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Research Detail

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M. Al-Amin
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M.M. Islam
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture (BINA), BAU Campus, Mymensingh 2202

S.N. Begum
Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture (BINA), BAU Campus, Mymensingh- 2202

M.S. Alam
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M. Moniruzzaman
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

A.K. Patwary
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Twenty eight rice germplasms were used for identification of salt tolerant rice genotypes at the seedling stage at the experimental farm and Biotechnology laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh during February 2009 to October 2009. Phenotyping for salinity screening of the rice genotypes was done using salinized (EC level 12 dS m-1) nutrient solution in hydroponic system. Genotypes were evaluated for salinity tolerance on 1-9 scale based on seedling growth parameters following modified Standard Evaluation Scoring (SES) of IRRI. Phenotypically, on the basis of SES and % total dry matter (TDM) reduction of the genotypes viz. PBSAL-614, PBSAL-613, PBSAL-730, Horkuch, S-478/3 Pokkali and PBSAL (STL)-15 were found to be salt tolerant; on the other hand Iratom-24, S-653/32, S-612/32, S-604/32, S-633/32, Charnock (DA6), BINA Dhan-6 and S-608/32 were identified as salt susceptible. For genotyping, ten SSR markers were used for polymorphism, where 3 primers (RM127, RM443 and RM140) were selected for evaluation of salt tolerance. In respect of Primer RM127, 7 lines were found salt tolerant and 11 lines were moderately tolerant and 10 lines were susceptible. Nine tolerant, 9 moderately tolerant and 10 susceptible lines were found when the primer RM140 was used and primer RM443 identified 8 lines as tolerant, 9 lines as moderately tolerant and 11 lines as susceptible. Thus, the salt tolerant lines can be used in further evaluation for salinity tolerance and the SSR markers used in this study are proving valuable for identifying salt tolerant genes in marker assisted breeding.

  Rice, Salinity Tolerance, SSR Markers, Seedling Stage, Polymorphism
  Biotechnology Laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh
  00-02-2009
  00-10-2009
  Variety and Species
  Rice

1. The present study were undertaken for determine the phenotypic performance of rice germplasm under salinized conditions at the seedling stage and to identify salt tolerant rice lines from twenty eight rice germplasm using SSR markers.

Plant materials The experiment was carried out at the experimental farm and Biotechnology Laboratory of the Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh from February 2009 to October 2009. In this study twenty eight rice germplasm were used for salinity screening at the seedling stage following International Rice Research Institute (IRRI) standard protocol. These germplasms were collected from different sources like, International Rice Gene bank (IRG) of International Rice Research Institute (IRRI), Philippines, Bangladesh Rice Research Institute (BRRI), Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Twenty eight rice germplasm (Horkuch, Charnock, Keshrail, Iratom-24, BRRI dhan-40, Binadhan-6, PBSAL(STL)-15, PBSAL-613, PBSAL-614, PBSAL-730, PBSAL-656, STM-1, PBRC(STL)-20, S-530/32, S-603/32, S-604/32, S-445/32, S- 611/32, S-612/32, S-546/32, S-653/32, S- 635/32, S-375/32, S-731/32, S-478/32, S- 608/32, S633/32 and Pokkali ) were evaluated for salinity screening. Phenotypic study of salinity tolerance at seedling stage The screening for salt tolerance were done using modified standard evaluation score (SES) of visual salt injury of the seedlings. Hydroponic system with IRRI standard protocol was used at the glasshouse to evaluate salt tolerance of rice germplasm. Nutrient solution was used in hydroponic system for screening salinity tolerance at the seedling stage. The modified standard evaluation score (SES) of IRRI was used to assess the visual symptoms of salt toxicity. This scoring discriminates the tolerant, moderately tolerant and susceptible rice lines. CTAB mini preparation DNA extraction The modified Cetyl Trimethyl Ammonium Bromide (CTAB) mini-prep method was used to extract DNA from the plant leaves. The following steps were followed in PCR-based DNA marker analysis. Healthy leaf samples were collected from 25-day old seedlings of each germplasm for isolation of genomic DNA. Leaf sample was washed in distilled water and ethanol. The collected leaf samples were then put into polythene bags with tags, placed on ice and stored in a –80oC freezer. The leaf samples were ground with pestle and mortar to collect DNA. Strict hygiene was maintained during the DNA extraction by autoclaving all glassware, micropipette tips, PCR tubes, distilled water, reagents and buffer solutions. Isolated genomic DNA contains a large amount of RNA and pigments, which cause over estimation of DNA concentration during spectrophotometer reading. Therefore, the DNA samples were evaluated qualitatively using agarose gel electrophoresis. For checking of DNA concentration, two methods can be applied: 1) ? DNA and 2) Spectrophotometry. For this study only ? DNA was used. After electrophoresis, the gel was taken out carefully from the gel chamber and transferred in a prepared ethidium bromide solution for staining. Staining was done for 20 minutes and then placed on the UV transilluminator in the dark chamber of the Image Documentation System. Amplification of microsatellite markers and evaluation of genotypes Ten primers of random sequence were selected for amplification of the DNA sequences. Primers were evaluated based on intensity of bands, consistency within individual, presence of smearing and potential for population discrimination. In this experiment, OSR14, OSR17 RM152, RM443, RM127, RM140, RM9, RM169, RM18 and RM21 primers were used for polymorphism. Out of ten primers, three polymorphic SSR markers viz., RM127, RM443 and RM140 were selected to evaluate 28 rice germplasm for salt tolerance. The PCR cocktail had total volume of 15.0 μl reaction mixture including 2 μl DNA based on salinity protocol, was placed in the PCR tubes and run in the DNA thermal cycler. The PCR tubes were set on the wells of the thermocycler plate and the machine was run according to the programme. The germplasms having similar banding pattern to Pokkali were considered as tolerant, banding pattern having similar to Iratom-24 were considered as salt susceptible and having banding pattern at the medium position between Pokkali and Iratom-24 were considered as medium tolerant In each marker, allelic bands were designated as T for tolerant, S for susceptible and MT for medium tolerant.

  Int. J. Agril. Res. Innov. & Tech. 3 (1): 52-59, June, 2013; Available online at http://www.ijarit.webs.com; ISSN: 2224-0616
  
Funding Source:
  

Salt tolerant rice lines were identified both phenotypically (salinity screening at the seedling stage) and genotypically using twenty eight rice germplasms. The tested markers RM127, RM140 and RM443 could be efficiently used to identify salt tolerant lines in rice and can also be used in marker-assisted selection (MAS) for breeding, quantitative trait loci (QTL) mapping, studying genetic diversity in germplasms and gene pyramiding in rice salinity breeding. Microsatellite marker analysis is promising to identify major gene locus for plant breeders to develop new cultivars. The selected salt tolerant rice lines could be further tested in saline areas to observe yield potentiality for developing high yielding salt tolerant varieties suitable for saline areas.

  Journal
  


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