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Characterization of Induced Sugarcane Somaclones and Their Sources Varieties Using Random Amplified Polymorphic DNA

Kuasha Mahmud*
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi, Pabna

K. M. Nasiruddin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh.

M. A. Hossain
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh

L. Hassan
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

Sugarcane somaclones and their sources varieties were analyzed by RAPD molecular markers to check the variation at molecular level based on 1.4% agarose gel electrophoresis (AGE). Six RAPD primers generated 237 bands with average 39.5 varied from 15 to 63 with size ranging 145 - 1000 bp among the four sugarcane varieties and their 12 somaclones. Genetic diversity or polymorphism information content (PIC) value ranged from 0.39 to 0.50 for all loci across the 4 varieties and their 12 somaclones based on RAPD markers. Dendrogram based on linkage distance using unweighted pair group method of arithmetic means (UPGMA) based on 6 RAPD primers indicated segregation of the 4 sugarcane varieties and their somaclones into two main clusters at linkage distance 36. Variety Isd 39 was observed in main cluster C1 while its (Isd 39) somaclones and other varieties (Isd 37, Isd 38 and Isd 40) and also their somaclones were found in main cluster C2 having different sub-clusters. Therefore, it may be concluded that RAPD markers can be used for identification of somaclonal variation and the relationship between sources varieties and their somaclones.

Keywords: Sugarcane, Somaclones, Variation, RAPD and UPGMA

Location: Biotechnology Laboratory, Bangladesh Sugarcane Research Institute (BSRI), Ishurdi, Pabna, Bangladesh

Start Date: 00-00-2010

End Date: 00-00-2011

Program Area: Crop-Soil-Water Management

Commodity: Sugarcane

Objectives:

The objectives were planned for four explant sources varieties viz., Isd 37, Isd 38, Isd 39 and Isd 40 in order to develop somaclones as well as to calculate the genetic variability in the somaclones compared to their sources varieties by the use of DNA markers such as RAPD.

Methodology:

The experiment was conducted at the Biotechnology Laboratory, Bangladesh Sugarcane Research Institute (BSRI), Ishurdi, Pabna, Bangladesh during 2010 to 2011 to obtain in vitro plant regeneration potentiality of BSRI released varieties Isd 37, Isd 38, Isd 39 and Isd 40. The leaf sheath explants were collected from 8 - 10 months old field grown sugarcane from BSRI experimental field. At first MS supplemented with green coconut water (10%) containing 3 mg/l of 2, 4-D was prepared for callus induction. After five weeks of explantation, the calli were inoculated for shooting on MS supplemented with concentration BAP (2 mg/l) + Kn (1 mg/l) and maintained by sub-culturing every two weeks and then regenerated shoots were inoculated for rooting by sub-culturing every two weeks on MS supplemented with 5 mg/l NAA. Rooted plantlets were acclimatized and transplanted to polybag and then field, respectively. For molecular studies, young meristem cylinder from 12 somaclones and their donor parents were taken from R0 regeneration and grinded using extraction buffer solution and amount of chemicals were important considerations for DNA isolation. DNA was extracted from sugarcane using the method modified and combined from the methods of Aljanabi et al. (1999) and mini-prep method adopted from Hossain et al. (2006) and Shahnawaz (2006). The DNA concentration was determined by Nano drop Spectrophotometer (2000/2000c, Thermo Scientific, USA) and was diluted to a concentration of 50 ng/μl. Samples were stored at –20⁰C for further use. Polymorphism was studied using RAPD as illustrated by Mondal et al. (2009) which supported by Williams et al. (1990). Six RAPD primers (Operon Technologies, Inc., Alameda, California, USA) were used. List of polymorphic primers and their sequences are subsequently given. The reaction mixtures 10 μl was amplified for each DNA sample in a Thermal Cycler (Genius, Techne, Cambridge Ltd.). Agarose gel (1.4%, w/v) was used for RAPD electrophoresis. The ethidium bromide at 10 mg/ml was added in gel for detection. Bands were viewed under ultraviolet transilluminator and documentation (FluorChem FC2, Cell Biosciences, USA) and also analysis system was used to make photographs. Besides, it was printed and saved on CD for lateral use. The presence and absence of a DNA fragment was considered as basis of polymorphism. DNA loci if present were scored as ‘1’ and if not were scored as ‘0’. The number of alleles per locus was determined and the polymorphic information content (PIC) values were calculated using the formula (Mondal et al. 2009). A dendrogram was constructed by using unweighted pair group method of arithmetic means (UPGMA) algorithm (Sneath and Sokal 1973) provided in the software (Statistica computer package).

Source of Information: Plant Tissue Cult. & Biotech. 25(2): 223-229, 2015 (December)

Article Link (Online Journal): http://www.banglajol.info/index.php/PTCB/article/view/26256/17637

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Genetic relationship among the 12 somaclones and their 4 parents (Isd 37, Isd 38, Isd 39 and Isd 40) generated some different cluster at the linkage distance range from 36 to 14 by all of the 6 primers. Dendrogram based on linkage distance using indicated segregation of the 4 sugarcane varieties and their somaclones into two main clusters C1 (Isd 39) and C2 at the linkage distance of 36. Besides, C2 produced different sub clusters. Formation of clustering and sub clustering in the tree diagram confirmed the presence of variability at DNA level among somaclones with respect to their parents.

Knowledge Management: Journal