In avocado, an economically important crop, fruits can store up to 70 % oil in the form of triacylglycerols (TAGs). While TAG synthesis in seed tissues mostly depends on an acyl CoA-dependent enzyme, diacylglycerol (DAG) acyltransferase (DGAT) to catalyze the conversion of DAG to TAG, the enzymes involved in non-seed tissues remains to be elucidated. Recent studies on oil palm suggested participation of an acyl-CoA-independent enzyme, phospholipid:diacylglycerol acyltransferase (PDAT), in TAG synthesis. Our research focuses on identifying acyltransferases involved in oil accumulation in mesocarp tissues of avocado. Furthermore, in ‘Hass’ avocado, where 20-60 % of the fruit are phenotypically small, even under favorable conditions, we are interested in determining the association between oil accumulation and fruit size. To this extent, we quantified gene expression levels for DGAT 1 and 2 and PDAT and the rate of oil accumulation in developing mesocarp (oilrich) and seed (non-oil rich) tissues of phenotypically 'small' and 'normal' fruits, using real-time PCR and gas chromatography, respectively. Candidate acyltransferase genes, highly expressed in mesocarp but not in seed, will be cloned and characterized. Understanding TAG synthesis in non-seed tissues will allow us to develop genetic tools necessary for generating bioenergy-rich crops.

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Triacylglycerol (TAG) is a main storage lipid in seeds and is utilized for seed germination. Humans commonly consume TAG in the form of vegetable oil. Currently, TAG contributes to 25% of the total calories consumed in the developed countries and dependency for biofuel production from plants lipid is also increasing day by day. By 2030 the expected consumption level of vegetable oil will be double and to fulfill this demand it is necessary to increase the oil production in plants. To accomplish this goal, it is pertinent to understand the regulation of TAG accumulation in plants. Avocado (Persea americana), a basal angiosperm, stores up to 70% oil in the form of TAG in nonseed tissue (mesocarp). Here, we use it avocado as a model system to understand TAG biosynthesis in tissues other than seed. In plants, TAG biosynthesis may involve acyl-CoA–dependent or –independent pathway catalyzed by diacylglycerol acyltransferases (DGAT) and phospholipid: diacylglycerol acyltransferases (PDAT), respectively. RNA-Seq and Q-PCR analysis of developing mesocarp of avocado revealed higher expression for DGAT1 and PDAT1, coinciding with the period of TAG accumulation. Therefore, we hypothesize that DGAT1 and PDAT1 are responsible for catalyzing the terminal step in TAG biosynthesis in Avocado mesocarp. Using the transcriptome data, we identified full-length coding sequences for DGAT1 and PDAT1. These acyltransferases are being cloned and will be characterized for their enzyme activity and substrate specificity, subsequent to their expression in yeast. Complementation of Arabidopsis dgat1 and pdat1 mutants and phenotypic characterization will also be carried out using Gateway-cloning techniques and transgenic lines will be assayed for TAG content in seeds. This study is expected to provide basic understanding of TAG accumulation in avocado mesocarp tissue.

Modern society’s demand for oil has resulted in depletion of resources and caused higher oil prices. Therefore, natural oil resources of plants are gaining the spotlight and are expected to increase twice that of current use by 2030. Plants are able to accumulate up to 90% oil by dry weight in the form of triacylglycerol (TAG) and it is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. In seed tissues, an acyl CoA-dependent enzyme, diacylglycerol acyltransferase (DGAT) participates in conversion of diacylglycerol (DAG) to TAG. However, TAG can also be formed by an acyl CoA- independent enzyme, phospholipid:diacylglycerol acyltransferase (PDAT). Avocados (Persea americana) store up to ~70% oil in mesocarp and I am interested in identifying the acyltransferase involved in oil biosynthesis in mesocarp tissues. Based on the transcriptome data available, I hypothesize that unlike in seeds, both DGAT and PDAT are associated with TAG biosynthesis in developing mesocarp of avocado. To test this hypothesis, I will determine 1) TAG content and composition and 2) expression levels for DGAT and PDAT genes in mesocarp and seed tissues of five stages of developing fruits of avocado (n=5). Total lipids will be extracted by isopropanol-chloroform method and analyzed for composition as methyl esters on GC-FID. Total RNA, for expression analysis, will be extracted by Trizol method and analyzed with gene-specific primers by real-time PCR. Statistical significance in change in oil content in association with gene expression during fruit development between mesocarp and seed tissues will be analyzed by ANOVA repeated measures. Comparison of temporal gene expression pattern of oil accumulation mesocarp, to that of seed, will allow us to differentiate the acyltransferase(s) specifically associated with TAG biosynthesis. The proposed research work will take the field of plant lipid biochemistry a step forward in understanding TAG synthesis in fruit tissue. Specifically, I will be able to clearly demonstrate the association of a particular acyltransferase to increasing lipid content in a non-seed (mesocarp) tissue. Understanding differences in oil regulation of a basal angiosperm (avocado) in relation to a monocot (oil palm) and a dicot (olive) also will provide additional insights into fundamental changes in TAG biosynthesis during the evolution of flowering plants. My research is part of a global project that includes a group in California, Florida and Israel. Data generated from my proposed work will be used to develop a joint Binational Agricultural and Research proposal. Therefore, my research work will subsequently contribute to development of ways to manipulation of extent and timing of oil accumulationa direct benefit to avocado growers. Furthermore, this study will be a pivotal step to understanding TAG synthesis that will lead to bioenergy crop.

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 / 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. / MT 2018

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