CITRUS RESEARCH BOARD

Transcription

1 CRB Project Plan Research Grant Proposal for FY CRB Project No. _ CITRUS RESEARCH BOARD PROJECT PLAN - RESEARCH GRANT PROPOSAL FOR FY-2011 Fiscal Year: Anticipated Duration of Project: long-term This Project is: New or X Ongoing (Year of ) Project Leader: _Mikeal L. Roose Name Location: Botany and Plant Sciences, University of California, Riverside, CA Address (University department, if applicable) Mailing Address (if different): Phone: FAX: _mikeal.roose@ucr.edu Cooperating Personnel: Project Title: Breeding of New Citrus Scion Varieties Keywords:, grapefruit, lemon, seedless, mutation Abstract (limit 200 words):(clearly and succinctly state what your project is about why you are doing it and expected out come and how the industry will use these outcomes) The objectives of this project are to develop new, orange, lemon and grapefruit-type cultivars suitable for California conditions. For cultivars, important traits are seedlessness, easy peeling, good flavor, high rind color, economic yield, and low tendency to alternate bearing. For grapefruit-type cultivars, the primary objective is to develop deeply red pigmented cultivars with sweet taste and low bitterness suitable for production in desert or inland valley regions. For lemon, the primary objective is a Lisbon type with very few seeds. The major approaches being used are hybridization-selection for s and grapefruit, and mutation induction to obtain seedless forms of existing s, oranges, and lemons. Promising selections are evaluated in field trials for tree size, yield, fruit quality, and disease susceptibility. Cultivars developed in the project are released to nurseries, generally under licenses from UC and thereby made available to growers. Problem and its Significance*: (include literature review) The California citrus industry is heavily dependent on oranges, with navels comprising the largest proportion of the total acreage. International and domestic markets are now demanding a wider variety of citrus fruit, particularly seedless, easy-peeling s. Mandarins make up a larger proportion of citrus production in most parts of the world than they do in California, but plantings have recently expanded considerably. Continued expansion of this market requires production of high quality fruit that meets the demands of marketers and consumers. In recent years California growers and researchers have imported many cultivars for evaluation under California conditions, but it is yet unclear whether these introduced cultivars will fill all the market

2 CRB Project Plan Research Grant Proposal for FY niches available. One major problem with this approach is that many imported cultivars are not well-adapted to California conditions. Examples of this are the problems experienced with red grapefruit cultivars originally selected in Texas and Florida, and Clementines selected in Spain and Morocco. These selections have been less successful in California than in the areas in which they were selected. An alternative approach to introduction and evaluation of existing cultivars is to develop new cultivars within California through breeding and selection within California. There are several comprehensive reviews of the overall field of citrus scion breeding. Of particular importance are several chapters in a recent book (Khan, ) on citrus breeding and biotechnology. Previously the field was reviewed by Soost and Roose (1996). There are active citrus breeding programs in many countries with significant citrus industries. All or nearly all are centered in public institutions, presumably because the long time frame for development of new citrus varieties discourages private investment in this activity. Many programs include both hybridization-selection approaches and mutation breeding for seedlessness, but some also use somatic hybridization (cell-fusion) to produce tetraploid hybrids that can be used as parents of crosses (Gmitter et al., ). This review is limited to methods similar to those used in our program. We exclude somaclonal variation, somatic hybridization for scion development, and transgenics, approaches that have not yet resulted in any released cultivars. Some important breeding programs are those at the USDA in Florida (McCollum et al., ), University of Florida (Gmitter et al., ), Texas (De Graca et al., 2006), IVIA in Spain, Italy (Russo et al., 2006), China, Argentina, Australia, Brazil, South Africa and others, but published descriptions of many of these programs are not available. In the past, Israel also had a large breeding program directed by Dr. Vardi, but this is now apparently less active. Hybridization. Essentially all programs are directed at producing seedless (or low-seeded) cultivars, but there are several major strategies toward this end. One approach, used by the UCR program and some others (Ollitrault et al., ), is production of triploids (plants with 3 sets of genes rather than two) by hybridization between diploids (2 sets of genes) and tetraploids (4 sets of genes). Triploids are typically very low-seeded because they have a high degree of male and female sterility. Tetraploid parents originate in several ways: 1) as spontaneous hybrids in crosses (particularly diploid x tetraploid crosses), 2) by treatment of buds of diploid varieties with colchicine, a type of mutagen, and 3) by somatic hybridization, a process in which cells of each "parent" are fused in culture and plants regenerated. Somatic hybrids will contain the full genome of each parent. Generally, major limitations to the triploid hybridization strategy are that few tetraploid parents have been available, the number of viable seeds per pollination is often low, and many triploids tend to be thorny. Another strategy to identify triploids, used in several Mediterranean countries, is selection for spontaneous triploids in diploid x diploid crosses by selecting small and abnormal seeds which often contain triploid embryos. Typically this requires "embryo rescue (tissue culture of small embryos). There is potential to produce triploids directly by somatic hybridization of haploid and diploid cells, but this has been limited by availability of suitable haploids in citrus. Tahiti lime is the best known triploid cultivar, but there are many others including Oroblanco, Melogold, the three "TDE" hybrids, Tacle, Clara, Mandared, Winola s and others. Seedless varieties are sometimes obtained by hybridization between diploid parents, particularly if one parent has male (e.g. satsuma) or female sterility (e.g. Mukakukishu). This approach limits choice of parents to those with suitable sterility. However, seedless varieties also occur occasionally among progeny of crosses between seedy diploid parents (e.g. Gold Nugget). Although seedlessness is preferred, some new varieties have been released that are seedy in mixed blocks, for example Sugar Belle (LB8-9) from the University of Florida ( Mutation breeding. Mutation breeding (Roose and Williams, ) can be used to obtain low-seed selections of existing genotypes. Generally, radiation has been used as the mutagen because it causes chromosome breakage that leads to rearrangements that cause sterility. Thus the "target" of the mutagen is not the specific genes involved in

3 CRB Project Plan Research Grant Proposal for FY fertility or seed development, but the entire genome, a much "larger" target that is easier to hit so that low seeded selections occur at quite high frequency in comparison with mutations that alter other traits such as fruit size, color, maturity date, etc. Budwood is typically irradiated with gamma-rays and trees are propagated from this for screening. A potential problem is that a dormant bud contains many different cells that will divide as it grows. Each cell can potentially have a unique mutation or no mutation, so that when the bud grows, there will be clusters of those daughter cells that may differ in their genetic makeup (a chimera). As branches form on the original trees, and additional trees are propagated from its buds, there is potential for some trees or branches to derive from nonmutant cells or express other unique mutations that may not be desirable. To minimize this effect, some programs have used additional generations of bud propagation before producing trees for evaluation. However, there are no published reports of such off-types occurring among trees in citrus mutation breeding programs. Examples of cultivars from mutation breeding include the Star Ruby grapefruit which was selected as a seedling after irradiation, and Rio Red grapefruit which is derived from a tree selected after irradiation (da Graca et al., 2006). Mor is a lowseeded selection of Murcott resulting from irradiation and released in Israel in Nova SL is a low-seeded form of Nova developed in South Africa in Wuhexueganlinghao and Fuyu are s released in China in 1998 and 2000 following irradiation. Many other selections are being field tested. New cultivars. Summaries of 65 cultivars released in various countries between 1985 and 2000 are given by Nicotra (2001), although some of the reported origins are incorrect (Nova SL is from South Africa, not Australia). Some other recent releases are noted above. References da Graca J.V., E. S. Louzada, and J. W. Sauls The origins of red pigmented grapefruits and the development of new varieties. Proc. Int. Soc. Citric McCollum, T., R. Niedz, and E. Stover.. Citrus scion breeding at the USDA/ARS U.S. Horticultural Research Laboratory. Gmitter, F. G. Jr, J. W. Grosser, W. S. Castle, and G. A. Moore..A comprehensive citrus genetic improvement programme. In: Khan, I. A. (ed.). Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, U.K., pp Khan, I. A. (ed.).. Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, U.K.. Nicotra, A Mandarin-like Hybrids of Recent interest for Fresh Consumption. Problems and Ways of Control. Ollitraut, P., Y. Froelicher, D. Dambier, F. Luro and M. Yamamoto.. Seedlessness and ploidy manipulations. In: Khan, I. A. (ed.). Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, U.K., pp Roose, M. L. and T. E. Williams.. Mutation breeding in Citrus. In: Khan, I. A. (ed.). Citrus Genetics, Breeding and Biotechnology. CAB International, Wallingford, U.K., pp Russo, G., G. Reforgiato-Recupero and S. Recupero New triploid hybrids of Citrus in Italy. Proc. Int. Soc. Citric Soost, R. K. and M. L. Roose Citrus, p In Janick, J. and J. N. Moore (eds.), Fruit Breeding, Vol. I: Tree and Tropical Fruits. John Wiley & Sons, Inc. Objectives*: (succinctly state each objective and milestone, ie the time expected to successfully complete an achieve each objective) The time and effort necessary to achieving specific objectives in a breeding program are difficult to predict with

4 CRB Project Plan Research Grant Proposal for FY precision because we lack adequate knowledge about the inheritance of many essential or desirable traits and how various traits are interrelated. Both hybridization and mutation breeding involve substantial elements of chance that determine whether hybrids or mutants with necessary genes or combinations of genes can be produced. The objectives and milestones below must be considered in this context. For each type of cultivar, the major milestones are 1) production of initial trees (hybrids or irradiated buds), 2) field planting of initial trees, 3) selection of candidate varieties from initial trees, 4) production of registered (or equivalent) source trees, 5) planting of replicated trials, 6) evaluation of replicated trials, 7) application for permission to release, and 8) official release of new cultivar. For each objective below, I give the current status (milestones achieved). The lower part of the table shows specific examples of selection of low-seeded cultivars from mutation breeding Objective Red grapefruit (anthocyanin) low acid Red grapefruit (carotenoid) Early-season Mid-season Late-season Red fleshed, early-season, midseason, late-season W. Murcott Daisy Fairchild Encore Milestones Method Initial trees produced Hybrid Hybrid Hybrid Hybrid Hybrid Hybrid Mutation Mutation Mutation Initial in field st selection Registered (VI) source Replicated trial planted Evaluation of trials Apply for release Release GN 2002-TDE Mutation Tango Mutation DaisySL Mutation FairchildLS Mutation exp.

5 CRB Project Plan Research Grant Proposal for FY Kinnow Nova Clementine pink gpft. and hybrid Cocktail gpft Lisbon lemon Eureka lemon Mutation Mutation Mutation Mutation Mutation 2006 Mutation 2002 Mutation ? ? exp , Project's Benefit to the Industry*: (How will the industry utilize your research results or product) This project is expected to develop new citrus cultivars that will expand domestic and international markets opportunities for California growers. Such opportunities may increase profits without reducing returns on most existing plantings. New cultivars are important in maintaining a competitive edge for California growers. California growers are given first access to new varieties developed in this program, and current licenses do not permit import of fruit from UC-patented varieties into the US. Research Collaboration* (be specific): Lindcove staff collaborate by providing fruit quality analysis and periodic evaluation of fruit quality on trees planted at Lindcove. Occasional off-station labor is also provided. Dr. Tracy Kahn provides evaluation data on a few advanced hybrids in the Citrus Variety Collection and other evaluation blocks, and Toni Siebert has assisted with tissue culture, pollination and grafting of irradiated buds. Growers collaborate in providing test sites in areas where UC field stations are not available (Willits and Newcomb, Arvin; Brokaw Nursery, Santa Paula; Rocky Hill Farms, Woodlake). Plans and Procedures* (use this section to describe your experimental design site location(s)and elaborate on objectives and milestones) 1) Hybridization new hybridization efforts will be focused primarily on early and very-late season triploid types. Parents will include Robinson, Lee, Clementines, and an unnamed tetraploid hybrid for earliness and Encore, Kara and a tetraploid pummelo x blood orange hybrid for mid/lateness. These may be adjusted later depending on the number of seeds obtained from crosses. A specific target trait within the hybridization scheme will be to introduce red/pink-flesh color as possible. If flowering duration is normal we expect to make about 500+ pollinations. In 2011, Dr. Federici and Ms. Kupper will again assist Mr. Williams in performing crosses to better meet objectives 1, 2 and 9. 2) Effects of new cultivars on seed content of adjacent varieties. As a regular part of the irradiation evaluation

6 CRB Project Plan Research Grant Proposal for FY program additional hybridizations will be performed to test the ability of pollen from irradiated selections to set seed in existing commercial varieties. These crosses include crosses with pollen from the non-irradiated parent variety as controls. Although the primary purpose of these pollinations is to test seed set, they also have potential as a source of new hybrids because most of the parents involved are high quality s or hybrids, although polyembryony will be a limiting factor. Seeds from pollination by low-seeded selections may give rise to low-seeded offspring, depending on the genetic mechanism responsible for reduced seed content in the parent. In -11 we expect to use the irradiated selections Encore IR6, Kinnow IR5, and Nova IR10 to pollinate onto Clementine and W. Murcott females. The low-seeded lemon selection Limoneira 8A IR1 will also be tested, although the hybrids produced from these crosses are less likely to be valuable. We will also test pollen viability of the many advanced selections from budwood irradiation since this will predict whether they produce seeds in other cultivars. 3) Conduct pollinations to determine potential for cross-pollination-induced seed set in promising irradiated selections. Selections to be pollinated include Encore IR6, Kinnow IR5, and Limoneira 8A IR1. Pollen sources will include W. Murcott, Clemenules and Fairchild, three cultivars with high pollen viability that are likely to be grown near the new selections. For the reasons listed above, hybrids obtained from these crosses are propagated and planted for evaluation. 4) Induction of seedless mutations by irradiation. Specific targets for additional irradiation will include some additional lemon varieties, both Lisbon and Eureka types, newly available Clementines, two diploid hybrids that have good fruit quality and are low seeded in isolation. A new effort is re-irradiation of Fremont IR2 and Fallglo IR2, selections which are promising but not sufficiently low-seeded. We expect to produce at least new trees at Lindcove from 1500 buds. Trees from earlier irradiation will continue to be propagated and planted in the field at UCR and Lindcove. 5) Propagation of existing hybrids. Continue propagation of seedlings from previous hybridization, planting most in the field in Riverside at moderate density. 6) Molecular-genetic tests. Test representative seedlings of each cross with DNA markers to verify hybridity, and, as possible, with flow cytometry to determine ploidy level. Advanced selections will be tested for markers to uniquely identify them. 7) Initial evaluation of hybrids. Evaluate all fruiting hybrids for fruit quality traits including maturity date, fruit size, fruit shape, fruit color, flavor, seediness, yield, tree size, diseases and other traits. 8) Initial evaluation of trees from irradiated buds. All field planted trees will be evaluated for vigor, seediness, fruit size, fruit quality and pollen fertility (which is related to seediness in solid blocks). Selections that show low seed number for two years will be propagated for larger-scale evaluation. 9) Advanced trials. Selected hybrids and low-seeded selections will be evaluated in advanced trials at 5 locations that represent the major production areas of California: UCR, CVARS, Santa Paula, Arvin, Lindcove. Trees at 2 additional sites (SCREC and Rocky Hill) will be used only to evaluate seed content. During -11 we expect identify new candidates and begin the propagation of new trees for 2 nd test evaluation trials. Data on fruit quality, particularly seediness, will be collected from fruiting trees at each location. Yield records will be collected at Lindcove, UCR, and other locations where picking can be arranged. Trees of DaisySL, Tango, FairchildLS, Kinnow IR5, and Encore IR6 at Lindcove were screened in spring to evaluate productivity when pollination is blocked. DaisySL, Kinnow IR5, and Encore IR6 will be screened again in Nova IR10 will be screened at UCR because there are no trees of this selection at Lindcove. Trees of 24 now eliminated selections have been or are being removed at some locations and will be replaced with new selections, as resources allow. Future plantings will not include the Rocky Hill and South Coast sites, but we will continue to record data on trees in these plantings until adequate data on the selections in these trials is available. New plantings will include 18 tree per variety at the remaining 5 locations, and we plan to expand the number of rootstocks to 3: Carrizo, C35 and Rich 16-6 trifoliate with 6 trees of each combination. A few trees of

7 CRB Project Plan Research Grant Proposal for FY lemon selections will be propagated on Macrophylla. This wider range of rootstocks will better test compatibility and rootstock effects on production and fruit traits. 10) Release of new selections. Release of KinnowLS has been approved, but release is pending development of an adequate bud supply at CCPP. Because growth of some trees from the CCPP source has been slow, an experiment to evaluate different bud sources of KinnowLS was started in June. A decision on release of KinnowLS buds will be made when we have results of this experiment, in approximately December. Final drafts of release requests for Encore IR6 and Nova IR10 are nearly complete and will be submitted to the Dean and patent disclosures to the Office of Technology Commercialization. They have already completed VI at CCPP and we expect to release them in January A new program to provide pre-release budwood of these and other advanced selections to California citrus nurseries has been approved and implemented. This will allow nurseries to increase the budwood supply in advance of release. After release, additional budwood will be available from registered trees at CCPP. No decision on patenting Encore IR6 and Nova IR10 has been made. We will continue to work with the California citrus industry through the CRB to explore mechanisms that limit acreage of new cultivars while making them available to all California growers. The probable timeframe for release of new varieties from crosses initiated under this project is about years from hybridization or mutation to release. New hybrids generally fruit after 4-6 years, evaluation of initial hybrid populations will require a minimum of 2-3 additional years, followed by propagation of trees for additional trials at several locations which requires at least 5 additional years. Although the time required for success in this project is clearly long, if work is not undertaken now, there will be no payoff in the future. Other Funding Sources for this Project (current, pending, potential; can this project be used as matching funds for other funding sources) We plan to request renewal of funding (about $20,000) from the California Citrus Nursery Board that would begin in January The PI received about $35,000 in total funding (for all projects) from the UCR Agricultural Experiment Station for -11. An unknown amount of additional funding, perhaps $30,000, may be available in late fall from gifts. Note that salaries for the 3 SRAs responsible for this project and project will require about $40,000 from these additional sources. We expect that part of the cost of greenhouse upgrades at Lindcove will be paid by UC-ANR who operate field stations. Technology Transfer* (include any potential intellectual property issues; steps necessary for grower utilization extension/communication component): Fruit of varieties under development is made available to growers for evaluation at field days at Lindcove and seminars and conferences at other locations. Providing growers with new cultivars is the most direct technology transfer resulting from this project. We develop and disseminate information on characteristics of cultivars developed in the program. Such information should be provided to growers by nurseries directly, through web sites, articles in the agricultural industry publications, or directly by UC personnel. We will cooperate with Farm Advisors to organize meetings/displays of new cultivars in various counties when the fruit are mature. It is likely that many cultivars developed in the breeding program will be patented by the University of California. We intend to continue to provide all California growers with equal access to such patented cultivars. We will continue to work with industry committees to develop appropriate release procedures. Budget Justification: Salaries include 12 months of SRA at 75% time (Tim Williams) mainly responsible for breeding, propagation and evaluation activities, 4 months of salary for an SRA (Claire Federici) responsible for assisting with hybridization, fruit collection, fruit evaluation, pollen testing, two months of salary for an SRA (Ricki Kupper) responsible for assistance with fruit collection, fruit quality studies, and pollination, and 6 months of salary

8 CRB Project Plan Research Grant Proposal for FY for an Agricultural Technician (Juan Alvarez) responsible for watering, assistance with fruit evaluation, and field planting. Supplies and expenses include pots, soil, supplies for marker analysis, truck rental to move trees to planting sites and other materials. Supplies and expenses also includes a contribution of $10,000 toward addition of screens and double doors on a greenhouse at Lindcove that is used mostly for this project. These modifications will be necessary to meet new CDFA standards for citrus propagation. Travel expenses include 12 overnight trips to Lindcove and other San Joaquin Valley sites for fruit collection, tree evaluation, and planting (average cost $100 per day per person plus $300 mileage). About 12 similar trips to CVARS, Santa Paula, and South Coast do not usually involve overnight stays, but incur mileage charges (about $100 per trip). An expected two trips to attend in-state meetings or present results to growers are also included. Propagation of trees at Lindcove is expected to cost about $10.50 per tree. Recharges cover initial propagation of about 930 trees and planting of about 100 trees at Lindcove and fees to maintain 14 acres at Lindcove, 13.5 acres at UCR, 5 acres at CVARS, and 4.5 acres at South Coast. Recharge rates at field stations have increased this year.

9 CRB Project Plan Research Grant Proposal for FY Project Budget Department Account Number: (if applicable) Salaries and Benefits: Year: Year: Year: Postdocs/Research Assistants $ 0 SRA's 61,599 Lab/Field Assistance 14,412 Benefits 36,944 Supplies and Expenses: 14,000 Equipment: Operating Expenses and Travel: 6,000 Lindcove Recharges: 13,000 Lindcove Packline: 0 Other: UCR and CVARS 16,600 SCREC 5,210 propagation at Lindcove 5,054 ANNUAL TOTAL: _172,819 Specifics regarding contract (i.e., split funding to more than one PI): Signatures Project Leader: Date: Date: Dept. Chair: Date: Campus Approval: Date: