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Phenotypic Screening and Molecular Characterization of 10 Rice (oryza Sativa) Landraces for Cold Tolerance

M. Shoroardi*
Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University

M. G. Mortuza
Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University

M. M. Islam
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture

T. Samiha
Department of Biochemistry and Molecular Biology, Bangladesh Agricultural University

Abstract/ Executive Summary:

Phenotypic screening of existing 10 familiar landraces performed at germination and seedling stages and their molecular characterization were conducted at Bangladesh Institute of Nuclear Agricultural (BINA) during the period of December 2014 to May 2015 to screen out better cold tolerant genotypes among 10 landraces where one susceptible, two moderately tolerant and two tolerant BRRI released varieties were used as check genotypes. The present study evaluated several parameters at the germination stage after 7 days of germination and at the seedling stage after 28 days of seedling growth for phenotypic screening. Modified hydroponic method was used at the seedling stage and the temperature treatments were maintained artificially at 15°C and 25°C respectively. Polymorphic microsatellite or SSR markers were selected for molecular characterization showing that attributes were positively regulated due to cold stress in BRRIdhan 36, LR 114- Badiabona and LR 54- Tor Balam. The findings of genetic diversity analysis for landraces LR 114- Badiabona and LR 54- Tor Balam appeared to resemble to that of tolerant genotype BRRIdhan 36. Based on phenotypic screening and UPGMA dendrogram study, landraces LR 114- Badiabona and LR 54- Tor Balam appeared superior as genetic material. Better cold tolerant rice variety may be developed from further breeding process.

Keywords: Modified hydroponic method, Polymorphism information content, Sustained water bath, SSR marker, UPGMA dendogram

Location: Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh

Start Date: 00-12-2014

End Date: 00-05-2015

Program Area: Variety and Species

Commodity: Rice

Objectives:

The specific objectives of the study include: to screen the rice genotypes phenotypically under normal and cold stressed condition at the seedling stage; to make DNA fingerprint and genetic relationship among 15 rice genotypes using SSR marker; to identify the cold tolerant rice genotypes.

Methodology:

Experimental site and period Experiments were conducted at the glasshouse, experimental field and Laboratory of Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh during the period of December 2014 to May 2015. Collection of germplasms A total of 15 rice genotypes were used. Ten experimental landraces viz. LR 171- Sokou Malsira, LR 150- Hekha, LR 167- Kala Dgopa, LR 114- Badiabona, LR 01- Dudh Kalam, LR 212- Naria Bochi, LR 25- Kathi Goccha, LR 37- Hamai, LR 54- Tor Balam and LR 101- Dud Sail were collected from the Seed Gene Bank of Biotechnology division, BINA, BAU Campus, Mymensingh-2202. Beside those, other 5 varieties were used as check varieties including one susceptible- BRRI dhan28, two moderately tolerant- BRRI dhan55 & BRRI dhan57 and two tolerant- BRRI dhan36 and BRRI dhan29 collected from BRRI, Joydebpur-1701. Experimental setup Modified Hydroponic system (Gregorio et al., 1997) was used at the glasshouse and the field to evaluate cold tolerance of the 18 rice germplasms using Peter’s solution (Yoshida et al., 1976). A special kind of seedling floats was prepared using cork sheet and a rectangular glass fiber tray with 12-L capacity and diameter of 23 inch was used to fit the floats. Peters water soluble fertilizer (Urea: TSP: MP=20:20:20) and ferrous sulphate (FeSO₄.7H₂O) were used as nutrient solution that mixed in plastic containers. The pH of the solution was monitored daily and maintained around 5.1 because +1.0 deviation of culture solution pH from 5.0 makes some nutrients toxic and others deficient. For the maintenance of cold environment, seedling floats were placed on the experimental tables in the field and ice crystals and cold water were dissolved with nutrient solution to reach the desired temperature level which was maintained at 15°C. The solution was replaced with the new one in every 8 day and the pH was maintained at 5- 5.5 daily. After breaking dormancy, seeds were washed and rinsed with tap water and then placed on petridishes with moistened filter papers and incubated for 48 hours at 30°C to germinate. In case of treatment, seeds were washed and rinsed with tap water and then placed on petridishes with moistened filter papers and kept in laboratory where the average temperature was maintained at 15-20°C. 20 pre-germinated seeds of each genotypes were placed in two rows and thus 5 genotypes can be placed per seedling float. Then the experimental trays are placed in the glasshouse as control and in the field for screening cold stress. Phenotypic screening of cold tolerance at the germination stage To conduct the phenotypic screening at germination stage, germination percentage and seedling survival rate are calculated. 20 seeds and 20 germinated seedlings were brought under test for each under control and stressed condition. The vigor of seed germination was recorded after 7 days for both control and treatment following the given formula: Germination (%) = Number of germinated grains/ Number of total grains given for germination x 100

Seedling survival rates were assessed after 7 days of growth at both normal growth and cold stressed condition which can be calculated as follows: Seedling survival rate (%) = Surviving Seedlings/ Number of budding seeds x 100. Phenotypic screening of cold tolerance at seedling stage Three seedlings of each genotype were selected randomly and the height of each plant was measured from base of the shoot to the tip of the main stem, root length from the shoot initiation to the root tip, total length from the root tip to the shoot tip, fresh weight and dry weight at the age 28 days of the seedling stage were also observed. The mean values were recorded and expressed in particular units. After imposition on cold stress, seedling growth scale was scored using standard evaluation system (SES) for rice. Extent of damage was determined at both normal and cold stressed condition at about 15°C on 1 to 9 scales standardized for rice (IRRI, 1996). 1 indicating dark green seedlings, 3 indicating light green seedlings, 5 indicating yellow seedlings, 7 indicating brown seedlings and 9 indicating dead seedlings. DNA fingerprinting of rice genotypes using SSR marker DNA extraction from 21-25 day old plant leaves was done at the Biotechnology Lab of BINA, Mymensingh using the mini preparation Cetyl Trimethyl Ammonium Bromide (CTAB) method. The simplified mini scale procedure for DNA isolation in Polymerase Chain Reaction (PCR) analysis developed in IRRI was followed. The quality of the isolated DNA in the protocol is sufficient for the PCR analysis (Zheng et al., 1995). Young vigorously growing fresh leaf samples from these seedlings were collected from 25 day old seedlings to extract genomic DNA. Microsatellite markers or SSRs are valuable for tagging and mapping of cold tolerance genes. Three random primers viz. RM140, RM215 and RM510 were selected to evaluate 15 rice genotypes for cold tolerance.

Source of Information: J. Environ. Sci. & Natural Resources, 10(1): 85-91, 2017 ISSN 1999-7361

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Achievement/Findings:

The present investigations helped to establish clearcut identity of all the landraces under consideration, which will be a great utility for the selection of cold tolerant landraces as genetic materials to release a new cold tolerant variety suited for the cold affected region of Bangladesh during winter season. Considering important morphological characters at both germination and seedling stages and from UPGMA dendrogram it is concluded that LR 114- Badiabona and LR 54- Tor Balam were the best among all the 10 landraces. These genotypes can be used for field trial for releasing new cold tolerant varieties based on further breeding program.

Knowledge Management: Journal