Tissue culture of mango var. 'Carabao'

The tissue culture system for mango was optimized for use in genetic engineering. The developed ripening genes have been cloned earlier and these will be used to transform the mango using the particle gun obtain plantlets with the developed ripening characteristics. The developed tissue protocol for mango may also be used for other purposes such as micropropagation of elite cultivars, in vitro conservation of the different mango varieties and 'carabao' strains and physiology- and breeding- related studies. Somatic embryos (1 deg, 2 deg, 3 deg, n deg) of mango var. 'carabao' were produced on a continuous basis using the protocol of Pateña, et al(2002). Since 2004 until 2007, immature mango fruits were collected for somatic embryogenesis from different sources: the IPB collection, the mango germplasm of the former Department of Horticulture, the Department of Agriculture Lipa Agricultural Experiment Station (DA-LAES) mango field genebank, and selected privately owned 'carabao' mango trees in Batangas, Laguna and Cavite [Philippines]. Primary (1 deg) somatic embryo (SE) induction ranged from 16-100% depending on the strain, time of collection and source. When subcultured, the somatic embryos proliferated, it was observed that a single somatic embryo was required to initiate the proliferation. Somatic embryos from Batches 3 and 4 (2006) showed a 94-100% proliferation rate and 17-75% germination rate. However, percent SE proliferation and germination decreased as the cultures aged (Batches 1 and 2, 2004-2005 collections). The embryos germinated in vitro and produced cotyledonary leaves initial shoots and true leaves similar to the germination of mature seeds in soil. Different media formulations were tested to improve plantlet regeneration, from the production of initial shoots to true leaf formation. It was noted that in every subculture cotyledonary embryo either produced new embryos on initial shoots the latter of which developed into complete plantlets. Proliferation of new embryos form cotyledonary embryos ranged from 2-29%. While initial shoot formation was 8-64%. Formation of true leaves from the initial shoots was 0-36.4%. In some cultures, abscission of true leaves from regenerated plantlets was observed. It was suspected that ethylene could have accumulated inside the culture vessel which caused the abscission and later browning of these leaves. However, this was ruled out using gas chromatography. Besides micrografting, several experiments were done on the transfer of regenerated plantlets to soil. Initial studies showed that plantlets survived for 10-17 days in potting medium while none survived in soil. Five other treatments with or without beneficial microorganisms (BIOTECH's BioGroe) were tested. Initial results showed that BioGroe had no promotive effect on the survival of the transplanted regenerants. Different acclimatization techniques and transfer systems were tested. Results showed that 'transfer system 2' combined with 'ex vitro' grafting proved successful in obtaining plants in soil. Plantlets were acclimatized in 2-4 weeks under controlled environment before 'ex vitro' grafting. In 'ex vitro' grafting, 3-3.5 cm scions from scions from plantlets derived from regenerated somatic embryos were used while the rootstocks were seedlings obtained from IPB's National Seed Foundation and from the PCTCL Collection. Of the 28 ex vitro grafted plantlets, 4 survived while 10 are still under observation after 2 months in the greenhouse. Ex vitro grafts produced new shoots in 14-27 days. It took us one year to develop the tissue culture requirements for SE induction and proliferation in mango.