Induction and establishment of transformed hairy root cultures of sarsaparilla (Hemidesmus indicus L.) R. Br.
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Date
2014
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College of Agriculture, Vellayani
Abstract
The study entitled Induction and establishment of transformed hairy root cultures of sarsaparilla (Hemidesmus indicus L.) R. Br. was conducted at the Biotechnology and Bioinformatics division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute during 2013-2014. The objective of the study was to induce and establish transformed hairy root cultures using wild strain(s) of Agrobacterium rhizogenes for determining the production of 2-hydroxy 4- methoxy benzaldehyde. Murashige and Skoog media (1962) was used for the experiments. Young shoots from the collected plants served as the explants for the initiation of cultures. The explants were surface sterilised using 0.1% HgCl2 at different intervals like 1- 5 minutes and standardised the time as 2 minutes since maximum percentage (86 %) of shoot bud initiation was obtained in explants sterilized for 2 minutes. The explants were inoculated into MS medium of full and half strengths with 3 %sucrose and agar (0.6% w/v) and augmented with various concentrations (1.0 - 2.5 mg/L) of 6-benzylaminopurine (BAP) alone or in combination with auxin (NAA/ IAA) and MS media devoid of hormones. The results showed that media containing only 2.0 mg/L BAP favoured maximum shoot bud initiation 2.52± 0.873 in a period of 3 weeks. The average length of shoots was ranging from 0.71 to 0.9 cm. Also young tender shoot tips showed maximum percentage of response (90%) with maximum number of shoot formation. For inducing multiple shoots MS media supplemented with different concentrations (0.25 1.5 mg/L) of BAP were used. Media containing 1.0 mg/L BAP was found to be the best for shoot multiplication with average number of shoots of 3.571 ± 0.272 of length 3.29cm length. The induction of hairy roots were done for the bioproduction studies. For the induction of hairy roots, various wild strains of Agrobacterium rhizogenes namely A4, R1022, LBA 9402, 15834, K599, NCIM 5140 maintained in Yeast extract mannitol (YEM) media were used. Infection of pre-incubated shoots was performed by wounding the internodal portion with a sterile scalpel blade containing bacteria scraped off from isolated colonies on YEM agar medium. The hairy roots emerged at the site of infection in the 4th week. Only A4 strain was found to be successful in inducing hairy roots. The initiated hairy roots were transferred to Petri-dishes containing MS basal agar medium (1% w/v agar). Of these roots, those showing bacterial infection were transferred to MS basal agar media supplemented with antibiotic (500 mg/L Streptomycin). After the decontamination of hairy roots, the roots were established in solid as well as liquid MS media devoid of hormones. The measurement of growth parameters of hairy root cultures over a period of 30 days with an interval of 5 days were done. The mean weight of roots and growth index were determined after each harvest. Growth was then expressed as Fresh Growth Index. The root biomass reached maximum (14.10±0.045g) on 25th day. After 25th day there was no further biomass increase which indicated that the roots reached stationary phase of growth. The roots appeared to be brownish at the centre portion of roots. The initial growth index was 0.083and it increased to 6.051 during the next 25 days, but after that the growth index decreased. The extraction of the commercially significant secondary metabolite, 2-hydroxy -4-methoxy benzaldehyde was done. The extracts were then concentrated using Rotavapor under vacuum. The concentrated samples of the extracts were used for chromatographic analysis. Thin layer chromatography was done for the identification of the compound. Well resolved bands were obtained in petroleum ether: acetone 10:1 for samples identical to the band of authentic compound 2- hydroxyl- 4-methoxy benzaldehyde. The relative flow of the compound was obtained as 0.4. The presence of 2- hydroxyl- 4-methoxy benzaldehyde was confirmed by HPLC. The retention time was found to be 4 .1 for the authentic compound and a peak was obtained in the chromatogram of the sample at same retention time. Further scaling up studies needs to be done for the pharmaceutical purposes and commercial product development.
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