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ThesisItem Embargo Genetic diversity analysis for nutrient efficiency and identification of nutrient responsive genes in cassava (Manihot esculenta Crantz)(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2023-02-10) Swathy Sivan; KAU; Arya, KThe present study entitled “Genetic diversity analysis for nutrient efficiency and identification of nutrient responsive genes in cassava (Manihot esculenta Crantz)” was carried out in the Department of Plant breeding and Genetics, College of Agriculture, Vellayani and Division of Crop Improvement, ICAR-CTCRI, Sreekariyam, during 2019-2022. The study was undertaken to screen and characterize N and K-efficient genotypes in cassava and identify the traits contributing to nutrient efficiency and study their gene expression. Thirty genotypes of cassava comprising of landraces, released varieties and pre-release accessions from ICAR-CTCRI and KAU were taken for the study. Field evaluation was done to identify low input nutrient efficient lines where analysis of variance revealed significant difference among the genotypes for all the characters studied except the girth of tuber. High GCV, PCV, heritability and genetic advance were observed for traits like tuber yield, N and K efficiency, plant height, number of leaves, number of branches, stem N and K content. Tuber yield was positively correlated to N and K efficiency, plant height, leaf and stem weight, stem girth, mean weight of tubers, number of nodes, girth and length of tubers, while negatively correlated to tuber starch and dry matter content. Path analysis showed that tuber length gave highest positive direct effect on yield followed by number of tubers, number of leaves, tuber starch content, plant height and nitrogen efficiency. Diversity studies grouped the genotypes into eleven clusters and the characters that gave the maximum contribution to divergence were tuber dry matter content, N and K efficiency. Root studies on thirty genotypes revealed that root traits like number of nodal roots, basal roots, adventitious roots, storage roots and root length displayed significant positive correlations with N and K efficiency. Evaluation of five highly nutrient efficient genotypes at the three levels of fertilizers (0%, 25% and 50% of the recommended dose of fertilizers) showed overall significance for traits like plant height, the number of leaves retained, stem weight per plant, girth of the stem, tuber yield, length of tuber, stem K content, tuber K content, total N and K uptake, N and K efficiency. The highest yielder was KBH 18 followed by 8S501-2. Maximum N efficiency was observed for KBH18 followed by 15S409, while maximum K efficiency was recorded for 8S501-2 followed by 15S409. Results showed that from 0 to 25 % there is an escalation in tuber yield as well as N and K efficiency, while the values at 25 and 50% are on par. All the selected four genotypes showed significant superiority over the K efficient check variety – Sree Pavithra – in terms of tuber yield, N efficiency and K efficiency. The study identified nine nutrient responsive genes in cassava which includes NRT1, NRT3, NLP1, GPT2, AMT1, TAR2 for nitrogen and KUP3, KUP4, KUP8 for potassium. Expression studies done using these genes in contrasting genotypes for N and K efficiency raised under field conditions showed significant upregulations and downregulations in their expression for efficient and less efficient genotypes. Allele mining for allelic variations in contrasting genotypes (15S409 and Export kappa) for two genes (AMT2 and NTR3) didn’t show much significant variation. Although SNP differences were observed for NRT3 gene, further functional studies are required to confirm this result. The study identified three main nutrient efficient genotypes viz., KBH18, which was the highest yielder, highest in N efficiency and third highest in K efficiency, 8S501-2, which was second highest in yield, highest in K efficiency, third highest in N efficiency and also exhibited early bulking and 15S409, which was the third highest yielder, second highest in N efficiency & K efficiency. The superior genotypes identified in the present study can be subjected to field trials for confirming their superiority and release as a variety.ThesisItem Embargo Gene action and gene expression analysis in yardlong bean(Vigna unguiculata ssp. sesquipedalis(L.) Verdcourt) for drought tolerance(Kerala Agricultural University, 2022) Rahana, S N; KAU; Beena, ThomasThesisItem Open Access Characterization and identification of black pepper accessions (Piper nigrum L.) for stress tolerance and quality(Department of plant breeding, College of Horticulture, Vellanikkara, 2019) Prakash, K M; KAU; Jiji, JosephBlack pepper (Piper nigrum L.), often described as the ‘King of spices’ is the most important spice crop, grown for its berries in the world. Indian pepper is preferred across the globe due to its intrinsic qualities. Foot rot is a devastating disease of black pepper. In the changing climate, drought can be a major threat in black pepper production. Hence, the present study was taken up at College of Horticulture, Vellanikkara and ICAR-IISR, Kozhikode to characterise and to identify superior accessions of black pepper for yield, quality and tolerance to biotic and abiotic stresses. Fifty accessions of black pepper in the bearing stage maintained in the National Active Germplasm Site of ICAR-IISR, Kozhikode formed the base material for the study. The accessions were characterised for fifty qualitative and fifty quantitative characters following the descriptor developed by IPGRI (1995). Wide variability was observed among the accessions for ten qualitative characters. Quantitative characters of shoot, leaf, spike and fruit also showed wide variability. Field tolerance to foot rot disease and pollu beetle infestation was observed among the accessions. Twenty accessions were selected from the base collection based on superiority of yield (> 450g green berries/vine) , field tolerance to foot rot disease infection (biotic susceptibility score 1) and pollu beetle infestation (biotic susceptibility score 1-3). They were further evaluated for biochemical principles of quality, tolerance to foot rot disease under artificial inoculation and tolerance to drought by physiological and biochemical analyses. Piperine, essential oil and oleoresin ranged from 3.61 - 6.96 per cent, 3.00 - 5.87 per cent and 7.10 - 11.18 per cent, respectively, across the accessions. The accessions with high value of piperine, essential oil and oleoresin were identified as 7293, 7211 and 7289 respectively. The two accessions identified viz. 7293 and 7252 contained more piperine than the highest of Panniyur 2 (6.6 per cent) reported among the released varieties . Artificial inoculation of selected accessions using Phytophthora capsici culture for screening for foot rot disease resistance based on over all disease severity index of both stem and leaf lesions showed that accession 7259 was moderately resistant. The selected accessions did not exhibit significant variation for various physiological and biochemical parameters at field capacity. However higher value of photosynthesis, chlorophyll content, chlorophyll stability index, relative water content and membrane stability index and low leaf temperature were observed for accessions viz. 7215, 7240, P 5 and 7241 after five days and ten days of moisture stress induction following field capacity compared to other accessions. Higher values of proline, SOD, catalase and peroxidase were also observed for these accessions. The visual scoring showed that accessions with higher values for most of physiological and biochemical parameters of drought tolerance viz. 7215, 7240, P5, and 7241 had lesser number of fallen leaves and more number of leaves retained at permanent wilting point (PWP). The accessions 7215 and 7240 took twenty days to reach PWP compared to eleven accessions which took only 16 days to reach PWP. Foliar nutrition with sulphate of potash, IISR - Power mix and Pink Pigmented Facultative Methylotrophs (PPFM) had positive effect on drought tolerance for the accessions (7215, 7240, P5 and 7241) having natural tolerance. The identified accessions with high yield , quality and tolerance to biotic or abiotic stress can be used for further breeding programme.ThesisItem Open Access Heterosis breeding in sesame (Sesamum indicum L.).(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2011) Gayathri, G; KAU; Dijee, BastianThe study entitled ‘Heterosis breeding in sesame (Sesamum indicum L.)’ was undertaken at the Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara. The objectives of the study were to collect and evaluate different genotypes of sesame for morphological traits and yield attributes, to identify useful parents producing heterotic crosses and developing hybrids in sesame. The study also intended to develop male sterile lines in sesame through interspecific hybridization with Sesamum malabaricum. Sesamum indicum and Sesamum malabaricum accessions were collected from Kerala and Tamil Nadu and evaluated for their morphological traits. Wide range of variation was noticed for characters like plant height, number of days to flowering and seed yield per plant which contributed maximum to genetic divergence. The genotypes studied were grouped into six clusters. High genotypic coefficient of variation (GCV) was recorded for number of capsules per plant, plant height, seed yield per plant and number of branches per plant. High heritability with high genetic advance as per cent of mean was recorded for number of days to flowering, plant height, number of branches per plant, number of capsules per plant and seed yield per plant. This indicates that the characters are governed by additive gene effects and selection for these traits will be effective. Association analysis revealed that seed yield per plant was correlated to plant height, number of capsules per plant and number of days to flowering. Path coefficient analysis indicated maximum positive direct effect by number of capsules per plant, capsule length, plant height and 1000 seed weight on seed yield per plant. In order to develop hybrids, fourteen parents were selected based on the per se performance of the genotypes. They were crossed in line X tester mating design. Forty eight hybrid combinations obtained were raised in the field along with the parents and evaluated for their heterosis and combining ability effects. Parental genotypes AVTS-06-5, AVTS-06-10, IVTS-06-12, KYM-1, Tilak and TMV-6 were identified as high combiners based on general combining ability (gca) effects. Two combinations viz. AVTS-06-5 X KYM-1 and IVTS-06-12 X TMV-3 had significant values of per se performance, specific combining ability (sca) effects and standard heterosis for seed yield per plant. They can be evaluated for their hybrid vigour over locations and seasons. The crosses AVTS-06-5 X TMV-3, AVTS-06-5 X TMV-6 and TCR 3279A X KYM-1 have been identified as potential cross combinations for isolation of promising segregants as the parents involved in these crosses had high significant gca effects for seed yield per plant but the hybrids recorded non significant sca effects. Interspecific hybridization between S.malabaricum and S.indicum was attempted to develop male sterile lines. Seed set was noticed in three interspecific hybrids which failed to germinate due to embryo abortion. Hence these embryos were rescued and raised in vitro to obtain the hybrids.ThesisItem Open Access Variability in asoka (Saraca asoca(Roxb.) de wilde)(Department of Plant Breeding and Genetics, College of Horticulture, Vellanikkara, 2010) Vidhu Francis, Palathingal; KAU; Radhakrishnan, V VAsoka (Saraca asoca) is a sacred tree among the Buddhists and Hindus. It is called sorrow-less tree as it removes the grief. The tree has immense medicinal properties. Its bark is mainly used for correcting uterine problems. The well-known Ayurvedic preparations of asoka bark are Asokarishtam and Asokaghrutham. Due to over exploitation of this tree for its bark, this has now become almost extinct. The International Union for Conservation of Nature and Natural Resources (IUCN) has listed this species under ‘globally vulnerable’ category. It is also enlisted among the 36 threatened and endangered medicinal plants of India. Due to its acute short supply compared to its demand, various development and research activities are being prioritized to conserve, utilize and improve this species. Therefore the present study was undertaken to assess morphological variations in existing germplasm and to study the reproductive biology of S. asoca. Collection of seeds and evaluation of seed and seedling traits, evaluation of therapeutical components and molecular characterisation of asoka were the other objectives of the study. Variability studies for morphological traits of asoka indicated that height of plant and stem girth have high correlation with bark yield as well as higher direct effect. These traits can be used for identifying better genotypes for higher bark yield. In discriminant function analysis, the selection index involving height of plant along with bark yield constituted for selection criteria among asoka genotypes. Using this selection index, accessions IC566463, IC566489, IC566488, IC566482 were selected as better accessions for higher bark yield. The 43 accessions maintained in germplasm of asoka at AICRP on M&AP were grouped into two major clusters based on morphological traits. These clusters further formed 6 clusters. Accession IC566488 grouped alone in one cluster indicated that this accession was different from all other accessions. In all the other 5 clusters, accessions from Thrissur was included. This indicated that accessions did not follow geographical distributions. Some of the accessions from Thrissur may have relationship with accessions from other districts. Reproductive biology of asoka was studied. It indicated that the reproductive traits like floral biology, anthesis, pollen morphology, its viability have variability among eight trees studied in KAU campus. Among the eight trees studied, ‘KAU8’ have better reproductive traits compared to the rest. Preliminary studies were carried out in pollination system and agents for pollination in asoka. The studies indicated that ants may be one of the pollinating agents. Seed and seedling behaviour of asoka were studied on seeds collected from 80 trees located in different districts of Kerala. Seed breadth, seed volume, height of plant and stem girth were identified as selection traits for better seedlings. Hence at seed stage, bigger sized seeds with higher seed volume will result into better seedlings. Vigorous seedlings is produced from tall seedlings with higher stem girth and in mature plants for higher bark yield, height of plant and stem girth can be selection traits. Thus the selection parameters were worked out in three stages in asoka. Based on seed and seedling selection parameters, OKL4, OKL2 from Odakkali, KKL2 from Kottakkal and TVM2 from Thiruvananthapuram were selected as better mother plants for higher bark yielding accessions. The biochemical constituents (phenol and tannin content) imparting medicinal properties were estimated among the different age groups of asoka. Both phenol and tannin contents were higher in bark compared to flower and leaves. About 50 per cent of therapeutical constituents are available in flowers and hence at non destructive level, flowers of asoka can be substituted for bark. The trees KAU8, KAU7, KAU6 and among the accessions IC566474, IC566467 and among the seedlings VKA6, VKA7, VKA8, KMK3 indicated higher phenol and tannin content compared to rest. Molecular characterisation among the selected asoka accessions representing the different districts of Kerala were studied. RAPD analysis was attempted among ten accessions selected. Dendrogram was constituted based on pooled RAPD data. The ten selected accessions were grouped into two major clusters as done in morphological grouping. In grouping at molecular level, the accession IC566488 kept apart as a single group indicating the same trend at its morphological level.The clustering pattern based on molecular characterisation did not follow geographical distribution of accessions.ThesisItem Open Access Cross compatibility analysis for production of hybrids in anthurium andreanum linden(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2010) MadhuKumar, K; KAU; Mayadevi, PThe present study, ‘Cross compatibility analysis for production of hybrids in Anthurium andreanum Linden’ was undertaken to identify suitable parents with commercial qualities and to determine the cross compatibility among the selected parents in anthurium. The present investigation was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during the period 2006-2009. The analysis of variance revealed significant variation among the 40 genotypes for the eighteen characters studied. This reveals the high genetic potential for the improvement in this crop. Variability studies indicated high phenotypic and genotypic coefficients of variation for the characters for anthocyanin content, pollen fertility, leaf size/leaf area, spathe size and spadix length. The high heritability coupled with high genetic advance values were found for characters plant height, leaf size/leaf area, internodal length, spathe size, total anthocyanin content, spadix length, inclination of candle with spathe, number of flowers per spadix, life of spadix, days to initiation of female phase, days to inter phase, duration of male phase, pollen fertility and pollen size. These characters are therefore controlled by additive gene action and amenable to genetic improvement through selection. Pollen fertility estimated using acetocarmine method indicated that most of the genotypes had low fertility values. Liver Red had the highest pollen fertility of 43.01 per cent followed by Lady Jane (36.14 per cent). Pollen emergence was low in the months from March to June, during which the average maximum and minimum temperatures were relatively high. Pollen emergence was highest during October to December months. A study of six qualitative characters such as colour of young leaf and petiole, spathe colour, spathe texture, candle colour and type of inflorescence axis also showed considerable variation among the genotypes studied. Plant height was found to have highly significant positive phenotypic and genotypic correlation with leaf size/leaf area, internodal length, total Anthocyanin content, spadix length, number of flowers per spadix and life of spadix. Number of flowers per spadix had significant positive genotypic correlation with plant height, leaf size/leaf area, internodal length, spathe size, spadix length, total anthocyanin content, life of spadix, pollen fertility and pollen size. Spadix length had significant positive genotypic correlation with plant height, leaf size/leaf area, internodal length, spathe size, number of flowers per spadix, life of spadix and pollen size. Days to initiation of female phase exhibited significant negative correlation with plant height, leaf size/leaf area, spadix length and pollen fertility. The environmental correlations were absent for almost all pairs of characters except for number of leaves/spadices per plant with spathe size. Path analysis revealed that spadix length, plant height, leaf size and life of spadix had high positive direct effect on number of flowers per spadix. Mahalanobis D2 analysis clustered the 40 genotypes into seven clusters. The maximum number of genotypes (17) were included in Cluster I, followed by cluster II (7), cluster III (5), cluster IV (5) and cluster V (4). Clusters VI and VII had one genotype each. Maximum divergence was shown between the Clusters II and VI, while the minimum divergence between clusters I and II. The intracluster distance was highest for the Cluster II. Among the 13 characters considered, life of spadix contributed maximum towards divergence followed by days to initiation of female phase. Grouping of genotypes into different clusters did not reflect the geographical origin of the varieties. Selection index analysis revealed that genotype Liver Red attained the maximum selection index value followed by PR x LR and PR x DT (1) and the minimum estimates were recorded for Rembolina, W x LJ and Corolix. The grouping of genotypes by selection indices followed almost the same pattern as their clustering pattern in the D2 analysis. Intervarietal hybridization was done to analyse the cross compatibility between 15 genotypes based on the percentage of candles bearing fruits, fruit set and seed germination. A total of 127 crosses were attempted based on the availability of receptive spadices and fresh pollen, out of which 80 were found to be successful. In almost all cross combinations the percentage of candles bearing fruits ranged from 50 to 100 per cent. Among the 15 genotypes, the maximum percentage of candles bearing berries was obtained for PR x OG (90.00 per cent) followed by AW (66.67 per cent) and PR x LR (65.00 per cent). The lowest value 5 per cent was obtained for Esmeralda and 11.11 per cent for Dragon’s Tongue. The number of fruits per candle ranged from 5 in Esmeralda x (PR x OG) to 85 in (PR x OG) x (OG x DT). The crosses LR x DT, (OG x DT) x (PR x OG), LR x FR and (PR x OG) x DT also recorded higher number of fruits per candle. The average number of fruits per candles was highest for Liver Red and lowest for Esmeralda. The percentage of fruit set was below 50 per cent for all the crosses. The lowest and highest percentage of fruit set was observed for Ceasor Violet and ‘Liver Red respectively. The berries obtained from different cross combinations took 4.5 to 7 months to mature. Most of the crosses had a high percentage of single seeded berries compared to double seeded berries except for the cross FR x LR. Among single seeded berries maximum seed size was for AW x (PR x OG). In a berry largest seed among the two seeded berries were observed for the crosses AW x (PR x OG) followed by [(PR x LR) x (PR x DT (2)]. The number of days taken for germination varied from four to nine days. The seeds obtained from most of the crosses showed germination except three crosses i.e., DT x LR, (KR x LR) x LJ and Esmeralda x (PR x OG) did not germinate at all. Percentage of germination was lowest in [PR x DT (2)] x LJ and highest in (PR x LR) x C. Seedlings of 57 out of 73 crosses that germinated survived for more than four months. The seedling survival percentage ranged from 38.46 per cent in LJ x (PR x OG) to 81.25 per cent in (KR x LR) x DT. The highest average survival was recorded by Liver Red and lowest was recorded for the genotype Lady Jane. Compatibility parameters estimated based on the performance of the fifteen genotypes as pollen parents. Highest percentage of candles bearing fruits was shown by PR x OG followed by Fla Red, Dragon’s Tongue and PR x LR. Lowest percentage were recorded for Flirt and KR x LR. Number of fruits per candle was the highest for the genotype PR x DT (2) followed by PR x OG, Dragon’s Tongue and Acropolis white. In Lady Jane, Flirt and KR x LR the number of fruits were relatively lower. Higher percentage fruit set was observed for the genotypes Dragon’s Tongue while it was lowest in Lady Jane. Percentage of seed germination was the highest for Fla Red and lowest for Lady Jane. Scoring of the compatibility reactions based on the percentage of fruiting candles, fruit set and seed germination on a scale ranging from zero to nine. After scoring the different genotypes, the crosses with highest compatibility score was for the crosses (PR x OG) x (OG x DT), (PR x LR) x C, FR x DT, AW x (PR x OG), (OG x DT) x (PR x OG), (PR x LR) x (PR x OG),PR x DT (2) x (PR x OG), (OG x DT) x LR, LJ x LR, (PR x OG) x DT, (PR x OG) x LR, (PR x OG) x (PR x LR) and (PR x LR) x FR. So these were the most compatible crosses. The performance of the variety as female parent was found to be different from its performance as male parent. The female parents PR x OG, PR x LR, Lady Jane, OG x DT and PR x DT (2) got the high varietal scores. Among male parents PR x OG, DT, OG x DT, FR and PR x LR got the high varietal scores. So PR x OG, OG x DT and PR x LR could be judged as the best male as well as female parents. Among the seedlings of the successful crosses maximum number of days for maturity of leaves was taken by the cross LJ x (PR x LR) and the least number of days was taken by C x (PR x OG). Number of leaves ranged from 4.50 in DT x FR to 6.33 in [(PR x OG) x (OG x DT)]. The leaf area was maximum for the cross LR x FR and it was minimum for LJ x (PR x OG). The colour of young leaf showed a range from brown to reddish brown to greenish brown to green. The colour of petiole also varied from brown to reddish brown to greenish brown to green.ThesisItem Open Access Genetic analysis of yield and leaf curl virus resistance in chilli (Capsicum spp.)(Department of Plant Breeding, College of Agriculture, Vellayani, 2010) Anandhi, K; KAU; Abdul Khader, K MPepper fruits (Capsicum spp.) are among the most consumed vegetables as fresh green or red and dried whole or ground forms in the world for its pungency. Leaf curl virus is an important biotic stress transmitted by the vector, Bemisia tabaci. Controlling the vector can be the only way to manage the disease and results in only partial control of disease. The incidence of disease is more in summer season and makes the cultivation uneconomical. There is an immediate need to develop leaf curl resistant varieties especially for summer cultivation. Hence the present investigation was undertaken at the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani during 2007-2009 with the objective of estimating the combining ability, heterosis and gene action involved in the inheritance of yield and leaf curl virus resistance. Five resistant C. frutescens accessions Mangalapuram Local, Thavanur Local, Kayamkulam Local, Mavelikkara Local and Nenmara Local were crossed in L x T pattern with three high yielding susceptible varieties Jwalamukhi, Jwalasakhi and Vellayani Athulya and 15 F1s were produced and evaluated along with the parents in randomized block design. Analysis of variance revealed highly significant genotypic difference for all the characters studied. The observations recorded were plant height (cm), number of branches, number of days to first flowering, plant spread (cm), duration of flowering (fruiting span), number of fruits per plant, fruit length (cm), fruit width (cm), pedicel - fruit ratio, fruit colour at intermediate stage, green fruit yield per plant (g), average fruit weight (g), number of seeds per fruit, hundred seed weight (g), duration of crop, vector population and virus disease scoring. Higher estimates of PCV and GCV were recorded for vulnerability index and green fruit yield per plant. High heritability coupled with high genetic advance were exhibited for days to first flowering, duration of flowering, number of fruits per plant, green fruit yield per plant, number of seeds per fruit, duration of crop and vulnerability index. Combining ability analysis showed that the line Mavelikkara Local and Nenmara Local were alone good general combiners for fruit yield along with leaf curl resistance. Four hybrids viz., Mavelikkara Local x Jwalasakhi, Nenmara Local x Vellayani Athulya, Kayamkulam Local x Jwalamukhi and Thavanur Local x Jwalamukhi exhibited significant sca effect for fruit yield. Considering per se performance, standard heterosis and sca effect two hybrids Mavelikkara Local x Jwalasakhi and Nenmara Local x Vellayani Athulya were found to be superior. Generation mean analysis was carried out using six-parameter model. Six generations viz. P1, P2, F1, F2, B1, B2 were built up among the crosses Mavelikkara Local x Jwalasakhi and Nenmara Local x Vellayani Athulya. Presence of additive, dominance and epistatic interaction for all the characters indicated that hybridization or recombination-breeding programme can be followed for future breeding.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.ThesisItem Open Access In vitro multiplication and DNA fingerprinting of selected hybrids and their parents in anthurium andreanum linden(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2007) Yasin Jeshima, K; KAU; Mayadevi, PAnthurium is the largest genus in the family Araceae, encompassing more than 800 species, native to tropical America, from Mexico, Costa Rica, Cuba to Brazil and Argentina. The spadix is composed of a multitude of flowers, which are perfect having two-carpelled ovary and four anthers. A few commercially grown plants are classified as "rat tail" anthuriums as their inflorescences have a long spadix and a small non-descript spathe. Anthuriums with colourful inflorescences have been grown for cut flowers. With the introduction of compact interspecific hybrids through breeding and the selection of somaclonal variants, the new commercially available types were developed. Propagation is not easy for anthuriums and is considered a long-term crop which will take long time for the propagator to multiply. In light of afore said views, an attempt was made to standardize in vitro multiplication and DNA fingerprinting of selected hybrids and their parents in Anthurium andreanum Linden. The explants after standardizing for the surface sterilization and survival were cultured on selected media with different hormone concentrations to get maximum callus induction. For callus induction the culture flasks were kept in dark at 25˚C and subcultured every third week. Calli were transferred to regeneration medium and embryogenic calli induction medium. Regenerants were selected and placed in rooting medium; further hardened and transferred to the field condition. Preconditioned embryos were suspended in calcium free half strength NN medium supplemented with 1.5 per cent sodium alginate and 0.5 M sucrose. This mixture was dispensed with a micropipette into 0.1 M calcium chloride. Twenty minutes after encapsulation beads were pre cultured on modified half strength NN liquid medium supplemented with 0.75 M sucrose and three per cent DMSO into 100 ml Erlenmeyer flasks for one day without agitation. Beads were then transferred to fresh medium of same composition and incubated in darkness at 4ºC for three days. Beads were desiccated in a sterile laminar air flow chamber. Dehydrated beads were transferred to 4 ml cryo vials and stored at – 80˚C. On rewarming over a water bath at 25˚C, the beads were transferred to culture medium for germination. Surface disinfection treatments were standardized for the different explants, irrespective of the explants and varieties and double sterilization was found to be effective. Among the explants, the highest number of sterile cultures was observed in double sterilization, followed by the treatment with 70 per cent Ethyl alcohol for 20 minutes. Majority of the contamination found in the cultures was due to the presence of systemic infection of Xanthomonas compestris pv dieffenbachiae. This directly influences the percentage of contamination occurred in the culturing condition and the size of explants which also play a major role in creating the bacterial contamination. Candle explants were found to exhibit more systemic infections than other explants and seed explants were found to be free from systemic infections. Leaf explants are highly vulnerable to exhibit systemic infections and are more sensitive; unable to recover even after treatments with antibiotics. The callus cultures exhibiting systemic infections can be recovered by kanamycin 50 mg per litre containing multiplication medium. In most studies of in vitro culture of anthurium, MS medium has been used. In the present study also, it was observed that Nitsch and Nitsch medium was better than MS medium for multiple shoot induction. Nitsch and Nitsch medium is especially suitable for morphogenesis, meristem culture and regeneration. As the genotype showed different nutrient requirements for their survival and growth, the present investigation was planned to standardize the media by screening with modified MS, half strength MS, modified NN and half strength NN medium. Modified NN with activated charcoal and coconut water showed better response. Half strength NN with coconut water and activated charcoal, modified MS with activated charcoal and coconut water and half strength MS activated charcoal and coconut water were also found to support the explants without hindering the survival. Addition of inositol and glycine along with folic acid was found to be essential but the presence of small amount was inefficient. In the present investigation no callus initiation was observed when inositol was reduced to half of the reported quantity. Various treatments were tried for callus multiplication. The maximum fresh weight of callus 4.2458 g was observed in PR X DT inoculated in NN medium with major nutrients at normal strength followed by 4.1325 g in OG X DT for the same composition in NN medium. From the economic point of view NN medium can be recommended for callus multiplication. Among the treatments, combination of 2, 4-D and zeatin was found to be the best. It stimulates callus formation and strongly antagonizes organized development. The low auxin requirement may be due to the high potency of the auxin which was used for callus initiation. The young developing leaf may be a rich source of endogenous auxins due to which lower exogenous application is required. Irrespective of the source of explant all the callus cultures were able to be converted into plantlets by redifferentiation. The number of days taken for regeneration ranges from 55.5 to 82. This variation is due to the varietal difference and difference in hormonal effect. Modified NN with activated charcoal and coconut water along with 22.2 μM BA, 11.42 μM IAA and 4.09 μM biotin was found to produce regenerants. Each genotype is varying with the response to change in media composition in producing somatic embryogenesis. The treatments with MS and modified MS media were found to be insignificant when compared with NN and modified NN media. Among these modified NN was found to be the best one. Within two weeks on embryo development medium, the globular embryos developed a bipolar shape. Embryos at this stage were comprised of cells larger than those at the globular stage. Bipolar embryos had an extended upper region that formed the cotyledon and the epicotyl, and a lower region that formed the radicle.The main difference between the mature embryos of monocotyledons in vitro and in vivo is the absence or presence of suspensor. The presence of single cotyledon which is the terminal structure and the shoot initials present at the sides or hidden creating a heart shape. When the cotyledon starts growing the embryo will have a single cotyledon at the terminal end which is some what cylindrical in shape. In anthurium tissue culture, no special rooting treatments were needed and the shoots developed in vitro were found to develop roots spontaneously even in the absence of additional growth hormones in the supporting medium. The spontaneous root formation was not due to the carry over effect of the hormones supplied in the previous cultures for shoot formation. Irrespective of the supporting medium the shoots were able to form roots even in sterile sand supplied with sterilized compost materials. For in vitro studies Completely Randomized Block Design (CRD) was followed for statistical analysis wherever necessary. Molecular characterization of twelve hybrids and their parents were carried out with RAPD using AP-PCR. Young leaf samples from each genotype were collected for DNA isolation.Young copper coloured leaf tissues were used immediately after collection for DNA extraction. Leaf samples were pre-chilled at -80ºC for half an hour with pestle and mortar and then pulverized in liquid nitrogen by rapid grinding to a fine powder.The frozen powder was used to extract the total genomic DNA using CTAB extraction buffer. The purity of the DNA was analysed by running in 0.8 per cent agarose gel with 1 X TAE buffer. The optimized PCR mixture with 50ng of template DNA for a final volume of 20μl was used in thermal cycling in a PCR machine. The amplified products were run in 1.6 per cent agarose gel with 1X TAE (Tris buffer, Glacial acetic acid and EDTA pH 8.0) buffer. A total of 114 AP-PCR bands were generated by the 25 primers, of which 74.56 per cent were polymorphic (88 bands) and 26 were monomorphic. Ten primers showed high level of polymorphism out of which seven were selected.Seven promising primers were identified for AP-PCR analysis based on performance in DNA amplification, production of highest number of polymorphic bands as well as intense bands and reproducibility viz. OPA 10, OPB15, OPA13, OPB20, OPB6, OPB8 and OPB18 primers were found to produce polymorphism in Anthurium andreanum Linden.A total of 50 scorable bands (average of 7.143 bands per primer were generated by the selected seven primers of which only 8 were monomorphic and the rest were polymorphic. The number of bands ranged form 4 to 11 with an average of 7.143 per primer. The reproducible bands were scored for their presence (1) or absence (0) for all the hybrids and parents. A genetic similarity matrix was constructed using Jaccards’s similarity co-efficient methods. From the cluster analysis based on the dendrogram, TR X MW was found to be extreamly different from the other accessions and its own parents showing the significance of hybridization. The hybrids like OO X KR, PR X DT, OG X DT, FK X LR and PR X MW are not closely related to either of their parents and hybrids were distinguished from others. Some hybrids like LJ X W and PR X MW; PR X LR and FK X DT shows 30 to 39 per cent similarity. This shows there is considerable variability among the genotypes selected and can be further utilized for crop improvement. Confirming that, they were quite different from the other hybrids and varieties. Pair wise genetic distances based on RAPD [(AP-PCR) (Nei and Li Genetic Distance GDNL)] genetic distance co-efficient values for twelve varieties and twelve hybrids ranged from 0.1935 to 0.7037 indicating the wider diversity. The AP-PCR profiles show the relatedness and diversity of the hybrids and varieties. The bands were found within 1.5kb from 100bp. Most of the bands were concentrated between 300bp and 1200bp.
