Specialty plants biotechnology group at ARS, FVSU. Left to right: Dr. Nirmal Joshee (Research Professional), Sadanand Dhekney (Ph.D. student India) and Dr. Anand K. Yadav (Professor).

Potential for Introducing Cold Tolerance into Papaya by Transformation with C-Repeat Binding Factor (CBF) Genes

This project was undertaken as a joint project between the groups at the University of Florida (UF TREC) and Fort Valley State University (FVSU). The idea for this research was to extend the range of a tropical fruit species papaya into the warm temperate zone of north Florida and south Georgia. Because of the absence of cold tolerance in papaya Carica papaya L., a proposal was developed that would 1) probe the genome of papaya and its cold-tolerant Andean relatives in the genus Vasconcellea for CBF (C-repeat binding factor) genes and 2) carry out genetic transformation of papaya with CBF genes. Michael Thomashow and his co-workers have characterized CBF genes as transcriptional activators that bind to the promoter regions of cold tolerance genes. Overexpression of CBF genes in Arabidopsis results in activation of cold-regulated (COR) genes, and has been linked with improved chilling tolerance. The University of Florida laboratory at Homestead, at the southernmost tip of Florida, has focused on tropical fruit biotechnology for several years, and has considerable experience working with several species in addition to papaya, including mango, avocado, litchi, carambola and longan.

The tropical fruit biotechnology research group at the UF TREC. From left to right: Dr. Simon Raharjo (post doctoral scientist Indonesia), Darda Efendi (Ph.D. student Indonesia), Isidro Suarez (Ph.D. student Colombia), Sadanand Dhekney (Ph.D. student India), Pamela Moon (senior biologist) and Professor Richard Litz.

The FVSU research emphasizes biotechnology to enhance cold hardiness in peach, papaya and guava and selected medicinal plants. Sadanand Dhekney, who completed a M.S. degree at the Tamil Nadu Agricultural University in Coimbatore, India, was recruited as a Ph.D. student at the University of Florida to work on this project. Sadanand determined that DNA sequences bearing homology to the CBF gene, although present in the cold-tolerant Vascaoncellea species, were missing in papaya. He developed a genetic transformation procedure for papaya based upon the co-cultivation of embryogenic cultures with genetically engineered Agrobacterium tumefaciens strain GV3101 that contained the CBF gene(s) under the control of the CaMV 35S promoter and the NPTII gene under the control of the CaMV 35S promoter. Embryogenic papaya cultures were successfully transformed, and plants were recovered. Regenerants are currently under evaluation.Sadanand A. Dhekney, Richard E. Litz, David A. Moraga and Anand Y. Yadav. Potential for introducing cold tolerance into papaya by transformation with c-repeat binding factor (CBF) genes, In Vitro Cellular & Developmental Biology-Plant, 43: in production.

Left to right: Touho Yoshida, Kazumi Ono, Motonobu Satoh, Hidekazu Sawada.

Nested PCR to Identify 14 Species of Animal Cells

It has been occasionally reported that cell lines derived from a certain source can be contaminated with another cell line. This cross-culture contamination is a serious problem for investigations using cultured cells. Therefore, it is very important to confirm the identities of cell lines as a quality control. So, we developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial DNA to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig and chicken, could be distinguished from each other by nested PCR. The 1st PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the 2nd PCR amplifies the DNA fragments with species-specific primer pairs from the 1st products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus the method developed in this study will provide a useful tool for the authentication of animal species. Kazumi Ono, Motonobu Satoh, Touho Yoshida, Yutaka Ozawa, Arihiro Kohara, Masao Takeuchi, Hiroshi Mizusawa, and Hidekazu Sawada. Species Identification of Animal Cells by Nested PCR Targeted to Mitochondrial DNA, In Vitro Cellular and Developmental Biology – Animal 43: 168-175, 2007.