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Open Access
Isolation and Characterization of cDNA encoding chalcone synthase from flower buds of orchid Dendrobium variety sonia 17
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2007) Anjana, G R; KAU; Soni, K B
The study entitled “Isolation and characterization of cDNA encoding chalcone synthase gene from the flower buds of orchid Dendrobium variety Sonia 17” was conducted at the Department of Plant Biotechnology, Vellayani, Thiruvananthapuram during the period from 2005 to 2007 with an objective of studying the isolation and characterization of cDNA encoding chalcone synthase gene involved in anthocyanin pigmentation in orchid flower buds. . Heterologous forward and reverse primers were designed based on the gene sequences of Oryza sativa, Fragaria ananasa and Phalaneopsis orchid using primer3 software. Total RNA was isolated from immature floral tissues 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). Reverse transcription-polymerase chain reaction was carried out to study the expression of gene. The RT-PCR amplified products representing chalcone synthase (CHS) gene was eluted and purified. The product was sequenced and studied the similarity of the same using homology search. All sequenced regions were subjected to BLASTN and BLASTX similarity search. Rice chalcone synthase specific primer produced an amplified sequence of 460 bp long and showed maximum similarity to the cDNA clone 5', mRNA sequence of. flower bud of Phalaenopsis violacea and flower bud of Phalaenopsis equestris Lambda ZapII cDNA Library in BLASTN similarity search.BLASTX analysis of the sequence showed similarity to maturase K protein of Aerangis kirki. The cDNA amplified with strawberry chs specific primer showed maximum similarity to the cDNA clone 5’, mRNA sequence of Phalaenopsis violacea flower bud and flower bud of Phalaenopsis equestris in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to LFY-like protein of Serapias lingua. The results of the nucleotide to nucleotide search (BLASTN) of the cDNA of orchid, amplified using chalcone synthase specific primer from orchid showed similarity to cDNA 5', mRNA sequence of Ipomoea batatas in the BLASTN similarity search. BLASTX analysis of the sequence showed similarity to retrotransposon protein of Oryza sativa (japonica cultivar-group). The result of the sequences obtained from the study shows similarity with the genes involved in the biosynthetic pathway of Phalaenopsis orchid flower fragrance.
Open Access
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; KAU; 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.
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
Molecular characterization of tomato (Solatium lycopersicum L.) with special reference to tomato leaf curl virus (ToLCV) resistance
(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikara, 2007) Anjali, Divakaran; KAU; Nazeem, M
Tomato (Solarium lycopersicum L.) is one of the major vegetable crops in the world. India ranks sixth in the production of tomatoes worldwide with a total area of 0.50 million hectares and productivity of 17.4 MT per hectare. Tomato leaf curl disease caused by the Tomato Leaf Curl Virus (ToLCV) and transmitted by whiteflies (Bemisia tabaci) is one of the most important diseases affecting this crop. The disease causes losses in yield to the tune of 70 to 100 per cent. ToLCV is severe under conditions prevalent in Kerala also. Identification of resistant sources of the disease and development of trait-related markers from these sources would be an important approach to overcome the problem of ToLCV. With this objective in mind, an investigation was undertaken at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara from the year 2005 to 2007 to characterize the reaction of tomato genotypes to ToLCV under conditions prevalent in the area and to identify molecular markers (RAPD and AFLP) linked to disease resistance.Fifteen genotypes were raised in sterile soil in earthen pots and field during the peak season of ToLCV infection (December - February) and their reaction to the disease was categorized based on the coefficient of infection. Out of 15 genotypes, eight were observed to be highly resistant to ToLCV under both pot culture and field experiments. Observations of biometric characters of the genotypes grown in pots and field were made. All genotypes showed significant difference in all the characters observed both in pot culture experiments and field study. Plant height was the most striking character of difference observed in the two different culture conditions. Genotypes were subjected to molecular characterization using RAPD and AFLP markers. Genomic DNA required for these assays was isolated by two protocols. The protocol suggested by Rogers and Bendich (1994) with modifications was found to be most appropriate for DNA isolation from tomato leaves. Forty random decamer primers were screened for RAPD assay. Thirty-six of these were used for further RAPD profiling of the tomato genotypes. Out of this, 12 primers displaying good and reproducible patterns were selected for molecular characterization. The primer OPS 8 recorded the highest resolving power. A total of 116 amplicons were generated by the 12 selected primers of which 71 were polymorphic. The dendrogram constructed separated the genotypes into two groups. ToLCV resistant genotypes Anagha and H-24 with 92 per cent similarity were found to be most related. RAPD analysis did not reveal any trait- related marker in the present study. AFLP assay was carried out with five combinations of Eco&l and Msel based primers. A total of 241 amplicons were detected, out of which 122 were polymorphic. Three markers linked to ToLCV susceptibility were obtained using the primer combination EAAG/MCAC. All genotypes studied showed genetic uniformity in RAPD and AFLP assay except with respect to a few primers. Trait-related marker was detected in a single primer pair in AFLP assay, while RAPD assay did not give any clear demarcation with respect to ToLCV resistance/susceptibility. The markers identified could be further exploited for obtaining nucleotide sequence information and level of specific gene expression in susceptible/resistant genotypes.
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
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; KAU; Valsala, P A
The present investigation entitled “Induction of variability in Vanilla planifolia Andrews through intra/interspecific hybridisation and embryo culture technique” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Kerala Agricultural University, Vellanikkara during the period from 2003-2005. Genetic variation in the germplasm of Vanilla planifolia is relatively limited because of the continuous clonal breeding of the existing gene pool. The present work was taken up with the main objectives of enhancing the spectrum of variability in vanilla through intra/interspecific hybridisation and thereby to get desirable recombinants of higher yields, richer vanilla flavour and disease resistant. The superior clones of V. planifolia (a 82, a 94, a 425, a 428, vv97/84) and V. tahitensis plants maintained at CPBMB were taken as parent plants in the hybridisation programme. Variation in the foliar and stem characters of parent plants was observed which could be manifested in the progeny. Floral morphology and biology of V. planifolia and V. tahitensis plants were studied and variation was observed in flowering season of both species. Pollen fertility was assessed in acetocarmine (1%) stain and was 61 per cent for V. planifolia and 74.69 per cent for V. tahitensis flowers. The viability of pollen was assessed in pollen germination reported by Ravindran (1979) and was 39 and 45.3 per cent for V. planifolia and V. tahitensis pollen grains. There was no significant variation in the pollen fertility and pollen viability percentages of both species. In vivo crossing was done as per the pollination technique of V. planifolia for inter and intraspecific crosses. The pod set percentage varied significantly with genotype. The pod development pattern was similar in both inter/intraspecific hybrid pods. Embryo culture was done as per the protocol reported by Mary et al. (1999) to get hybrid progenies. Seeds from pods of different ages were cultured. The time taken for embryo germination and intensity of embryo germination varied significantly with the maturity and genotype of pods. Seeds from hybrid pods (a 82 x a 94) of 6 weeks maturity germinated early compared to seeds from half mature (14, 18, 20, 22 and 24 weeks) and mature (28 and 36 weeks) pods. The germinated seedlings were multiplied and planted out with the already available protocol. Variability in the seedling progenies was assessed at monthly intervals for morphological characters. The hybrids obtained from intraspecific crosses a 82 x a 94 and vv 97/84 x a 94 were found vigorous with respect to plant growth. The hybrid progenies from interspecific cross V. t x V. p showed remarkable variation in leaf shapes and four leaf shapes broadly ovate, oblong lanceolate, lanceolate and linear lanceolate were observed in the progenies against narrow lanceolate in V. t parent and oblong-elliptic in V. p parent. Simple and branched stem type were observed in the progenies of interspecific cross (V. t x V. p) and intraspecific crosses (a 82 x a 94 and vv 97/84 x a 94) against normal simple stem in the parents. Single and double aerial roots were produced from single node in the progenies of interspecific cross (V. t x V. p) and intraspecific crosses (a 82 x a 94 and a 425 x a 94) against single root origin in the parent plants. Variation was also observed for number of leaves, plant height, leaf size, leaf colour and internodal length. Molecular characterisation of selected parents and hybrid progenies was done using RAPD technique. Heirarchical cluster analysis of RAPD data revealed variability between parents and hybrid progenies of intra/interspecific crosses. The variation in the parents and progenies of interspecific cross (V. t x V. p) ranged from 8 - 40 per cent. The intraspecific crosses a 82 x a 94, a 94 x a 82, a 425 x a 94 and vv 97/84 x a 94 showed variation range of 16 - 36 per cent, 4 - 31 per cent, 10 - 34 per cent and 12 - 28 per cent respectively. The results obtained from morphological and molecular characterisation of parents and progenies confirmed that variability was induced in vanilla through intra/interspecific hybridisation and embryo culture technique.
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.

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