. For improving the in vitro growth of hybrid monopodial orchid seedlings refinement of this medium by supplementing with IAA (8 mg l-1) and NAA (2 mg l-1) was beneficial. Significant differences among the combinations were observed with respect to number of days taken for germination initiation, number of days taken for development of protocorms, chlorophyll, first leaf, first shoot and first root primordia and for deflasking. Significant differences in seedling morphology were observed among the 36 hybrid combinations at deflasking. These were kept in humidity chamber for acclimatization for one month, transferred to net house for hardening and maintained there for further growth. In the present study, RAPD was employed for studying the genetic diversity and for the fingerprinting of 20 monopodial orchid hybrids, making use of arbitrary primers to amplify random DNA sequences in the genome. To identify the promising primers for RAPD analysis, 70 decamer primers of kit A, B, C and D were screened using the DNA of hybrid H-2. Based on the performance in DNA amplification, eight decamer primers were identified for RAPD analysis. Primers that produced highest number of polymorphic bands which were intense and reproducible were selected. They were OPB-07, OPB-15, OPB-l7, OPC-04, OPC-05, OPC-08, OPC-15 and OPD-02. a total of 57 scorable bands (average of 7.125 bands per primer) were generated by the selected eight primers of which six were monomorphic and the remaining 51 were polymorphic (89.47%). The estimation of Jaccard’s coefficients and construction of dendrogram by using UPGMA revealed the presence and extent of genetic similarities among the 20 monopodial orchid hybrids. The overall similarity coefficients ranged from 0.40 to 0.84. Cluster analysis revealed that at 0.69 similarity coefficient, the 20 monopodial orchid hybrids got divided into six groups. Among the 20 hybrids, H-18, H-7, H-13, H-17, H-19 and H-20 stood separately in clusters II, III A, III B, IV, V and cluster VI respectively. This substantiates the moderately broad distribution of genetic variability, which can be attributed to the broad genetic base in their ancestry. Cluster I A contained four hybrids viz., H-1, H-10, H-3 and H-9. This grouping is justified by the presence of a common parent i.e., Arachnis Maggie Oei Red Ribbon in their parentage. Moreover, two common species viz., Arachnis hookeriana and Arachnis flos-aeris are involved in their ancestry. All these support their belonging to the same cluster. The three hybrids such as H-5, H-6 and H-8 fell in cluster I B. RAPD technique is relatively simpler, quicker, less expensive and non-radioactive than other molecular characterization techniques. The results of present investigation proved that it can detect sufficient polymorphisms in genetic distance studies in monopodial orchids.