MSc

Banana is an important fruit crop widely grown throughout the world. With the increasing demand and vast export potential coupled with the farmers desire to grow banana on a large area, in vitro propagated plants are becoming increasingly important as planting material for rapid multiplication of economically important commercial varieties. In vitro propagation has many advantages, such as higher rates of multiplying clean (pest and disease-free) planting material and the small amount of space required to multiply large number of plants.

A major problem associated with micropropagation is the occurrence of genetic variation resulting from in vitro cultures, i.e., somaclonal variation amongst sub-clones of one parental line. The molecular basis of such variation is not well explained till date.
The study entitled “Morphological and molecular analysis of genetic stability in micropropagated banana (Musa spp) var. Nendran” was carried out at the Centre for Plant Biotechnology and Molecular Biology (CPBMB), College of Horticulture, Vellanikkara, during the period from 2010 to 2012 with the objective of evaluation and characterization of the variation in tissue culture derived banana plants regenerated in different subcultures through in vitro organogenesis.
Micropropagation of banana was carried out using male bud and sucker as explant source. Adventitious shoots were induced from both the sucker derived and male bud derived explants. The culture establishment rate and multiplication frequency were significantly low in male bud derived explants. The plantlets derived in specific subcultures (3, 6, 8, 10, 12, 14 and 16) were planted out and subjected for further evaluation. Considering vegetative characters during the field performance;
plants derived from later subcultures were found inferior to others in both male bud and sucker derived plants. The fruit characters of the male bud derived plants were comparable to the mother plant though bunch weight and size was less in later subculture (10th).
The CTAB procedure reported by Rogers and Benedich (1994) for the extraction of nucleic acids was used for the isolation of genomic DNA from tissue culture derived banana plants. The young unfurled leaves from healthy plants were collected early in the morning and used for the genomic DNA isolation. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis.

The PCR conditions were optimized for Inter Simple Sequence Repeats (ISSR) assay. DNA isolated from each subculture were bulked and amplified using 10 selected primers. The amplification pattern was scored and analysed for quantifying the variation among the plants derived from different subcultures using male bud and sucker as explant. The computer package NTSYS-pc was used for cluster analysis. Plants derived from different subcultures were found grouped into two distinct clusters. Plants derived from upto 12th subculture were grouped in first cluster (<10 % variation) and those derived from 12th subculture onwards were found grouped in second cluster. The maximum genetic variation detected through ISSR assay was 15% for both male bud and sucker derived plants and the variation was relatively more in the plants derived in later subcultures (12th onwards).
Changes in DNA methylation (addition of -CH3 to cytosine) has been hypothesized as an underlying mechanism of tissue culture induced variation due to the high frequency of quantitative phenotypic variation, the activation of transposable elements, heterochromatin-induced chromosome breakage events etc. An attempt was made to detect the extent of methylation pattern in micropropagated banana plants using Methylation Sensitive Amplification Polymorphism (MSAP) assay.

In the study, four MSAP primer combinations were used to amplify the fragments cleaved by restriction enzymes MspI and HpaII (isoschizomers). For male bud derived plants a total of 39.4 percent methylation sites were detected while for sucker derived plants it was only 23.3 percent. Variation in methylation pattern was observed more in later subcultures (10th onwards) than initial. Percentage of internal methylation was relatively more compared to hemimethylation for both male bud and sucker derived plants.

From the study, it was observed that the plants derived from initial subcultures showed lesser variation (<10 %) compared to later subcultures (12th onwards). The methylation pattern detected through MSAP assay was observed as an important factor inducing variation in tissue cultured banana. The ISSR and MSAP primers which detected polymorphism could be further utilized in quality control of tissue culture derived banana plants. Since the banana genome is sequenced recently, the information generated could be exploited to detect the hotspots in banana genome.