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

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.