Molecular Characterization of BananaVar. Nanjanagudu Rasabale andIdentification of Fruit Ripening SpecificProducts
2008
Banana is a major fruit crop and India is the place of origin and diversity for
banana. The cultivar under consideration for the present study is an endangered one
namely “Nanjanagudu rasabale” (NR) having genotype AAB. The present study has
established genetic relationship of NR, among other 21 commercially important
banana cultivars of South India, by using genetic markers. Analyses using 50
Randomly Amplified Polymorphic DNA (RAPD) and 12 Inter Simple Sequence
Repeats (ISSR) primers resulted in the amplification of totally 641 bands of 200 –
3100 bp, of which 382 bands were polymorphic. Based on these data, a genetic
similarity matrix was established and a dendrogram for each set of primers was
developed by UPGMA. The Genetic similarity coefficients in RAPD analysis ranged
from 0.3177 to 0.7818 and in ISSR analysis from 0.1800 to 0.8462. The presence of
a specific RAPD (OPC 5800) band was observed for an endemic cultivar - NR. A
group of 8 cultivars was identified having highly diverse relationship from one
another and this information would be useful for generating 2X and 4X breeding
populations for further application in breeding secondary hybrids.
Since edible banana plants are sterile hybrids, obtaining variants through
biotechnological methods would greatly assist in the generation of genetically diverse
and agronomically improved clones for which in vitro regeneration is a pre-requisite.
Thus aiming, the leaf explants were cultured first on medium with a high level (22.4
μM) of benzyladenine (BA), second on indole-3-butyric acid (IBA) supplemented
medium, and third on reduced BA medium under incubation in the dark. The highest
adventitious shoot regeneration in 24% of the explants, with the number of shoots
ranging from 2-3 per explant, occurred in the explants incubated at the first step in
medium with 24.6 μM and 0.198 μM of IBA. Addition of various levels of (10-50
μM) spermine, spermidine, and putrescine to cultures with secondary embryogenesis
showed that about 50% of embryogenic calli rapidly produced secondary embryos
only in the presence 40 μM spermine but not in other treatments. The shoot cultures
were checked for their performance on solid medium (SM) and partial immersion
system (PIS). The rate of shoot multiplication was higher in PIS than in SM. A
micropropagation protocol was also developed where high levels of Benzylamino
purine (BAP) up to 53.28 μM and Kinetin (Kn) up to 55.80 μM showed direct
correlation between the two resulting in a highest number of 80 shoot buds per
segment in BAP (31.08 μM) treatment. The plantlets were analysed for their genetic
stability using 50 RAPD and 12 ISSR genetic markers that resulted in 625 distinct
bands showing homogeneous patterns. The absence of any genetic variation indicates
that the micropropagation protocol developed in this study as well as for developing
stable regenerants of NR for rapid in vitro multiplication is appropriate and applicable
for clonal propagation.
Fruit ripening and softening involve depolymerization of complex cell wall
components. More than one class of enzymes and other proteins are known involved
in this process. In the present study the regulation of fruit softening studied at
molecular level and demonstrated the simultaneous activities and gene expressions of
expansins (the cell wall loosening proteins) and other cell wall hydrolyzing enzymes.
Two types of expansin genes, MaEXPA-1 and MaEXPA-2, were found to be banana
fruit-ripening-specific and their expressions significantly and differentially altered by
ethylene inducers/inhibitors. Activities of pectin methyl esterase (PME),
polygalacturonase (PG) and pectate lyase (PEL) in banana cv. NR fruit were
measured over a period of 10 days after ripening was initiated with ethylene.
Ethylene-stimulated activities of the above three enzymes was differentially
suppressed by GA, MH, SA and IAA whereas ABA, ethrel and smoking stimulated
the activities of all hydrolases, except polygalacturonase. These treatments appear to
play a major role in up- / down-regulation of the activities of various cell wall
hydrolases.
To gain a better insight on the molecular regulation of banana fruit ripening,
the mRNA Differential Display technique coupled with silver-staining was used and
specific transcripts differentially expressed during ripening were first identified.
Using 71 primer combinations and four populations of mRNA (Pre-climacteric;
Climacteric 1; Climacteric II and post climacteric), a total of 120 transcripts were
cloned into T/A cloning vector and sequenced. DNA sequence analyses revealed
significant homology to transport protein, sucrose phosphate synthase, heat shock
protein, transcriptional regulator, senescence protein. These proteins can be
associated to biological processes like primary, secondary and RNA metabolism,
signal transduction and stress responses or defense. The RNA dot analysis showed the
expression of most of the up- and down-regulated genes are specific to fruit and
ripening. Since banana lacks information about the molecular regulation of ripening,
the results of the present finding provide better insight for the characterization of the
changes in gene expression that accompanies the ripening process. The enormous
data developed through these studies form the basis for developing chemical
formulations for controlled ripening of banana fruit, probably with extended shelf life.
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