Annual Report of Progress
to the
MISSISSIPPI SOYBEAN PROMOTION BOARD
for 1996

Objectives & Significant Accomplishments

1. Completed the development of a unique protocol (set of procedures) for regenerating soybean in tissue culture. Thirteen public lines were tested and all could be regenerated via this protocol. This was a critical goal to achieve prior to development o f a gene transfer protocol. This regeneration protocol is patentable and a MSU patent disclosure form is in preparation.
2. Determined which chemical to use to aid in the selective growth of soybean cells containing the introduced foreign DNA. This should enhance the probability of regenerating transgenic soybean plants (those that contain the introduced foreign DNA).
3. Completed and confirmed the development of a gene transfer protocol by delivery of foreign DNA into soybean cells in culture. This process is also patentable and will be included in the regeneration patent disclosure form.
4. A manuscript detailing our unique regeneration protocol has been completed and will be submitted for internal review shortly. Two additional manusucripts are in preparation detailing a different soybean regeneration protocol and our DNA introduction p rotocol. Publication of these scientific articles should dramatically increase the stature of our lab in the soybean transformation arena and may lead to future funding from additional sources. (An attempt to acquire supplemental funding for this project from the USB in 1996 was unsuccessful.)

Accomplishment of Individual Objectives

Last year we had begun the development of a unique regeneration protocol for soybean. We utilized 13 public lines (12 varieties and one plant introduction). Recently, we have completed the protocol which utilizes hypocotyl tissue excised from germinated s eedlings. Greater than 70% of all hypocotyl sections placed in culture were capable of producing shoots using our regeneration protocol (Table 1). In addition, 4 - 15 shoots could be produced per responding hypocotyl section. Coupling the shoot initiation protocol with a developed rooting protocol, we can regenerate intact soybean plants in as little as 3 - 4 months (shoot initia tion and elongation: 8 - 10 weeks; rooting and plantlet elongation: 3 - 6 weeks). Our regeneration protocol is depicted in Figure 1. This type of adventitious regeneration protocol was required so DNA could be introduced into cells prior to regeneration i nto intact plants. This was important because the gene gun can only successfully introduce foreign DNA into individual cells and small cell clumps, but not evenly over large targets (seeds, seedlings, shoot tips...).

Since the DNA is scattered randomly over the surface of the upright hypocotyl sections, many soybean cells do not contain any introduced foreign DNA (they are normal soybean cells). Therefore, growth of those normal cells would need to be suppressed to al low identification of the cells which contain the foreign DNA. This would allow selective growth of transformed soybean cells along with subsequent plant regeneration from them. To aid in the selective growth of transformed soybean cells, a gene coding fo r resistance to a chemical (antibiotic or herbicide) is included as part of the foreign DNA that is introduced into plant tissues. Therefore, those that received the introduced foreign DNA will contain the resistance gene and would be able to grow in the presence of the corresponding antibiotic or herbicide.

We had three resistance genes: two were for resistance to the antibiotics kanamycin and hygromycin, and one was for resistance to the herbicide glufosinate (sold as Ignite, Finale, Liberty, etc.). Therefore, we screened soybean tissues on media containing different amounts of each chemical (separately) to determine which one would work as the best inhibitor of growth in normal (untransformed) soybean tissues. We determined that glufosinate (at 5.0 mg/L) was the best chemical for selection. Soybean cells ( normal) could not grow on media containing this amount of glufosinate, but soybean cells that contained the resistance gene would be able to grow.

Objective 2. Continue to develop genetic transformation protocols for soybean via gas-mediated microprojectile bombardment using the Biolistic^¨ PDS-1000/He^¨ device (gene gun).

The protocol for introducing foreign DNA into soybean cells has been developed. We are using the only commercially available gene gun (called PDS-1000/He^¨ device). In optimization, of this protocol, we had to determine the following: size and amount of gold particles per shot (DNA is coated onto gold which delivers the DNA into soybean cells), amount of DNA per shot, number of shots per set of target tissues, helium pressure used (the velocity which forces the DNA-coated gold into soybean tissues), along with gap and target distances (distances from helium entry to DNA-coated gold and then to targeted soybean tissues, respectively).

Optimization of the variables listed above were accomplished by scoring for the presence of foreign DNA in the soybean tissues after being shot (bombarded) with the gene gun. To easily accomplish this, the foreign DNA that was introduced contained a gene which allowed quick visual detection of its gene product in cells. (The tissues could be scored three days after bombardment.) The gene we used is called GUS (short for beta-glucuronidase). Each soybean cell that contained the GUS gene would turn blue when exposed to a specific chemical substrate (Figure 2). By counting the number of blue spots on the soybean tissues, we could determine which gene gun settings yielded the greatest numbers of blue spots (the greatest numbers of soybean cells that received the foreign DNA).

Objective 3. Screen (potential) transformants for presence of introduced foreign DNA.

It took most of 1996 to refine and confirm the regeneration, selection, and transformation protocols. Confirmation of transformation required destructive sampling which meant that those soybean tissues had to be killed for testing purposes. In addition, t he foreign DNA initially used for introduction into soybean tissues did not contain the glufosinate resistance gene, so there was no way to selectively grow soybean cells which contained the introduced foreign DNA. With the recent completion of Objective 1., we have begun to utilize foreign DNA which allowed selective growth of transgenic soybean cells on tissue culture media containing glufosinate. To date, we have some soybean tissues that may be transgenic because they have survived on this selective m edia for two months. [Normal (untransformed) soybean tissues usually die within this same time period.] However, plants have not been regenerated from these tissues yet.

Soybeans in Mississippi Mississippi Agricultural and Forestry Experiment Station  Mississippi State University Extension Service Division of Agriculture, Forestry and Veterinary Medicine

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