Abstract
The prevalent of iron deficiency anemia (IDA) and vitamin A deficiency (VAD) are in Bangladesh. Since these are food-based problem, therefore, a biofortification strategy of BRRI dhan29, the most popular rice variety in Bangladesh with iron and provitamin A was taken. BRRI dhan29 has basically low iron (3-4 mg/kg) in its polished grains and no provitamin A at all. Four experiments were designed to achieve the broad objective, biofortification of BRRI dhan29 with iron and provitamin A. A binary plasmid vector, pGPTV-bar-fer was used for transformation of Ferritin gene to enhance iron content in its endosperm. Five day old young and healthy calli grown in continuous light were used for inoculation and selection was done with PPT(5.0mg/l). Two batches of transformation were done and a total of 6 and 14 tranformants were obtained in first and second batch respectively. Selection of positive plants in T₁ population was done though 0.5% Basta spraying. Chi-square test revealed that Ferritin gene followed 3:1 segregation. The iron content in transgenic polished grains ranged between 7.89 and 9.49 mg/kg whereas in non-transformed grains it was only 4.32mg/kg. For increasing the transformation efficiency and better expression, Ferritin genes from soybean (GmH1) and French bean (PvFer) were cloned with TOPO cloning vector and used in construction of transformation vector with endosperm specific promoters GluB-4 and Glb-1. As the backbone, pCAMBIA0380 and pCAMBIA1380 were used. Finally eight plasmid vectors were successfully constructed. The effect of different milling times on its iron content were determined using three genotypes viz. IR68144-2B-2-2-3-1-120, Areumbyeo, and PSBRc28. Positive correlations of milling degree, transparency and whiteness with milling time were observed but the negative correlation was observed in case of grain iron content. A simple and rapid method for rice grain iron estimation was standardized. Four staining reagents viz. 1-phenanthroline, 2,2-dipyridyl, ferrozine, and ferene were evaluated in this study. Ferene was found most sensitive in color development with iron. The method was further validated with three genotypes at eight different polishing levels. The correlation coefficients between ICP-OES and spectrophotometric methods were highly significant (p<0.001). An effort for introgression of Golden rice genes to BRRI dhan29 was undertaken through marker assisted backcrossing. A total of 108 and 104 polymorphic SSR markers were identified for background selection of GR1 and GR2 events respectively. Hybridization was done with three events of GR1 and six events of GR2 with BRRI dhan29 taking it as female parent. Three steps of selections viz. foreground selection with gene specific primers, back ground selection of transgene carrier chromosome, and overall background selection were carried out. Seeds were produced from different BC₁F₁ populations. For the selection of golden rice gene in the seeds a simple technique using 2.5% NaOCl was introduced. Foreground results revealed that all the BC₁F₁ and BC₂F₁ populations followed 1:1 segregation. The highest 87.65% of recipient parent's allele conversion occurred in BC₂F₁ population. Based on phenotypic acceptability, four top ranked plants viz. BR29-309-BC2-64-129, BR29-309-BC2-9-178, BR29-309-BC2-64-22, and BR29-309-BC2-9-65 were selected for further backcrossing.