# Presentation: 261758

## Development of in Vitro Culture and Genetic Transformation Systems for Switchgrass 1992-2002

**B. V. Conger**
**University of Tennessee, Knoxville**

## Regeneration from Callus Cultures

**Various Explants**

## Slide 3

## Establishment of 1000 Regenerants in the Field

**983 ‘Alamo and 27 ‘Cave-in-Rock’**

## Slide 5

## Node Culture

**Potential Micropropagation System**

## Slide 7

## Slide 8

## Inflorescences from Node Cultures

**Source of Axenic Explants**
**Potential *****in vitro *****Fertilization System**

## Slide 10

## Regenerable Suspension Cultures

**Initiated from *****in vitro***** Produced Inflorescences**

## Slide 12

## Multiple Shoot Regeneration System

**Initiated from Mature Caryopses of Both Lowland and Upland Types**
**Thidazuron Required**

## Slide 14

## Genetic Transformation

## Microprojectile Bombardment

## Slide 17

## Slide 18

## Southern Blot Hybridization

**Southern Blot Hybridization**
** ****for the *****bar***** and *****uid*****A genes**

- Southern blot hybridization of seven transgenic plants for the *uid*A gene. Lanes 1 & 10 pAHC25.  Lanes 2 – 8 transgenic plants 1 – 7.  Lane 9  Alamo control plant.  All DNA was digested with *Kpn *I.

- Southern blot hybridization of six transgenic plants for the *bar* gene.  Lane 1 pAHC25.  Lanes 2 –7 transgenic plants 1 – 6.  Lane 8 Alamo control.  All DNA was digested with *Kpn* I, which linearizes the plasmid.

-   1        2        3       4        5       6       7       8

-    1      2      3    4     5     6     7     8     9    10

- 9.7kb

- 9.7kb

- 6.5kb

## Slide 20

## Slide 21

## Southern Blot Hybridization for the bar and sgfp genes

- Southern blot hybridization of two transgenic plants for the *bar* gene.  *Lane 1* psGFP-BAR  (10 ng), *lane 2 pst* I digest of control DNA, *lane 3 Bgl* II digest of control DNA,* lane 4* uncut control DNA, *lanes 5* and* 8 Pst* I digests of Tr1 and Tr2, *lanes 6* and* 9* *Bgl* II digests of Tr1 and Tr2, *lanes 7* and *10* uncut Tr1 and Tr2, *lane 11* psGFP-BAR (20 ng).

-    1  2  3  4   5   6  7  8  9  10  11

- 1.7 kb

- 0.6 kb

- Southern blot hybridization of five transgenic plants estimating copy number of the *gfp* gene.  *Lane 1* psGFP-BAR (1 pg), *lane* *2* control plant DNA, *lanes 3, 4, 5, 6* and *7* transgenic plants DNA, *lane 8* psGFP-BAR (2 pg).  All DNA was digested with *Kpn *I which linearized the plasmid.

-      1      2       3       4       5       6       7       8

- 8 kb

- 22 kb

## Slide 23

## GFP Expression in T1 and T2 Plants

**GFP Expression in T****1**** and T****2**** Plants**

**T****1**

**T****2**

## Slide 25

## Agrobacterium

## Slide 27

## Slide 28

## Slide 29

## Slide 30

## LIGNIN ANTISENSE

**Cobombardment**
**With**
**Phosphomannose Isomerase (PMI)**
**and**
**Green Fluorescent Protein**

## Lignin Antisense

**Lignin Antisense**

**Phosphomannose Isomerase**

**Green Fluorescent Protein**

## GFP expression in a young leaf

**GFP expression in a young leaf**

**CPR assay for PMI**

## ANTHER 

**AND  MICROSPORE**
**CULTURE**

## Slide 35

## Slide 36

## Slide 37

## VARIETYTRIALS

## Slide 39

## METABOLIX CORPORATION

- Transfer Genes for Polyhydroxybutrate (PHB) Synthesis into Switchgrass
- Plastid Transformation
- Betaine Aldehyde Dehydrogenase (BAHD) as a Selectable Marker, an Alternative to Spectinomycin Resistance Converts Betaine Aldehyde to Glycine Betaine
- Nuclear Transformation Will Probably be Attempted Initially
- Orchardgrass May be a Better Choice?

## PROPOSED NEW TASKS

**Refinement of Embryogenic Suspension Cultures**
***Improvement and Optimization of Gene Transfer Protocols**
***Development of Alternative Selectable Marker Gene Systems**
***Transformation for Agronomically Important Genes**
**	****	****1) Lignin biosynthesis**
**	****	****2) Male sterility (*****barnase *****gene)**

## New Tasks Continued

***Investigations on Gene Flow**
***Inheritance  of Transgenes**
**Production of Haploid Plants**
**Existing Field Experiments**
***Studies Emphasizing Transformation**