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Engineering Provitamin a Synthesis Pathway with β-carotene Metabolism in Rice Endosperm of a Restorer Line Br827r

S. Rehana
Weed Management Laboratory, Department of Agronomy, 1Bio-Technology and Genetic Engineering Discipline, Khulna University, Khulna 9208

N. Baishakh
School of Plant Environmental and Soil Sciences, Lousiana State University, 214 M.B. Sturgis Hall Baton Rouge, LA 70803

K. Datta
Laboratory of Translational Research on Transgenic Crops, Department of Botany, University of Calcutta, Kolkata-700073

N. Oliva
Plant Tissue Culture Laboratory, International Rice Research Institute, DAPO 7777, Metro Manilla

E. Abrigo
Plant Tissue Culture Laboratory, International Rice Research Institute, DAPO 7777, Metro Manilla

M.A. Mazid
525/A, Senpara Parbata, Mirpur 14, Dhaka 1216

Shah-E-Alam
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh 2202

M.R. Uddin
Department of Agronomy, Bangladesh Agricultural University, Mymensingh 2202

Swapan K. Datta
Department of Crop Science, Institute of Agriculture, Visva Bharati University, Santiniketan- 731235 (West Bengal)

Abstract/ Executive Summary:

Vitamin A deficiency (VAD) is a serious public health problem in South Asia particularly in Bangladesh. Indica rice as a major staple in the country completely lacks vitamin A or compounds with provitamin A activity after milling. A combination of transgenes has been introduced enabling biosynthesis of provitamin A in the endosperm of a restorer line using biolistic system of transformation. The rice seed-specific glutelin promoter (Gt-1 P) was used to drive the expression of phytone synthase (psy), while lycopene b-cyclase (lcy) and phytoenedesaturase (crtI), fused to the transit peptide sequence of the pea-Rubisco small subunit, were driven by the constitutive cauliflower mosaic virus promoter (CaMV35s P). Transgenic plants were recovered through selection with CaMV35sP driven hph (hygromycinphosphotransferase) gene. Molecular analysis demonstrated stable integration and expression of the transgenes. The variable segregation pattern in T₁ generation indicated single to multiple insertions of the transgenes in the genome. This is the first report of the development of a transgenic restorer line with carotenogenic pathway into the endosperm for use of hybrid rice improvement.

Keywords: β-carotene, Biolistic transformation, Provitamin A, Restorer line, Hybrid rice.

Location: South Asia particularly in Bangladesh

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Rice

Objectives:

To develop a transgenic β-carotenoid restorer line, a new tool for improving hybrid rice.

Methodology:

An elite indica restorer line (BR827R) was selected for transformation on the basis of its superior grain quality. Altogether three different plasmids were used for the co-transformation experiments. The vector pBall3 contained the daffodil phytone synthase (psy) gene (Burkhardt et al., 1997) under control of an endosperm-specific Gt1 promoter and a bacterial phytone desaturae (crtI) gene fused to a transit peptide sequence of a pea-rubisco small subunit (Misawa et al., 1993) to direct the expression of this bacterial gene into the plastids by constitutive 35S promoter. In order to yield the plasmid pTCL6 under control of the 35S promoter and nopaline synthase terminator, lycopene β-cyclase (lcy) cDNA (Al-Babili et al., 1999) was subcloned from pCyBlue with the KpnI-BamHI site of pGL2 (Gritz and Davies, 1983); to the selectable marker gene, plasmid pGL2 containing the selectable marker gene hph for hygromycinphosphotransferase under CaMV 35S promoter (Datta et al., 1990). Rice immature embryos were used as target explants for cotransformation (Figure 3) of the above-mentioned vectors using the PDS-1000He particle gun. Selection started 16-20 hours after bombardment on fresh callus induction medium containing 40-mg/L hygromycin as described earlier (Datta et al., 1998). The putative primary transgenics and the subsequent seed progenies were grown in the containment greenhouse of IRRI, following a day night temperature regime of 29/22±2 ºC and 70-85% relative humidity. Genomic DNA was isolated from 1-month-old plants using the micro prep method and 50-100 ng of template DNA was used for PCR analysis with gene-specific primers as described earlier (Baisakh et al., 2001). Plant genomic DNA was extracted from the freshly harvested leaves of transgenic and non-transgenic control plants for southern analysis, following the modified CTAB method (Murray and Thomson, 1980). Ten micrograms of DNA were digested overnight with EcoRI-HindIII for psy and lcy, BamHI for crtI and run in 1% TAE-agarose gel. Southern membrane transfer, hybridization and exposure were done as previously described (Datta et al., 1998). PCR-amplified fragments of the three genes were radiolabelled with (α-³²P)- dCTP and used as hybridization probes.

Source of Information: Archives of Agriculture and Environmental Science 3(2): 103-108 (2018)

Article Link (Online Journal): DOI: 10.26832/24566632.2018.030201

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

This investigation concluded that a restorer (r) line BR827R for hybrid rice production was used to explore the potential for transformation of Indica rice adapted in Bangladesh. Rice immature embryos were transformed with pBaal3, pTCL6 and pGL2 using the particle gun transformation system. The transgenic plants were confirmed by PCR and Southern Blot analysis. Hybridization with psy, crtI , lcy and hph probes suggested the integration of the respective genes in the genome of the transgenic BR827R plants.

Knowledge Management: Journal