Browsing by Author "Kesavachandran, R"

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DNA fingerprinting of selected chilli (Capsicum spp.) varieties
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Manibala Kumari; Kesavachandran, R
Chilli (Capsicum spp.) is considered as one of the most important commercial spice crops and is a widely used universal spice, named as the wonder spice. It is raised over an area of 18 lakh ha. in the world, with a production of 29 lakh t. India is not only the largest producer but also the largest consumer of chilli in the world. The study entitled “DNA fingerprinting of selected chilli (Capsicum spp.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2012-2013. The objectives of the study were to characterize the released chilli varieties of KAU using different molecular markers- RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Six chilli varieties, namely Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi collected from CoH, Vellanikkara and CoA, Vellayani and maintained at CPBMB, CoH were used for the study. Morphological parameters of six chilli varieties were taken such as stem colour, branching habit, leaf size, leaf colour, fruit colour, fruit shape and fruit surface. DNA extraction was done by CTAB (Rogers and Bendich, 1994) method. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A 260 /A ) 1.80 - 1.91 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assays. Thirty RAPD, 30 ISSR and 30 SSR primers were screened with bulked DNA of Ujwala, Anugraha and Jwalamukhi variety for amplification and those which gave reliable distinct banding patterns were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analyses were separated on two per cent agarose gel and the amplification patterns were recorded. Genomic DNA from each variety was amplified with ten selected primers of RAPD, ISSR and SSR primer pairs. The amplification patterns were scored and depicted to develop DNA fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 8.33 (S 12) to 12.9 (OPAH 06) for RAPD primers and 8.66 (SPS 03) to 14.33 (ISSR 07) for ISSR primers, indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.80 (S 12) to 0.86 (OPAH 06) for RAPD primers and it was 0.82 (SPS 03) to 0.88 (UBC 840) for ISSR primers. Distinct bands were used to develop DNA fingerprint of chilli varieties (Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi) through RAPD, ISSR and SSR analyses. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint for each variety with RAPD, ISSR and SSR data was also developed. The amplification patterns observed in RAPD, ISSR and SSR analyses were scored and analyzed for quantifying the variability among the varieties. The computer software NTSYS-Pc was used for cluster analysis (Rohlf, 2005). Maximum variability observed was 41 per cent for the variety Vellayani Samrudhi. The varieties Ujwala and Anugraha indicated 91 per cent similarity. The fingerprint developed was sufficient to provide varietal identity and the analysis could reveal variability/ relatedness among the six varieties.
Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers.
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Choudhary Rakeshkumar Sheshrao; Kesavachandran, R
The present investigation on “Genetic analysis of plantain ecotypes of banana (Musa spp.) using RAPD and ISSR markers” was undertaken in the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2009-2011 with an aim to characterize the variability in plantain ecotypes of banana (Musa spp.) using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Twelve plantain ecotypes collected from BRS, Kannara was used for the study. Standardisation of DNA was done with the CTAB method. Optimum PCR conditions for both RAPD and ISSR were standardised with various quantities of DNA, dNTPs, MgCl2, primers and Taq polymerase. Initially 60 RAPD and 40 ISSR primers were screened against genomic DNA of two plantain ecotypes (Big Ebanga and Njockkon) for their ability to amplify DNA fragments. Of these, 16 RAPD and 14 ISSR primers were selected for further detailed RAPD and ISSR profiling. All selected primers produced robust amplification patterns. The PCR products obtained were separated on 1.4 per cent and 1.6 per cent agarose gel respectively stained with ethidium bromide. A total of 138 bands were obtained by using 16 RAPD primers. The number of bands produced by the primers varied from 5 (OPS 40) to 14 (OPS 31 and 37) and the molecular weight of bands varied from 2.876 to 0.564 Kb. The average number of bands was 8.63 and average percentage of polymorphism was 3.25. The total percentage of polymorphism was 37.68. The Polymorphic Information Content (PIC) value for 16 primers varied between 0.79 (OPS 40) and 0.92 (OPS 37) with mean of 0.87. The Resolving power (Rp) of the random primers ranged between 9.33 (OPS 12) and 24.33 (OPS 37) with an average of 15.49. The Marker Indices (MI) of primers varied from 0.83 (OPS 7) to 7.28 (OPS 31) with a mean of 2.86. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Njockkon and Changalikodan, occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. A total of 111 bands were obtained by using 14 ISSR primers. The number of bands produced by the primers varied from 5 (UBC 835, 820) and 11 (UBC 857) and the molecular weight of bands varied from 1.584 to 0.564 Kb. The average number of bands was 7.93 and average percentage of polymorphism was 4.14. The total percentage of polymorphism was 52.25. The PIC value for 14 primers varied between 0.77 (UBC 835) and 0.90 (ISSR 6) with an average of 0.85. The resolving power of the ISSR primers ranged between 7.83 (UBC 820) and 16.83 (ISSR 6) with an average of 12.49. The Marker Indices (MI) of primers ranged from 0.77 (UBC 835) to 8.01 (UBC 857) with a mean of 3.55. In the dendrogram, the 12 plantain ecotypes were grouped into three major clusters. The ecotypes Changalikodan and Zanzibar; Manjeri Nendran (a) and Manjeri Nendran (b) occurring in the first cluster were the most closely related with 94 per cent similarity. The Principal Component Analysis (PCA) showed a similar result to that of clustering. The combined dendrogram was also derived from pooled data from RAPD and ISSR analysis and morphological data. The dendrogram generated revealed grouping of plantain ecotypes into clusters more or similar to earlier dendrogram with a few exceptions. The study revealed that variability exists among the plantain ecotypes of banana.
In vitro callus induction and its exploitation in Coscinium fenestratum (Gaertn.) Colebr.
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1999) Sindhu, M; Kesavachandran, R
The present investigations were carried out in the Plant Tissue Culture and Biochemistry Laboratories, College of Horticulture, Vellanikkara, Thrissur during the year 1997-1999. The study was undertaken with the objective to standardise the in vitro techniques for callus induction, proliferation and regeneration. It was also envisaged to identify and quantify the active principle in in vitro cultures. Surface sterilisation treatments were standardised for the different types of explants from the field. For immature fruits and leaves, the sterilisation treatment standardised were found to be with 0.05 per cent cetrimide immersion for 5 minutes followed by 0.1 per cent HgCh for 5 minutes with reference to least percentrage of contamination and higher rate of establishment and growth of the explants obtained. The exudation of polyphenols from the cut surfaces of shoot tip, nodal and internodal segments was minimised by pre-treatment with antioxidants. The pre-treatment followed was with Cetrimide 0.1 per cent for 5 minutes, Emisan 0.1 per cent for 5 minutes, ascorbic acid 0.01 per cent + citric acid 0.01 per cent for 10 minutes and mercuric chloride 0.1 per cent for 3 minutes. Though calli were obtained from leaf segments as well as leaf segments with petiole bases, leaf segments were found most suitable for callus cultures and produced profuse calli. The best treatment for callus induction was found to be solid Vz MS medium for leaf bit cultures. The treatment with IAA 2 mg rl and BA 1 mg r' on Vz MS solid medium was the best for callus induction from leaf segments and leaf segments with petiole bases. Similarly a combination of auxins such as IAA 2 mg rl and 2,4-D 1 mg rl on Vz MS solid medium also readily induced callusing from leaf segments and leaf segments with petiole bases. The above treatments were superior with respect to callus index and the number of days taken for callus initiation. Callusing was obtained from immature fruits when cultured on solid Y2 MS medium with phosphate ions reduced to 25 per cent supplemented with lAA 2 rng rl and BA 1 mg r'. The shoot tips, nodal and internodal segments exhibited recalcitrancy and there was no cell growth from these explants. There was neither any proliferation nor any noticeable cell growth of calli in the liquid medium. Light stimulated the growth of yellowish friable callus and berberine synthesis in the initial stages. But in the later stages, continuous illumination proved to be detrimental and its growth was retarded. The calli did not respond to regeneration treatments being neither organogenic nor embryogenic. The wavelength of marker berberine was recorded as 228 nm. Berberine was detected in calli produced from leaf explants of different treatments in solid Y2 MS media supplemented with growth regulators such as BA 0.25 mg i', BA 0.5 mg r', Kin 0.25 rng r', lAA 2 mg rl + BA 1 mg r' and lAA 2 mg r' + 2, 4-D 1 mg r'. Age of the callus had a profound influence on berberine production. Employing special techniques for synthesis of berberine in in vitro cultures such as administration of osmoregulants, incorporation of media additives, increasing concentration of agar, addition of stress inducers and modification of carbon source in solid Yz MS medium failed to sustain the growth of callus. Incorporation of abscissic acid at low concentration of 0.25 mg r' sustained the callus development and berberine was detected by thin layer chromatography. The quantity of berberine recovered was 0.095 ug/g callus tissue. Administration of spermidine to liquid Yz MS medium neither caused any noticeable cell browning nor any cell growth. The medium became deep yellow due to the release of the alkaloid. Spermidine at 60 ug concentration in the liquid Yz MS production medium had a berberine yield of 5.0 15 ug/g callus. Berberine was detected from the field grown plant samples also. The quantity was the highest in the tender leaves, that is 0.320 ug from 25 g tender leaves which is equivalent to 0.013 ~g/g leaf. The berberine content in the stem was found to be 0.010 ug/g stem, that is 0.304 ~g/30 g stem. The stem extract also contained another compound whose Rfvalue was 0.66 and it gave a brownish spot under visible light. The highest berberine recovery obtained was in callus obtained from half strength MS liquid medium than in half strength MS solid medium. The in vitro derived callus had higher amounts of berberine than the samples from the field grown plant. The highest berberine yield was obtained when phosphorus ion sources were reduced to 25 per cent in the Yz MS liquid medium supplemented with IAA Zmgl" and BA lrngl'. The recovery was 10.079 ~g/g callus tissue. The next highest yield of berberine (5.015 ug/g callus) was obtained when 60 ug of spermidine was added to the 25 ml of the Yz MS liquid medium supplemented with IAA Zmgl" and BA Irngl".
Incidence of tapping panel dryness in rubber in the small holdings of Meenachil Taluk
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1994) Sebastian Michael; Kesavachandran, R
The study was conducted to find out the extent of incidence of TPD and related parameters in the rubber growing small holdings of Meenachil taluk. Fifty four small holders who sell their produce as latex and representing different parts of the taluk were selected for the study. Holdings with trees tapped either on BO 1 or Bo 2 panel only were included in the study. The study revealed that the small holders have high preference for the clone RRII 105 due to its inherent capacity for faster growth, higher yield and disease resistance. Rainguarded tapping is not being practiced on a large scale, as only 35.19 per cent of the holdings resorted to this practice. Tapping intensity in the surveyed holdings was invariably 1/2S d/2, without any rest on Sundays. Planting density varied from 460 ha-1 to 550 ha-1 and density of trees under tapping varied from 300 ha-1 to 500 ha-1. Planting density upto 550ha-1 was found not to have any effect on bark thickness or incidence of TPD. The mean incidence of TPD, in terms of complete dry cuts, was 7.56 per cent for the clone RRII 105. During the initial four years of tapping, the incidence was below five per cent in the holdings surveyed. In the present study, positive correlation was observed between dry rubber yield per unit area and incidence of TPD. Mean bark thickness showed highly negative correlation with incidence of TPD. Also, bark thickness was found to have highly significant positive correlation with annual mean drc. But no correlation was observed between annual mean drc and TPD incidence. Bark consumption showed highly significant correlation with TPD incidence, and non significant correlation with yield. This indicates induction of TPD by recovery tapping without concomitant increase in yield. Thus comparable incidence of TPD was encountered in rainguarded and nonrainguarded holdings, without realizing comparable yield. Low incidence of TPD was observed in plantations with slower growth. High incidence of TPD was generally observed near house holds and cattle sheds, in low lying areas, banks of rivers and canals etc.
Induction of somaclones in vetiver (Chrysopogon zizanioides (L) Roberty)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Resmi, S K; Kesavachandran, R
Malformation in Kodampuli (Garcinia Cambogia Desk.)
(Kerala Agricultural University, 1994) Sarah T George; Lila Mathew, K; Kesavachandran, R; Rajeevan, P K
Management of recalcitrancy in in vitro cultures of cashew (Anacardium occidentale L.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2009) Jusna Mariya, P L; Kesavachandran, R
Propagation studies in cocoa
(Department of Horticulture (Plantation Crops), College of Horticulture, Vellanikkara, 1979) Kesavachandran, R; Sivaraman Nair, R
A study on the different aspects of propagation of cocoa was undertaken at the College of Horticulture from May 1978 to July 1979 to standardise the criteria for selecting the pods, seeds and seedlings for raising the nursery, to find out the optimum size of polythene bags and the suitable medium for raising the nursery and also to standardise the best vegetative propagation methods for cocoa. The results had indicated that the volume and weight of the pods varied within the three classes of pods namely large, medium and small. There was not much variation in the number of seeds among the three classes of pods and the mean number varied between 30 to 42. The number of seeds were found to be highest in pods harvested in February and March followed by April. The highest percentage of germination was recorded in March followed by February, January, December and April. The size of the pod and the position of seeds (pedicel end, middle and distal end) had no significant influence on the germination and the growth of the seedlings. However, the large and medium sized pods are found to produce better seedlings. Based on the studies the following recommendation are made i) Large and medium sized pods weighing more than 350g each with not less than 400 cc volume should be selected for raising the nursery during the month of February and March. ii) The seed should be sown on the same day of harvest but it can be stored under room conditions upto six days. The percentage of germination will be decreased to 66 per cent by the sixth day. iii) A selection criterion for selecting the seedlings when they are three months old is recommended. The seedlings should have atleast 30cm height and 10 or more number of leaves when they are three months old. For raising three to five month old seedlings, the optimum size of bag is found to be 30 x 20 cm and the best medium for raising cocoa nursery is a mixture soil, sand and farm yard manure in the proportion 1:1:2. Considering the pattern and extent of root and shoot growth of the seedlings, planting the seedling when they are three to four months old is suggested. For higher percentage of rooting and optimum number of roots and higher root length, a ‘quick dip’ method for 60 sec in 4000 ppm NAA or 6000 ppm IAA is recommended for producing rooted cuttings. A ‘mist chamber’ method is suggested for rooting the cuttings. Forkert method of budding is recommended for cocoa either on eight to nine months old root-stocks or by green budding on three to four months old root-stock. The best time for budding is February and March on older root-stocks and April and May for green budding.
Standardisation of in vitro techniques for rapid multiplication of holostemma annulare k schum
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1996) Sophia John, A; Kesavachandran, R
Studies were conducted on standardization of in vitro techniques for the rapid multiplication of Holostemma annulare K. Schum. At the Plant Tissue Culture Laboratory of the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during 1993-1995. Surface sterilization was standardized for explants from different sources. For two to three month old explants from the glasshouse, treatment with 0.1 per cent mercuric chloride for 5 min or 10 min was found to be better. A combination of sterilants was necessary for mature explants taken either from the glasshouse or field. Explants collected in the months of January and February gave the lowest contamination rate. Early release of buds and further growth of nodal segments and shoot tip explants was better in MS media supplemented with BA. Cultures in medium containing KIN were short, robust, darker and with lesser number of buds and shoots than those in medium containing BA. Extremely low concentrations of TDZ could stimulate axillary bud proliferation. Additives like silver nitrate and activated charcoal could drastically reduce callus production in culture, but the shoot growth was also reduced with these additives. Nodal segments were better in respect of early release of buds, more number of longer shoots, nodes and buds than shoot tips. Higher temperature proved better than lower temperature for the growth of cultures. Also exposure to light was favourable for healthy growth of shoots. Proliferation rate was higher at higher concentrations of BA but the shoots were very swollen, weak and had to be subcultured as a clump into media containing lower concentrations of BA for healthy growth of shoots. Shoots could be proliferated at extremely low concentrations of TDZ. MS basal with full concentration of salts was better for better growth of shoots. When the best treatment in each subculture was given in sequence approximately 2 crores 37 lakh nodes could potentially be obtained over a period of 225 days. Maximum rooting, early rooting and more number of longer roots could be obtained in solid. MS basal media when shoots were kept for in vitro rooting. Ex vitro rooting of shoots was successful when they were treated with IBA 1000 mg1-1 as quick dip followed by planting in plastic pots filled with sand in the initial stages for early rooting and then transplanted to plastic or mud pots filled with cocofibe for vigorous growth of root and shoot portions. TDZ produced the highest callus index at relatively lower concentrations. The callus produced was hard, green in colour and compact. 2, 4-D proved better than NAA for obtaining more regenerative callus among the auxins tried. Leaf segments (with or without petiole attached) oriented with the abaxial surface touching the solid medium supplemented with 2,4-D and exposed to light alone produced embryoids after one or two subcultures into MS medium with lower concentrations of 2,4-D. The embryoid production could be triggered if the calli were subcultured to liquid MS basal medium and when further transferred to solid media alone produced elongation of such embryoids. But the original explants had to be raised in MS medium supplemented with either TDZ or KIN as cytokinin for the embryoids to form subsequently. Encapsulated beads were successfully formed with nodal segments using 2.5 per cent sodium alginate and 75 mM calcium chloride with a complexation time of 30 min and the beads could be stored successfully for 15 days at room temperature and upto 40 days at 40 C. The peroxidase isozyme pattern of the leaves and roots from in vitro plantlets and in vivo plantlets were similar having the same number of bands