Methods for Agrobacterium-mediated transformation of sugar cane

Patent metadata

Field	Value
Publication number	US-8952219-B2
Application number	US-201013378497-A
Country	US
Kind code	B2
Filing date	Jun 24, 2010
Priority date	Jun 25, 2009
Publication date	Feb 10, 2015
Grant date	Feb 10, 2015

Abstract

The present invention provides methods for producing a transformed sugar cane tissue or cell thereof, said methods comprising: a) inoculating a sugar cane tissue or a cell thereof with an Agrobacterium inoculation suspension, said Agrobacterium comprising a nucleic acid of interest, to obtain an Agrobacterium -inoculated sugar cane tissue or cell thereof; b) co-cultivating said Agrobacterium -inoculated sugar cane tissue or cell thereof on a surface in the absence of co-culture media for a time period sufficient to reduce original weight of said Agrobacterium -inoculated sugar cane tissue or cell thereof; and c) selecting a transformed sugar cane tissue or a cell thereof comprising said nucleic acid of interest. The transformation methods of the invention provide for increased transformation frequency and recovery of transgenic sugar cane plants.

First claim

What is claimed is:

1. A method of producing a transformed sugar cane tissue or cell thereof, said method comprising:
a) inoculating a sugar cane tissue or a cell thereof with an Agrobacterium inoculation suspension, said Agrobacterium comprising a nucleic acid of interest, to obtain an Agrobacterium -inoculated sugar cane tissue or cell thereof; and
b) co-cultivating said Agrobacterium -inoculated sugar cane tissue or cell thereof on a surface in the absence of co-culture media and in a desiccating environment for a time period sufficient to reduce original weight of said Agrobacterium -inoculated sugar cane tissue or cell thereof by at least 35%, thereby producing a transformed sugar cane tissue or cell thereof.

2. The method of claim 1 , wherein said co-cultivating comprises culturing said Agrobacterium -inoculated sugar cane tissue or cell thereof on at least one layer of dry paper.

3. The method of claim 2 , wherein said co-cultivating comprises culturing said Agrobacterium -inoculated sugar cane tissue or cell thereof on two or more layers of dry paper.

4. The method of claim 2 , further comprising changing the paper periodically during co-cultivation.

5. The method of claim 3 , further comprising changing the paper periodically during co-cultivation.

6. The method of claim 4 , further comprising changing the paper each day during the co-cultivation.

7. The method of claim 5 , further comprising changing the paper each day during the co-cultivation.

8. The method of claim 1 , wherein said inoculation suspension is substantially removed from said Agrobacterium -inoculated sugar cane tissue or cell thereof prior to co-cultivating in the absence of co-cultivation media.

9. The method of claim 1 , wherein said co-cultivating comprises culturing said Agrobacterium -inoculated sugar cane tissue or cell thereof on said surface in the absence of dry paper.

10. The method of claim 1 , further comprising selecting a transformed sugar cane tissue or cell thereof comprising the nucleic acid of interest, wherein said nucleic acid of interest comprises an expression cassette comprising a nucleic acid that confers resistance to a selection agent, and wherein said selecting comprises culturing said Agrobacterium -inoculated sugar cane tissue or cell thereof in a medium comprising said selection agent, and selecting a transformed sugar cane tissue or cell thereof comprising said nucleic acid of interest.

11. The method of claim 1 , wherein said sugar cane tissue or cell thereof is obtained from a sugar cane stalk or tiller.

12. The method of claim 1 , wherein said sugar cane tissue or cell thereof is embryogenic callus tissue.

13. The method of claim 11 , wherein said sugar cane tissue or cell thereof is obtained from a leaf roll segment or a leaf sheath segment excised from said stalk or tiller.

14. The method of claim 11 , wherein said sugar cane tissue or cell thereof is obtained by pre-culturing a segment of said stalk or tiller for a period of time to produce embryogenic callus tissue prior to contacting said sugar cane tissue or cell thereof with said inoculation suspension comprising Agrobacterium .

Assignees

• De Lucca Paulo Cezar
• Dong Shujie
• Geijskes Robert Jason Christopher
• Dunder Erik Martin
• Sainz Manuel Benito
• Syngenta Participations Ag
• Univ Queensland

Inventors

• De Lucca Paulo Cezar
• Dong Shujie
• Geijskes Robert Jason Christopher
• Dunder Erik Martin
• Sainz Manuel Benito

Classifications

• C12N15/8205 Primary

  Agrobacterium mediated transformation · CPC title