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NOVEL TRIPLOID CITRUS FRUITS BY INTERPLOID HYBRIDIZATION;
POTENTIAL FOR LIME IMPROVEMENT
Jude W. Grosser*, Zenaida Viloria, and Frederick G. Gmitter, Jr., University of Florida, IFAS, Citrus Research and Education Center, Lake Alfred, FL 33850 ABSTRACT
Our citrus improvement program has relied heavily on interspecific hybridization of complementary parents as needed to package appropriate traits. Seedlessness is also highly desirable in new citrus fruits, thus we are using ploidy manipulation to generate seedless triploids. The most efficient method for generating unique triploids is interploid crossing of teteraploids with diploids, using monoembryonic citrus as females. The availability of quality tetraploids is essential to the success of this approach, and we have invested a great effort to build our tetraploid germplasm. We have produced quality autotetraploids by several methods, including the screening of seedling populations, in vitro colchicine treatments, and as byproducts from somatic fusion experiments. Crosses of these with selected diploid females of different citrus species generate triploid interspecific progeny. We have also produced numerous allotetraploid somatic hybrids via protoplast fusion, using our model system of fusing embryogenic cell culture protoplasts with leaf protoplasts of a second complementary parent; many of these are interspecific hybrids. We now have many quality tetraploids that are flowering, and we have an evolving program of interploid hybridization that has generated more than 16,000 triploids to date, mostly interspecific. Significant progress has been made in three primary areas: hybrid mandarin improvement by combining mandarin germplasm with sweet orange and/or grapefruit germplasm with emphasis on flavor, shelf-life and convenience to eat; hybrid pummelo/grapefruit improvement with emphasis on flavor, red pigmentation, disease resistance and reduced furanocoumarins; and acid-citrus fruit improvement (lemon and lime types) with emphasis on disease resistance, cold-hardiness and oil quality. This latter work has demonstrated good potential for applying these technologies to lime improvement in Mexico, as several high quality seedless lime-like hybrids have been produced. We have also shown that canker resistance from kumquat can also be incorporated into such hybrids. Examples of unique new seedless hybrids in each category will be provided, with focus on the lime-like hybrids. Key words: Seedlessness, Ploidy, citrus breeding
TECO M A N , CO LI M A.,, 4 , 5 y 6 DE N O V I EM BRE 2 0 10 INTRODUCTION
Ploidy manipulation has become an important component of scion improvement programs, especially to facilitate the production of improved seedless cultivars. Seedlessness has become a primary breeding objective of all citrus fresh fruit improvement programs, as seedless fruits are much preferred in the marketplace. Somatic hybridization via protoplast fusion technology provides the opportunity to combine complementary elite diploid scions into INTERPLOID HIBRIDIZATION
Flowering somatic hybrids are being used as breeding parents in interploid crosses with selected complementary diploid parents to generate triploid progeny (Grosser and Gmitter, 1990; 2005; Grosser et al. 2000). Autotetraploids, often a by-product of somatic hybridization experiments and also produced by other in vitro techniques, are also used as parents in interploid crosses. However, more variation in triploid progeny is generally observed when using allotetraploid parents. In citrus, two types of embryos can be produced in seed, either of nucellar (genotypically equivalent to the mother tree) or zygotic origin. Oranges, grapefruit, lemons, and many mandarins produce seed that generate multiple embryos predominantly of nucellar origin (referred to as polyembryonic), making them difficult to use as female parents in conventional crosses. Citrus accessions that produce monoembryonic seed generally contain zygotic embryos, and of course these are the preferred seed parents for breeding. The number and diversity of high quality, monoembryonic diploid parents is limited, though some that have been used by various programs around the world include 'Clementine' selections, , 'Fal glo', 'Fortune', and 'Temple' for mandarin breeding. In the UF-CREC citrus breeding program, we are also utilizing a number of good new monoembryonic parents from our diploid citrus breeding program, many shown previously to possess good general combining ability, producing families with relatively high percentages of hybrids that yield fruit with acceptable quality. For grapefruit/pummelo improvement, pummelos selected high quality pummelos are being used as seed parents. In general, interploid crosses of diploid seed parents with tetraploid pollen parents areproblematic because fully developed seeds containing viable embryos are not usually recovered. In most cases when fully developed seeds are occasionally recovered, these TECO M A N , CO LI M A.,, 4 , 5 y 6 DE N O V I EM BRE 2 0 10 generally grow into seedlings that are tetraploid, presumably the product of an unreduced gamete from the seed parent being fertilized by the pollen parent. The problem of abnormal seed development from 2x X 4x crosses has been attributed to an unfavorable embryo:endosperm ratio, resulting in endosperm failure and subsequent embryo abortion. In vitro embryo rescue is required to circumvent this for efficient triploid embryo recovery (Viloria and Grosser, 2005; Viloria et al., 2005). If a tetraploid tetraploid seed parent is used, this problem does not exist, as is the case with the unreduced gametes coming from diploid seed parents. Unfortunately, the number of high-quality monoembryonic tetraploids is currently quite limited. Moreover, production of monoembryonic tetraploid somatic hybrids is difficult, because the fusion technology requires the use of an embryogenic callus or suspension culture arising from a nucellar parent to provide the necessary totipotency, and in general most somatic hybrids derived from such a donor parent reveal the dominance of nucellar embryony. To date it has not been possible to generate embryogenic callus cultures of monoembryonic citrus types. However, very recently we have combined nucellar parents with monoembryonic parents, with the latter being the leaf-derived protoplast donor. A few of these hybrids have proven to be monoembryonic and are being utilized in interploid crosses as females (for example 'Succari' sweet orange + 'Hirado Buntan Pink' zygotic pummelo). We are still waiting for several other such hybrids to pass through juvenility to flowering. Our program has generated more than 16,000 triploid citrus hybrids from interploid crosses, with a few thousand of these being fathered by somatic hybrids (Gmitter and Grosser, 1990; 2005; Viloria and Grosser, 2005; Viloria et al. 2005; Grosser et al., 2000). Our first triploid hybrids are now overcoming juvenility, and seedless triploid fruits have been obtained in all three categories to provide proof of concept. For mandarin improvement, additional breeding objectives beyond seedlessness include easy-peeling, good external/internal color, good flavor, a range of maturity dates, and good shipping ability/shelf-life. Our program is continuing efforts to generate improved breeding parents, and we recently reported 9 new somatic hybrids and 5 autotetraploids produced by protoplast fusion, with focus on the zipper- For grapefruit/pummelo improvement, primary breeding objectives include improved resistance to citrus canker, fruit quality (flavor and color), and extended maturity seasons. Also of interest is the potential to reduce or eliminate furanocoumarins in new grapefruit-like cultivars. Furanocoumarins are the chemicals found in all commercial grapefruit cultivars that interact negatively with prescription drugs, preventing many elderly consumers from enjoying TECO M A N , CO LI M A.,, 4 , 5 y 6 DE N O V I EM BRE 2 0 10 grapefruit. We have found that several of our pummelo breeding parents and triploid progeny have little or no furanocoumarins (Chen et al, submitted). For acid lemon/lime improvement, primary breeding objectives include improved cold-hardiness and disease resistance (citrus canker, citrus tristeza virus, and witches broom), and potential new industrial oils. We have previously shown that kumquat-derived resistance to citrus canker can be transferred to triploid lime-like hybrids when using canker resistant 'Lakeland limequat' as a parent (Viloria et al., 2004). A few hundred acid-fruit triploid hybrids were grafted to rootstocks and planted in the field during 2005/2006. Many of these are now fruiting, and several interesting lime-like hybrids have been identified, with two exhibiting the small 'Mexican' lime size. Two very large-sized lime-like hybrids were also recovered. A few of these hybrids are one third sweet orange and one third lemon, yet they still show predominantly lime characteristics and aroma. These results, based on a relatively small effort, clearly demonstrate the potential of this technology for developing improved seedless lime cultivars, and such a program in Mexico could significantly benefit the Mexican Horticultural characteristics of triploid trees are also important, and these are also naturally affected by parentage. We are observing significant differences in important traits such as the level of thorniness and the length of juvenility. For example, a small population of 'Clementine' derived triploids from unreduced gametes are slower to bear fruit and much more thorny than triploid hybrids from a cross of 'Sugar Belle' ('Clementine' x 'Minneola') with the 'Nova' mandarin + 'Succari' sweet orange somatic hybrid. Thus, it is advantageous to conduct a broad range of interploid crosses using elite parents to identify the best parental combinations to achieve specific breeding objectives. LITERATURE CITED
Grosser, J.W. and F.G. Gmitter Jr. 1990. Protoplast fusion and citrus improvement. Pp. 339- 374. IN: Janick, J. (Ed.). Plant Breeding Reviews. Timber Press, Inc, Portland, Oregon, USA. TECO M A N , CO LI M A.,, 4 , 5 y 6 DE N O V I EM BRE 2 0 10 Grosser, J.W., P. Ollitrault, and O. Olivares-Fuster. 2000. Somatic hybridization in citrus: An effective tool to facilitate variety improvement. In Vitro Cellular & Developmental Biology- Grosser, J.W. and F.G. Gmitter, Jr. 2005. 2004 SIVB Congress Symposium Proceedings "Thinking outside the cell": Applications of somatic hybridization and cybridization in crop improvement, with citrus as a model. In Vitro Cellular & Developmental Biology-Plant. 41:220- Grosser, J.W., H.J. An, M. Calovic, D. Lee, C. Chen, M. Vasconcellos, and F.G. Gmitter Jr. 2010. Production of New Allotetraploid and Autotetraploid Citrus Breeding Parents: Focus on Zipperskin Mandarins. HortScience 45(8):1-4. Viloria, Z. and J.W. Grosser. 2005. Acid citrus fruit improvement via interploid hybridization using allotetraploid somatic hybrid and autotetraploid breeding parents. Journal of the American Society for Horticultural Science. 130:392-402. Viloria, Z., D.L. Drouillard, J.H. Graham, and J.W. Grosser. 2004. Screening triploid hybrids of 'Lakeland' limequat for resistance to citrus canker. Plant Disease. 88:1056-1060. Viloria, Z., J.W. Grosser, and B. Bracho. 2005. Immature embryo rescue, culture and seedling development of acid citrus fruit derived from interploid hybridization. Plant Cell Tissue and TECO M A N , CO LI M A.,, 4 , 5 y 6 DE N O V I EM BRE 2 0 10

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