Production and Molecular Characterization of Wide Cross Derivatives In Rice

Abstract

Cultivated rice (Oryza sativa L.) is an important staple food for more than half of the people all around the world. Rice has a very narrow gene pool which can be broadened through transgression of genes from wild species for improving the cultivated rice. A comprehensive wide hybridization program was started at Department of Genetics, Hazara University, Mansehra, Pakistan. Four commercial varieties viz., Bas-385, JP-5, Fakhre Malakand, Swat-1 and two wild species of rice i.e. Oryza rufipogon and O. longistaminata were used in this endeavor. Seed viability of the germplasm was recorded and its dormancy was broken down for synchronized flowering. Interspecific crosses were made between commercial varieties and Oryza rufipogon. The pollinated spikelets were sprayed immediately with 75 ppm GA3 to enhance pollination. A mixture of growth regulators (100 ppm GA3, 25 ppm NAA and 5 ppm Kinetin) was applied twice a day for 7 days continuously to prevent embryo abortion. The F1 hybrids were confirmed through phenotypic markers such as color of the basal leaf sheath, stigma and awn color, pollen fertility, meiotic analyses, seed proteins, response to bacterial blight, phenotypic trait analysis in F2 and resistance pattern to bacterial blight in the F3 progeny. Total seed protein profile was also helpful in confirmation of the F1 hybrid. Results regarding the seed viability revealed significant results ranging from 78 - 92%, the lowest (78%) being recorded for the commercial variety Swat-1. Results of the seed dormancy, showed that O. rufipogon and O. longistaminata remained dormant and did not show any sign of germination at 32°C. The dry heat treatment of seed at 50°C, after remaining hull, improved the germination. The optimum temperature for germination was 35 ± 50 °C x and 40 ± 50 °C for the commercial varieties and wild species, respectively, in the lab. The quantitative traits also showed considerable diversity i.e., medium to high variance was recorded for culm length, culm diameter, culm number, peduncle length, leaf width, leaf area, flag leaf width, flag leaf area, ligule length, auricle length and anther length. Dendrogram analyses of the phenotypes sortout all genotypes into two main clusters A and B. Cluster A was comprised of commercial varieties along with their wild proginator O.rufipogon. Cluster A contained further two sub clusters A1 and A2. The sub cluster A1 consisted JP-5 and Fakhre Malakand that belong to O. sativa ssp. Japonica, whereas A2 consisted Indica type varieties along with O. rufipogon. The group B consisted O. longistaminata only. The total seed protein analyses showed 19 bands providing genetic distance estimates from 0–45%. Dendrogram constructed on the basis of total seed protein also sortedout all the genotypes into A and B main clusters. Where cluster A constituted the commercial varieties while cluster B consisted of wild species. Cluster A was further sub divided into two sub groups, A1 and A2. Sub cluster A1 was comprised of Japonica type varieties whereas A2 was comprised of Indica type varieties, the cluster B consisted of wild species only. Maximum genetic distance was observed between Bas-385 and O. longistaminata. Varities JP-5 and Fakhre Malakand showed 100% homology on the basis of total seed proteins. Cytogenetic analyses showed that all the genotypes had normal meiosis with remarkably high degree of chromosome pairing at M1. All the parents had a consistent chromosome number of 2n = 24, exhibiting one to one synapsis and one by one disjunction. Molecular analyses showed the presence of bacterial blight resistant gene Xa-21 in O. longistaminata. Oryza rufipogon, O. longistaminata, Bas-385 and Fakhre xi Malakand exhibited xa-13 gene. Varieties JP-5 and Fakhre Malakand showed the presence of xa-5 gene through PCR analysis. Resistance response to bacterial blight showed that the wild species were highly resistant to all the three local isolates of bacterial blight. Among the commercial varieties, Bas-385 proved moderately susceptible to Xoo-2 & Xoo-3, and moderately resistant to Xoo-1. Swat-1 proved to be highly susceptible to Xoo-2 and Xoo-3 and moderately susceptible to Xoo-1. The varieties JP-5 and Fakhre Malakand were moderately resistant to Xoo-1 and Xoo-2 and moderately susceptible to Xoo-3. Results of the interspecific crosses reveled that seed setting was generally very low ranging from 5.3 to 8.9% while its germination percentages ranged from 0–17.2%. Among the cultivated varieties only Bas-385 produced viable F1 hybrids when crossed with O. rufipogon. The F1 hybrids manifested heterosis for most of the quantitative traits. The syndatic behavior of chromosomal compliments of O. sativa and O. rufipogon, were analyzed in F1 hybrids. The frequently observed univalents, trivalents and laggards proved the hybrid nature of PMC’s. At metaphase I, all hybrids had more than 11.6 bivalents and 23.3 chiasma/PMC. Although on average all the hybrids showed normal meiosis, they produced 50-60% sterile pollen resulting in 85-95% spikelet sterility. Sixty three randomly selected F2 individual plants were evaluated on the basis of 14 quantitative traits. Basic statistics showed a considerable variability among the F2 genotypes for different traits. The response to bacterial blight showed that most of the F3 plants were resistant to bacterial blight isolates. Lines No, 1, 5, 10, 11, 12, 14, 20, 23, 24, 27, 29 and 30 proved highly resistant to all the three isolates of bacterial blight. Other lines showed differential responses to different Xoo isolates. The results showed that considerable xii variability with respect to the bacterial blight resistance was available in the F3 progeny, wherein the highly resistant germlines were selected as promosing lines for their potential use in genetic improvement of the cultivated rice.