Development of a protocol for the proliferation of in vitro axillary buds in avocado (Persea americana) cv. 'edranol'

Mansoor, Faatimah
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Seed recalcitrance in avocado (Persea americana) has meant that avocado genetic material cannot be conserved in orthodox seed banks. Thus, biotechnological approaches have been considered for the long-term conservation of this species’ genetic material, through the cryopreservation of tissue culture-generated axillary buds. A study was conducted to develop a system for the proliferation of in vitro avocado cv. ‘Edranol’ axillary buds for the purpose of cryopreservation. Experiments were conducted to optimise avocado mother plant establishment and pretreatment. It was determined that potting soil mixes comprising of either 1:1:1 pine bark, perlite, river sand or 1:1:1 peat, perlite, river sand were suitable to culture healthy avocado mother plant seedlings. With these soil mixes approximately 2 shoots per plant developed after 11 weeks of transplanting and between 2.9 ± 0.31 and 3.37 ± 0.32 secondary shoots were produced after 5 months. Additionally, the mother plants produced well extended shoots (7.30 ± 1.29 cm; 8.77 ± 1.39 cm) with a sufficient number of axillary buds (7.75 ± 0.39; 6.33 ± 0.53), which were subsequently used as nodal explants. After surface decontamination, the establishment of an aseptic culture in vitro was successfully achieved. Six semi-solid tissue culture media were tested for the proliferation of in vitro axillary buds. Four media comprised of half (½) and full strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962), with either 0.5 or 1mg/l 6-Benzylaminopurine (BAP). Two media were based on the P. indica medium as proposed by Nel et al. (1983), and comprised of half strength MS macronutrients, full strength MS micronutrients, 2mg/l BAP and 1mg/l GA3. All media were supplemented with 3g/l Gelrite and 30g/l sucrose at pH 5.6-5.8. Physiological measurements were taken six weeks after establishment, the first, the second and the third subculture. Tissue browning, death and contamination were observed in explants cultured on the media containing 0.5mg/l BAP, suggesting that this concentration of BAP was not suitable for cv. ‘Edranol’. Additionally, hyperhydricity appeared to be associated with the media containing ½ MS, which could be attributed to mineral deficiencies. Overall, there was no significant difference in the number of shoots and axillary buds developed across all the media tested, suggesting that endogenous auxin levels were higher than the concentration of cytokinin used in the media tested. In support of this, strong apical dominance and callus formation was observed. An increase in tissue browning, death and hyperhydricity on all the media tested, coupled with a decrease in shoot length, suggested a decline in the vigour of explants in vitro. 1MS + 1mg/l BAP was selected as the most appropriate medium for the initiation of cv. ‘Edranol’ cultures, producing between 3.2 ± 0.2 and 4.9 ± 0.5 axillary buds per explant. However, hyperhydricity, browning and death were observed in explants cultured on this medium. Overall, the in vitro axillary bud explants did not behave predictably or uniformly. Thus, the system was not optimised, indicating that further study is needed for the mass multiplication of axillary buds to be used for the cryo-conservation of avocado genetic material. It is recommended that future experiments will be needed to further test tissue culture media, with a focus on the optimisation of the nutrient and plant growth regulator concentrations. Additionally, the recalcitrance of explants to the in vitro environment may have been influenced by the physiological state of the mother plants, indicating that research should be focused on the effect which the mother plants may have on the endogenous responses of the in vitro explants.
A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree Master of Science Johannesburg, 2018
Mansoor, Faatimah (2018) Development of a protocol for the proliferation of in vitro axillary buds in avocado (Persea americana) cv. 'edranol', University of the Witwatersrand, Johannesburg, <>