ZEESHAN NADEEMROLL NO. 26

BS. BIOTECHNOLOGY 7THIBB, PU

DEVELOPMENT OFSELECTABLE MARKER FREE,

INSECT-RESISTANT,TRANSGENIC MUSTARD (Brassica

juncea)PLANTS USING

CRE/LOX MEDIATED RECOMBINATION

BY

ARPITA BALA, AMIT ROY, AYAN DAS, DIPANKAR CHAKRABORTI AND SAMPA DAS

INTRODUCTION» Brassica juncea (Indian Mustard) an important crop» Lipaphis erysimi (a sap sucking hemipteran pest)

OBJECTIVES

» Development of Insect resistance by: Expressing Allium sativum leaf agglutinin (ASAL)

gene (replacement of chemical pesticides and cry toxins)

» Removal of Selectable Marker Genes (hpt) by: Using Cre/lox mediated recombinantion

CRE LOX MEDIATED RECOMBINATION

VECTOR CONSTRUCTS

RESULTS…

Southern Blot Analysis of Expression of ASAL and Cre Genes

Multiplex PCR Analysis of Marker Gene Excision

Western Blot Analysis of ASAL Expression in F1 Hybrid Plants

Agglutination and Thermal Stability Assay of Expressed ASAL Protein from F1 Hybrids

In-Planta Insect Bioassay

Insect Bioassay Setup

PCR Analysis of Completely Marker Free F2 Mustard Plants

CONCLUSION

Efficient expression of insect resistant ASAL gene

in Brassica juncea plantseven after the elimination of selectable

marker geneby Cre/lox mediated recombination