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Cloning and transformation of mustard (Brassica juncea L.) with At-DWARF4. (MSc. Thesis Duong Van Hay, E-mail: tahaduong@gmail.com)
Thứ hai, 07-09-2015 | 14:12:30

DIVISION OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY. INDIAN AGRICULTURAL RESEARCH INSTITUTE, NEW DELHI-110012

 

MSc. Thesis Abstract

Mr DUONG VAN HAY; IAS; VIETNAM

e-mail: tahaduong@gmail.com

Chairman          Dr. Prasanta K Dash

Co-chair           Dr. Rhitu Rai

 

 

ABSTRACT

 

Phytohormones play critical roles in plant growth and development. Brassinosteroids (BRs) are essential phytohormones required for optimum physiological processes of plant and their deficiency causes distinctive dwarf phenotypes with markedly shorter siliques in Arabidospsis. Homeostasis of BRs in plants is maintained by DWF4 enzyme that mediates multiple 22α-hydroxylation steps in brassinosteroid biosynthesis. Arabidopsis plants over-expressing DWF4 protein show increase in inflorescence, number of branches and siliques; thereby increase number of seeds/plant. This suggests that it will be possible to enhance seed yield in mustard by engineering DWF4 biosynthesis in plants. Thus, DWARF4 gene was isolated form Arabidopsis by gene specific PCR. The PCR generated approximately 2.9kb gene was cloned in pENTR entry vector by gateway cloning procedure. Authenticity of the cloned gene was checked by sequencing 5’ and 3’ end of the gene and by comparing homology with genomic sequence of DWARF4 available on TAIR database. Subsequently, the gene was mobilized into binary (destination) vector pK7FWG2 using LR clonase and by freeze thaw method Agrobacterium strain GV 3101 was transformed. Colony PCR with gene specific primer was used to verify the presence of DWARF4 gene in transformed Agrobacterium.  Mustard cultivar Pusa jaikisan was transformed with DWARF4 and regenerated on MS+Kan selection medium. Average transformation efficiency of Pusa jaikisan was 7.5% and we obtained six putative transgenic plants.  Confirmation of the presence of marker gene (NPT II) was accomplished by PCR and all the PCR positive plants are being rooted for development of transgenic plant.

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