Browsing by Author "Keshavachandran, R"

Now showing 1 - 12 of 12

Agrobacterium mediated genetic transformation of ginger (zinbiger officinale rosc.)
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2006) Suma, B; Keshavachandran, R
Investigations on genetic transformation in ginger (Zingiber officinale Rosc.) variety Rio-de-Janeiro using Agrobacterium tumefaciens strain EHA 105 harbouring antibiotic resistant selectable marker genes (npt II) and GUS reporter genes were carried out at the Department of Plantation Crops and Spices and Plant Tissue Culture Laboratory, CPBMB, College of Horticulture, Vellanikkara, Thrissur during the period from 1999 to 2005. Axenic shoot bud cultures of ginger variety Rio-de-Janeiro was raised under in vitro condition to generate explants with reduced contamination for transformation. Half strength MS medium with BA 3 mg l-1 was found to be the best for establishing shoot bud cultures. In order to standardise a regeneration protocol, MS medium supplemented with varying concentration of auxin and cytokinin were tried on different explants. Embryogenic calli were induced from bud explants of ginger supplemented with MS + 1.0 mg l-1 2,4-D + 0.5 mg l-1 BA, followed by plant regeneration on MS medium + BA 3 mg l-1 + 2,4-D 0.5 mg l-1. Bactericidal effect of antibiotics towards different strains of Agrobacterium and sensitivity of ginger tissues to different antibiotics were also studied to standardise the optimum level of antibiotics. Cefotaxime at a concentration of 300 mg l-1 was selected for eliminating the bacteria after co-cultivation. Kanamycin 100 mg l-1 was used to discriminate between transformed and untransformed cells. Agrobacterium strains were collected, recombinants were made and the presence of the construct confirmed in the native strains of Agrobacterium before starting transformation experiments. Agrobacterium strain EHA 105 p35SGUSINT was used for standardising the optimum conditions by comparing the levels of transient GUS expression in inoculated buds. A suitable transformation protocol would include 3 days preculture of explants, bacterial dilution of 1:20 (v/v), infection time of 5 min, co-cultivation of 48 h and post cultivation on callus induction medium with 100 mg l-1 kanamycin + 300 mg l-1 cefotaxime in darkness for 2 weeks and then under 16/8 h photoperiod. Use of acetosyringone in the co-cultivation medium (200 µm) and vir induced Agrobacterium strain (200 µm), increased the efficiency of transformation. Histochemical GUS assays were employed to study and compare the transient GUS expression, stable expression from putative transgenics. Further confirmation was made by PCR assays. The regeneration protocol as well as transformation protocol could be effectively used for further transformation.
Biochemical evaluation of root tubers and in vitro induced callus of adapathiyan (Holostemma ada-kodien K. Schum.)
(Kerala Agricultural University, Vellanikara, 2001) Karmarkar, S H; Keshavachandran, R; Augustin, A
Holostemma ada-kodien is a laticiferous climber belonging to the family Asclepiadaceae. The root tubers of the plant are medicinally important and are useful in opthalmopathy, orchitis, cough, burning sensation, stomachalgia, fever and to cure 'tridosha'. The medicinal properties of Holostemma are due to the presence of terpenoid sugars and amino acids. This study deals with the preliminary biochemical analysis of root tubers and in vitro induced callus of Holostemma. Comparative analysis of amino acids shows no alteration in the primary metabolism in the callus. The metabolism of production of terpenoid compounds in the callus however shows alteration.
Gene expression analysis in relation to fusarium wilt resistance in banana (Musa spp.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Jusna Mariya, P L; Keshavachandran, R
Banana is one of the important fruit crops of India. Banana is susceptible to several fungal pathogens, nematodes, viruses and insect pests. The greatest threats to global banana production is Fusarium wilt or Panama wilt caused by Fusarium oxysporum f. sp. cubense. Control of the pathogen is difficult and mainly involves the use of disease free suckers. Although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult due to its triploid nature and sterility factors of banana. The study entitled "Gene expression analysis in relation to Fusarium wilt resistance in banana (Musa spp.)" was carried out at the Centre for Plant Biotechnology and Molecular Biology, Vellanikkara during the period 2009-2013 with an objective to identify differentially expressed genes in disease resistant genotype of banana, Palayankodan using the molecular technique called suppression subtractive hybridization (SSH). Total RNA and mRNA were isolated from healthy and inoculated plants (with Fusarium oxysporum f.sp. cubense) and were used respectively as 'driver' and 'tester' in SSH reaction. The reactions were performed utilizing the PCR select" cDNA subtraction kit provided by CLONTECH, USA. Control subtraction was carried out first using PCR select" cDNA subtraction kit, which gave satisfactory and expected results. For experimental subtraction, the double stranded cDNAs synthesized from Zug mRNA from normal 'driver' and treated 'tester' were digested with RsaI enzyme. Two tester populations were created and each ligated to two different adaptors. This was followed by two hybridization reactions and finally a selective PCR amplification. Only differentially expressed cDNAs were amplified exponentially. This was confirmed by analyzing the PCR products on agarose gel, which showed a smear ranging from 0.9 to 1.3 kb in the subtracted sample and was different from smear pattern of unsubtracted ones. The cDNA fragments from subtracted sample were cloned in pJET and pGEMT vectors and sequenced. Fifty clones were sequenced and analysed after vector and adaptor editing. In silica analysis using bioinformatics tools revealed that some of the cloned sequences showed similarity with known sequences which play important roles during disease resistance conditions directly or indirectly. These included resistance gene candidate NBS type protein, mitogen activated protein kinase, phytoene desaturase, glycerol 3-phosphate dehydrogenase, neutral invertase, 1- aminocyclopropane-l-carboxylase synthase, superoxide dismutase, MADS-box protein, ubiquitin 2, actin, NADPH oxidase, phytoene synthase, ACC synthase, sucrose phosphate synthase, phosphatidic acid phosphatase-like protein, ORF III like polyprotein, bHLH transcription factor like protein, cytochrome oxidase, isochorismatase hydrolase, basic helix-loop-helix family protein, constitutive triple response I-like protein, granule bound starch synthase, alpha amylase precursor, rop protein, GTPase family protein, S-adenosyl-L-methionine synthase protein, ADP-glucose pyrophosphorylase glucose-l-phosphate adenylyl trans, ethylene signal transduction factor and ribosomal protein. Clones were classified into 6 major groups based on function of protein. Sequences had conserved domains for the above mentioned proteins. Genes involved in defense, signal transduction, metabolism, hypothetical protein, transcription factor and translation. For further exploitation of these sequences it is necessary to clone full length cDNA. ESTs thus generated in the present study will be of great use in future for further downstream applications.
Genetic transformation and hairy root culture in ada-kodien
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 2001) Karmarkar, Shirish Hari; Keshavachandran, R
Genetic transformation for hairy root induction and enhancement of secondary metabolites in Aswagandha (Withania somnifera (L) Dunal)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2006) Smini Varghese; Keshavachandran, R
Genetic transformation in Artemesia annual L. for hairy root induction and enhancement of secondary metabolites
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Shaneeja, V M; Keshavachandran, R
Genetic transformation in sarpagandha(Rauvolfia serpentina(L.) Benth.) for enhancement of secondary metabolite production
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2010) Thangasuja, S; Keshavachandran, R
The present study entitled “Genetic transformation in sarpagandha (Rauvolfia serpentina (L.) Benth.) for enhancement of secondary metabolite production” was carried out at the Centre for Plant Biotechnology and Molecular Biology of the College of Horticulture, Vellanikkara. The objective of the study is to genetically transform Rauvolfia serpentina (L.) Benth. using Agrobacterium rhizogenes by inducing hairy roots so as to enhance the secondary metabolite production. A viable protocol for in vitro propagation of Rauvolfia serpentina was developed. Shoot tip and nodal segment explants from net house grown plants were used for the study. MS media supplemented with BA 1 mg l-1and NAA 0.1 mg l-1 gave maximum regeneration response and also induced multiple shoots (4 shoots/explant) from shoot tip and nodal segment explants. The shoots obtained were best rooted on half strength MS medium supplemented with IBA 0.2 mg l-1and NAA 0.2 mg l-1. The in vitro rooted plantlets were successfully hardened and planted out with 95 per cent survival. Genetic transformation for induction of hairy roots in R. serpentina was attempted with six strains of Agrobacterium rhizogenes, namely ATCC 15834, ATCC 11325, TR 7, A4, TR 107 and MTCC 532. Cefotaxime (500 mg l-1) was found effective for elimination of A. rhizogenes strains from the explant tissues. Different explants such as leaf segments, shoot tips and nodal segments were used for the genetic transformation. The influence of various parameters like co-culture period, acetosyringone treatment and etiolation of shoot tip explant on transformation were studied but none of the treatments showed positive response on induction of hairy roots in R. serpentina. Co-cultivation of shoot tip explant with TR107 strain for two days showed root induction, but the induced roots failed to further proliferate on MS basal medium. Virulence of the different strains and viability of the methodology were checked by inducing hairy roots in Nicotiana tabacum. Among the various explants used for transformation in N. tabacum, leaf segments showed a greater percentage of transformation followed by nodal segment and shoot tip explant. Suspension method of inoculation showed higher percentage of transformation with leaf as explant in ATCC 15834, ATCC 11325 and TR7 strain whereas direct inoculation method showed higher percentage of transformation with leaf as explant in A4, TR 107 and MTCC 532 strain. Among the six strains used for transformation, ATCC 15834 showed the highest frequency of transformation followed by TR7, while TR107 and MTCC 532 failed to infect the explants. The hairy roots induced showed high degree of lateral branching, negative geotropism, fast growth rate and were able to grow in the absence of growth regulators. Opine analysis confirmed the transformation in ATCC 15834, TR7 and A4 hairy root cultures of N. tabacum. Molecular detection through PCR further confirmed the integration of T-DNA from the soil bacteria into hairy root genomes. TR107 induced roots of R serpentina elicited a negative result in PCR amplification as well as opine analysis. Growth rate of hairy roots in liquid culture was faster than in solid media. Half MS with 3.0 per cent sucrose was found to be superior for promoting hairy root growth followed by MS with 3.0 per cent sucrose and B5 with 3.0 per cent sucrose. ATCC 15834 induced hairy roots, showed faster growth producing more biomass as compared to other strains and control. HPLC technique was used for the quantitative analysis of nicotine with the help of class LC10 software (Shimadzu, Japan). Analysis of roots of field grown and in vitro plants as well as hairy root cultures showed that field grown roots had the highest nicotine content followed by hairy roots and attempts were made to enhance the secondary metabolite in hairy root cultures. Enhancement of secondary metabolite production was studied using techniques such as addition of osmoregulants, elicitation and precursor feeding. Addition of 1 per cent sorbitol increased the biomass along with an increase in nicotine content by 2 fold and with further increase in the concentration, the growth rate as well as the nicotine content decreased. Polyethylene glycol at 5 per cent increased the nicotine concentration but there was no increase in biomass production. Yeast Extract at 2 and 5 per cent augmented the nicotine yield but failed to increase the biomass. Addition of the precursor, L-arginine at 50 ppm increased the biomass production along with an increase in nicotine content by 2.6 fold, and with further increase in L- arginine concentration, the nicotine yield dropped down.
Hairy root induction in adapathiyan (Holostemma ada-kodien K. Schum.)
(Kerala Agricultural University, Vellanikara, 2001) Karmarkar, S H; Keshavachandran, R; Nazeem, P A; Girija, D
Holostemma ada-kodien, commonly known as adapathiyan is a laticeferous climber belonging to the family Asclepiadaceae. The root tubers of the plant are useful to cure various ailments of eye and many other human diseases. Due to the indiscriminate collection of root tubers, the plant population in the natural habitats has declined drastically and consequently it has been listed out as vulnerable and rare in the FRLHT red list of medicinal plants. The present study reports the hairy root induction in Holostemma useful in conservation of the plant and also to explore possibilities for in vitro production of the active chemicals in Holostemma, which would be a good alternative to meet its ever-increasing demand. The procedure for induction of hairy roots is given in detail.
In virto multiplication and standardisation of hardening techniques in pineapple (Ananas comosus (L.) Merr.)
(Department of Pomology and Floriculture, College of Horticulture,Vellanikkara, 1993) Prabha, J; Keshavachandran, R
Studies were conducted on in vitro multiplication and standardization of hardening techniques in pineapple (Ananas comosus (L.) Merr.) at the Tissue Culture Laboratory of the All India Co – ordinated Floriculture Improvement Project attached to the Department of Pomology, College of Horticulture, Vellanikkara during 1991 – 1993. Surface sterilization treatment was standardized for crown explants. Among the different treatments tried, treatment with Emisan 0.1 per cent for 30 minutes followed with mercuric chloride 0.1 per cent for 10 minutes was found to be the best. Explants collected in the months of January and February gave the least contamination and maximum survival percentage. MS medium with BA 5.0 mg/1 inche alone or in combination with NAA 1.0 mg/1 inche gave maximum establishment of the explants. The globular structures were formed at maximum intensity within the shortest time of 5.96 days in MS medium supplemented with BA 5.0 mg/1 inche and NAA 1.0 mg/1 inche. Among the three cytokinins tried, the fastest response and the highest intensity of globular structures was obtained with BA followed by KIN and 2ip. Maximum shoot proliferation (11.9 per culture) was obtained with basal MS medium in which cent per cent of the cultures developed vigorous dark green shoots. Rooting of the in vitro derived shoots was obtained in in vitro as well as ex vitro conditions. Hundred per cent in vitro rooting was obtained in basal MS medium as well as in media supplemented with various concentrations of IBA and NAA. The fastest rooting (in 8.54 days) was obtained in the basal medium. Rooting was also faster in liquid medium compared to solid medium. In solid medium, early root initiation and the maximum length of roots were observed with 0.65 per cent agar concentration. Among the ex vitro rooting treatments tried, treatment with the rooting powder Rooton resulted in the fastest rooting and the maximum length of the roots. Profuse rooting of the shoots was obtained without using growth regulators by keeping them in a mist chamber. Treatments were standardised for successful transfer of the plantlets to the outside environment. Hundred per cent survival of the plantlets was obtained by immersing the roots of the plantlets in sterile water for 18 hr prior to transplanting. Among the different containers tried, plantlets grown in plastic pots, in general showed maximum vigour with respect to the number of leaves, height and width of the largest leaf, followed by those in mud pots and poly bags. The maximum percentage increase in these parameters was observed for the plantlets in pro-trays. Potting mixes such as cocopeat, soilrite, biofibe and vermiculite were found to be better in inducing vigorous growth of the plantlets. Plantlets grown in plastic pots with cocopeat or plastic bags with soilrite mix, in general, grew more vigorously. A nutrient starter solution of NPK fertilizer solution once a week or one fourth strength basal MS salts was found to be sufficient to induce healthy growth of the transplanted plantlets in the early stages of growth. To induce better growth of the plantlets in the later stages, application of the NPK fertilizer solution twice a week or Hoagland’s solution once a week was found to be better. Encapsulated beads were successfully formed with the differentiating globular bodies formed from the primary explants. The globular bodies could be encapsulated using 2.5 per cent sodium alginate and 75 mM calcium chloride with a complexation time of 30 minutes. The plantlets after 90 days of growth in the greenhouse with a minimum height of 10 cm and 12 leaves were successfully transferred to soil.
Molecular characterization and development of trait related markers for aphid resistance in cowpea (Vigna unguiculate (L) Walp.)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2007) Ramya Haridas; Keshavachandran, R
Cowpea is one of the most important grain legumes that has been grown throughout the tropics. It is a rich source of proteins (25 percent). Their ability to fix nitrogen is of great advantage on small farms. However their production is greatly limited by many pests, especially aphids. Keeping in view of the importance of this crop, a study was conducted on ‘Molecular characterization and development of trait related markers of aphid resistance in cowpea (Vigna unguiculata (L) Walp.)’. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology and Radio Tracer Laboratory, College of Horticulture during the period 2005-2007. The objectives of the study were to identify the sources of resistance to aphids and to develop markers for aphid resistance in cowpea. The cowpea accessions which were susceptible, moderately resistant and resistant were identified based on the average count of aphids. Five susceptible (VS 1177, VS 1179, VS 1173, VS 1208, VS 1034) and five resistant (VS 1230, VS 1231, VS 1201, VS 1248, VS 1263) accessions were used for the present study. These 10 accessions were subjected to molecular characterization using RAPD and AFLP markers. For RAPD and AFLP analysis, the protocol for genomic DNA isolation from cowpea was standardized. The protocol suggested by Doyle and Doyle (1987) was found to be the most appropriate. Thirty random primers were screened and ten were selected for RAPD profiling of cowpea accessions. The primer OPA 4 was found to have the highest resolving power. A total of 75 scorable amplification products were generated by 10 random primers of which 48 bands were polymorphic. Specific bands were generated for the resistant accessions VS 1230 and VS 1201 with OPA 2, VS 1248 and VS 1263 using OPA 3 and VS 1248 alone with OPA 10. In the dendrogram, the 10 accessions were grouped into two major clusters of 8 and 2 accessions each. The resistant accessions VS 1230 and VS 1231 were the most closely related with 90 percent similarity. Another two resistant accessions VS 1248 and VS 1263 were also grouped together in the dendrogram. The cowpea accessions were subjected to AFLP analysis with 5 primer combinations of EcoR1 and Mse1. A total of 237 scorable amplification products were produced by the primer pairs of which 93 bands were polymorphic. The highest resolving power was obtained for EACT+MCAA. Specific bands were produced for EAAG+MCAA in resistant accessions VS 1230, VS 1263 and VS 1248. The dendrogram obtained for AFLP showed that VS 1177 and VS 1179 were most closely related at 91 percent similarity. Similarly the resistant accessions VS 1263 and VS 1248, VS 1230 and VS 1201 were grouped in different sub clusters. Thus RAPD and AFLP markers were utilized to characterize cowpea accessions for aphid resistance. The specific bands identified in the resistant accessions could be treated as trait related markers for aphid resistance. Further studies should be conducted on screening the cowpea accessions with more number of primers to develop more specific markers with reference to the aphid resistance. The studies should also be focused on converting these RAPD and AFLP markers linked to aphid resistance to a more specific amplification, a technique called Sequence Characterized Amplified Regions (SCAR).
Standardisation of in vitro techniques for rapid multiplication of Trichopus zeylanicus Gaertn
(Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara, 1997) Seema, B J; Keshavachandran, R
Studies were conducted on standardisation of in vitro techniques for rapid multiplication of Trichopus zeylanicus Gaertn at the Plant Tissue Culture Laboratory of the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during 1995-1997. Surface sterilization was standardised for different explants. Treatment with 0.1 per cent mercuric choloride for 5 min was found to be the best for all the explants. Explants collected during January to April showed lower contamination and maximum survival. Soaking seeds in water for one hour was found to reduce the number of days for germination but lower germination percentage. Young, purple shoots were observed to show maximum establishment and growth. Establishment percentage and maximum number of buds was observed to be highest in SH media supplemented with BA compared to 2iP and KIN. Also exposure to light was favourable for better establishment of buds. Proliferation rate was higher at higher concentration of BA but shoot development was better at lower concentration of BA. Addition of adenine sulfate increased the proliferation rate of buds and development of shoots but supplements like yeast extract and casein hydrolysate were not effective in promoting shoot growth. Tender leaf and petiole explants were found to respond better than mature explants and percentage of callus initiation and callus index was higher in combinations of NAA and 2,4-D with BA. Direct organogenesis was observed in 1/2 MS supplemented with BA and NAA. Regeneration of healthy and longer shoots were obtained in MS medium supplemented with KIN. Somatic embryogenesis was observed in media containing BA and BA + coconut water, and embryoid germination was obtained in MS medium. Maximum rooting was obtained by culturing shoots in media containing brassinolide for one week and thereafter transfer to IBA. Earlier rooting was obtained in liquid medium. Keeping in 1/2 MS with reduced sucrose and increased light intensity in the culture room for two weeks before transfer to hardening unit resulted in better survival of plantlets.
Variability in Chakkarakolli (Gymnema sylvestrwe R.Br.) using morphological, biochemical nad molecular markers
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2005) Smitha Nair; Keshavachandran, R
Plants represent an unparalleled source for drug development. Plant based natural products play a dominant role in the pharmaceutical industry. Plant based remedies are available for a number of different health problems. In today’s scenario, diabetes is one of the most common non-communicable diseases globally and is also the fourth major cause of death in most developed countries. So the demand for natural alternatives to blood sugar control is ever increasing. Several indigenous medicinal plants have been indicated in the Ayurveda system of medicine to possess antidiabetic activity. Native to the forests of South India, Gymnema sylvestre R.Br. is known for its hypoglycaemic property from the time immemorial. Practitioners of Ayurveda first used Gymnema to treat diabetes almost 2000 years ago. Keeping in view the importance of this medicinal plant, a study was conducted to identify the variability in Gymnema accessions collected and maintained at the Centre for Plant Biotechnology and Molecular Biology. The present study was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during the period 2003-2005. The objectives of the study were to characterise the variability in Gymnema germplasm accessions using morphological, biochemical and molecular markers. The morphological characterisation of 93 Gymnema accessions based on vegetative characters indicated the existence of a wide variation. All the 93 accessions were then characterised using biochemical markers. The total saponin content in the leaves was chosen as the marker constituent since saponins are the major bioactive components in Gymnema. The saponins were estimated based on TLC-Densitometry technique. The techniques for detection of saponins through TLC could be standardised. The saponin content in the leaves of different accessions ranged between 0.6 per cent and 5.4 per cent. Based on the morphological and biochemical characters, variants were identified in Gymnema germplasm collection. Eighteen such plants showing high variation were subjected to molecular characterisation using isozyme and RAPD markers. Isozyme analysis was carried out using three enzyme systems viz. malate-dehydrogenase, esterase and RUBISCO. Eight isozyme loci could be clearly identified of which five were polymorphic. Ten alleles were identified over the five polymorphic loci giving an average of two alleles per polymorphic locus. A mean observed heterozygosity of 19.8 per cent and a mean expected heterozygosity of 17.2 per cent were obtained over the heterozygous loci studied. The cluster analysis grouped the 18 accessions in to two major clusters of 17 and one accession. Molecular characterisation using RAPD markers was conducted to appraise the extent of diversity among the 18 accessions of Gymnema. The protocol for genomic DNA isolation from Gymnema was standardised. The protocol suggested by Rogers and Bendich (1994) with slight modification was found to be most appropriate. The protocol for RAPD assay in Gymnema was standardised. Sixty random primers were screened and 15 were selected for RAPD profiling of Gymnema accessions. The primer OPAH 12 was found to have the highest resolving power. A total of 123 amplification products were generated by 15 primers, of which 90 were polymorphic. In the dendrogram, the 18 accessions were grouped in to two major clusters of 16 and two accessions. The accessions Peringottukurushi 137 and Kuzhalmannon 89, occurring in the third sub-cluster were the most closely related with 85 per cent similarity. A combined dendrogram was also derived from the results of morphological and molecular studies. The present study revealed the existence of sufficient genetic variation in the Gymnema germplasm collection. This variability can be used to identify useful genotypes that could be used as cultivars for the extraction of standard drugs. More precise techniques like HPLC and AFLP could further be used to get a better idea about the extent of variability in Gymnema.