Browsing by Author "Valsala, P A"

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Cloning and characterization of fusarium wilt resistance gene analogs in banana (Musa spp.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Ramesh; Valsala, P A
Development of a molecular marker for bacterial wilt resistance in brinjal ( Solanum melongene L.) varieties Surya and Swetha.
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Somya, P P; Valsala, P A
The study entitled ‘Development of a molecular marker for bacterial wilt resistance in brinjal (Solanum melongena L.) varieties Surya and Swetha’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the important problems of brinjal cultivation in warm humid tropics. The loss due to this varies from 30-100 per cent. Use of chemicals and field sanitation are not sufficient for controlling the disease. Worldwide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant brinjal varieties for cultivation. The Surya and Swetha are two among them and have received bacterial wilt resistance from SM-6 an Annamalai collection. This investigation was taken up to develop a molecular marker for bacterial wilt resistance in Surya by RAPD through bulk segregant analysis as reported by Michelmore et al (1991). It also aimed to test the suitability of the same for identifying bacterial wilt resistance trait of resistant variety Swetha. The genotypes used for the study were Surya, Pusa Purple Long (susceptible variety released from IARI), Swetha and F2 population of the cross between Surya and Pusa Purple Long. To raise segregating F2 population F1 was raised by controlled crossing of Surya with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population. Two different methods viz., stem puncturing and soil drenching with root wounding were compared for the delivery of inoculum of R. solanacearum for bacterial wilt incidence and stem puncture method was found as the best. So stem puncturing method was used for phenotyping of genotypes for bacterial wilt incidence. The F2 population along with Surya, Pusa Purple Long and F1 were phenotyped for bacterial wilt incidence. This was done through artificial inoculation with Ralstonia solanacearum by stem puncture method. Confirmation was done by ooze test. The genotypes were classified according to classification of Mew and Ho (1976). The variety Surya was resistant and Pusa Purple Long was susceptible. F1 population showed 90 per cent susceptibility while F2 population showed 83 per cent susceptibility. They were classified as susceptible. Five resistant and five susceptible genotypes from F2 were selected for bulk segregant analysis. Genomic DNA for RAPD analysis was isolated by Rogers and Bendich method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis.PCR reaction mixtures and conditions for DNA amplification were standardised. Ninty two, 10-12 bp primers belonging to OPA, OPB, OPC, OPF, OPE, OPU, OPH, OPAH, OPAG, OPL, OPM, RY, RN, RA, SC, RF, AG 8, WG 44, GLE11, RF, R10, R6, and PUC101 were initially screened with resistant genotype Surya and susceptible genotype Pusa Purple Long to select primers with polymorphism and good amplification. Out of ninty two primers tested thirty were reported as bacterial wilt specific. The PCR products were electrophoresed and twenty two primers were selected for BSA based on amplification power and polymorphism. They were RY 01, RY 02, RY 03, RN 19, OPF 5, OPL 04, OPA 04, OPA 6, OPA 9, OPA 24, OPA 26, OPA 34, OPA 36, OPS 9, OPS 10, OPS 16, OPS 17, PUC 101, RA 12-41, and RF. Among these only the primer RY 02 recorded polymorphism between resistant and susceptible variety with an amplicon of 1.2kb. In bulk segregant analysis DNA of Surya, Swetha, Pusa Purple Long and bulk DNA from resistant genotypes and susceptible genotypes were amplified with selected primers and products were electrophoresed. All primers produced only monomorphic band. None has produced unique polymorphic bands capable of differentiating resistant and susceptible genotypes. This may be due to low polymorphism at genomic level.
Evaluation of turmeric types for yield and quality
(Kerala Agricultural University, 1995) Alice Kurian; Valsala, P A
Genetic transformation of black pepper (piper nigrum L) for phytophthora foot rot resistance/tolerance
(Department of Plantation Crops and Spices, College of horticulture, Vellanikkara, 2007) Lissamma Joseph; Valsala, P A
Investigations on "Genetic transformation of black pepper (Piper nigrum L.) for Phytophthora foot rot resistance/tolerance" were carried out at the Department of Plantation Crops and Spices, and Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2001-2006. Three selected black pepper varieties Panniyur 1, 4 and 6 were utilized for the study. Axenic cultures of the selected varieties were raised from nodal segments as well as ripe seeds for embryogenesis and transformation studies. Among the varieties average number of multiple shoot/explant was high for variety P6 in ½ MS medium with BA and IAA 1.0 mg l-1. Somatic embryogenesis was induced on ripe seeds of the variety P6 at the micropylar region in ½ MS medium with inositol 1.0 g l-1. Further development of the somatic embryo observed in liquid shaking cultures of SH basal. Growth regulators like BA,2,4-D ,dicamba and thidiazuron did not give rise to embryogenesis in the different explants of black pepper. Multiple shoot induction from cotyledonary nodal explants and zygotic embryo explants were observed in all the varieties in ½ MS with 1.0 mg l-1 BA and IAA. Direct regeneration from leaf segments was also observed in the same media combination. Agrobacterium tumefaciens strains EHA105, AGL.1.1303, GV2260 and LBA4404 were used for the transformation work. Strain EHA105 contains the plasmid p35SGUSINT with gus A gene and npt II gene. The AGL.1.1303 contains the plasmid harbouring antibiotic resistant selectable marker genes (npt II and hpt IV) and GUS and GFP reporter genes. The GV2260 contain the plasmid pGV2260 with osmotin gene and npt II gene. The LBA4404 contains the plasmid pBZ100 containing alfalfa glucanase gene, rice chitinase gene and npt II gene. Sensitivity studies of black pepper tissues to various antibiotics resulted in selecting the optimum threshold level of antibiotic to be used in the screening medium. Kanamycin 25 mg l-1, 50 mg l-1, and 100 mg l- were selected as the cut off level for the selection of transformants from zygotic embryo, leaf segments and nodal segments respectively. Cefotaxime at 250 mg l-1 was selected for the effective elimination of Agrobacterium after infection. Genetic transformation was standardized with Agrobacterium strain EHA105 using leaf disc and zygotic embryo explants. Tentative protocol for transformation in black pepper include Agrobacterium inoculum density 0.9, infection time 10 min and co-cultivation period of 48 h. Acetosyringone at 50-100 µM favoured transformation. Transient gus assay revealed faint blue staining on the infected leaf explants. Explants, leaf segment, cotyledonary node and zygotic embryo were used for transformation with Agrobacterium strains AGL.1.1303, GV2260 and LBA4404. There was explant specificity for the different Agrobacterium strains used. With LBA4404 zygotic embryo explants gave maximum survival in the screening medium containing 50 mg l-1 kanamycin and 250 mg-1 cefotaxime. Direct gene transfer using gene gun attempted with pBZ100 and cotyledonary node explants. Bombarded explants were found surviving in the screening medium with kanamycin for four months. However molecular analysis of selected transformants through PCR revealed that npt II gene integration has not happened in the tissues subjected to PCR analysis.
In situ green manure production as mulch material for ginger
(Kerala Agricultural University, 1997) Alice Kurian; Sreekandan Nair, G; Valsala, P A
In vitro micropropagation protocol for vanda hybrids with clonal fidelity analysis
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2016) Rosemol Baby; Valsala, P A
Vanda orchids are one of the most sought after orchids in the international as well as domestic flower markets both as cut flower and potted plants. It is a monopodial orchid with vividly coloured, loosely arranged large beautiful flowers which has a long shelf life. Presently, many Vanda hybrids are becoming prominent even in the home gardens. However the present scenario of importing these hybrids from Thailand, Singapore and Malaysia to meet the Indian demands throws light on the need for developing an efficient propagation method for Vanda orchids. One of the major limiting factors for its spread and large scale cultivation in India is the non-availability of good quality and true to type planting material at a reasonable price. As the demand is more for the true to type plants, micropropagation is mostly recommended for orchid propagation. Hence this study was undertaken to develop an efficient micropropagation protocol for two Vanda hybrids namely Dr.Anek and Sansai Blue and to check the variability between the parents and regenerated plantlets. The different explants tested to initiate the cultures were leaf, root, stem and inflorescence segments. Initially the surface sterilization procedure was standardized for the explants. The results of the experiment showed that treating the explants with 0.1 per cent carbendazim for 20 minutes, followed by 70 per cent ethanol for 5 minutes and 0.1 per cent mercuric chloride for 5 min effectively reduced the microbial contamination with highest percentage of explant survival.Trial was made to initiate cultures using eight reported media compositions. The study showed positive results for inflorescence segments inoculated on to 1/2 MS + 10 mg l-1 BA+ 2 mg l-1 TDZ+30 g l-1 sucrose+7.5 g l-1agar + 250 mg l-1 cefotaxime as observed as direct shooting of the dormant buds. About 80 per cent and 60 per cent culture establishment was brought about in Dr. Anek and Sansai Blue respectively in 9 weeks. The established cultures successfully produced multiple shoots on MS+4.5 ml l-1BA +30 g l-1 sucrose + 7.5 g l-1 agar + 250 mg l-1 cefotaxime both when inoculated with and without the stalk in about 100 days of inoculation of explant. The micro-shoots from cultures without stalk were further transferred to hormone free basal MS media for elongation. Elongated shoots of about 4 cm were transferred to rooting media with a composition of MS + 0.5 mg l-1 NAA + 1 mg l-1 IAA +30 g l-1 sucrose +7.5 g l-1agar + 250 mg l-1 cefotaxime for better rooting of the regenerants. The percentage of rooting was observed to be 72.41 per cent for Dr. Anek and 70.37 per cent in Sansai Blue. The rooted plantlets with ample number of healthy roots were planted outm in small earthen pots with charcoal, coconut husk and brick pieces. These were successfully hardened in net house of 50 per cent shade and showed a hundred percent plantlet survival.Good quality DNA isolated from the mother plants and their respective clones using Rogers and Bendich procedure were analyzed for the clonal fidelity. ISSR analysis was done using 5 UBC (University of British Columbia) primers such as UBC 808, UBC 811, UBC 826, UBC 835 and UBC 841. An average of 8 to 9 bands was obtained from all primers in Dr. Anek and Sansai Blue. Out of 5 primers, UBC 808 and UBC 835 generated polymorphic bands in two clones of Dr. Anek. For Sansai Blue, all five primers generated monomorphic bands for all the mother plants and their respective clones analyzed. The per cent polymorphism in Dr. Anek was calculated to be 1.11 per cent whereas for Sansai Blue, there was no polymorphism detected revealing the true to type nature of the clones. The results showed that the identified protocol for in vitro regeneration of selected Vanda hybrids is a viable protocol since there were no changes in the banding pattern observed in tissue culture plants as compared with that of mother plant. Hence it can be concluded that the developed micropropagation protocol can be used for commercial production of Vanda hybrids without much risk of genetic instability. ISSR markers were effective to evaluate the genetic stability of the clones regenerated from the mother plants by the identified protocol.
In Vitro pollination in kacholam (Kaempferia galanga L.) for seed set
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2002) Vineel Vasudev Bhurke; Valsala, P A
In vitro pollination, embryo rescue and germination studies in ginger
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1997) Bindu, R; Valsala, P A
Investigations were carried out for the refinement of in vitro pollination techniques in ginger developed at the Plant Tissue Culture laboratory, Department of Plantation Crops and Spices, College of horticulture, mainly to identify the conditions required for the germination of in vitro produced seeds of ginger. The study was carried out from 1994 to 1996. Investigations were carried to study the effect of season, cultivars and position of flowers in the inflorescence on pollen fertility and viability. The results showed that pollen fertility and viability were influenced by the season and genotype but not by the position of flowers on the inflorescence. The pollen viability was high in inflorescences produced during the early and mid period of the flowering seasons. So scheduling the pollination works for the early and mid period of the flowering seasons may lead to more seed set. The cultivars SG-66 and Rio-de-Janeiro exhibited more pollen fertility and viability. So chances of seed set will be more in crosses involving these cultivars as male parents. Crossing studies showed that Rio-de-Janeiro as female parent can be crossed with Kuruppampady, SG-66, Nadia and as a male parent can be crossed with Kuruppampady, Nadia, SG-66 and Bajpai. So the high yield potential of Rio-de-Janeiro can be transferred to cultivars suitable for dry ginger production. Selfing studies showed that the cultivars viz., Rio-de-Janeiro, Kuruppampady, SG-66, Nadia, Bajpal, SG-603 and SG-543 can be selfed by the in vitro pollination and fertilization techniques. The in vitro fertilized ovules developed into mature seeds in the medium of ½ MS with 2, 4-D 0.1 to 1.0 mg 1-1 and BAP 5 to 20 mg 1-1. The effect of 2, 4-D could be replaced by NAA 0.5 to 2 mg 1-1 or IAA 0.05 to 0.2mg 1-1. The results of seed viability test with tetrazolium salt showed that seeds of 40 and 80 DAP are viable so seeds from 40 DAP onwards can be subjected to germination studies. The studies on germination of ginger seeds showed that primary treatments like water soaking, incubating on moist filter paper, moist sand or basal medium (both solid and liquid state) did not favour germination of ginger seeds. Incubating the seeds in ½ MS + 6 per cent sucrose along with 2, 4-D 0.1 to 1.0 mg 1-1 or NAA 0.5 to 2.0 mg 1-1 and BAP 5 to 20 mg 1-1 had no influence on germination of ginger seeds. The combination of NAA 0.5 mg 1-1 or IAA 0.05 to 0.2 mg 1-1 with 2iP 2.5 to 5 mg 1-1 had no influence on seed germination. GA3 5 to 10 mg 1-1 and ethylene 5 to 10 mg 1-1 also did not favour seed germination. Seed treatments like chemical and mechanical scarification, stratification, washing the seeds in running water and subjecting the embryos to stress condition by dehydrating hydrated seeds for 12 h or soaking the seeds in 12 per cent each of Mannitol and PEG-4000 solution did not influence germination. Embryo rescue involving transfer of embryo along with endosperm to culture media with varying combinations of auxins and cytokinin also did not promote development of embryo to seedling.
In vitro shoot regeneration and micrografting in nutmeg (Myristice fragrans houtt.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2008) Liffey Zachariah Antony; Valsala, P A
Induction of genetic variability in ginger (zingiber officinale rosc.) through in vitro fertilization
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Rethidevi, A; Valsala, P A
Investigations on ‘Induction of genetic variability in ginger (Zingiber officinale Rosc.) through in vitro fertilization’ were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2004-2005. Molecular characterization studies of the eight ginger cultivars -Z-0-78 (V1) Kodakara Local (V3), Kuruppampady (V4), Mahima (V5), Maran (V6), Rejatha (V7), Rio-de-Janeiro (V8) and Varadha (V9)- by RAPD using nine random primers revealed that the genetic variability among the cultivars very narrow in the range of 5 to 19 per cent. The cultivar Rio-de-Janeiro was distinct with maximum variability. The variability among the released varieties from IISR ranged from 5 to 10 per cent. The flowering season in ginger extended from August to November. The cultivars showed variability with respect to duration for initiation of flowering and flowering duration. The mean pollen fertility and viability among the ginger cultivars were 35.76 per cent and 8.11 per cent respectively. The autotetraploids had high pollen fertility and viability compared to diploids. The range of pollen viability in autotetraploids was from 12.03 to 15.68 (Z-0-78) and in diploids it was from 3.68 to 8.90 per cent. Among the diploids the released varieties Varadha, Mahima and Rejatha had more pollen fertility and viability. Attempts were made to refine the in vitro pollination technique with respect to basal media combination, growth regulators, vitamins and gelling agent. Studies conducted in the cross Varadha X Z-0-78 using placental pollination technique. The basal medium of half MS with 2X vitamins was superior to B5 medium. As gelling agent phytagel 0.18 per cent was superior to agar 0.65 per cent. Among the various plant growth regulator combinations tried the combination of picloram and BA was found to be best. The medium of half MS with 2X vitamins + 3 per cent sucrose + BA 2.5 mgl-1+ picloram 0.2 mgl-1 + 0.18 per cent phytagel was the best with respect to maximum ovule swelling, percentage of cultures with ovule development and percentage setting per culture. Seed set and seed development in various crosses involving autotetraploids and diploids were assessed. The parental combination between autotetraploids (Z-0-78 X Z-0-86) produced seeds with very good swelling in maximum number of cultures with maximum setting per culture. The parental combination Z-078 X Varadha and its reciprocal also favoured very good ovule swelling. The parental combination Z-0-86 X Varadha as well as Varadha X Mahima and their reciprocals produced seeds with good swelling. The size increase of the developing ovules after in vitro pollination was monitored from the day of pollination to 60 DAP. The size increase upto 15 DAP was rapid. At 60 DAP the ovules attained four-fold increase in size. The pollinated ovules cultured, in the course of maturation turned pink and later blackened. The seeds showed complete endosperm filling at 10 DAP. The soft and jelly like endosperm became hard by 20 DAP. Viability test with tetrazolium showed that the seeds were viable up to 50 DAP. Embryo was found to be seated towards the chalazal end and endosperm constitutes the major portion of the seed. Germination studies were conducted in a number of plant growth regulator combinations. The 60 days old seed derived from the parental combination of Kuruppampady X Z-0-78 showed radicle emergence in the medium of half MS + 3 per cent sucrose + 0.65 per cent agar + IBA 3.0 mg l-1. The seeds of parental combination Z-0-78 X Varadha germinated through somatic embryogenesis in the medium of B5 + 3 per cent sucrose + 0.18 per cent phytagel + 2,4-D 0.2 mg l-1 + BA 2.5 mg l-1 105 days after in vitro pollination. The germination was 10 per cent It was found that the seed coat was pushed apart by the callusing embryo and it developed globular somatic embryoids. Embryoids developed root and shoot poles in the same medium. The somatic embryoid is in the initial stage of development. Embryo culture studies with embryos excised from seeds of 15 to 60 DAP in various media combinations did not give positive response. Prolonged culturing may give positive results.
Induction of variability in Vanilla planifolia Andrews through intra/inter specific hybridisation and embryo culture technique
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Blessy Paul; Valsala, P A
Molecular characterization of chilli(Capsicum annuum L.) genotypes for tagging bacterial wilt resistance gene
(Department of Plant Biotechnology and Molecular Biology, College of Horticulture,Vellanikkara, 2008) Rashmi Kumari; Valsala, P A
Investigation on “Molecular characterization of chilli (Capsicum annuum L.) genotypes for tagging bacterial wilt resistance gene” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2006-2008. Bacterial wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al. is the most important problem of chilli cultivation in warm humid tropics. The loss due to this varies from 30-100 per cent. Use of chemicals and field sanitation are not sufficient for controlling the disease. World wide approach is to use resistant varieties. KAU has developed and released bacterial wilt resistant varieties viz., Ujwala, and Anugraha for cultivation. The present investigation was taken up to develop a molecular marker for bacterial wilt resistance gene through molecular characterization of resistant and susceptible genotypes. The genotypes used for the study were Ujwala, Pusa Jwala, Anugraha and F2 population of the cross between Ujwala and Pusa Jwala. The genotypes were phenotyped for bacterial wilt incidence through artificial inoculation with Ralstonia solanacearum. The varieties Ujwala and Anugraha were resistant. Pusa Jwala, F1 and F2 progenies were susceptible. The molecular marker techniques used were RAPD and SCAR. Genomic DNA was isolated by Rogers and Bendich (1994) method. Forty seven decamer primers belonging to six Operon primer series viz., OPE, OPAH, OPN, OPP, OPS and OPY were used for primer screening. Twenty two primers which gave more than seven bands were used for molecular characterization of selected genotypes through bulk segregant analysis. OPS 1 primer amplified a DNA fragment of 1.24 kb in resistant parent and resistant bulk. Co-segregation analysis was also done with OPS 1 primer with individuals of susceptible and resistant bulk. The polymorphic band produced by OPS 1 primer in resistant parent and resistant bulk was eluted and cloned in pGEM-T vector, and was transformed into E. coli JM 109 cells. Recombination of the insert was confirmed through RAPD reaction with OPS 1 primer. The cloned fragment was sequenced to obtain the nucleotide sequence information and was named as Chilli seq 1. The sequence obtained after vector sequence deletion was named as Chilli seq 2 and it contained only 440 bp in place of 1240 bp. It was subjected to nucleotide blast search. It revealed significant levels of homology with Capsicum annuum ethylene responsive element binding protein C2, lipid transfer protein III gene, clone A1-4 PR-protein gene and ripening regulated protein DDTFR10/A gene of chilli deposited in the public domain. The sequence was also subjected to various sequence analysis using bioinformatics tools, which include ORF finder, SOPMA, NEB cutter, Hydropathy plot, NASTATS and AASTATS tools of Biology Workbench. Based on the end sequences of the cloned RAPD fragment, SCAR primers were designed. The efficiency of SCAR primer to distinguish resistant and susceptible genotypes was tested but no polymorphism was detected. The SCAR primer OPS 11240 amplified a fragment of 396 bp in all the genotypes tested. Full sequence data of the cloned RAPD fragment was not available for SCAR primer designing. More efforts needed to get full-length sequence data.
Molecular docking of antiviral properties of Glycosmis pentaphylla (Retz.) Correa
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2017) Brinda, O P; Valsala, P A
Glycosmis pentaphylla (Retz.) Correa, a tropical shrub, locally known as ‘panaV, is widely used in Ayurveda, for cough, rheumatism, anaemia and jaundice. Although, various parts of this plant are used against different diseases ranging from ulcer to cancer and also as an antiviral agent, there is no scientific evidence for proving its antiviral potential. Molecular docking facilitates the screening of large number of phytocompounds for their capability to interact with and deactivate the disease effecting proteins, within a short period of time. Hence the present study entitled “Molecular docking of antiviral properties of Glycosmis pentaphylla (Retz.) Correa” was undertaken with the objective to characterize the active ingredients in Glycosmis pentaphylla and to identify the compounds offering antiviral properties to this plant, through docking studies. Separate dry samples were prepared from root, stem and leaf and were powdered. The finely ground powder was converted into a hydroalcoholic extract and was concentrated using rotary evaporator. The concentrated extract was subjected to LCMS/MS analysis through outsourcing. The mass to charge ratio obtained through LCMS/MS analysis was compared with the masses of various compounds identified from this plant though literature review. Identified 23 phytochemicals from leaves and 14 from stem and root were subjected for molecular docking against viral protein targets of chikungunya, hepatitis, dengue and influenza using Discovery Studio 4.0. The protein targets were identified on the basis of information provided in the Chikungunya Drug Target Database. Protein targets for other viral diseases were selected based on the literature. Twelve proteins were selected for chikungunya. Two target proteins each were selected for Hepatitis B, hepatitis C and dengue and one for influenza. Suitability of phytocompounds to be developed as a drug was identified by screening with Lipinski’s and Veber’s rule and ADMET analysis. The ligands with good interaction with the protein target were identified based on the minimum difference between CDOCKER energy and CDOCKER interaction energy. Two phytocompounds, isovaleric acid and avicequinone C, have shown acceptable interaction with the protein targets of all selected viral diseases. These two phytocompounds were found to have acceptable ADMET values (out of the seven parameters five were falling in the acceptable range). Isovaleric acid and avicequinone C were able to establish good interaction with majority of protein targets for the selected viral diseases with minimum difference between CDOCKER energy and CDOCKER interaction energy. They were capable to form hydrogen bonds with the involvement of aminoacid residues along with the establishment of good binding energy. The isovaleric acid and avicequinone C molecules with strong capability to interact and deactivate the disease causing proteins are the basis for the antiviral properties of Glycosmis penlaphylla. Futher isolation and purification of these molecules and wet lab analyses on animal models have to be done to develop antiviral drugs from this plant.
Refinement of in vivo and in vitro pollination techniques in turmeric (Curcuma domestica Val.)
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2001) Vijayasree, P S; Valsala, P A
Response of turmeric Curcuma domestic Val. to in vivo and in vitro pollination
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1999) Renjith, D; Valsala, P A
Seed set in ginger (Zingiber officinale rosc.) through in vitro pollination
(Kerala Agricultural University, 1996) Valsala, P A; Sreekandan Nair, G; Nazeem, P A
Different in vitro pollination techniques viz., stigmatic, srylar, intra-ovarian, placenta!, modified placental pollination and test-tube fertilization were tried in ginger flowers. The pollen grains suspended in ME, medium were used for pollination. The pollinated pistil or its part was cultured in the medium of 1/2 MS + NAA 0.5 mg 1' + BAP 2.5 mg 1 ' + coconut water 15 per cent v/v. Seed development was obtained in placental, modified placental pollination and test tube fertilization. The seed germinated under in vitro condition on supply of appropriate combination of 2,4-D, BAP and NAA
Sex determination in nutmeg (Myristica fragrans Houtt.) through molecular and biochemical markers
(Centre for Plant Biotechnology, College of Horticulture, Vellanikkara, 2010) Maddela Sudhamayee; Valsala, P A
Nutmeg (Myristica fragrans Houtt.) is an important tree spice of southern India, Malaysia and Indonesia yielding two products of commercial value, ‘nutmeg’ and ‘mace’. Nutmeg of commerce is the dried seed and mace is the dried aril. It is a spreading evergreen tree of the family Myristicaceae and is dioecious with long pre bearing period of 5 to 7 years. Presently, the dioecy in nutmeg is overcome by vegetative propagation or top working. Even though several vegetative methods have been reported, the large scale adoption of these methods is constrained due to insufficient number of orthotrops. So seedling continues to be the major propagating material. In the present study, an attempt was made to identify sex in nutmeg through molecular and biochemical markers so as to identify sex at seedling stage itself. Molecular marker used was RAPD and biochemical marker was isozymes of acid phosphatase and glutamate oxaloacetate transaminase (GOT). Based on bearing pattern and floral morphology, five typical male and female plants of age 10-15 years were selected for RAPD analysis. DNA isolation technique was standardised using CTAB method. Good quality DNA with UV absorbance ratio (A260/A280) 1.80- 1.84 was used for analysis. Sixty seven decamer primers were screened and four primers showing the polymorphism has been selected for further RAPD analysis. PCR amplification with selected primers viz. OPD 15, OPA 27, OPF 05 and OPK 01 was carried out with samples of bulk DNA from five male and female, DNA of individual male and female and negative control. Among them OPK 01 amplified reproducible female specific band (1.1 kb) in bulked and individual samples. The polymorphic female specific band amplified by OPK 01 primer was eluted and cloned in pDrive vector and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed white clones were sequenced at Bioserve, Hyderabad. The sequence obtained after VecScreen (Nut seq 816 bp) was analysed with various bioinformatic tools like Blastn, Blastp, GenScan, ORF Finder, Transeq, GOR and Protparam. Three open reading frames identified in the sequence Nut seq816 sing NCBI tool “ORF Finder”. The +2 ORF strand encodes two domains for amino asparate transaminase (GOT) and cystathionine beta- lyases with E value 3.6. The other ORFs didn’t encode any domain. GenScan predicted no gene in the Nut seq816. Based on sequence information two pairs (SP1 and SP2) of SCAR primers (24 bp) were designed using primer3 programme. The efficiency of SCAR primer to distinguish male and female plants was tested by PCR amplification of DNA from five male and females. The SCAR primers amplified around 300 bp single band in five females. SCAR primer was again checked with four occasional fruiting males and it amplified 300 bp band in one occasional fruiting male. Expression of SCAR primers was tested in ten seedlings and got amplified in four samples. Leaf samples from identified male and female trees were used for the biochemical marker analysis. Polyacrylamide gel electrophoresis (PAGE) for isozyme assay was standardized with standard protein and nutmeg leaf protein. Acid phosphatase assay recorded four zones of activity and all were monomorphic. Glutamate oxaloacetate transaminase showed polymorphism and need protocol refinement. Accuracy of SCAR primer SP1 to distinguish male, female and occasional fruiting male has to be done with more samples. Commercialization of the technique can be explored based on the accuracy and cost benefit analysis.
Standardisation of in vitro pollination and fertilization for generating genetic variability in Zingiber officinale (Rosc.)
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1994) Valsala, P A; Sreekandan Nair, G
Investigations were carried out to standaridse in vitro pollination and fertilization technique for seed set in ginger, Zingiber officinale (Rosc.) at the Plant Tissue Culture Laboratory of Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during 1990-1993. Investigations to improve flowering showed that early planting (March- April) is an important factor, which induce flowering in ginger, when the crop is grown from rhizome bits of standard size i.e., 15 g. Cultivar difference was also found to exist with respect to flowering. But increasing the size of seed bit from 15 g to 200 g resulted in early flowering of all the seven cultivars. Maintaining the plants as biennial registered still earlier and more profuse flowering of all the cultivars. In biennial plants flowering started as early as in 1st July, while it was only on 17th September in annual plants from 15 g. In annuals the flowering season was for two months, while it was extended for four months in biennials. The application of growth regulators did not significantly influence the flowering of different ginger cultivars. The study of floral biology of ginger flowers showed that, an inflorescence of ginger took 30-32 days from initiation to blooming. The number of flowers per inflorescence ranged from 20 to 31 and the blooming period ranged from 15 to 22 days. The anthesis started by 3.00 pm and continued upto 4.30 pm. Anther dehiscence did not occur simultaneously with flower opening and took place only after ½ to 11/2 h after anthesis. The ginger flowers are characterized by a spiny stigma and a long style of mean length of 3.46 cm. The ovoid ovary measured a mean length of 2.71 mm and diameter of 2.59 mm and recorded a mean ovule number of 24.43. The ovules recorded a mean length of 535.13 μm and breadth of 323.41 μm at the middle. The pollen fertility with acetocarmine stain in the studied cultivars ranged from 14.15 per cent in Nadia to 35.28 per cent in SG-66. Attempt to develop a medium which will support pollen germination and tube growth resulted in the identification of ME3 medium with osmoticum as 12 per cent PEG. The maintenance of the medium at pH reactions of 4.0 to 8.0 not significantly influence pollen germination and tube growth. The maximum pollen tube length registered was 1042.17 μm. The pollen germination started within 3 h of incubation and continued till 24 h. The histological examination of ovules of flowers on the day of anthesis revealed the presence of viable egg cell. The flower buds for in vitro pollination were collected and surface sterilized prior to anthesis (3.00 pm). The bacteria destroying the cultures were identified as Psuedomonas solanacearum. Dipping the unopened flower buds in streptocyclin 500 mg 1-1 for 1 h followed by wiping with 70 per cent alcohol and rinsing with mercuric chloride 0.1 per cent for 3 min gave satisfactory microbial sterilization of flower buds for in vitro pollination. Initial experiments for culture establishment showed that ginger ovary would develop under in vitro condition in ½ MS, SH or Nitsch medium when supplemented with growth regulators and CW. There was no ovary development with out hormones. Among the various methods of pollination tried, ovules developed in placental pollination, modified placental pollination and ovular or test tube fertilization. In all the three cases, pollen grains along with ME3 medium were applied over the ovules. The observation of ovules after placental pollination under fluorescence microscopy revealed that pollen germination starts within 3 h of pollination and pollen tube growth is sufficient to fertilize the ovule. Histological examination of ovules 4 DAP showed eight celled pro-embryo and 40 DAP showed well developed embryo and endosperm rich in starch and oil grains. The aforesaid successful pollination techniques are suitable for selfing and crossing in ginger under in vitro condition. Among the successful methods of in vitro pollination tried placental pollination is the best as it registered maximum number of seeds per culture with minimum effort. The mean seed set per culture in this method in four favourable media combinations was 61.56 per cent. The mean number of well developed seeds per culture was 6.87 at 80 DAP. The flower buds collected on the day of anthesis and one day after anthesis were suitable for in vitro pollination. Studies were made to standardize media supplements for enhancing ovule development. The ovules developed at all levels of sucrose concentration 3.0, 6.0, 9.0 and 12.0 per cent levels. Considering the increased seed set, 6.0 to 8.0 per cent is the optimum. The auxins as well as cytokinins alone induced ovule development but combinations proved to be better. The combination of NAA 0.5 to 1 mg 1-1 with varying concentration of BAP from 2 to 10 mg 1-1 had shown positive effect. The effect of BAP in the ovule development could be replaced by KIN (2.0, 5.0 mg1-1) or 2iP (2.5 mg1-1). Similarly the effect of NAA (0.5 to 2.0 mg1-1) could be replaced by 2,4-D (0.05 to 1.0 mg1-1) or IAA (0.05 to 0.2 mg1-1). The GA levels (2.0, 4.0, 6.0 and 10.0mg1-1) did not favour ovary and ovule development. The IAA precursor, typtophan (0.1,0.5 and 1.0 mg1-1) also behaved in the same manner. Other supplements like CW (10 to 12 % v/v) and CH (200 to 500 mg1-1) enhanced ovule development along with cytokinin and auxin. The extract from 15 days old inflorescence of ginger at a concentration of 0.3 to 3.0 mg1-1 promoted ovule development with hormones. The aminoacid L-glutamine (25 to 500 mg1-1) did not inhibit ovule development while YE (250 to 1000 mg1-1) inhibited ovule development. The solid as well as liquid form of favourable media combinations supported ovule development after in vitro pollination. The ovary and ovules developed at 260C as well as 280C but the lower temperature was best for the visual assessment of ovary and ovule appearance. With respect to light intensities, they developed in dark, diffused light and light intensities of 500 and 1000 lux. The in vitro produced seeds and fruits of ginger grew rapidly at the initial stage of 20 Dap and later growth was slow and comparatively little. The colour of the seeds at the initial stage was creamy white and in the course of development about 14.95 per cent of cultures develop, purple red colour within a period of 30.65 days. The whole seeds of a culture turned black within a period of 60 to 90 DAP. In the culture condition about 40 per cent of ovaries developed orange colour of ripening within a period of 35 to 65 days. They turned black by 90 DAP. The fruit of ginger is a thick walled three valved capsule with small black arillate seeds. Eighty days after pollination they recorded a mean diameter of 6.5 mm and a maximum of 9.5 mm. The ginger seeds recorded a mean length of 2.20 mm and breadth of 1.60 mm 80 DAP. The arillate seeds showed two seed coats, the outer being thick and the inner one being thin. The seed coat encloses a cavity and in the cavity endosperm with embedded embryo is seen. The in vitro produced seeds of ginger germinated when 80 days old seeds were incubated initially in the medium of ½ MS with 2,4-D 8 mg 1-1 for two months and then in hormone combination of BAP 9 mg1-1 and 2,4-D 0.1 mg1-1.
Tagging of bacterial wilt resistance gene in Solanum melongena var.insanum by molecular markers
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2012) Chavan Pradeep Uttamrao; Valsala, P A
The study entitled ‘Tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by molecular markers’ was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara during the period 2009-2011.Bacterial wilt caused by Ralstonia solanacearum (Smith 1896, Yabuuchi et al., 1995) is an important problem in cultivation of tropical and subtropical crops like potato, brinjal, chilli, tomato etc. World wide approach to control the disease is to use resistant varieties. Gopimony (1983) reported Solanum melongena var. insanum (wild variety) is resistant to bacterial wilt and controlling gene is monogenic and dominant.This investigation was taken up for tagging of bacterial wilt resistance gene in Solanum melongena var. insanum by RAPD through bulked segregant analysis as reported by Michelmore et al., (1991). The genotypes used for the study were resistant variety Solanum melongena var. insanum I.C. number 421463, susceptible variety Pusa Purple Long and segregating F2 population for bacterial wilt incidence. To raise segregating F2 population F1 was raised by controlled crossing of Solanum melongena var. insanum I.C. number 421463 with pollen grains of Pusa Purple Long. Then F1 plant was selfed to get F2 population.The parents and F2 genotypes were screened for bacterial wilt incidence by stem puncture method of artificial inoculation using R. solanacearum inoculum. In all cases death of plants were confirmed by ooze test. The genotypes were classified against bacterial wilt incidence according to classification of Mew and Ho (1976). The variety Solanum melongena var. insanum I. C. number 421463 was resistant, Pusa Purple Long and F2 were susceptible. Ratio of susceptible to resistant in F2 generation was 2.7:1. So it can assume that bacterial wilt resistant character in resistant parent can be homozygous and recessive.Genomic DNA for RAPD analysis was isolated by modified CTAB method (1994). Good quality DNA with an absorbance ratio of 1.8-2.0 was used for RAPD analysis. PCR reaction mixtures and conditions for DNA amplification were standardized. Ninty eight primers belonging to series A, OPA, OPAG, OPAH, OPB, OPC, OPF, OPP, OPU, RY and RN were initially screened with DNA of resistant and susceptible parents to select primers with polymorphism. Out of ninty eight primers tested thirty, were reported as bacterial wilt specific. Seventeen primers were selected for BSA based on polymorphism. None of wilt specific primers showed polymorphism.Bulked segregate analysis was done with seventeen selected primers for polymorphism. The genotypes used for the study were susceptible parent, resistant parent, five resistant and five susceptible F2 progenies. Among the tested primers only OPP-14 has recorded polymorphism between resistant and susceptible genotypes in BSA. It has produced 470 bp polymorphic amplicon in resistant parent and resistant bulk. Co-segregation analysis was done with OPP-14 primer with individuals of susceptible and resistant bulk. In co-segregation analysis OPP-14 specific amplicon got amplified not only in resistant parent, resistant bulked but also in three susceptible F2s also. The polymorphic band produced by OPP-14 primer in resistant parent and resistant bulk was eluted from resistant parent and cloned in pGEM-T vector, and was transformed into E. coli JM 109 cells. Recombination of the insert was confirmed through colony PCR reaction with universal T7 and SP6 primers. The cloned fragment was sequenced to obtain the nucleotide sequence information and was named as W-3. The sequence obtained after vector screening was named as “Sol-3”, was subjected to Blastn search. Blastn search revealed significant levels of homology with AC237888 Solanum tuberosum clone RH183L22 present in NCBI database. The sequence was also analysed using EMBOSS Transeq and Blastp. Analysis revealed homology with ABC948993 polyprotein (Oryza australiesis). ORF analysis revealed the longest ORF of 108 bp was on +3 frame which revealed significant level of homology with putative retrotransposon protein in Solanum demissum.Two sets STS primers were designed using Primer3Plus. The efficiency of STS primer set BWRGB-2 to distinguish resistant and susceptible genotypes was tested and it amplified a fragment of 200 bp in all the genotypes tested
Validation of apomixis and transcriptome analysis for detection of the genes related to apomixis in black pepper (piper nigrum L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2017) Rohini Rajkumar Bansode; Valsala, P A