Tissue culture propagation of banana has gained attention due to its potential to provide genetically uniform, pest- and disease-free planting materials. In India, a large number of verities of banana are multiplied in vitro for commercial cultivation. Among the delicious dissert bananas in India, Musa acuminata, var. NanjanaguduRasabale (NR) (group “Silk” having genotype AAB) which is grown in Mysore district of Karnataka, has very high commercial demand due to its inviting aroma and white fluffy sweet pulp. This variety is highly susceptible to bacterial and viral diseases reducing the area of cultivation to only 5 hectares and hence recently considered as an endangered one. In order to save this cultivar from extinction, tissue culture methods were developed for its conservation (Bhagyalakshmi and Singh, 1995; Venkatachalam et al. 2006). However, it is necessary to ascertain the genetic fidelity of such cultures.

Analyses of patterns of specific enzymes provide a convenient method for detection of genetic changes but they are subjected to ontogenic variations. Such methods have limited applications due to their limited numbers and only those DNA regions coding for soluble proteins can be sampled. Tissue and environment independence in expression of DNA based markers have made them more reliable over morphological and isozyme markers. Among various DNA-based markers, though Restriction Fragment Length Polymorphism (RFLP) can be used for screening genetic stability of tissue cultured plants, the method involves use of expensive enzymes, radioactive labeling and extensive care, therefore, appears unsuitable. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers, on the other hand, require only small amount of DNA sample without involving radioactivity tests and are simpler as well as faster. RAPD has proven to be quite efficient in detecting genetic variations even in closely related organisms such as two near isogenic lines (NIL). ISSR technique is also very simple, fast, cost-effective, highly discriminative and reliable. At present, RAPD and ISSR markers have been successfully applied to detect the genetic similarities or dissimilarities in micropropagated material in various plants. The aim of the present study was to assess the impact of the protocol and long term in vitro effects on the induction of somaclonal variation in banana var. NR.

Plant material

A healthy and high yielding banana clone of var. NanjanaguduRasabale (classified under “Silk” with genotype AAB) was collected and maintained as mother plant at the back yard of authors’ laboratory. The rhizomes of sword suckers were used to excise the meristem and establish shoot cultures.

Culture medium and incubation conditions

The aseptic shoot cultures used in the present study were established as reported in an earlier study (Bhagyalakshmi and Singh, 1995) and were maintained on M1 medium containing ¾ strength Murashige and Skoog (Murashige and Skoog, 1962) basal salts (MS) with additional 1g l^-1 of potassium nitrate and 1 ml l^-1 of vitamin mixture (Novak et al. 1989) supplemented with ascorbic acid (100 mg l^-1), BAP (1 mg l^-1), Indole Butyric Acid (IBA, 0.2 mg l ^-1) and gelrite® (Sigma, USA) (2.5 g l^-1). The pH was adjusted to 5.8 before autoclaving at 121ºC and 15 lbs for 20 min. Cultures were maintained at 25 ± 1ºC under a 16 hrs light (320 µmol.m^-2 S^-1) / 8 hrs dark photoperiod. The in vitro multiple shoot cultures were established and maintained on M2 medium (Table 1).

Plant material for DNA extraction

For testing the long-term effects, the plantlets obtained after repeatedly sub-culturing in M2 medium for 150 times (about 10 years), each after 4 weeks period, were used. Eleven plantlets, including certain morphological variants were chosen from over 4000 micropropagated plantlets (Figure 1) for studying DNA fingerprints.

Preparation of template DNA

The DNA was extracted by using the GenElute^TM Plant Genomic DNA Mini prep kit supplied by Sigma (USA). The RNA contamination in all the samples was removed by digesting the extract with 100 RNase-A (100µg ml^-1; Bangalore Genei, Bangalore, India) for 30 min at 37ºC. 

DNA amplification

RAPD and ISSR amplifications were performed routinely using PCR mixture (25 µl) which contained 25 ng of genomic DNA as template, 1 x PCR buffer (Fermentas GMBH, Germany), 200 µM dNTPs (Fermentas GMBH, Germany), 1 unit (U) of Taq DNA polymerase (Bangalore Genei, India), 1 μM of each primer with various concentrations of MgCl₂ (Fermentas GMBH, Germany) (Table 2) depending on the primer (Sigma-Aldrich, India). In the case of RAPD, PCR was performed at initial denaturation at 93ºC for 4 min followed by 40 cycles of 1 min denaturation at 94ºC, 1 min annealing at 36ºC and 2 min extension at 72ºC with a final extension of 72ºC for 10 min using a thermal cycler. For ISSR primers, PCR was performed at initial denaturation of 94ºC for 4 min followed by 40 cycles of 1 min denaturation at 94ºC, 1 min at 2ºC lower than specified annealing temperature for each primer and 2 min extension at 72ºC with a final extension of 72ºC for 10 min using a thermal cycler.

The PCR products obtained were separated on 2% agarose gel, stained with ethidium bromide (0.001%) and documented in a gel documentation system. The size of the amplification products was estimated from 100 bp DNA ladder.

In the present study, two PCR based techniques, RAPD and ISSR were adopted for evaluation of clonal fidelity of banana plantlets. Use of GenElute^TM Plant Genomic DNA Mini prep kit from Sigma (USA) for extraction resulted in sufficient quantity and quality of DNA for genetic analyses. A comparison of RAPD and ISSR patterns of 11 plants chosen from 4000-plus plantlets (Figure 1) and a control plant (mother plant) was carried out in order to confirm the genetic fidelity. Of the 50 arbitrary RAPD primers initially screened, 30 produced clear and scorable amplification products generating 377 scorable band classes, ranging from 200 bp to 3100 bp in size. The number of bands for each primer varied from 6 (OPJ 08) to 22 (OPM 20) with an average of 12.6 bands per RAPD primer (Table 2). The screening with 5 ISSR primers generated 47 scorable band classes, ranging in size from 200 bp to 2200 bp. The number of bands for each primer varied from 7 (UBC 811) to 13 (UBC 834) with an average of 9.4 bands per ISSR primer. A total of 5088 bands (numbers of plantlets analysed x number of band classes with all the ISSR and RAPD primers) were generated, giving rise to monomorphic patterns across all the plantlets analysed. Samples of the monomorphic band classes obtained for RAPD (Figure 2 and Figure 3) and ISSR (Figure 4) markers are shown.  In all these analyses, no polymorphic bands were observed.

Reliable monitoring of variability in DNA sequences of plants has been achieved using PCR-based molecular markers like RAPD, SSR, ISSR and AFLP. Absence of genetic variation using RAPD has been reported in several cases such as micropropagated shoots of Pinusthunbergii (Goto et al. 1998), axillary bud proliferation of chestnut root stock hybrids (Carvalho et al. 2004). Contrarily, occurrence of somaclonal variations were found in micropropagated plants of Pinustremuloides (Rahman and Rajora, 2001) and Actinidiadeliciosa (Palombi and Damiano, 2002) where, PCR based screening with the use of RAPD and ISSR markers were very useful.

In the present study, we adopted the use of two PCR-based techniques, RAPD and ISSR, for the identification of genetic variations in tissue-culture-derived banana plantlets because of their simplicity and cost-effectiveness. The use of two types of markers, which amplify different regions of the genome, allows better chances for identification of genetic variation in the plantlets. Although this study has not detected any genetic change, it is possible that some changes might have occurred that go undetected as there is a possibility of point mutations occurring outside of the priming sites. In conclusion, this study has screened a large number of random primers that are common for higher plants, and some of them are prevalent in monocots. Since there were no changes in the banding pattern observed in tissue culture plants as compared with that of mother plant, we conclude that that our micropropagation protocol developed for banana, var. NanjanaguduRasabale, might be applicable for a considerable length of time without much risk of generation of genetic instability.

The authors thank Dr. V. Prakash, Director, CFTRI for his encouragement in the research activities.

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