Theses

Browse

Search Results

Now showing 1 - 7 of 7

Open Access
Response of immature inflorescence for in vitro regeneration on coconut (cocos nucifera L)
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2006) Siny, C V; KAU; Nazeem, P A
Investigations on 'Response of immature inflorescence for in vitro regeneration in coconut (Cocos nucifera L.)' were carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara during 2005 to 2006. Y3 medium was found to be the best basal medium for in vitro culture of immature inflorescence of coconut. Inflorescence of length 40 to 50 cm was found to posses male flowers at pollen mother cell stage of microsporogenesis. Wiping the spathe with 70 per cent ethyl alcohol before excising the immature inflorescence parts could effectively control contamination with 100 per cent culture establishment. The young inflorescence parts could survive up to 12 minutes treatment with 0.1 per cent HgCl2. Among the different explants tried, anthers at premeiotic stage and immature rachillae were found to be the best for callus induction and embryo formation. When the explants were inoculated the exudation of polyphenols from the explants adversely affected their survival. Polyphenol exudation was checked by incorporating PVP 0.1 per cent and activated charcoal 0.2 per cent in the medium and by incubation under dark condition. 2,4-D at 15 to 30 mgl-1 was found to be the most effective auxin for callus induction and proliferation. Y3 basal medium with growth regulator combinations of 15 mg l-1 2,4-D, 0.5 mg l-1 picloram, 1mg l-1 NAA and 0.1 mg l-1 TDZ was identified as the best medium for callus induction and embryogenesis of immature anther. Sucrose at 5 per cent concentration was identified as the optimum concentration for callus induction. Pretreatment of inflorescence at 4°C for 24 hrs or 30 hrs doubled the callus induction and reduced the browning of explant. Callus induction was observed from rachillae tissue in Y3 medium containing15 mg l-1 2,4-D, 1 mg l-1 picloram, 1 mg l-1 IAA and 0.1 mg l-1 TDZ.
Open Access
Amplification and sequencing of spacer region between two tRNA genes and its flanking region in the chloroplast genome of Centella asiatica L.
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Manju Elizabeth, P; KAU; Rajmohan, K
The study entitled “Amplification and sequencing of spacer region between two tRNA genes and its flanking region in the chloroplast genome of Centella asiatica L.” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2005-2006 with the objective of isolating a spacer region and its flanking regions from the chloroplast genome of Centella asiatica to develop a species specific vector for the chloroplast transformation. Heterologous primers were designed based on the chloroplast genome sequences of Arabidopsis thaliana, Nicotiana tabacum and Panax ginseng using Pimer3 software for the spacer regions trnG-trnfMet, trnE-trnT, trnT-trnL and rps16-trnQ and were amplified on genomic DNA of Centella asiatica. The entire isolated regions were sequenced except trnT-trnL spacer region. All sequenced regions were subjected to BLASTN and BLASTX similarity search. The trnG-trnfMet spacer region (270bp) showed maximum similarity to the same region in Panax ginseng (GI: 51235292, AY582139.1) chloroplast genome. The spacer region between genes rps16 and trnQ (1749bp) showed maximum similarity to rps16 gene and its intron in Centella asiatica (GI: 6692894, AF110603.1) chloroplast genome and to rps16 gene, spacer region after rps16 and starting region of trnQ gene in Panax ginseng (GI: 51235292, AY582139.1) chloroplast genome. Primers for flanking regions of rps16-trnQ spacer were designed manually based on the primers of this spacer and the chloroplast genome of Panax ginseng. The right flanking region amplified (1582bp) with primers Hf and Hr showed maximum similarity to trnQ gene, spacer region after trnQ, psbK gene, spacer region after psbK and psbI gene in Panax ginseng chloroplast genome. The left flanking region of rps16-trnQ spacer sequence amplified (1227bp) with primer combination Ff and Fr showed similarity to rps16 gene, spacer region after trnK gene, trnK gene and spacer region after matK gene of Panax ginseng chloroplast genome. The sequence amplified (1089bp) with primers Gf and Fr showed similarity to rps16 gene, spacer region after trnK of Panax ginseng and to matK gene of Centella erecta (GI: 2281236, US8599.1). The spacer region between rps16 and trnQ gene and its flanking region isolated can be used in developing a Centella asiaticaL. specific vector for chloroplast transformation.
Open Access
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; KAU; Keshavachandran, R
The present study entitled ‘Genetic transformation for hairy root induction and enhancement of secondary metabolites in Aswagandha (Withania somnifera (L.) Dunal) was carried out at the Centre for Plant Biotechnology and Molecular Biology and Biochemistry Laboratory of the College of Horticulture, Vellanikkara and the Biochemistry Laboratory of Aromatic and Medicinal Plants Research Station, Odakkali. The study was undertaken to standardize the in vitro regeneration protocol in W. somnifera from different explants, to standardize the genetic transformation using Agrobacterium rhizogenes, to standardize the biochemical techniques for the estimation of secondary metabolites in roots and also to enhance the secondary metabolite production in the hairy root cultures of W. somnifera. An efficient method for in vitro plant regeneration was developed in W. somnifera. Different explants such as hypocotyls, cotyledonary segments, leaf segments, shoot tips, nodal segments and roots from in vitro germinated seedlings were used for the study. The seeds showed early and high germination under dark compared to 16 h photoperiod. Multiple shoot formation was observed from hypocotyl segments placed directly and upside down. Maximum regeneration response was obtained from hypocotyls, cotyledonary segments and leaf segments in MS + BAP 1.0/ 1.5 mg l-1 + IAA 0.5 mg l-1. Shoot buds produced from hypocotyls and cotyledonary segments showed good multiplication in MS + BAP 0.4 mg l-1 + IAA 0.5 mg l-1. Cotyledonary segments derived callus regenerated in MS + BAP 2.0 mg l-1. Shoot tips and nodal segments taken from axenic seedlings showed highest frequency of multiple shoot formation in MS + BAP 2.5 mg l-1 and IAA 0.5 mg l-1. Regeneration from basal callus, produced by shoot tips and nodal segments was also obtained in the same culture cycle. The roots taken from in vitro seedlings and in vitro rooted plantlets showed high callusing but failed to regenerate by direct organogenisis. However, somatic embryo like structures were produced from seedling roots in MS + BAP 1.5 mg l-1 and IAA 0.5 mg l-1. The shoot/ shoot buds produced good elongation in MS + GA3 0.5 mg l-1. In vitro flowering of cotyledonary segment derived shoots was also obtained in this combination. The shoots were successfully rooted in half MS + 0.25 per cent activated charcoal by pulse treatment with IBA 1000 mg l-1 for five seconds. The plantlets were successfully hardened and transferred to large pots in the green house. Genetic transformation was carried out in W. somnifera using three different A. rhizogenes strains like A4, ATCC 15834 and MTCC 2364 for inducing hairy roots. The explants such as hypocotyls, cotyldonary segments, leaf segments, shoot tips and nodal segments were used for genetic transformation. Here the influence of different parameters such as type of explants, type of bacterial inoculum, co-cultivation periods and acetosyringone effects on transformation frequencies were studied. Among the three A. rhizogenes strains, the strains A4 and ATCC 15834 produced successful transformation. Of this two successful strains ATCC 15834 showed a greater potential for transformation. Among the various explants used, only the leaf segments and shoot tips produced hairy roots. Leaf segments showed a greater percentage of transformation than the shoot tips. Though A4 strain produced successful transformation in W. somnifera by direct inoculation of bacteria from single cell colonies as well as in the suspension form, the strain ATCC 15834 produced transformation only in the suspension form. A co-cultivation period of one day was found to be the best for leaf segments, whereas shoot tips responded more under two day co-culture period. The acetosyringone (100 M) enhanced the transformation percentages with A4 strain, whereas no such influence was observed with ATCC 15834 strain. The hairy root cultures established on MS + 250 mg l-1 cefotaxime showed phenotypic variations in growth habit. The hairy roots normally produced high lateral branching with plagiotropic growth habit and showed sigmoid growth pattern. Among the four liquid media tested, half MS was found to be superior in promoting hairy root growth followed by MS, B5 + 2.0 per cent sucrose, B5 + 3.0 per cent sucrose respectively The confirmation of transformation by opine detection was found to by unsuccessful in W. somnifera because of the presence of interfering substances which produced spots near the positions of agropinic acid including control. Because of the low concentration of DNA, Southern hybridization technique failed to produce band corresponding to hairy root samples. However, the transformation was confirmed in A4 and ATCC 15834 induced hairy roots by PCR and dot blot analysis. A Thin Layer Chromatographic method was employed for withanolide estimation. Withaferin A was used as the standard in estimation studies. Silica gel60 F254 plate chloroform- methanol (9.8: 0.2) was used as the solvent system. The spot was observed under UV at 254 nm and also the sensitivity was improved by using vanillin (0.05 g) + boric acid (1.0 g) + H2SO4 (2.0 ml) + Methanol (100 ml) spray reagent. Withaferin A produced magenta spot, which changed to bluish violet on further charring. Field root possess more withaferin A followed by hairy roots and in vitro roots contained the least. Enhancement of secondary metabolite production was studied using techniques such as addition of osmoregulants, precursor feeding and elicitation. The withaferin A content in the hairy root biomass and the culture medium were estimated. The osmoregulant PEG (2.0 % and 5.0 %) and methionine precursor (1mM and 2mM) failed to enhance the withaferin A content. With the addition of yeast extract (2.5 and 5.0 g l-1) a reduction in withaferin A content was observed in the root biomass. However, the biotic elicitor Aspergillus homogenate (250 and 500 l /125 ml) elicited a positive influence on the biosynthesis of withafein A in the hairy root cultures.
Open Access
Agrobactrium tumefaciens mediated genetic transformation in Kudangal (Centella asiatica L. Urban)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Nanditha Krishnan, V; KAU; Soni, K B
A study on “Agrobacterium tumefaciens mediated genetic transformation in kudangal (Centella asiatica L. Urban.)” was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2004-2006. Centella asiatica is an important medicinal plant of India and is used in many ayurvedic formulations. Centella asiatica contains a blend of compounds including triterpenes (asiatic acid, madecassic acid and asiaticoside) that appear to have antioxidant, tissue regenerative and memory enhancing properties. The present study was undertaken with the main objective of evolving a protocol for Agrobacterium tumefaciens mediated genetic transformation in Centella asiatica, which could further be utilized for the metabolic engineering of Centella to enhance the level of secondary metabolites. Callus was induced from leaf and node explants of Centella on MS medium supplemented with growth regulators. MS medium supplemented with Kn 2 mg l-1 and NAA 4 mg l-1 was proved to be the best in terms of callus induction percentage (85.7) from leaf explant in 25.50 days. With node explants, the maximum callus induction (86.67%) was obtained on MS medium supplemented either with Kn 2 mg l-1 and NAA 3 mg l-1 or with Kn 1 mg l-1 and NAA 3 mg l-1 in 23.67 and 22.00 days respectively. Of the various regeneration treatments, 16.67 per cent regeneration from callus was obtained on MS medium supplemented with Kn 2mg l-1, BA 4mg l-1, NAA 0.25 mg l-1 and ADS 20 mg l-1. Two strains of Agrobacterium tumefaciens viz., LBA4404 and EHA105 harbouring the plasmid pCAMBIA2301 were used for genetic transformation. As the plasmid harbour nptII and gus reporter genes, the sensitivity of Agrobacterium strains and Centella callus to different concentrations of kanamycin was evaluated. The lethal dose of kanamycin to Agrobacterium and Centella callus were 350 and 125 mg l-1 respectively. The effective dose of cefotaxime for the elimination of bacterial strains LBA4404 and EHA105 was 75 mg l-1 and the lethal dose of cefotaxime to Centella callus was 150 mg l-1. Genetic transformation was achieved by co-cultivating callus and node with bacterial suspension. Conditions like infection and co-cultivation time, type of the explant, selection agent and suitable Agrobacterium strains were optimized. The Agrobacterium strain, EHA105 with pCAMBIA2301 was more efficient for transformation in Centella. The most effective infection time was 20 minutes, followed by a co-cultivation period of four days. The survival of tissues transformed by the strains LBA4404 and EHA105 on the selection media were 80.65 per cent and 66.67 per cent respectively. Maximum transformation efficiency of 50 percent was obtained when callus was co-cultivated with EHA105 (pCAMBIA2301) for four days. The transformation efficiency was increased when acetosyringone 100 µM was added to infection and co-cultivation media. Transformation was confirmed by histochemical GUS assay of putative transformants. This study provides a protocol for genetic transformation in Centella which can be used for transferring desirable genes.
Open Access
Agrobacterium rhizogenes mediated genetic transformation in Koduveli (Plumbago spp. Linn.)
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Roshna Bhaskar; KAU; Reghunath, B R
Investigations on “Agrobacterium rhizogenes mediated genetic transformation in Koduveli (Plumbago spp. L.)” were carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2003-2006. Two plant species viz., Plumbago zeylanica and Plumbago rosea having great medicinal value by virtue of plumbagin, a naphthoquinone, present in them were selected for the study. Standardisation of rapid in vitro propagation of these medicinal plants was attempted in the present study. Enhanced release of axillary buds from nodal explants, with the highest shoot proliferation of 5.5 was obtained in MS medium supplemented with BA 1.0 mg l-1 + IAA 0.05 mg l-1 for P. zeylanica . In P. rosea the highest shoot proliferation of 5.0 was recorded in MS medium supplemented with BA 1.50 mg l-1 + IAA 0.05 mg l-1. Among the different basal media tested full strength MS medium was found to be the best for shoot proliferation. In indirect organogenesis, the highest callus index (300) was recorded in MS medium with BA 2.00 mgl-1 and 2,4-D 0.50 mg l-1 for P. zeylanica. In P. rosea also maximum callus index (300) was obtained in the same medium. In P. zeylanica BA 1.75 mg l-1, IAA 0.05 mgl-1 and adenine sulphate 20 mg l-1 in MS medium was identified as the best medium for shoot regeneration from leaf derived callus. Whereas in P. rosea BA 2.00 mg l-1, IAA 0.05 mg l-1 and adenine sulphate 20 mg l-1 in MS medium obtained the highest rate of shoot regeneration from callus. Coconut water (100 ml l-1) was found to increase the rate of shoot regeneration in P. rosea. Rooting of in vitro raised shoots was achieved by sub culturing them on MS medium containing IAA 0.50 mg l-1. In vitro root culture was carried out successfully in semi solid MS medium containing 1.50 mg l-1 NAA. Genetic transformation was carried out by co-culture method. Agrobacterium rhizogenes strains A4, MTCC 532 and MTCC 2364 were utilized for transformation. YEP medium was used for culturing the bacteria. The strain A4 was the best for transforming Plumbago spp. There was no significant difference between the two species P. zeylanica and P. rosea in their response to transformation. Nodal explants recorded the highest transformation percentage followed by, leaf segments. Calli and root explants did not respond to transformation. Bacterial density of one (O.D600) during transformation resulted in high transformation percentage. Co-culturing for two days resulted in high transformation percentage, whereas co culturing for more than two days resulted in bacterial over growth on the tissues and low transformation per cent. After two days of co-culture, the bacteria were killed by transferring the tissues to MS medium containing cefotaxime or ampicillin 500 mg l-1. The transformed tissues induced hairy roots in a period of seven to ten days. The hairy roots produced by transformation were negatively geotropic and possessed numerous root hairs. Transformation was confirmed by opine analysis, which was carried out by high voltage paper electrophoresis. Opines were detected only in transformed samples and not in normal roots. The hairy roots induced in P. zeylanica and P. rosea is a potential source for production of plumbagin in vitro.
Open Access
Differential expression of genes involved in anthocyanin pigmentation in red banana and green-red clones
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Rehna Augustine; KAU; Rajmohan, K
The study entitled “Differential expression of genes involved in anthocyanin pigmentation in Red banana and Green-red clones” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2004 to 2006 with an objective of contrasting the expression of genes (chalcone synthase, dihydroflavonol 4-reductase and UDP: glucose 3-oxy-glucosyl transferase) involved in anthocyanin pigmentation in Red banana and Green-red clones. Experiments were also conducted to differentiate the clones based on total sugar concentration and protein profile. Reverse transcription-polymerase chain reaction was carried out to study the expression of genes. Heterologous primers designed based on the gene sequences of Oryza sativa and Vitis vinifera were used for RT-PCR. Total RNA from tissue culture shoot, leaves and fruit from three different stages of Red banana and Green-red clones were isolated using hot phenol method which gave an yield of 80 - 200 µg g -1 of the tissue and a A260/A280 ratio ranging between 1.6 –2.0. Messenger RNA was purified from the total RNA using the mRNA purification kit from GENEI (Bangalore). The RT-PCR amplified products of the two clones, representing chalcone synthase (CHS), dihydroflavonol 4- reductase (DFR), and UDP: glucose flavonoid 3-oxy-glucosyl transferase (UFGT) were eluted and purified. The cDNA fragments were cloned to pCRII vector (TA Cloning Kit, Invitrogen. Inc., USA) and sequenced. The nucleotide to nucleotide BLAST of cDNA clone of 183 bp from Red banana showed similarity with the chalcone synthase mRNA sequence of rice (Acc. No. X89859). cDNA from Green-red clone showed no significant similarity to any other chalcone synthase gene during homology search. The translated query vs. protein database (blastx) search exhibited similarity with shikimate 3- dehydrogenase, an enzyme in the biochemical pathway for aromatic amino acids. The cDNAs synthesized from Red banana and Green-red clone with gene specific primers for dihydroflavonol 4- reductase were having 354 bp and 325 bp length respectively. The homology search (blastn) revealed no similarities with any of the nucleotide sequences specific for dihydroflavonol 4- reductase. The cDNAs amplified from Red banana and Green-red clone with UDP: glucose 3-oxy-glucosyltransferase specific primers were of 361 bp and 345 bp, respectively. The homology search using blastn showed similarity with mRNA sequence for UDP: glucose 3-oxy-glucosyltransferase in grapes. In Red banana the blastx search revealed similarity with a glucosyltransferase from Synechocystis sp. There was no similarity for UDP: glucose 3-oxy-glucosyltransferase cDNA of Green-red clone in the NCBI database. SDS-PAGE analysis showed no difference in the protein profile of Red banana and Green-red clone. Total sugar content in the peel, pulp, peel together with the pulp of red banana and Green-red clone did not showed any significant difference. The expression analysis of the key genes of the general pathway showed no significant difference in both the clones. All the three genes selected for the study were present in both Red and Green-red clones. The genes isolated were not totally identical in the two banana clones. Further studies are needed to get a better insight into the cause of colour change.
Open Access
In vitro regeneration and agrobacterium mediated transformation in tomato (Lycopersicon esculentum mill.) in relation to disease resistance against groundnut bud necrosis virus
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2006) Ramjitha, P; KAU; Wilson, D
A study on in vitro regeneration and Agrobacterium mediated transformation in tomato (Lycopersicon esculentum Mill.) in relation to disease resistance against groundnut bud necrosis virus (GBNV) carried out in the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2004 to 2006. Irrespective of explants of tomato 0.08 per cent mercuric chloride for seven minutes and one per cent sodium hypochlorite for 15 minutes were found to be the most effective treatment for surface sterilization. Highest callus induction was obtained from cotyledonary leaves (78.00%) and leaves (68.00%) in MS medium supplemented with BA 2 mg l-1 and IAA 1 mg l-1 and regeneration from cotyledonary leaves (79.42 %) and leaves (78.00%) was highest in medium containing 2.5 mg l-1 BA and 1 mg l-1 IAA. The MS medium with 2.5 mg l-1 BA and 1 mg l-1 IAA was the best regeneration (74.62%) medium for the nodal segments. Rooting was obtained on MS medium with 1 mg l-1 IAA. Among the rooted plants 66.8 per cent of plants were successfully established ex vitro. Experiments were conducted to evaluate the tomato calli to different doses of antibiotics viz. kanamycin and cefotaxime. It was observed that kanamycin 75 mg l-1 induced bleaching of callus. At concentration of 100 mg l-1 and above there was complete browning of callus. The callus was not seriously affected by cefotaxime up to 150 mg l-1. The Agrobacterium strain LBA 4404 containing plasmid pCAMBIA 2301 was used for the experiment. Effect of kanamycin and cefotaxime on bacterial growth was studied. It was observed that no colonies were produced after 350 mg l-1 kanamycin and 75 mg l-1 Cefotaxime. The transformation efficiency was high with the infection process for 15 minutes using the bacterial suspension. Highest transformants was recovered only with the wounded callus in dark for three days. The transformation efficiency was very high with the addition of 200 µM acetosyringone. The bacteria after transformation were killed with 75 mg l-1 Cefotaxime. The selection of transformants was done by using 100 mg l-1 kanamycin. Out of the total kanamycin resistant tissues 95 per cent showed GUS activity. The distribution of GUS expression (blue spots and blue patches) on the surface of the callus was even. The transformation efficiency obtained was 96.6 per cent based on the GUS staining. DAC-ELISA was done for the immunological detection of GBNV in tomato. A polyclonal antibody for tospo virus was used for detecting the GBNV virus in tomato. The results of the experiment revealed that the antibody gave high reactivity towards the virus isolates. The molecular characterization of the coat protein gene of the GBNV was done by RT-PCR, with the primer pair derived from the N gene sequence of GBNV. The amplicon (800 bp) was visualized and documented using gel-documentation system.

Theses

Browse

Thesis Type

Type

Author

Date

Subject

Search Results