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METHOD OF MONOCOT TRANSFORMATION USING MATURE EMBRYOS

IP.com Disclosure Number: IPCOM000240134D
Publication Date: 2015-Jan-05
Document File: 6 page(s) / 49K

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The IP.com Prior Art Database

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Madhusudhana Janga: INVENTOR [+3]

Abstract

A mature embryo based method for production of transgenic monocot plants, wherein the method does not involve callus induction or wounding of the embryo prior to the transformation step, is provided.

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TITLE

METHOD OF MONOCOT TRANSFORMATION USING MATURE EMBRYOS

ABSTRACT

A mature embryo based method for production of transgenic monocot plants, wherein the method does not involve callus induction or wounding of the embryo prior to the transformation step, is provided. 

INTRODUCTION

The ability to directly transform agronomically important plant species at a usable frequency and across a wide range of genetic diversity is important for the development of commercial products with improved traits such as disease resistance, herbicide tolerance, and increased nutritional value.  Efficient transformation techniques are particularly important for grain crops (e.g., maize, rice, and soybean), which are considered staple foods.  Since a majority of grain crops are monocotyledonous, it would be of great benefit to improve the ability to genetically engineer monocot species.

MATERIALS AND METHODS

Gene constructs and Agrobacterium Preparation

Gene constructs:

Two constructs were made to test transformation using mature embryos.  One construct contained 3X 35S CaMV enhancer, Maize Ubiquitin 1 (ZmUbi) promoter, Maize Ubiquitin 1 (ZmUbi Intron), a glyphosate N-acetyltransferase (GLYAT), and potato proteinase pin II terminator.  The other contained ZmUbi promoter, ZmUbi Intron, a glyphosate N-acetyltransferase (GLYAT), and the pinII terminator. 

Similarly another GLYAT version was operably linked viz., ZmUbi promoter, ZmUbi Intron, the glyphosate N-acetyltransferase (GLYAT), pin terminator and to 3X 35S enhancer, ZmUbi promoter, ZmUbi Intron, the glyphosate N-acetyltransferase (GLYAT), pin terminator.

Preparation of Agrobacterium suspension:

Agrobacterium strain LBA4404 carrying a GLYAT gene was streaked out from a - 80° C frozen aliquot onto a plate containing AB medium (Chilton et al., 1974 Proc. Natl. Acad. Sci. USA 71, 3672–3676) and cultured at 28°C in dark for 3 days. 

AB media was prepared sequentially by adding filter sterilized Stock Solution A (50 mL), Stock solution B (50 ml), Spectinomycin; 100mg/ml (Sigma Chemicals, Bangalore) 1 µL; Rifampicin; 25mg/ml (DUCHEFA; Netherlands) 1 µL; Tetracycline 5 mg/ml (Sigma Chemicals, Bangalore) 1 µL into 900mL of autoclaved media conatiing 5 g glucose (Duchefa, Netherlands) and 15 g DIFCO agar (BD Biosciences, USA).  20 mL of this media was poured in 90mm Petri plates and used for streaking bacterial cultures.

Stock Solution A: 20X AB salts: Dissolved 20 g NH4Cl (Hi-Media Labs; Mumbai), 6 g MgSO4.7H2O (DUCHEFA; Netherlands), 3 g KCl (Hi-Media Labs; Mumbai), 265 mg CaCl2. 2H2O (DUCHEFA; Netherlands) and 50 mg FeSO4 .7H2O (Sigma Chemicals, Bangalore) in 900 ml distilled water and made up the volume to 1,000 ml (Chilton et al., 1974).  Autoclaved the contents at 121°C for 15 min and stored at 4°C until further use. 

Stock Solution B: 20X AB buffer: Dissolve 20 g NaH2PO4 (Hi-Media Labs; Mumbai) and 60 g K2HPO4 (Hi-Media Labs; Mumbai) in 900 ml distilled water and made up the volume to 1,000 ml....