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Characterization of progenies from intergeneric hybridization between oryza sativa l. and porteresia Coarctata (roxb.) tateoka

Tasmia Islam
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Sudip Biswas
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Umme Habiba Mita
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

R.H. Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M. Sazzadur Rahman
Bangladesh Rice Research Institute, Gazipur, Bangladesh.

M. Ansar Ali
Bangladesh Rice Research Institute, Gazipur, Bangladesh.

K.M.S. Aziz
International University of Business Agriculture and Technology, Uttara, Dhaka-1230, Bangladesh

Zeba I. Seraj
Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Porteresia coarctata (Roxb.) Tateoka is an endemic halophyte growing all over the coastal belt of Bangladesh, propagating through rhizomes and setting a few ricelike grains. So exploiting the genetic potential of this wild rice as salt tolerant donor in possible wide crosses with rice (2n = 24) could be useful. We attempted intergeneric hybridization between Oryza sativa L. and P. coarctata. The survival rate of hybrid progenies in embryo culture was low but among them 2 hybrid plants were successfully matured from the intergeneric cross between the cultivated induced tetraploid of rice, Latisail (2n = 4x = 48) and P. coarctata (2n = 48). The hybrid plants could be successfully established in soil and were not like either of the parents in morphology although some of their features were similar to their maternal parent, Latisail (4x). Both of the hybrids were investigated through physiological analysis under salinity stress and molecular analyses with rice specific SSR markers. Molecular analysis of the F1 DNA with only 3 SSR markers, RM581, RM20224 and RM25271, out of 36 others tested, showed bands specific to both of the parents, while all had common bands with the maternal parent. Dendrogram analysis of the hybrids with the 36 SSR markers, show that P. coarctata forms a different clade and is clearly separated from Latisail and the hybrids. The putative hybrids however made a subgroup with Latisail. These observations could be possibly explained if chromosome loss of the paternal parent had occurred or may be it was a pleotropic effect of intergeneric hybridization. Physiological screening of the hybrid progenies at the F₂ generation in seedling stage showed better result in leaf damage score (LDS) and salinity tolerance than their maternal parent Latisail (4x) at 150 mM salt stress for 10 days. F₂ plants from one of the hybrid plants (H-2) showed better performance but there was a large variation in response from each of the individual progenies. So, it is likely that some of the salt tolerant characteristics of the pollen parent might have been transferred to the recipient Latisail (4x). For introgression of better salt tolerant loci from P. coarctata, more wide hybrids will need to be produced and repeatedly crossed with P. coarctata.

Keywords: Porteresia coarctata, Induced‐tetraploid, Intergeneric hybridization, SSR marker

Location: Cox’s Bazar district, Bangladesh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Rice

Objectives:

To determine Characterization of Progenies from Intergeneric Hybridization Between Oryza sativa L. and Porteresia coarctata (Roxb.) Tateoka

Methodology:

The materials used in hybridization were comprised of rice cultivar Latisail (2n = 4x = 48) and wild rice, Porteresia coarctata (2n = 48). P. coarcatata was collected from Cox’s Bazar district, Bangladesh. Two accession of P. coarctata one from Bakkhali and another from Teknaf used in this research were propagated in normal soil in pots for 1 - 2 years in the net house of the Plant Biotechnology Lab, University of Dhaka. Latisail (2n = 2x = 24), a Bangladeshi rice variety was germinated in tissue culture and meristematic zones treated with cotton soaked in colchicine (Blakeslee et al. 1937). New shoots which were much thicker and larger in size were separated and placed in auxin-containing media with colchicine for root growth. Rooted plants were acclimated in hydroponics and then grown in soil. Seeds were collected over several generations (Seraj ZI unpublished data). Latisail (4x) and P. coarctata was allowed to grow until panicle maturity. Immediately prior to flowering, the panicles of the donor P. coarctata were cut and soaked in water for 3 hrs (between 11 a.m. and 2 p.m.) and emasculation of the recipient Latisail (4x) was accomplished at the same time. Emasculated panicles were covered with oil paper to prevent external pollination. Pollen were collected in a Petri plate by smoothly sliding one panicle with another. The pollen were then dusted on to the stigma of the recipient plant by a small brush and the panicles were then covered again. Immature seeds were harvested in day 10 to 12 and mature seeds were harvested in day 21 from crosses after pollinations. Gibberellic acid (75 ppm) was sprayed every 24 hrs after crossing for 6 consecutive days. This was done particularly to avoid spikelet shattering in the maternal rice parent, Latisail (4x). After harvesting, seeds were sterilized and germinated in semi-solid MS (1/4th strength) medium. The hybrids germinated in more than 14 days and took more than 4 - 6 weeks to grow to about 10 - 12 cm. Thereafter, the seedlings were transferred to hydroponics (Yoshida et al. 1976) for 3 - 4 weeks and then transplanted to soil. Mature hybrid plants were confirmed for the presence of P. coarctata genome by rice specific SSR marker' based PCR. Genomic DNA was isolated from (0.5 - 1.0 g) pooled leaf tissue of hybrids at F1 generation with parents Latisail (4x) and P. coarctata using the modified CTAB method (Doyle 1987) and was quantified using the Nanodrop spectrophotometer (Nanodrop 1000). The quality of the DNA was assured by checking in 0.8% agarose gel in TAE buffer. A total of 36 pairs of SSR primers were used to amplify DNA from the leaves. The distribution of the selected SSR primers was even throughout the 12 rice chromosomes. The 36 markers were selected on the basis of the genome' wide distribution from a previous list of highly polymorphic 83 markers identified in earlier studies on different rice landraces (Seraj ZI, unpublished data). PCR analysis was carried out in a 15 μ l reaction mixture containing 100 ng of plant DNA, 100 μM of each dNTP, 2.4 ng of forward and reverse primers each, 1.0 unit of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 1.6 mM MgCl₂, DMSO 2.6% and 1× PCR buffer MgCl₂ (Invitrogen). The mixture was then denatured at 95ºC for 5 mins, followed by 35 cycles of denaturation at 95ºC for 1 min, 1 min of annealing at 55- 60ºC (depending on primer’s Tm), 1 min of extension at 72ºC with a final extension step at 72ºC for 7min. The 36 RM markers were obtained commercially from idt' 1st base, Singapore. The amplified PCR products were resolved and visualized in non denaturing 10% polyacrylamide gel electro-phoresis. The gels were stained in EtBr and visualized with alpha imager gel documentation system. For the microsatellite DNA fingerprinting of the hybrids along with parents Latisail (4x) and P. coarctata, polymorphisms were scored according to their molecular weight on polyacrylamide gels by the Alpha Ease FC imaging system (www.alphaimager.com). The genotyping was done using the Powermarker Software (Liu et al. 2005). The selected dataset is genotype data, the "unknown" is gametic phase for the data type, and the missing numeric is −9/−9. Cluster analysis was based on similarity matrices using the Unweight Pair Group Method with arithmetic mean (UPGMA) (Sneath et al. 1973) and the relationship between cultivars was visualized as a dendrogram using Power marker and MEGA5. The UPGMA tree was constructed by using the frequency?based distance for the "shared allele" (Chakraborty et al. 1993). The phenotypic screening for the salinity tolerance at seedling stage was done by the method described by (Amin et al. 2012). Screening was done on F2 hybrid plants. In the screening, female parent Latisail (4x), tolerant control Pokkali and sensitive control IR29 were also used. The seedlings were allowed to grow in Yoshida (Yoshida et al. 1976) culture solution until they reached the four?leaf stage (14 to 18 days after germination). NaCl stress was applied gradually starting from 5 to 15 dS/m at 24 hourly increments of 2 dS/m. An increase of leaf number and length was measured after an interval of 4 days up to 16 days or until 90% of the leaves of the sensitive control were damaged. The tolerance related traits (Leaf damage score or LDS, chlorophyll content and electrolyte leakage) of all stressed plants were then measured. The level of salinity tolerance was evaluated mainly based on the value of LDS, which is based on the percentage of the leaf damage. The plants were scored according to the protocol mentioned by (Amin et al. 2012). The chlorophyll content and electrolyte leakage of the stressed and control hybrids shoots as well as Latisail (4x) were measured at this stage (Yasmin et al. 2016). All statistical analyses were done using Data Analysis Tool Pak of Microsoft Office Excel 2007 and R package. The F test was performed to verify equal variance of the independent set of samples and the test and ANOVA was performed based on the result assuming equal variance or unequal variance as applicable to compare significant differences (p > 0.05) between the stressed and control hybrid plants along with Latisail (4x).

Source of Information: Plant Tissue Cult. & Biotech. 27(1): 63?76, 2017 (June)

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Achievement/Findings:

The results suggest that in the future massive hybridization programme using Latisail (4x) as female can be undertaken and maybe the hybrids produced repeatedly crossed with P. coarctata with the hope of obtaining hybrid seeds with novel recombinants from the halophyte genome. More crosses between P. coarctata and other local cultivars grown in the moderately saline coastal belt will also be attempted in the light of finding more salt tolerant new cultivars and attempts made to advance generations in order to stabilize the genomes for attaining homozygosity.

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