Progress 09/01/00 to 08/31/05
Outputs
During this reporting period, in vitro protocols for guava plant regeneration which were developed and/or standardized earlier for organogenesis and somatic embryogenesis, were further refined / tested for their reproducibility. These protocols for both organogenesis and somatic embryogenesis were also tried to regenerate additional guava cultivars including Golden, Allahabad, and Ruby Supreme. The former two cultivars responded very well to the standardized protocols. However, a modification was needed for the third cultivar which will be tested during the 2006 experiments. In vitro plant regeneration through somatic embryogenesis was repeated three times with good reproducibility / uniformity. The somatic embryos from guava explants were used to successfully develop synthetic seeds followed by their germination. The resulting emblings were transferred to greenhouse containers for few weeks and then established directly in the ground in the specialty plants house
where they are well established and are growing for the last several months. Initiation of somatic embryogenesis using other guava cultivars will be attempted during the 2006 season. The putative transgenic guava plant which was transferred to the soil in the specialty plants house in September 2003, began producing fruits in May 2005. The fruit size, shape and flesh color were similar to that of the mother plant. In order for genetic transformation, attempts will be devoted to co-cultivate embryogenic tissues of guava with Agrobacterium during the 2006 fruiting season, the most appropriate time of the year due mainly to unlimited availability of immature zygotic embryos to produce adequate embryonic mass.
Impacts
Developing suitable biotechnology for in vitro plant regeneration and genetic transformation of guava will enable scientists to integrate cold hardiness genes into popular guava varieties. Thus, this will enhance cold hardiness of guava germplasm to genetically improve it for adaptation to at least the mild temperate climatic areas of the United States such as Georgia and the rest of the southeastern USA.
Publications
- Biswas, B. K., A. Yadav, N. Joshee, N. and Anand K. Yadav. 2005. In Vitro Plant Regeneration and Genetic Transformation to Enhance Cold Hardiness in Guava: A Nutraceutical Fruit. International Guava Symposium, December 5-8, Lucknow, India, Abstracts Book 31-32.
- Biswas, B. K., A. Yadav, N. Joshee, N. and Anand K. Yadav. 2005. In Vitro Plant Regeneration and Genetic Transformation to Enhance Cold Hardiness in Guava: A Nutraceutical Fruit. ActaHort (Communicated)
- Biswas, B. K., A. Yadav, N. Joshee, N. and Anand K. Yadav. 2005. Development and Application of Biotechnology in Guava: A Nutraceutical Fruit. Acta Hort. (Communicated).
- Biswas, B. K., N. Joshee, A. Yadav, and A. K. Yadav 2005. Development and Application of Biotechnology in Guava: A Nutraceutical Fruit. Acta Hort. (Communicated).
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Progress 01/01/04 to 12/31/04
Outputs
Suitable in vitro plant regeneration protocols like organogenesis and/or embryogenesis are necessary to develop cold tolerant guava germplasm through Agrobacterium-mediated gene transfer. During 2004, standardization of in vitro regeneration of Psidium guajava through organogenesis and somatic embryogenesis were carried out using mature tissues (nodes, shoot tips, internodes, leaves, anthers, and the zygotic embryos of different ages) from outdoors guava trees. Somatic embryogenesis in guava was obtained from immature zygotic embryos by culturing them on BJY medium, a modified MS especially for guava since there was no embryo induction achieved on other media even with hormone supplements cultured for 4 weeks. Direct somatic embryo induction started from all over the explant surface after 4 weeks of cultures and continued for 5 months when all embryos were isolated for germination and synthetic seed development on 3% alginic acid sodium salt alone or with charcoal.
Direct germination of embryos was observed on the induction medium alone or with BA or GA3 for 2 weeks. The emblings were normal and healthy. The somatic embryos were used as fresh explants to produce numerous somatic embryos when cultured on the same medium. These protocols will be used for co-cultivation to introduce cold genes into guava. For standardization of gene transfer protocols for guava nodal explants, they were cocultivated with Agrobacterium containing binary vector pBI 121 having selectable markers (nptII and GUS) with CaMV 35S promoter gene. Standardization of co-cultivation duration, presence of inducing (induce activity of vir genes) compounds (acetosyringone), was performed for effective genetic transformation and the gene transfer frequencies. The resultant plant showed kanamycin resistance and was transferred to soil. During 2005, efforts will be made for use of gene gun and cocultivating Agrobacterium with embryogenic tissues of guava. Introduction of cold
tolerance genes (CBF1, CBF2, CBF3) will be attempted in organogenic and embryogenic explants to develop cold hardy guava plants using protocols developed at FVSU.
Impacts
Developing suitable biotechnology for in vitro plant regeneration and genetic transformation of guava will enable scientists to integrate cold hardiness genes into popular guava varieties. Thus, this will enhance cold hardiness of guava germplasm to genetically improve it for adaptation to at least the mild temperate climatic areas of the United States such Georgia and the rest of the southeastern USA.
Publications
- Joshee, N. M. Mutua, F. Zee, and A. K. Yadav. 2004. In Vitro Shoot Proliferation in Guava as Influenced by Genotype. Acta Horticulturae Volume number 632, the International Society for Horticultural Sciences. http://www.actahort.org/books/632/632_36.htm (Hard Copy in press).
- Biswas, B. K., N. Joshee, and A. K. Yadav. 2004. Can Agricultural Biotechnology Help Guava Growing in Temperate Climate. HortScience 39(4):861.
- Biswas, Bipul K., N. Joshee, and A. K. Yadav. 2004. Guava (Psidium guajava) - a Suitable Fruit Tree for Agroforestry. 1st World Congress of Agroforestry, Book of Abstracts 1:110.
- Biswas B. K., N. Joshee, and A. K. Yadav. 2004. Developing Biotechnology to Regenerate and Improve selected Nutraceutical Plant. In Vitro, 2004 World Congress on In Vitro Biology Abstract Issue 40:23-A, P-33.
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Progress 01/01/03 to 12/31/03
Outputs
Suitable in vitro plant regeneration protocols like organogenesis and/or embryogenesis are necessary to develop cold tolerant guava germplasm through Agrobacterium-mediated gene transfer. During 2003, standardization of in vitro regeneration of Psidium guajava through organogenesis and somatic embryogenesis were carried out using mature tissues (nodes, shoot tips, internodes, leaves, anthers, and the zygotic embryos of different ages) from outdoors guava trees. Multiple shoot buds were obtained from mature nodal explants of guava cultured on MS supplemented with BAP, KIN and AdS, and well developed shoots were excised and rooted. The plantlets were acclimatized and planted in outdoors soil where they have been growing well for many months. Somatic embryogenesis in guava was induced in immature zygotic embryos. Direct somatic embryos induced from all over the explant surfaces in 4 weeks and continued for 24 weeks, till all embryos were isolated for further studies
(germination or synthetic seed production). Production of synthetic seeds of guava was successful. Direct germination of embryos were carried out. Standardization of Agrobacterium-mediated gene transfer protocols was attempted with a binary vector pBI 121 having nptII and GUS as selectable markers and CaMV 35S as a promoter gene. The transformed guava plant showing kanamycin resistance was transferred to soil in the greenhouse. The GUS gene expression will be analyzed by flurometry. Efforts will be made for cocultivating Agrobacterium with embryogenic tissues of guava. Introduction of cold tolerance genes (CBF1, CBF2, CBF3) will be attempted in organogenic and embryogenic explants to develop cold hardy guava plants using protocols developed at FVSU.
Impacts
Developing suitable biotechnology for in vitro plant regeneration and genetic transformation of guava will enable scientists to integrate cold hardiness genes into popular guava varieties. Thus, this will enhance cold hardiness of guava germplasm to genetically improve it for adaptation to at least the mild temperate climatic areas such Georgia and the rest of the southeastern United States.
Publications
- Joshee, N. M. Mutua, F. Zee, and A. K. Yadav. 20042. In Vitro Shoot Proliferation in Guava as Influenced by Genotype. pp 000-000 in Xxxxxx (editor), Acta Horticulturae, Vol # 5xx, International Society for Horticultural Science (in press).
- Biswas, B. K., N. Joshee, and A. K. Yadav. 2003. Developing Biotechnology to Regenerate and Improve selected Woody Fruit Plants. Woody Plants Biotechnology Symposium, University of Tennessee, Knoxville, TN, 16-17 Oct 2003.
- Biswas, B. K., N. Joshee, and A. K. Yadav. 2004. Guava (Psidium guajava L.) - a Suitable Fruit Tree for Agroforestry. 1st World Congress of Agroforestry, held in Orlando, FL, from 27 June to 02 July 2004 (abstract).
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Progress 01/01/02 to 12/31/02
Outputs
This project focusing on developing an efficient in vitro plant regeneration system, amenable to gene delivery techniques and formulating reliable protocols for gene transfer into guava has progressed well. Dr. B. K. Biswas, a Post-Doctoral Research Associates was employed. An undergraduate student was hired to assist in the micropropagation research on guava. Standardization of in vitro propagation protocols for selected cultivars of Psidium guajava for either organogenesis or somatic embryogenesis was carried out using nodes, shoot tips, internodes, and leaves for explants. In order to standardize genetic transformation protocols for guava, nodal explants has been cocultivated with Agrobacterium containing a binary vector pBI121 having npt II gene driven by nos promoter conferring resistance to kanamycin. After standardizing transformation protocols with reporter genes, we plan to transfer the cold tolerance genes (CBF1, CBF2 and CBF3) to develop cold hardy
transgenic guava plants. We also plan to finger print all guava germplasm (38 individual guava plants growing in the plant house) using RAPD or AFLP molecular markers using genomic DNA. Guava plants of 6 cultivars received from the USDA, Hilo, HI, were successfully established along with other cultivars in the specialty plants house.
Impacts
Developing biotechnology for in vitro regeneration and genetic transformation of guava will enable scientists to integrate cold hardiness genes into popular guava varieties. This will enhance cold hardiness of guava germplasm to genetically improve it for adaptation to at least mild temperatate climatic areas such Georgia and the Southeast.
Publications
- Opoku-Fianko, Nora, M. Mutua, N. Joshee, and A. K. Yadav. In vitro shoot proliferation and root induction in guava (Psidium guajava L. Cv. Beaumont). National Institute of Science, Student Poster Presentation Conference, University of South Carolina, Columbia, SC, 26 March 2002.
- Mutua, Mercy K., Nirmal Joshee, and Anand K. Yadav. In vitro micropropagation of Datura metel, a medicinal plant. National Institute of Science, Student Poster Presentation Conference at the University of South Carolina, Columbia, SC, 26 March 2002.
- Joshee, N., M. Mutua, and A. K. Yadav. 2002. In Vitro Shoot Proliferation in Guava as Influenced by Genotype. International Hort Congress Abstracts p. 513.
- Joshee, N. M. Mutua, F. Zee, and A. K. Yadav. 2002. In Vitro Shoot Proliferation in Guava as Influenced by Genotype. pp 000-000 in Xxxxxx (editor), Acta Horticulturae, Vol # 5xx, International Society for Horticultural Science (Accepted for Publication).
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Progress 01/01/01 to 12/31/01
Outputs
This new project focusing on developing an efficient in vitro plant regeneration system, amenable to gene delivery techniques and formulating reliable protocols for gene transfer into guava either by Agrobacterium Ti plasmid apprach or microprojectile bombardment technique, had an encouraging start. Dr. Ramana Gosukonda, a Post-Doctoral Research Associate was transferred to the guava project to carry out biotechnology research on in vitro micropropagation and genetic transfrmation. An undergraduate student worker was hired to assist in the micropropagation research on guava. Necessary equipment and laboratory supplies were procured and a plant tissue culture room became fully operative to house cultures of guava and other species. Preliminary work with explants from three guava cultivars at FVSU and tissue culture media using different supplements was initiated for shoot multiplication from mature tissues and somatic embryogenesis from immature tissues. Further,
greenhouse investigations were conducted on different antibiotics to eliminate Agrobacterium from three guava genotypes. Rooting of micropropagated young shoots was carried out successfully. Also, we received three cold hardy genes necessary for conducting research on genetic transformation to enhance cold tolerance of guava. Guava plants of 6 cultivars rooted by layering that we received from the USDA, Hilo, HI, were successfully established in the greenhouse.
Impacts
Developing biotechnology for in vitro regeneration and genetic transformation of guava will enable scientists to integrate cold hardiness genes into popular guava varieties. This will enhance cold hardiness of guava germplasm to genetically improve it for adaptation to at least mild temperatate climatic areas such Georgia and the Southeast.
Publications
- Gosukonda, R.M., Singh, M. and Yadav, K.A. 2001. Optimum levels of Antibiotics to Eliminate Agrobacterium and In Vitro Selection in Guava. HortScience 36(3):535.
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