M S Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
T Biswas
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
M S Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
M S Hossain
Bangladesh Jute Research Institute, Dhaka, Bangladesh
Hibiscus cannabinus, In vitro, RAPD, Somaclonal variation, Polymorphism
Bangladesh Jute Research Institute (BJRI).
Variety and Species
Sample collection: Seeds of three varieties of kenaf viz., HC-2, HC-3 and HC-95 were collected from Bangladesh Jute Research Institute (BJRI). Among them, HC-2 (1977) and HC-95 (1995) are high yielding varieties and resistant to nematode (Khatun, 2009). In vitro grown plants from seeds were used as the source of plant material. Hormone treatment and plantlet regeneration from callus: Approximately 8-10 mm2 segments of hypocotyls, petiole and cotyledonous petiole from in vitro grown plants on MS (Murashige and Skoog, 1962) medium were excised and placed on callus production medium (MS medium containing NAA, BAP). Two treatments were applied to produce somaclones. Treatment 1 refers to the use of NAA and BAP at a concentration of 2.00 and 0.50 mgl-1, respectively whereas treatment 2 comprised of the use of NAA and BAP at a concentration of 1.00 and 1.00 mgl-1 respectively. Somaclones produced by treatment-1 were considered as clone-1, whereas somaclones produced by treatment-2 were considered as clone-2. All cultures were kept in a culture room at a 16h photoperiod (2000-3000 lux illumination) and 25°C controlled temperature. After 3-4 weeks, calli were initiated. Three segments of calli (approximately 8-10 mm2 each) were sub-cultured on the same medium at an interval of 2 weeks. After 6-7 weeks, shoots and roots were initiated from the calli. Finally, a total of twenty seven individuals [three mother plants and two clones (clone 1 and 2) from each mother plant with three replications] were subjected to RAPD analysis to detect somaclonal variation. Genomic DNA extraction: Genomic DNA was isolated from leave tissues of twenty seven individuals using phonol, chloroform and isoamylalcohol extraction and ethanol precipitation method. In brief, approximately 50 mg of fresh, vigorous, young actively growing leaf tissues were taken into a microcentrifuge tube, cut into small pieces, homogenized and digested with RNAse in extraction buffer (50 mM Tris-HCl, 25 mM EDTA, 300 mM NaCl, pH=8.0 and 1% SDS ). DNA was purified by successive extraction with 600 µl of phenol: chloroform: isoamylalcohol (V: V: V = 25: 24: 1). DNA was precipitated first using about 0.1 volume of sodium acetate with 2.5 volumes of absolute ethanol and peletted by centrifugation. DNA was reprecipitated by adding two volumes of 70% ethanol and peletted by centrifugation. The pellets were then air-dried and resuspended in an appropriate volume of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH= 8.0). DNA quality was checked by electrophoresis and quantified using a spectrophotometer (SpectronicR GenesisTM). Primer selection: Initially, twenty five decamer primers (Merck, India) of random sequence were screened on a sub sample of three randomly chosen individuals from each variety, to test their suitability for amplifying kenaf RAPDs that could be accurately scored. Primers were evaluated on the basis of intensity or resolution of bands repeatability of markers and consistency within individual and potential to different cultivars (polymorphism). Finally, three primers were selected for the analysis of the whole sample set of the three varieties. PCR amplification and agarose gel electrophoresis: PCR amplification was done following the procedure (Williams et al., 1990) with some modificatoins. PCR reactions were performed on each DNA sample in a 10µl reaction mix containing: 1µl of 10x Ampli Taq polymerase buffer, 1µl of 2.5 mM dNTPs, 0.6 µl of 50 mM MgCl2, 1 unit of Ampli Taq DNA polymerase (Merck, India), 50 ng template DNA, 2 µl of 10 µM primer and a suitable amount of sterile deionized water. DNA was amplified in a thermal cycler (Eppendorf Mastercycler Gradient). The reaction mix was preheated at 94ºC for 3 min followed by 40 cycles of 1 min denaturation at 94ºC, 1 min annealing at 36ºC band elongation at 72ºC for 2 min. After the last cycle, a final extension of 7 min at 72ºC was added to allow complete extension of all amplified fragments. The amplified product from each sample was separated electrophoretically on 1.4% agarose gel in a 1XTBE buffer at 120V for 1½ h. Two molecular weight markers (1 kb and 20 bp DNA markers) were run alongside the RAPD reactions. The gels were stained with ethidium bromide and bands were observed and photographed by a gel documentation system (Bio- Rad). RAPD data analysis: Fragments were scored as 1 if present and 0 if absent. The scores were then pooled for constructing a single data matrix. This was used for comparing the frequencies of all polymorphic RAPD markers and estimating Nei’s (1973) gene diversity (h), gene flow (Nm), coefficient of gene differentiation (GST), genetic distance (D) and constructing a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among populations using POPGENE (version 1.31) (Yeh et al., 1999) computer program. The similarity index values (SI) between the RAPD profiles of any two individuals were calculated from RAPD markers according to the formula: Similarity index (SI) = 2 Nxy/Nx+Ny, Where, Nxy is the number of RAPD bands shared by individuals X and Y respectively, and Nx and Ny are the number of bands in individual x and y, respectively.
Progressive Agriculture 28 (2): 100-108, 2017
Journal