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Novel approaches for Molecular Analyses, Micropropagation and Curing of Vanilla (Vanilla planifolia)

Sreedhar, R. V. (2009) Novel approaches for Molecular Analyses, Micropropagation and Curing of Vanilla (Vanilla planifolia). PhD thesis, University of Mysore.

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Abstract

Vanilla is the most popular flavor both by monitory and tonnage basis finding vast applications in food, pharmaceutical, beverage and cosmetic industries. Natural vanilla flavor is obtained from careful curing of vanilla beans and it is the second most expensive spice traded in the world market. In a study for assessing the diversity among Indian vanilla clones and to create a database for the available germplasm, analysis of isozymes as markers was found to be inappropriate due to inconsistencies in zymograms of enzymes. On the other hand, genetic markers such as RAPD and ISSR were found highly suitable. The genetic diversity among 25 accessions collected from 13 major locations was studied. The PCR-amplification patterns within accessions were similar for a given primer indicating that the morphological difference observed had no genetic background. Molecular analysis among different accessions also yielded identical PCR band profiles in both RAPD and ISSR analyses. These results clearly indicate that V. planifolia cultivated in India appears to share the same genetic background and therefore, the genetic diversity is either extremely low or non-existing. Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Through an investigation using RAPD and ISSR markers, micropropagated multiple shoots of vanilla established and multiplied in vitro for over 10 years were assessed which established that micropropagation protocol used in this study can be carried out for a considerable length of time without any risk of genetic instability. Vanilla shoot multiplication in semi-solid (SS), complete immersion system (CIS) and partial immersion system (PIS) were evaluated for biomass, shoot multiplication and elongation aiming at developing an improved micropropagation protocol. GrowtekTM bioreactor, functioning on the principle of PIS, was found to be most suitable for micropropagation of vanilla. For automation of the micropropagation system, different medium contact periods were provided to shoot cultures in CIS in which the 30 min contact three times a day appeared most congenial. Similarly a bioreactor developed with intermittent bathing of the cultures with nutrient medium was congenial for shoot multiplication of vanilla. A combination of red soil: sand: vermicompost in equiproportion was found to be the best for greenhouse hardening. Field-evaluation showed that the micropropagated plants were early to flower and high yielders than the conventionally propagated ones. The vanilla shoots cultured under completely immersed condition showed hyperhydricity syndrome (HHS). A study focusing on unraveling the major structural, biochemical and molecular changes occurring during HHS was carried out. The HHS was associated with severe damages at cellular and sub-cellular levels, increase in free polyamines and accumulation of water, and decrease in quantities of chlorophyll, protein and drastic changes in reducing and non-reducing sugars. The onset and progression towards hyperhydricity (HH) showed higher activities of antioxidant enzymes, indicative of shoots’ defensive efforts against oxidative stress. Thirty one HHassociated cDNAs identified by DDRT-PCR were cloned and sequenced whose electronic homology searches using BLASTX analysis resulted in the identification of 23 cDNA clones showing homology with various stress, apoptosis, DNA repair and carbohydrate breakdown related proteins expressed differentially during HHS. BLASTN analysis yielded 18 fragments having homology with different stress linked cDNA clones. A partially characterized transcriptome of hyperhydric condition in V. planifolia has been developed which paves the way for a better insight into gene expression during this common physiological disorder. Vanilla beans derived from the micropropagated plants along with the beans available commercially were cured following different biotechnological approaches for development of an efficient curing technique. In this study, effects of pre-treatments on the flavor formation during accelerated curing at 38 oC for 40 days were studied. Use of naphthalene acetic acid (5 mg/L) or ethrel (1%) with blanching pre-treatment resulted in 3-fold higher vanillin on 10th day. All major quality parameters analyzed were found comparable to commercial sample. In another experiment, food-grade elicitors were used in combination with pre-treatments for the accelerated curing of beans. When acetone dried red beet elicitor - a rich source of peroxidase was used, 2.65% vanillin was formed in 10 days, which was 1.7-fold higher than in control beans of this study and 3.2-fold higher than the conventional curing. HPLC analysis of elicitor-treated samples showed the formation of almost all the major compounds found in the conventionally cured beans (cured for 3-6 months) with better sensorial properties. These observations appear useful for developing a rapid process for the curing of vanilla beans.

Item Type: Thesis (PhD)
Uncontrolled Keywords: Vanilla, micropropagation, molecular analysis
Subjects: 500 Natural Sciences and Mathematics > 10 Plants > 05 Tissue Culture
500 Natural Sciences and Mathematics > 10 Plants > 06 Trees And Shrubs
Divisions: Plant Cell Biotechnology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 15 May 2012 09:52
Last Modified: 06 Jul 2015 12:38
URI: http://ir.cftri.com/id/eprint/10761

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