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BREEDING OBJECTIVES. Objectives for gene transfer to papaya include resistance to PRSV, delayed ripening, fungal, oomycete, insect resistance and cold tolerance and were reviewed by Fitch (2016) and Dhekney et al. (2016).

PROTOCOL. Papaya leaves were transformed with Agrobacterium, but plants were not recovered (Pang and Sanford, 1988). Fitch et al. (1990) described a procedure that involved embryogenic cultures that was developed from earlier walnut research (McGranahan et al., 1988). Immature ‘Sunset’ and ‘Kapoho’ zygotic embryos, 90–105 days post-pollination, were excised, and one cotyledon was removed from each embryo to expose the apical meristem. The embryos were plated directly on induction medium consisting of modified ½ MS medium containing 50 μM 2,4-D. Four- to 23-day-old zygotic embryo cultures provided one tissue type for bombardment. One week prior to bombardment, 40–100 zygotic embryos were transferred to each Petri dish. Embryos were oriented with apical meristems exposed and radicles embedded in the induction medium. Embryos were arranged in a 2.5-cm ‘doughnut’ configuration to avoid damage in the central 1-cm area from the projectile ‘blast’. The second tissue bombarded was derived from 2-day-old hypocotyl sections from 14-day-old in vitro germinated seedlings of ‘Kapoho’. Six-month-old somatic embryo cultures derived from hypocotyl sections (Fitch, 1993) comprised the third tissue type bombarded. A fresh weight amount of 500 mg was plated onto filter paper on induction medium and bombarded.

An Agrobacterium binary vector, pGA482GG/cpPRV-4 construct, was bombarded (Klein et al., 1988) into the embryogenic zygotic embryos. The resistance gene, the PRSV coat protein gene of a mild PRSV strain, HA 5-1 (Yeh and Gonsalves, 1984) and plant transformation promoter and terminator regions in the HindIII site of the vector polylinker made up the primary section of the construct (Fitch et al., 1990). A 35S promoter, 5′-untranslated region, PRSV coat protein gene fragment fused to CaMV fragment, and a 35S terminator for the polyA tail completed the HindIII insert. Outside of the insert, right and left borders of the T-DNA fragment, the cos site from the bacterial phage lambda, and the NOS-NPTII fusion gene and GUS (β-glucuronidase) reporter gene are within the Agrobacterium border region. The gentamicin and tetracycline resistance gene sites are present outside of the binary insert and only necessary for Agrobacterium manipulations.

Cultures were transferred to induction medium 8–10 days after bombardment and subcultured on induction medium containing 200 mg/l cefotaxime. Cultures were grown in the dark for 3 weeks followed by transfer to maturation medium containing 75 mg/l kanamycin sulfate. Four weeks later, the selection concentration was increased to 150 mg/l kanamycin sulfate. Thereafter, all cultures were subcultured monthly until selectively growing somatic embryo lines were observed, and confirmation assays commenced.

Other genes were transformed into papaya to function as alternative selection agents, e.g. phosphinothricin (bialaphos) herbicide resistance (Cabrera-Ponce et al., 1995), green fluorescent protein gene and a phospho-mannose isomerase (PMI ) gene (Zhu et al., 2004, 2005). Table 6.1.2 lists the non-virus transformation projects, e.g. aluminium tolerance was sought with overexpression of a citrate synthase gene (de la Fuente et al., 1997) and delayed ripening with gene silencing of ACC oxidase and synthase.

ACCOMPLISHMENTS

PRSV resistance. The first transgenic, virus-resistant, commercialized papaya in the USA was Line S55-1 (Fitch et al., 1992; Gonsalves, 1998). PRSV-resistant transgenic plants inoculated with virulent PRSV showed no symptoms after 2 weeks while controls had typical infection symptoms (Fitch et al., 1992). These results were confirmed (Lius et al., 1997; Ferreira et al., 2002), and the crop was commercialized in the USA in 1998, 7 years after resistance was demonstrated in the greenhouse (Gonsalves, 1998). International deregulation followed in 2003 in Canada and 2011 in Japan (Fitch, 2016). ‘Rainbow’ papaya continues to be the staple for the Hawaii papaya industry that is sustained with hybrid seed produced by crossing resistant parents that surround and protect non-transgenic parents from ever present PRSV (Matsumoto et al., 2016).

Transgenic ‘Kamiya’ papaya was also recovered following Agrobacterium-mediated gene transfer (Fitch et al., 1993, 1998); however, these transgenic lines contained multiple copies of the resistance gene, although fruit were normal-looking; others were apparently tetraploids and had fruit that were unsuitable to market (Fitch, 2002a, b).

Guangdong, China deregulated transgenic papayas carrying the replicase gene that was cloned from PRSV (Chen et al., 2001), and PRSV-resistant papayas were commercialized in 2006. ‘Huanong#1’ fruit are sold only in Guangdong Province, South China. The cultivar was tested for resistance in other provinces and found to be susceptible to other strains of PRSV (Wu et al., 2018). There are three groups of isolates in South China, and 27.1% of the isolates were identified as recombinants based on their coat protein gene nucleotide sequences. Resistance broke down in ‘Rainbow’ inoculated with Taiwan and Thai strains of PRSV. Transgenic papayas from Hawaii, Thailand, Taiwan and elsewhere were not resistant to PRSV in Hainan (Zhao et al., 2016). Divergence among PRSV populations in Hainan was noted, signalling failure of their own transgenic papayas. Hawaiian transgenic papayas are not immune to even the local strain(s) of PRSV as breakdown of resistance has been reported in young plants that have not adequately developed gene silencing resistance (Gaskill et al., 2002; Tripathi et al., 2008; J. Sugano, University of Hawaii Extension Agent, Hawaii, 2018, personal communication).

In Florida, the USDA, EPA and the FDA have approved the ‘X17-2’ selection, but release has not occurred (Davis and Ying, 2002; Federal Register, 2008; M. Davis, Florida, 2018, personal communication).

Delayed ripening. In many countries, transport of fruit to markets results in losses from ethylene-induced ripening in enclosed containers where refrigeration can sometimes be compromised.

RNA interference of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1 and ACO2) genes from ‘Eksotika’ (Sew et al., 2007; Sekeli et al., 2014) prolonged the shelf life of transgenic ‘Eksotika’ papaya fruit in Malaysia. From sequence databases showing significant differences in the 5ʹ and 3ʹ untranslated regions (UTR), two ACO cDNAs from ‘Eksotika I’ and ‘Eksotika II’, about 1400 bp long, were full length, were included in constructs, and transformed into ‘Eksotika’ papaya using Agrobacterium. A total of 176 putative transgenic lines from 15,000 embryogenic cultures were selected on kanamycin medium, verified for gene insertion by PCR, regenerated into plants and field tested. Two to three weeks of additional shelf-life was reported, and total soluble solids ranged from 11° to 14° Brix, comparable to control non-transgenic fruit (Sekeli et al., 2014). Trees were observed in field tests, but commercialization was not reported.

Transgenic papayas exhibiting delayed ripening to extend postharvest shelf life were also developed using an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene, c.800 bp in length, from ‘Solo’ papaya (Laurena et al., 2002; Magdalita et al., 2002). Embryogenic cultures were transformed with the construct using Agrobacterium. Ripening of fruit from field-grown plants required 4–7 days rather than 2 days for non-transgenic fruit (Mendoza et al., 2008). Food safety data were collected and were comparable to control non-transgenic fruit (Cabanos et al., 2013). Magdalita et al. (2013) transformed ‘Solo’ with the ACC oxidase antisense gene from ‘Solo’ and a partial sequence from Philippine yellow ‘Solo’, an 800 bp fragment, using Agrobacterium. Concerns about transgenic fruit in the Philippines resulted in a moratorium on further research (P.M. Magdalita, Philippines, 2018, personal communication).

Cold tolerance. Cold-tolerant papayas could enable growers in subtropical regions to avoid adverse effects on production during suboptimal temperatures. A C-repeat binding factor (CBF) in Arabidopsis thaliana imparts cold tolerance (Gilmour et al., 1998; Thomashow, 1999). Genes associated with cold tolerance may stimulate synthesis of cryoprotective proteins or other protective gene products. CBFs are transcriptional activators that bind to the promoter regions of cold tolerance genes, and overexpression of a CBF 1 gene in tomato resulted in enhanced chilling tolerance (Hsieh et al., 2002). Papayas were determined to be devoid of CBF orthologue sequences found in the cold-tolerant V. pubescens (Dhekney et al., 2007). Embryogenic cultures of ‘Sunrise’ papayas were transformed with Agrobacterium using a binary vector containing CBF 1/CBF 3 (Gilmour et al., 1998) controlled by the CaMV 35S gene promoter. Selection with 300 mg/l kanamycin resulted in regenerated plants that were shown to contain the resistance transgenes by Southern hybridization. The plants were morphologically abnormal (S. Dhekney, Wyoming, 2018, personal communication).

Other than using heated enclosures (D. Raquinio, California, 2016, personal communication; Garcia et al., 2017; Guerra, 2017; Pinillos et al., 2017; Romero et al., 2017a,b), cold-tolerant papayas could be an economic crop since the more stable female papayas are grown and marketed in colder temperatures (Fukamachi and Kato, 2006; R.E. Paull, Hawaii, 2018, personal communication). Babaco (Van Droogenbroeck et al., 2004) and V. pubescens, both of which are cold tolerant, have been cultivated for canned fruit and juice (Carrasco et al., 2017). It will be interesting to see how papaya crossed with babaco, Vasconcellea × heilbornii (Manshardt and Hoover, 2017; R. Manshardt, Hawaii, 2018, personal communication), will perform. While F1 crosses of V. pubescens with papaya resulted in PRSV-resistant but sterile fruit, bridge-crosses with V. parviflora and backcrosses have some fertility with papaya (Kanchana-udomkan et al., 2018); genes for cold hardiness could be transferred to papaya if markers for the cold tolerance genes could be identified. It is conceivable that breeding with wild collected selections (Pesqueira et al., 2017) and comparisons of the genomes of the cold-tolerant Vasconcellea species and papaya and wild papayas found growing in the coldest ranges of their habitats could extend the range of papaya.

Papayas have been transformed with genes either from papaya or other sources, and these studies have been summarized in Table 6.1.4.