Progress 09/15/04 to 09/14/07

Outputs
Objective: To develop genetic transformation protocols for Tifton 85 bermudagrass and seashore paspalum. Explants of Tifton 85 obtained from young inflorescences, shoot apices and nodal segments were tested for their callus initiation and regeneration response on various media. An extensive screening of various auxins such as 2,4-D (1-6 mg l-1), dicamba (1-5 mg l-1), IAA (1-5 mg l-1) either alone or in combination with kinetin (1, 2 and 3 mg l-1) or BAP (0.01, 0.1 and 1 mg l-1) was performed. For seashore paspalum, four turf ecotypes were maintained vegetatively in the greenhouse to produce stolons and immature inflorescenses, used as alternative targets for production of embryogenic callus. Objective: To introduce a gene construct coding for constitutive expression of the most effective Bt endotoxin into Tifton85 and seashore paspalum and identify transgenic grasses with stable expression of the insecticidal protein. A synthetic cry 1Fa gene was made according to the published codon optimized sequence by GeneArt (Regensburg, Germany). The synthetic cry 1Fa was subcloned under control of the constitutive ubiquitin promoter and nos polyadenylation signal. The vector backbone sequence was removed by restriction digest and gel purification. This minimal cry 1Fa expression cassette was co-introduced with a minimal constitutive nptII expression cassette into embryogenic Tifton85 and seashore paspalum callus by biolistic gene transfer. Selection parameters were optimized on the basis of eliminating non-transgenic callus and suppression of non-transgenic shoots. Objective: To evaluate the FAW resistance of transgenic grasses in small field plots in Puerto Rico and Ona, FL and with laboratory bioassays using plant tissue. Small field plots. A small plot study was conducted at the Fortuna substation of the University of Puerto Rico. The objective of the study was to asses plant spread of a commercial type of Seashore paspalum SeaIsle 1 from the University of Florida, and two cultivars of Zoysia (Manila and Meyer). During and after establishment, turfgrasses were monitored for FAW damage. Plugs (approximately 10 cm2) of SeaIsle 1 and Manila and Meyer were transplanted in well-prepared plots (in the summer of 2005). The maintenance of the turfplots included irrigation and weeding as needed. Percentage spread was taken bi-weekly using a marked cm2 quadrat. Laboratory bioassays. Larval feeding studies were conducted on plants obtained under greenhouse-growing conditions. FAW larvae used in this experiment were from two different cultures. Larvae shown to carry the mitochondrial marker of corn strain (JS3) were from a population collected from sweet corn in Miami-Dade County in 2005. This culture was maintained on a pinto bean artificial diet according to the procedures of Guy et al. (1985). Larvae shown to carry the mitochondrial marker of rice strain (Ona) were from a culture of individuals collected in Ona in July 2002 and were maintained on bermudagrass and stargrass grown in Gainesville, FL.

Impacts
Young, immature inflorescence explants of Tifton85 proved to be an excellent source for obtaining embryogenic callus, as opposed to apical meristems and nodal segments. Embryogenic callus with demonstrable morphogenetic competence was obtained on MS basal medium containing 30 g l-1 sucrose, 4 mg l-1 2,4-D, 0.01 mg l-1 BAP and 200 mg l-1 casein hydrolysate. The regenerants were rooted on hormone free MS basal medium supplemented with 30 g l-1 sucrose, successfully established in soil under greenhouse conditions and did not show any phenotypic differences compared to wild type Tifton 85 plants. Successful callus induction and plant regenertion was achieved for all the studied seashore paspalum ecotypes and both explants. Two superior ecotypes were selected with more than 60% of the immature inflorescenses and more than 10 % of the stolons producing embryogenic, regenerable callus. The number of shoots regenerated per callus was two to five times higher from immature inflorescenses compared to stolon derived callus. However, the advantage of using stolons is that stolons are a readily available tissue, whereas immature inflorescenses are only seasonally available and the identification of the correct developmental stage of inflorescenses (length of 5 to 10 mm) for production of embryogenic callus is very tedious. Callus was bombarded with the cry 1Fa gene, selected and rengenerated. Transgenic plants have been obtained. A portion of these transgenics are displaying high resistance to fall armyworm.

Publications

• Luciani, G., F. Altpeter, J. Yactayo-Chang, H. Zhang, M. Gallo, R.L. Meagher, and D.S. Wofford. 2007. Expression of cry1Fa in bahiagrass enhances resistance to fall armyworm. Crop Science (47)2430-2436.
• Neibaur, I. E., F. Altpeter, and M. Gallo. 2008. The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (Paspalum vaginatum Swartz). In Vitro Cellular & Developmental Biology Plant (submitted).

Progress 10/01/05 to 09/30/06

Outputs
A minimal cry expression cassette was co-introduced with a constitutive nptII expression cassette into embryogenic Tifton85 and seashore paspalum callus by biolistic gene transfer. Selection parameters were optimized on the basis of eliminating non-transgenic callus and suppression of non-transgenic shoots. Gene transfer rate to seashore paspalum callus, as evaluated by transient reporter gene assays, were successfully enhanced with an alternative auxin form in the culture medium. Nevertheless, stably transformed seashore paspalum plants have yet to be recovered following gene transfer into a large number of calli and selection on antibiotic containing culture medium. Therefore, in addition to seashore paspalum, we began to introduce the cry expression cassette into bahiagrass (Paspalum notatum Flugge) an alternative, commercially important turf and forage grass in the subtropical region. This has allowed us to evaluate our cry gene against fall armyworm in bioassays. Six transgenic lines from cultivars Argentine and Tifton 9 were generated. PCR and Southern blot analysis confirmed the stable integration of the synthetic cry gene into the bahiagrass genome. RT-PCR analysis confirmed transgene expression of the six transgenic lines. ELISA using a cry protein specific antibody revealed high level of transgene expression in two of the transgenic lines. Leaves from these two transgenic lines showed significant effects on insect mortality when fed to neonate larvae of the fall armyworm in contrast to wild-type leaves and transgenic lines not expressing the transgene at a level that would be detectable with ELISA. On average of four independent experiments with 10 larvae each survival of neonates when fed leaves from the two transgenic plants was 2.5% and 17.5 % respectively, whereas the majority of the neonates survived when reared on leaves from wild-type. Further studies will evaluate if these lines are able to control later instars and can control insect populations in field conditions. For Tifton85, bombarded calli are still on selection medium and we have recently begun evaluating Agrobacterium-mediated transformation as an additional gene transfer system. The cry expression cassette has been subcloned into a T-DNA vector and was introduced into the Agrobacterium strain AGL 0. We have begun to use Agrobacterium-mediated gene transfer to insert cry into Tifton85 stolon explants.

Impacts
Improved seashore paspalum cultivars have been recently commercially released after many years of research on various ecotypes and represent a very attractive grass for environmentally friendly, high quality golf courses and home lawns especially in coastal areas of the southern U.S. and the Caribbean. Tifton 85 bermudagrass is a high yielding, improved forage grass that produces high weight gains in cattle because of good digestibility and protein content. FAW is a devastating pest on Tifton85 and seashore paspalum in the tropical and subtropical US and the Caribbean. Therefore, improvement of FAW resistance in both grasses will significantly enhance their productivity and persistence.

Publications

• Luciani, G., Yactayo-Chang, J., Altpeter, F., Zhang, H., Gallo, M., Meagher, R., Wofford, D.S. 2006. Expression of a synthetic Bacillus thuringiensis endotoxin to enhance resistance against fall armyworm in bahiagrass. Florida Genetics Symposium, Gainesville FL, November 1st- 2nd 2006.

Progress 10/01/04 to 09/30/05

Outputs
The overall goal of this project is to produce transgenic turf and forage grasses that are resistant to the insect pest fall armyworm (FAW) (Lepidoptera: Noctuidae), Spodoptera frugiperda (J. E. Smith). Seashore paspalum is a relatively new turfgrass, which due to its high salt tolerance can tolerate poor water quality and therefore is well suited for coastal areas of the Southern US and the Caribbean. `Tifton 85' bermudagrass is one of the most important forage grasses in the Southern US. Improvement of FAW resistance in both grasses will significantly enhance their productivity and persistence. We are generating transgenic'Tifton85' bermudagrass and seashore paspalum with stable expression of a synthetic insecticidal Bacillus thuringiensis (Bt) Berliner δ-endotoxin gene that was previously shown to cause FAW toxicity. Successful callus induction and plant regenertion was achieved for four seashore paspalum ecotypes. Two superior ecotypes were selected with more than 60% of the immature inflorescenses and more than 10% of the stolons producing embryogenic, regenerable callus. The number of shoots regenerated per callus was two to five times higher from immature inflorescenses compared to stolon derived callus. However, the advantage of using stolons is that stolons are a readily available tissue, whereas immature inflorescenses are only seasonally available and the identification of the correct developmental stage of inflorescenses (length of 5 to 10 mm) for production of embryogenic callus is very tedious. An efficient regeneration system via somatic embryogenesis from immature inflorescences of hybrid bermudagrass cv. Tifton 85 has been established. Embryogenic callus with demonstrable morphogenetic competence was obtained on MS basal medium containing 30 g l-1 sucrose, 4 mg l-1 2,4-D, 0.01 mg l-1 BAP and 200 mg l-1 casein hydrolysate. Regenerants were rooted on hormone free MS basal medium fortified with 30 g l-1 sucrose, and successfully transplanted under greenhouse conditions. So far biolistic gene transfer has not been successful for stable introduction of the cry expression cassette into seashore paspalum. As an alternative we plan to evaluate Agrobacterium-mediated gene transfer. The cry expression cassette is currently being subcloned into a T-DNA vector for Agrobacterium-mediated gene transfer. Biolistic as well as Agrobacterium-mediated transformation is underway in `Tifton85'.

Impacts
Improved seashore paspalum cultivars have been recently commercially released after many years of research on various ecotypes and represent a very attractive grass for environmentally friendly, high quality golf courses and home lawns especially in coastal areas of the southern U.S. and the Caribbean. `Tifton 85' bermudagrass is a high yielding, improved forage grass that produces high weight gains in cattle because of good digestibility and protein content. FAW is a devastating pest on `Tifton85' and seashore paspalum in the tropical and subtropical US and the Caribbean. Therefore, improvement of FAW resistance in both grasses will significantly enhance their productivity and persistence.

Publications

• Jain, M. K. Chengalrayan, M. Gallo-Meagher, and P. Mislevy. 2005. Embryogenic callus induction and regeneration in a pentaploid hybrid (Cynodon dactylon c C. transvaalensis) bermudagrass cv. Tifton 85. Crop Sci. 45:1069-1072.

Progress 10/01/03 to 09/30/04

Outputs
Although turf and forage grasses provide much income, large sums are spent for pest control. The overall goal of this project is to produce transgenic turf and forage grasses that are resistant to the insect pest fall armyworm (FAW) (Lepidoptera: Noctuidae), Spodoptera frugiperda (J. E. Smith). We propose to generate transgenic 'Tifton85' bermudagrass and seashore paspalum with stable expression of a synthetic Bacillus thuringiensis (Bt) Berliner endotoxin gene that was previously shown to cause FAW toxicity and evaluate transgenic plants under field conditions for FAW resistance. Objective 1 is to assay the Bt endotoxins, Cry 1Fa and Cry1Ca, against newly established cultures of the rice strain of FAW. Objective 2 is to develop genetic transformation protocols for `Tifton 85' bermudagrass and seashore paspalum. Objective 3 is to introduce a gene construct coding for constitutive expression of the most effective Bt endotoxin into `Tifton85' and seashore paspalum and identify transgenic grasses with stable expression of the insecticidal protein. Objective 4 is to evaluate the FAW resistance of transgenic grasses in small field plots in Puerto Rico and Ona, FL and with laboratory bioassays using plant tissue. Since this project was initiated on 9/15/04 (two weeks), there is currently no progress to report.

Impacts
This research will result in the collection of important data on FAW rice strain, and the development of an efficient genetic transformation protocol for two highly valuable grasses. The proposed study will result in the generation of Bt-transgenic, FAW-resistant turf and forage grasses that can be effectively used in an integrated pest management approach.

Publications

• No publications reported this period