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Screening Sugarcane Somaclones and Their Parent Varieties Against Red Rot (colletotrichum Falcatum) and Assessment of Variability by Rapd and Ssr Markers

Kuasha Mahmud
Principal Scientific Officer
Biotechnology Division, Bangladesh Sugarcrop Research Institute, Ishurdi, Pabna.

K. M. Nasiruddin
Professor
Dept. of Biotechnology, Bangladesh Agricultural University, Mymensingh.

M. A Hossain
Chief Scientific Officer
Biotechnology Division, Bangladesh Sugarcrop Research Institute, Ishurdi, Pabna.

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

Abstract/ Executive Summary:

The experiment was conducted to find out red rot disease causing pathogenic reactions of somaclones (R₀) and their respective parents at BSRI farm in the cropping season 2011-2012. A total number of sugarcane genotypes 24 including 4 sugarcane varieties viz. Isd 37, Isd 38, Isd 39 and also Isd 40 were inoculated to screen red rot resistant levels (R = Resistant, MR = Moderately Resistant) to susceptible levels (S = Susceptible, MS = Moderately Susceptible, HS = Highly Susceptible) after 7 months of planting. As a result, Isd 37 variety (source/parent) and its somaclones CC–37–12 and CC–37–86 were found to be resistant while 7 somaclones showed moderately resistant reaction. Furthermore, somaclones of Isd 38 variety viz. CC–38–2 as moderately resistant, CC–38–10 as moderately susceptible and also CC–11(38)–8 as susceptible reaction were recorded while Isd 38 variety showed resistant reaction. On the other hand, Isd 39 and Isd 40 source varieties with their somaclones were found as resistant reaction against red rot pathogen. Some somaclones showed different reaction from their source varieties such as moderately resistant somaclones were obtained from resistant source variety Isd 37 while somaclones CC–38–2 as moderately resistant, CC–38–10 as moderately susceptible and somaclone CC–38–8 as susceptible were obtained from resistant source variety Isd 38 against red rot pathogens respectively. Besides, some somaclones showed similar reaction from their resistant source varieties Isd 37, Isd 39 and also Isd 40. It revealed that reaction against red rot pathogen, induced somaclones showed variation with their source varieties. Red rot resistance somaclones were isolated and assessed for the presence of variability through RAPD and SSR markers. Cluster and sub cluster formation verified the presence of variability in the red rot resistance somaclones with respect to the parent.

Keywords: Fungus, Inoculation, Red rot disease, Resistant, Sugarcane, Susceptible

Location: Bangladesh Sugarcrop Research Institute (BSRI) experimental field at Ishurdi, Pabna.

Start Date: 00-00-2011

End Date: 00-00-2012

Program Area: Pest Management

Commodity: Sugarcane

Objectives:

To find out the level of resistance of induced somaclones and their sources varieties against red rot pathogen (Colletotrichum falcatum Went.).

Methodology:

The leaf sheath explants were collected from 8-10 months old field grown sugarcane from Bangladesh Sugarcrop Research Institute (BSRI) experimental field at Ishurdi, Pabna. At first MS medium supplemented with green coconut water (10%) containing 3 mg l^-l of 2, 4-D was prepared for callus induction. After five weeks of explantation, the calli were inoculated for shooting on MS medium supplemented with concentration BAP (2 mg l^-l) + Kinetin (1 mg l^-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 medium supplemented with 5 mg l^-l NAA. Callus culture derived somaclones (R₀) and their parent varieties were planted at BSRI Biotechnological experimental field on February, 2011. Isolates of Colletortichum falcatum of different cane growing zones were used for inoculation. The pathogen was collected from different zones and maintained in the oat meal slant. Artificial inoculation with different isolates was done on 7-8 months old induced somaclones along with sources varieties. Eight to ten days old sporulating cultures of the fungus were used for inoculation. Inoculation was made with individual as well as with mixed isolates of Colletotrichum falcatum. Ten healthy canes of each somaclone and their sources varieties were inoculated by spore suspension (spore conc.10⁶ ml^-l) of red rot disease causing fungus (colletotrichum falcatum) with the help of hypodermic syringe method. After two months of inoculation, data on disease development in the canes were recorded. On the basis of disease index the somaclones and their parents were categorized as Resistant (R), Moderately Resistant (MR), Moderately Susceptible (MS), Susceptible (S) and Highly Susceptible (HS) to this disease. Red rot inoculation result sheets (Appendix VI) were used for screening somaclones and their parents as Resistant (R), Moderately Resistant (MR), Moderately Susceptible (MS), Susceptible (S) and Highly Susceptible (HS) to red rot disease. For molecular studies, young meristem cylinder from 2 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. The DNA concentration was determined by Nano drop Spectrophotometer (2000/2000c, Thermo Scientific, USA) and was diluted to a concentration of 50 ng μl^-1. Samples were stored at -20⁰C for further use. Polymorphism was studied using Random Amplified Polymorphic DNA (RAPD). Six RAPD and nine SSR primers (Operon Technologies, Inc., Alameda, California, USA) were used. The reaction mixtures 10 μl was amplified for each DNA sample in a Thermal Cycler (Genius, Techne, Cambridge Limited). Agarose gel (1.4% and 2%, w/v) were used for RAPD and SSR electrophoresis. The Ethidium Bromide at 10 mg ml^-1 was added in gel for detection. Bands were viewed under ultraviolet trans-illuminator and documentation (FluorChem FC2, Cell Biosciences, USA). 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’. Dendrograms were constructed by using Unweighted Pair Group Method of Arithmetic Means (UPGMA) algorithm provided in the software (Statistica computer package 2006, STA_CLU).

Source of Information: SAARC J. Agri., 13(2): 173-182 (2015)

Article Link (Online Journal): http://dx.doi.org/10.3329/sja.v13i2.26578

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Some somaclones showed similar reaction from their resistant source varieties Isd 37, Isd 39 and also Isd 40. Besides, it is possible to get variation using tissue culture technique containing 2, 4-D with MS media. It revealed that in case of reaction against red rot pathogen, induced somaclones showed variation with their source varieties. Cluster and sub cluster formation using results of RAPD and SSR markers verified the presence of variability in the red rot resistance somaclones with respect to the parent.

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