In Vitro Cell. Dev. Biol.—Plant 40:442–449, September– October 2004 q 2004 Society for In Vitro Biology 1054-5476/04 $18.00+0.00

DOI: 10.1079/IVP2004547

GENETIC TRANSFORMATION OF PERENNIAL TROPICAL FRUITS MIGUEL A. GO´MEZ-LIM1* 1

AND

RICHARD E. LITZ2

CINVESTAV Unidad Irapuato, Km. 9.6 Carretera Irapuato-Leo´n, Apartado Postal 629, Irapuato GTO 36500, Mexico 2 Tropical Research and Education Center, University of Florida, Homestead, Florida 33031 (Received 17 September 2003; accepted 10 February 2004; editor E. C. Pua)

Summary Genetic transformation provides the means for modifying single horticultural traits in perennial plant cultivars without altering their phenotype. This capability is particularly valuable for perennial plants and tree species in which development of new cultivars is often hampered by their long generation time, high levels of heterozygosity, nucellar embryony, etc. Most of these conditions apply to many tropical and subtropical fruit crops. Targeting specific gene traits is predicated upon the ability to regenerate elite selections of what are generally trees from cell and tissue cultures. The integrity of the clone would thereby remain unchanged except for the altered trait. This review provides an overview of the genetic transformation of perennial tropical and subtropical fruit crops, i.e., citrus (Citrus spp.), banana and plantain (Musa groups AAA, AAB, ABB, etc.), mango (Mangifera indica L.), pineapple (Ananas comosus L.), avocado (Persea americana Mill.), passion fruit (Passiflora edulis L.), longan (Dimocarpus longan Lour.), and litchi (Litchi chinensis Sonn.). Key words: avocado; banana; genetic transformation; mango; tropical fruits.

plants in the USA from 1987 to 2000, less than 1% (approximately 40) were fruit trees (www.aphis.usda.gov/biotech).

Introduction Tropical fruits are important in the diet of people in lessdeveloped countries, and are increasingly important as exports from many of these countries. Conventional breeding of perennial tropical fruit cultivars has been limited by their long juvenile period (up to 20 years), low fertility, high levels of heterozygosity, various levels of ploidy, polyembryony, complex intraspecific incompatibility relationships, and severe inbreeding depression. Plant breeders often do not have access to adequate genetic diversity within local germplasm collections. Genetic diversity within many tropical fruit crop species is unexplored, and most cultivars are either seedlings from uncontrolled pollinations or dooryard selections. The production of many tropical fruit crops is based on a rather limited number of cultivars, which are poorly characterized for genetic traits. Biotechnologies that could increase the efficiency of tropical fruit crop improvement are essential to generate improved cultivars with novel traits. For example, genetic mapping could provide breeders with the tools to make rapid progress in crop improvement. Functional genomics could provide insights into genetic regulation of plant function and novel means for isolating genes for manipulation in transgenic plants. Older biotechnologies, including somatic hybridization, in vitro mutation induction and selection, etc., have rarely been applied to these tropical fruit species for crop improvement. These tools have only begun to be employed for many tree crops, and work with tropical crops lags far behind that with herbaceous species. Of the nearly 6000 field releases of transgenic

Genetic Transformation of Tropical Trees The difficulty in regenerating many tree species from elite or mature phase selections is one of the most serious obstacles for applying gene transfer technologies to these plants. In addition, few genotypes of a particular species have been transformed and, in many instances, these genotypes are not commercially important. Evaluating the performance of transgenic fruit tree cultivars requires approximately 12 years or until fruiting and flowering have been observed, depending on the species. Thus, molecular breeding represents a highly efficient approach for developing improved, perennial tropical fruit cultivars. The following discussion of tropical fruit crops outlines some of the research in progress, and is not meant to be an exhaustive review. Citrus (Various Citrus) Citrus species are the most widely grown fruit, and world production exceeds 102 600 000 Mt (FAOSTAT, 2004). Breeding imperatives have focused on rootstock and scion cultivar improvement. Rootstocks should be tolerant of biotic and abiotic stresses associated with roots, show good graft-compatibility with scions, show polyembryony, and support good yields. Scion breeding has attempted to retain the characteristics of existing superior selections but with improved fruit quality, yield, disease resistance, earlier or later maturing fruit, etc. Breeding goals of fruit for processing and for fresh consumption are different. Application

*Author to whom correspondence should be addressed: Email mgomez@ ira.cinvestav.mx

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of biotechnology to citrus has recently been reviewed by Moore et al. (2004). Genetic transformation studies involving citrus have been based upon organogenic and embryogenic regeneration pathways. Induction of citrus embryogenic cultures was described by Maheshwari and Rangaswamy (1958), and many later studies defined the optimum conditions for maintenance of embryogenic cultures and somatic embryo development (Kochba and SpiegelRoy, 1977a, b). The earliest citrus transformation experiments involved direct uptake of DNA into embryogenic protoplasts (Kobayashi and Uchimiya, 1989; Vardi et al., 1990; Niedz et al., 1995). Particle bombardment was used to transform embryogenic suspension cultures of ‘Page’ tangelo (Yao et al., 1996), and Hidaka et al. (1990) described Agrobacterium tumefaciens-mediated transformation of embryogenic cultures. Cervera et al. (2000) characterized 70 transgenic Carrizo citrange plants and observed altered ploidy level, T-DNA rearrangements, and low uidA expression, possibly due to multiple inserts or transgene silencing. The current protocol has involved the transformation of cells in seedling stem pieces and regeneration of shoots from organogenic cultures. Moore et al. (1992, 1993) transformed Citrus with A. tumefaciens EHA101 containing pMON9793 with npt II and uidA, and transformation of Poncirus trifoliata, trifoliate orange, using a similar technique was reported by Kaneyoshi et al. (1994). The recovery of transformed grapefruit plants has been described using A. tumefaciens strain EHA101 with pGA482GG (Luth and Moore, 1999) and strain C58C1 with pBin35SGUS (Yang et al., 2000), both containing nptII and uidA. Pen˜a et al. (1995a, b) reported efficient transformation of Poncirus £ sweet orange hybrid Carrizo citrange and sweet orange ‘Pineapple’ from internodal stem segments inoculated with A. tumefaciens strain EHA105 with 35SGUSINT. In order to overcome poor rooting of transformed shoots, they were micrografted onto Troyer citrange. Bond and Roose (1998) transformed epicotyl segments of ‘Washington’ navel orange using A. tumefaciens strain C58C1 with p35SGUSINT. Transgenic key lime was recovered by Pen˜a et al. (1997) using A. tumefaciens strain EHA105 with p35SGUSINT. The procedure required co-culture of stem pieces on feeder plates consisting of tomato cells on medium containing high auxin levels. The regenerated shoots were micrografted onto Troyer citrange seedlings. Yu et al. (2002) refined Agrobacterium-based transformation, and utilized epicotyl segments that were cut in half longitudinally to increase the wounded area of the explants. Ghorbel et al. (1999) transformed three citrus types using this procedure with A. tumefaciens strain EHA105 (pBin 19-sgfp), which contains a gene for green fluorescent protein (GFP). Recovery of transformed citrus in the mature phase has been reported. Cervera et al. (1998) partially rejuvenated mature sweet orange by grafting buds onto seedling rootstock. Internodal stem pieces were inoculated with A. tumefaciens strain EHA105 with p35SGUSINT, and then co-cultivated on tomato cell culture feeder plates. Some of the transformed regenerants flowered, and fruit were produced after 14 mo. In another study, Carrizo citrange was transformed to express the Arabidopsis LEAFY (LFY) or the APETALA1 (AP1) genes, which promote floral initiation (Pen˜a et al., 2001). The regenerants displayed an abnormal phenotype, but plants expressing AP1 had fertile flowers and bore fruit in the first year. Perez-Molphe-Balch and Ochoa-Alejo (1998) reported transformation of Mexican lime following inoculation of stem segments

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with a wild strain of A. rhizogenes containing a binary vector plasmid pESC4 that contained nptII and uidA. Shoots were regenerated directly or from the hairy roots. Genetic transformation of Citrus has focused on enhancing disease resistance and improving fruit quality. Several groups have transformed citrus with the CTV-CP gene (Gutierrez-E. et al., 1997; Dominguez et al., 2000; Ghorbel et al., 2000) or with an untranslatable version of this gene (Yang et al., 2000). Cervera et al. (2000) transformed Carrizo citrange with the yeast gene HAL2, which seems to be implicated in salt tolerance. Yang et al. (2000) also transformed grapefruit with the Galanthus nivalis agglutinin gene, an insecticidal gene. A gene for a pathogenesis-related (PR) protein from tomato (PR-5) has been expressed in transgenic sweet orange, and regenerants showed increased tolerance to Phytophthora citrophthora (Fagoaga et al., 2001).

Banana (Musa groups AAA, ABB, AAB, etc.) Bananas and plantains together are a major staple food crop of the tropics and the dessert banana is a valuable export commodity. The annual production of banana and plantain is approximately 69 000 000 Mt (FAOSTAT, 2004), and is exceeded among fruit only by Citrus. Most of the producers grow the crop(s) on a small scale for domestic consumption. Fusarium oxysporum Schlecht f. sp. cubense (E. F. Smith) Snyd and Hans (Panama disease or wilt) and Mycosphaerella fijiensis Morelet (black Sigatoka) have spread rapidly throughout the major production areas during the past generation. Despite the importance of banana and plantain for food security, conventional breeding has been ineffective for addressing these and other production problems because all important Musa cultivars are sterile triploids. World production is based upon very few cultivars that have been vegetatively propagated for centuries; somatic mutations have provided the only genetic variability. The genome size is approximately 873 Mb or six times that of Arabidopsis thaliana (Arumuganathan and Earle, 1991). Genetic transformation of Musa has focused on disease resistance and the control of fruit ripening, particularly of the ‘Cavendish’ type of cultivars. Genetic transformation of Musa has been reviewed by Smith et al. (2004a), and can be achieved in several ways, most of which are based on manipulation of embryogenic cultures. Embryogenic cultures are usually induced from immature male (Shii et al., 1992; Escalant et al., 1994) and female flowers (Grapin et al., 2000). Cote et al. (1996) and Dhed’a et al. (1991) optimized conditions for maintenance and somatic embryo development from suspension cultures. May et al. (1995) wounded the meristems of in vitro plantlets by microprojectile bombardment followed by co-cultivation with A. tumefaciens (LBA4404 strain). Although transformed plants were regenerated, this procedure has not been widely utilized because of low transformation rates and chimeras. Other successful transformation procedures have involved direct DNA uptake into protoplasts by electroporation (Sagi et al., 1994) and particle bombardment of embryogenic cultures (Sagi et al., 1995; Becker et al., 2000). The most commonly used procedure has involved Agrobacteriummediated transformation of embryogenic suspension cultures. Ganapathi et al. (2001) infected embryogenic suspension cultures of ‘Rasthali’ (ABB) with A. tumefaciens strain EHA105, and

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transformed plants were regenerated; this procedure has now been adopted by other groups. Embryogenic cultures have been transformed with genes for resistance to banana bunchy top virus (BBTV) and banana bract mosaic virus (BBrMV) (Becker et al., 2000), i.e., genes important or essential for BBTV replication and a gene encoding the BBrMV-CP. Several banana genes have been cloned (Clendennen and May, 1997; Medina-Suarez et al., 1997), including some coding for defense proteins (Clendennen et al., 1997), which should be useful for addressing disease problems. Banana has been transformed with genes encoding antimicrobial peptides, and extracts from leaves of transformed plants strongly suppress growth of Mycosphaerella fijiensis (black Sigatoka) (Sagi et al., 1998). Musa has been transformed with genes coding for plant defensins (Broekaert et al., 1995; Remy et al., 1998). Bananas have also been suggested as an appropriate vehicle for edible vaccines (Mor et al., 1998) and bananas containing malaria epitopes have been generated and are currently in field evaluation (Y. J. Pen˜a-Ramirez and M. A Go´mezLim, unpublished results). In other field trials of transgenic banana, delayed fruit ripening has been obtained using sense suppression of genes involved in ethylene biosynthesis (Balint-Kurti et al., 2001). Transformation of bananas has also facilitated the testing of novel promoters such as the constitutive banana actin 1 promoter (Hermann et al., 2001), and promoters derived from the BBTV DNA-1 to 25, which are expressed mainly in vascular tissue (Dugdale et al., 2001). Mango (Mangifera indica L.) Mango is one of the most important tropical crops, with world production of approximately 26 million Mt (FAOSTAT, 2004), which is exceeded only by Citrus, Musa (bananas and plantains), table grapes, and apples. The species consists of two ecogeographic races that can be distinguished on the basis of their seed type, i.e., monoembryonic/subtropical and polyembryonic/tropical (Mukherjee, 1998). The rapid growth of the mango industry that has occurred in the past 25 years has largely been based upon modern selections of monoembryonic mangoes, i.e., ‘Haden’, ‘Keitt’, ‘Kent’, and ‘Tommy Atkins’, which were identified from (uncontrolled) openly pollinated seedlings in Florida, and now form the basis for the international trade of fresh fruit. Mango production in the humid tropics is often based upon polyembryonic selections that originated in South-East Asia, e.g., ‘Carabao’, ‘Cambodiana’, ‘Nam doc Mai’, ‘Manila’, etc. With very few exceptions, mango cultivars have not emerged from breeding programs (Iyer and Degani, 1998). Mango breeding has not had a high priority, because it has been very difficult to achieve breeding objectives in an expeditious manner. The mango has a 7year juvenile period and the time required to evaluate seedling trees can be up to 12 years. The species is an allotetraploid (2n ¼ 4x ¼ 40) (Mukherjee, 1950) with a genome size about three times that of A. thaliana (439 Mb) (Arumuganathan and Earle, 1991). The frequency of fruit set has been estimated to be approximately 0.001% (Mathews and Litz, 1992), which is too low to permit manual, controlled pollinations. Genetic transformation of mango has been based upon embryogenic cultures derived from the nucellus of young fruit (Litz et al., 1982; Litz, 1984). DeWald et al. (1989a, b) and Litz et al. (1993) optimized the conditions for induction, maintenance,

maturation, and germination. Genetic transformation of mango has recently been reviewed by Litz and Gomez-Lim (2002). Mathews et al. (1992, 1993) transformed embryogenic cultures of ‘Hindi’ and of a ‘Keitt’ zygotic embryo-derived embryogenic line, respectively. These two studies utilized different disarmed, engineered strains of A. tumefaciens: strain C58C1 containing the plasmid pGV 3850::1103 with nptII (Mathews et al., 1993) and strain A208 containing the plasmid pTiT37-SE::pMON9749 with uidA and nptII (Mathews et al., 1992) under the control of the CaMV 35S promoter. Transgenic ‘Keitt’ plants were regenerated. Genetic transformation of mango has involved a two-step selection (Mathews et al., 1992). Only a single breeding objective of mango has been addressed using genetic transformation, i.e., control of fruit ripening. Two cDNA clones, mango 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, have been identified (Go´mez Lim, 1993), and a cDNA for mango alternate oxidase has been isolated (Cruz Hernandez and Go´mez Lim, 1995). Cruz-Hernandez et al. (1997) transformed ‘Hindi’ utilizing A. tumefaciens strain LBA4404 containing pBI121 with nptII, uidA, and each of the following: mango ACC oxidase, ACC synthase and ACC alternative oxidase cloned in the antisense orientation and under the control of the 35S constitutive promoter. Since mango is a climacteric fruit, regeneration of plants in which ethylene production in mature fruit is inhibited could resolve the problem of premature ripening (jelly seed) and major post-harvest losses due to spoilage. The feasibility for genetic manipulation of mango will allow the manipulation of fruit quality, the alteration of tree architecture, greater resistance to insect pests and diseases and the generation of parthenocarpic mango fruit (Yao et al., 2001). Some of these goals are being pursued in the laboratories of the authors. Pineapple (Ananas comosus L.) Pineapple world production is .14 700 000 Mt annually (FAOSTAT, 2004), and 30% of the crop is exported either as fresh fruit or as processed products. Pineapple is native to South America and its center of origin is in the Orinoco and Rio Negro River basins. It has a diploid number of 2n ¼ 2x ¼ 50 (Brown et al., 1997), although triploid, tetraploid, and heteroploid cultivars exist (Collins, 1960). The main objective of cultivar development is improvement of the processing cultivar ‘Smooth Cayenne’, specifically with respect to nematode resistance, pineapple mealybug wilt virus resistance, resistance to fungal diseases (Espinosa et al., 2002), flowering, fruit ripening control, and blackheart resistance (Botella et al., 2000; Graham et al., 2000; Rohrbach et al., 2000). Although pineapple is a major fruit crop, there have been few molecular genetic studies, and very few genes been isolated. Much of the work involving genetic transformation of pineapple is proprietary, and has not been published. The current status of pinapple biotechnology has been reviewed by Smith et al. (2004b). Pineapple has been transformed by microprojectile bombardment and by co-cultivation with A. tumefaciens. Nan et al. (1996) used microprojectile bombardment to transform embryogenic suspension cultures and obtained low-frequency transformation. Firoozabady and Gutterson (1998) and Isidron et al. (1998) recovered transformed plants from embryogenic cultures using A. tumefaciens. Graham et al. (2000) transformed ‘Smooth Cayenne’ leaf bases using A. tumefaciens, and recovered transformed plants from organogenic cultures. Espinosa et al. (2002) transformed morphogenic callus of

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‘Smooth Cayenne’ with Agrobacterium strains AT2260 and LBA4404 and regenerated plants. Maize ubiquitin and CaMV 35S promoters have been shown to function well in various pineapple tissues (Firoozabady and Gutterson, 1998; Graham et al., 2000). The breeding objectives of pineapple that have been addressed using genetic transformation include disease resistance, control of fruit ripening, and control of blackheart disorder. Espinosa et al. (2002) described a complete protocol for transformation of pineapple with PR genes using A. tumefaciens containing constructs pHCA58 and pHCG59, respectively. Plasmid pHCA58 contained a class I bean chitinase gene under the control of a hybrid OCSCaMV 35S-rice actin I promoter (pA5) and tobacco ap24 gene with the CaMV 35S promoter. The plasmid pHCG59 contained the chitinase gene under the control of the hybrid pA5 promoter and a class I tobacco b-1,3-glucanase gene with the CaMV 35S promoter. Plants were regenerated. Although pineapple fruits are non-climacteric, both ethylene biosynthetic genes are up-regulated in the flesh of pineapple fruits during ripening (Cazzonelli et al., 1998, 1999). An ACC synthase gene that may be involved in floral initiation has been cloned (Botella et al., 2000), and its silencing in pineapple could suppress flowering until it is induced artificially. This might facilitate synchronization of fruiting and ripening, and enable mechanized harvesting. Transformed pineapple plants containing genetic constructs to inactivate this gene as well as the ripening-related ACC synthase are under field evaluation (Botella et al., 2000). Blackheart is a fruit defect caused by exposure of pineapples to temperatures , 208C, which stimulates polyphenol oxidase (PPO) activity. Stewart et al. (2001) cloned a PPO gene from pineapple fruits under conditions that produce blackheart. The PPO gene has been silenced in transformed plants and transgenic plants are under field evaluation. Avocado (Persea americana Mill.) World production of avocados is . 2 800 000 Mt (FAOSTAT, 2004), and Mexico accounts for approximately 35% of the total production. The international trade in fresh avocado fruit is very important, and Chile and Mexico are the leading exporters. The species is genetically heterozygous with a 7 – 8-year juvenile period and high rates of flower abscision and immature fruit drop. There are three races or subspecies of avocado: Mexican (subtropical), Guatemalan (highland tropical), and West Indian (lowland tropical). The chromosome number is 2n ¼ 2x ¼ 24 (Garcia, 1975) and the size of the genome is about six times that of A. thaliana and similar to that of banana (883 Mb) (Arumuganathan and Earle, 1991). Major breeding objectives include resistance to Phytophthora root rot (PRR) caused by Phytophthora cinnamomi Rands. in rootstock cultivars, control of tree size, extended shelf-life of fruit, and development of a replacement for ‘Hass’ with many of the best qualities of this cultivar. Avocado breeding programs in various countries have been moderately successful, although cultivars have been derived from uncontrolled open pollinations. There are no genetic barriers among the avocado subspecies/races, and many cultivars are hybrids involving two or more subspecies. ‘Hass’ and ‘Fuerte’, which dominate world trade, are Guatemalan £ Mexican hybrids. Genetic transformation of avocado has been reviewed recently (Litz and Witjaksono, 2002; Litz et al., 2004c). Zygotic embryos

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(Pliego-Alfaro and Murashige, 1988; Raviv et al., 1997; Witjaksono and Litz, 1999a, b) and the nucellus (maternal) (Witjaksono et al., 1999) have been used for induction of embryogenic cultures. Conditions for optimizing growth of embryogenic cultures in suspension, somatic embryo development, and plant recovery have been defined (Witjaksono and Litz, 1999a, b). Genetic transformation of avocado has been based upon highly embryogenic suspensions (Witjaksono and Litz, 1999a, b). In the first report, Cruz-Hernandez et al. (1998) utilized a twostep selection procedure, involving incubation of the putative transformed tissue in increasing concentrations of kanamycin. This study utilized A. tumefaciens strain LBA4044 with binary vector pBI121 containing nptII and uidA under the control of the 35S CaMV promoter. Transformation of somatic embryos was confirmed; however, plants were not regenerated. The breeding objectives of avocado that have been addressed using genetic transformation include disease resistance and control of fruit ripening. In order to address some of the disease-related problems of avocado, embryogenic cultures have been transformed with various genes for PR-related proteins (Litz and co-workers). (1) Embryogenic cultures have been infected with A. tumefaciens strain EHA101 harboring pHGAFP which contains the antifungal protein gene (AFP) together with uidA, the gene for resistance to hygromycin and the CaMV 35 s promoter. (2) Glucanase and chitinase have been cloned in pGPTV, together with uidA, bar (Basta resistance), and the CaMV 35S promoter (Witjaksono, S. Raharjo, M. A. Go´mez-Lim and R. E. Litz, unpublished data). Transformed plants with the AFP gene have been regenerated, and are currently in greenhouse trials. In addition, a complex transformed somatic hybrid consisting of ‘Fuerte’ with b-1,3glucanase, chitinase, bar, and uidA þ ‘Hass’ with AFP, nptII, and uidA was selected on medium containing Basta and kanamycin sulfate, and plants (tetraploid) have been regenerated (S. Raharjo, Witjaksono, M. A. Go´mez-Lim and R. E. Litz, unpublished data). The regenerants will be screened as potential rootstocks. Avocado has also been transformed to extend the shelf-life of avocado fruit, and to extend the on-the-tree storage of mature fruit. Avocado fruit are climacteric. Fruit of Mexican, Guatemalan, and Guatemalan £ Mexican types do not ripen on the tree, and can be stored in this way for a few months (Whiley, 1992). Tropical avocado fruit ripen on the tree, and have a poor shelf-life. Embryogenic cultures have been genetically transformed using A. tumefaciens strain EHA101 with pAG4092 harboring SAM hydrolase, a gene that blocks ethylene biosynthesis, in a construct containing nptII and under the control of an avocado fruit-specific cellulase promoter (Efendi, 2003). Plants have been regenerated. Longan (Dimocarpus longan Lour.) and Litchi (Litchi chinensis Sonn.) The litchi and longan originated in South-East Asia and southern China. The leading litchi-producing countries are China, Taiwan, India, Madagascar, and Thailand (Menzel, 1992), and significant production also occurs in South Africa, Australia, and the USA. Longan is important in Thailand, China, Taiwan, Australia, and the USA. Neither longan nor litchi has benefited from conventional breeding. There is little genetic diversity within germplasm collections of these species (Menzel, 1992; Ding et al., 2001). The chromosome number of longan is 2n ¼ 2x ¼ 30 (Choo and

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Ketsa, 1992), and that of litchi is 2n ¼ 2x ¼ 28, 30, or 32 (Menzel, 1992). Trees are very heterozygous, and are vegetatively propagated. Litchi and longan have not become major crops because of their relatively brief harvest period and the rapid decline of fruit quality and appearance after harvesting. The fruit are non-climacteric. Some of the important cultivars are very susceptible to anthracnose caused by Colletotrichum gloeosporioides Penz., and chemical control is becoming unsustainable. Litchi and longan trees are irregular bearers, and high yields and regular bearing are necessary in order to exploit the potential of local and international markets. In China, the most valued litchi and longan fruit have small aborted seeds, known as ‘chicken tongues’, a trait that is cultivar-related. Longan and litchi biotechnology have been reviewed recently (Litz et al., 2004a, b). Somatic embryogenesis has been described from mature tissue of elite cultivars of longan (Litz, 1988) and from zygotic embryos of litchi (Zhou et al., 1996; Yu and Chen, 1997; Yu et al., 2000) and longan (Lai et al., 1998, 2000). Conditions for optimizing maintenance of embryogenic suspension cultures (Lai et al., 1995; Yu and Chen, 1998) and for somatic embryo development have been described. Zheng et al. (2001) transformed somatic embryos derived from longan zygotic embryos by inoculating them with the R1601 wild strain of A. rhizogenes. Secondary somatic embryos were induced from the hairy roots and transformed plants were recovered. Embryogenic litchi cultures derived from zygotic embryos have been genetically transformed with the antifungal protein gene (Witjaksono and R. E. Litz, unpublished data). Cultures were infected with A. tumefaciens strain LBA4404 containing pBI121 with nptII, uidA, and the antifungal protein gene, with the CaMV 35S promoter. Although transformed somatic embryos were recovered, plants have not been regenerated. A major objective of longan and litchi transformation is the recovery of parthenocarpic fruit (Yao et al., 2001); however, a de novo regeneration pathway for litchi from elite selections must first be defined. Passionfruit (Passiflora edulis and P. edulis Sims f. flavicarpa Deg.) Passion vines are vigorous, wood-stemmed, tendril climbers, with a 90-d juvenile period. Passion fruit is grown commercially throughout the tropics and subtropics. Brazil is the leading producer of the yellow passion fruit, P. edulis Sims f. flavicarpa Deg., and 35% of Brazilian production is processed. The main commercial species and horticultural hybrids of passion fruit are 2n ¼ 2x ¼ 18. Breeding programs have focused on P. edulis, the purple passion fruit, and P. edulis flavicarpa, the yellow form. The main breeding objectives include: (1) improved yield, (2) fruit quality, and (3) disease resistance. The most important diseases include Brown spot (Alternaria spp.), Phytophthora blight (Phytophthora ssp.), Fusarium wilt (Fusarium oxysporium f. passiflorae), Xanthomonas campestris pv. passiflorae, and passionfruitwoodiness virus (PWV). De novo regeneration of passionfruit has been by the organogenic pathway from explants derived from seedlings. Organogenesis can occur either from callus (Mourad-Agha and Dexheimer, 1979; Kantharajah and Dodd, 1990; Monteiro et al., 2000) or directly from the explant (Kawata et al., 1995; Faria and Segura, 1997; Otahola, 2000). Plant regeneration without a callus phase has been optimized for P. edulis flavicarpa. The application of biotechnology to passion fruit has been reviewed by Vieira and Carneiro (2004).

Transformation of P. edulis mediated by A. tumefaciens has been reported (Silva, 1998; Hall et al., 2000). Agrobacterium tumefaciens strain 1065, carrying uidA and nptII, has been used to infect leaf and root segments. Manders et al. (1994) utilized A. tumefaciens strain GV3111SE with pMON200 that contains nptII, and obtained transformed plants. Stable transformation of passion fruit with nptII and uidA has also been described by Silva (1998), who used the LBA4404 strain. The primary breeding objectives that are being addressed using genetic transformation include resistance to bacterial and virus diseases. Particle bombardment has been used to transfer the bactericide attacinA gene driven by the 35S – 35S promoter to yellow passion fruit (Vieira et al., 2002), with the objective of resistance to X. campestris pv. passiflorae. Braz (1999) transformed passion fruit with a sequence derived from the replicase and capsid protein (cp) genes isolated from PWV. Preliminary results suggest that this strategy can be used to control this virus disease. Conclusions Genetic transformation of perennial tropical fruits has generally depended on embryogenic systems, and therefore regenerants of the woody species, i.e., mango, avocado, longan, litchi, citrus, etc., must pass through a period of juvenility before they can be properly evaluated. The two alternatives that have been utilized to overcome this limitation include: (1) invigorating plant material through grafting of mature buds onto juvenile stock plants (Cervera et al., 1998) and (2) constitutive expression of either the LEAFY or APETALA 1 genes from A. thaliana to shorten the juvenile phase and promote precocious flowering (Pen˜a et al., 2001). Both of these innovations could stimulate more transformation attempts with these species. The major hindrances that have stymied genetic transformation studies with tropical/subtropical fruit, however, concern lack of regeneration protocols for elite (mature phase) selections, e.g., litchi and many tropical/subtropical fruit and nut species not included in this review, and the relative absence of molecular studies involving species other than citrus and bananas. The latter reflects the state of the science in many developing countries where these fruit crops are grown on a large scale and the relative severity of production and post-harvest problems of the crop. Biotechnology studies involving fruit crops everywhere are generally underfunded, and national and international agencies should perhaps consider more support for research with these plants. Acknowledgments M.A.G.-L. gratefully acknowledges financial support from CONACYT. This paper is Florida Agricultural Experiment Station Journal Series no. R-10211. References Arumuganathan, K.; Earle, E. D. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 9:208–218; 1991. Balint-Kurti, P.; Firoozabady, E.; Moy, Y.; Mercier, R.; Fong, R.; Wong, L.; Gutterson, N. Better bananas – the biotech way. Infomusa 10:vi; 2001. Becker, D. K.; Dugdale, B.; Smith, M. K.; Harding, R. M.; Dale, J. L. Genetic transformation of Cavendish banana (Musa spp. AAA group) cv.

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