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Genetic transformation is the only practical solution for improving existing elite selections of perennial species for specific horticultural traits. The transformation of mango has been reviewed by Litz and Gómez-Lim (2002, 2005; Litz, 2008; Litz et al., 2009).

PROTOCOL. The genetic transformation of mango was first reported by Mathews et al. (1992, 1993), who used embryogenic cultures of ‘Hindi’ and of a ‘Keitt’ zygotic embryo-derived embryogenic line, respectively. These two studies utilized different disarmed, engineered strains of Agrobacterium tumefaciens: (i) strain C58C1 containing the plasmid pGV 3850::1103 with the selectable marker gene for neophosphate transferase (NPTII) which confers resistance to kanamycin (Mathews et al., 1993); (ii) strain A208 containing the plasmid pTiT37-SE::pMON9749, a cointegrate vector, with genes for NPTII and the scorable marker β-glucuronidase (GUS or uidA) (Mathews et al., 1992). A subsequent report by Cruz-Hernández et al. (1997) utilized A. tumefaciens strain LBA4404 containing NPTII, GUS and genes that mediate a horticulturally useful trait (see Accomplishments section below) in binary plasmid pBI121. Mathews and Litz (1990) in an earlier, preliminary study had demonstrated that 12.5 μg/ml kanamycin is toxic to embryogenic suspension cultures; whereas, much higher levels (200 μg/ml kanamycin) are toxic to embryogenic cultures that are grown on semisolid medium.

Genetic transformations of mango have followed a two-step selection procedure (Mathews et al., 1992, 1993; Cruz-Hernández et al., 1997). Embryogenic suspension cultures in their logarithmic phase of growth are separated by passing them through sterile filtration fabric (1000 μm pore size). The large fraction (>1000 μm) is gently abraded with a sterile brush and then incubated with acetosyringone-activated A. tumefaciens for 3 days in liquid maintenance medium, with subculture into fresh medium at 24 h intervals. The cultures are then transferred onto semisolid maintenance medium supplemented with 200 mg/l kanamycin and 500 mg/l cefotaxime. After 10 months on this selection medium, the embryogenic cultures are transferred to semisolid maintenance medium containing 400 mg/l kanamycin. Proliferating cultures are subcultured in liquid maintenance medium containing 100 mg/l kanamycin, and somatic embryo development is initiated by subculture onto semisolid maturation medium. Mathews et al. (1993) reported the recovery of transgenic mango plants derived from a ‘Keitt’ zygotic embryo embryogenic culture and which had been transformed with pGV 3850::1103 containing the selectable marker nptII. Genetic transformation was confirmed by: (i) growth in selection medium containing inhibitory levels of kanamycin; (ii) positive histochemical reaction for GUS with X-GLUC (Jefferson, 1987); and (iii) Southern hybridization.

ACCOMPLISHMENTS. The mango is a climacteric fruit; consequently, ethylene is a critical regulator of the biochemical processes that occur during ripening. Certain of the rate-limiting genes that mediate ethylene production in mango have been cloned (see Section 2.3 Gene cloning). Cruz-Hernández et al. (1997) described the genetic transformation of embryogenic ‘Hindi’ mango cultures with mango ACC oxidase, ACC synthase and ACC alternative oxidase cloned in the antisense orientation and under the control of the 35S CaMV constitutive promoter in the pBI121 binary vector. The constructs were transferred individually into A. tumefaciens strain LBA4404 by electroporation. Embryogenic cultures were transformed by the two-step procedure described above. Although the phenotype of the transformed lines was not reported, the genetic transformations were confirmed by the GUS reaction, growth in the presence of inhibitory levels of kanamycin, Southern-blot hybridization and NPTII amplification by PCR. Successful regeneration of plants and inhibition of ethylene production by mature mango fruit could resolve the production problem of premature ripening (jelly seed) and postharvest loss due to spoilage.