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p f\J . ti-\ f5 y _J-(5 3 qqoyo ID- ~ 1 iJ FINAL REPORT Covering Period: 8/21/90 to 8/20/95 (includes no-cost extension) Submitted to Human Capacity Development Bureau for Global Programs, Field Support and Research U.S. Agency for International Development SOMATIC EMBRYOS FOR GERMPLASM STORAGE AND CLONAL PROPAGATION Principal Investigator: Dennis J. Gray Grantee Institution: University of Florida Collaborator: Magaly Dufour Institution: Centro Agronomico Tropical de Investigation Y Ensenanza (CATIE) Project Number 10.278 Grant Number: DHR-5600-G-00-0057-00 Al.D. Grant Project Officer: Dr. Joel Cohen Project Duration: 8/21/90 to 8/20/95 (includes no-cost extension) REC'D IN R&D/R MAY 7 1996 2 Table of Contents: Title page ............................................................................................................................................. 1 Table of contents ................................................................................................................................ 2 Executive summary ............................................................................................................................ 3 Research objectives ............................................................................................................................ 4 Methods and results ........................................................................................................................... 4 Grape ....................................................................................................................................... 4 Coffee ...................................................................................... ,.................... "" ......................... 17 Impact, relevance and technology transfer .................................................................................... 23 Project activities/outputs ................................................................................................................ 23 Project productivity ........................................................................................................................... 24 Future work ........................................................................................................................................ 25 Literature cited ................................................................................................................................. 25 3 Executive Summary: The purpose of this project was to develop alternative methods of germplasm conservation and propagation for crops such as grape and coffee. Genotypes, including cultivars, of these crops currently can be conserved only as plants growing in a plantation or vineyard setting. This method of conservation is so expensive as to have stifled conservation of these crops as well as others like them. Preserving both the genetic diversity and the genetically-fixed phenotypes of such crops is of paramount importance in order to insure an adequate genetic base for future improvement and to meet changing environmental stresses. Successful implementation of this project would eliminate the primary expense of germplasm conservation by allowing cultivars to be maintained as clonal somatic embryos (instead of intact plants), which then could be kept more efficiently in conventional seed germplasm repositories. Many other crops and native species cannot currently be conserved for similar reasons; this represents a critical problem as native plants are destroyed due to population growth. During this project, we advanced toward the goal of storable somatic embryos by accomplishing the following: 1) We increased the number of genotypes from which useful cultues can be recovered. 2) We developed a highly-refined culture system for initiating and manipulating embryos. 3) We refined methods to control embryo maturation and plant recovery. 4) We evaluated various methods to dehydrate and/or cryopreserve embryos for prolonged storage. These improvements have moved us closer to a useful system. As a direct result of this project, a significant literature base has emerged that both has disseminated this new information and has effectively increased the awareness and promoted the idea of clonal germplasm conservation to a wide international audience. This latter conclusion is based on the numerous contacts and reprint requests recieved from developing countries and the emergence of similar research goals, as judged from surveying the literature and reviewing grant proposals, which cite our AID-supported publications. 4 A) Research Objectives: The purpose of this project was to develop technology in order to utilize somatic embryos as "synthetic seeds" for clonal germplasm conservation and propagation of two model species (coffee and grape). This project is of strategic importance to insure the conservation of most clonally-propagated species, by drammatica1ly decreasing the cost of long-term maintenance. Many of these species already are being lost due to population growth and destruction of natural environments in conjunction with near total lack of adequate funds to accomplish conventional conservation. Research in this specific area or related areas of technology are underway worldwide. Recently, an entire book was devoted to the subject of using somatic embryos as synthetic seeds (Redenbaugh, 1993) that contains an overwhelming documentation of similar research being conducted by others. The book also contained two chapters (Gray and Compton, 1993; Gray et al., 1993) that resulted from and were directly supported by this project. This specific project was not supported by other organizations. B) Methods and Results: Accomplishments are separated based on crop type (grape or coffee). Published or accepted manuscripts are referenced and attached to this report. Grape: For grape, emphasis was placed on: 1) development of methods to produce embryogenic cultures from additional species and varieties, 2) refinement of culture conditions to optimize growth of embryogenic cultures, and 3) evaluation of culture pretreatments to cause better embryo maturation and desiccation tolerance. Development of methods to produce embryogenic cultures: The ability to produce embryogenic cultures from many grape species and varieties has been limited by apparent genotypic specificities. Of approximately 30 described species in the genus Vltis, embryogenesis has been reported in only three: V. longii, V. mpestris and V. vinifera, or hybrids between these and other species. It is fortunate that V. vinifera is responsive, since it is the major grape of commerce. Genotypic specificity also exists within these species, since only certain varieties will respond. Our methodology to induce and maintain embryogenic cultures of V. rotundifolia (muscadine grape) is presented in the attached AID-supported publication (Gray, 1992). Thus, this report extends the embryogenic response to a new species, V. rotundifolia, which is grown extensively in the Southeastern US and contains approximately 100 named varieties. Varieties are variously used for fresh fruit, juice, jelly and wine production. Our approach has been to utilize a combination of ovule and embryo culture to induce embryogenic cultures. Although resulting cultures arise from zygotic embryos and not desirable clonal tissue, this methodology demonstrates that embryogenesis is possible in this genus. We refined the methodology and demonstrate the technique for additional varieties. Reports describing this research were published (Gray, 1992; Gray and Hanger, 1993). Both papers acknowledge Al.D. funding. 5 Other research to develop embryogenic culture ~stems: Leaves from in vitro micropropagation cultures of V. rotundifolia varieties mentioned above and V. vinifera 'Cabernet Sauvignon', 'Grenache', 'Ruby Cabernet' and 'Thompson Seedless' were cultured on Nitsche's medium with 5 µ.M 2,4-D and 1 µ.M BA in attempts to induce embryogenic cultures. To date, we have found that 10 percent of 'Thompson Seedless' leaves produce embryogenic cultures and a single embryogenic culture of 'Cabernet Sauvignon' has been recovered. We were able to produce embryogenic cultures from leaves of V. rotundifolia 'Carlos' by utilizing a unique three-step culture system. In this system, leaves obtained from micropropagation cultures were first cultured on Nitsche's medium with 5 µM 2,4-D and 1 µM BA for four weeks, then transferred to medium with 5 µ.M NAA and 1 µ.M BA for four weeks prior to transfer to basal medium. Apparently, the second medium promotes cell divisions, resulting in the survival of more embryogenic cells. The three-step medium culture system was tested on additional germplasm. We first developed a cultivar co11ection of potted stock plants that could be maintained in the greenhouse. These stock plants then were utilized as source material for in vitro micropropagation cultures. Cultures of 15 cultivars were established, leaves of which were utilized as explants in somatic embryo initiation experiments. Methodology for obtaining these cultures will be published (Compton & Gray, 1994; Myerson et al., 1994). Table 1 lists the micropropagation cultures that were be used. Subsequently, leaves from established cultures were placed into the three-step culture system. The following cultivars produced embryogenic cultures in this system: Carlos, Dixie, Autumn Seedless, Cabernet Sauvignon, Thompson Seedless and Niagara Seedless. Optimization of media for growth of perennial embryogenic cultures:

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