Towards the formation of somatic embryos from avocado leaf explants

Oyerinde, Rebecca Opeyemi
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Avocado (Persea americana Mill.) is the most important and the only tree that produces edible fruits from the many economically-valuable trees of the Lauraceae. Beyond the fruits, other parts of the plant are highly sought after for further use such as home remedies and beauty products. The increasing demand for avocado outweighs its production and this demand cannot be met by its natural means of propagation, which is characterized by particular difficulties such as flowering pattern, long juvenile period and diseases. The advances in plant biotechnology have facilitate do ther propagation methods, such as micropropagation, to bypass the difficulties associated with natural propagation. Somatic embryogenesis is a micropropagation method that has been used to generate in vitro avocado plantlets in the past. However, this has been achieved mainly by the use of immature zygotic embryos. Considering that zygotic embryos are a result of sexual reproduction, their genotypes are not truly known. Somatic embryogenesis, in some cases, involves the formation of callus as an intermediary phase before plant regeneration. The use of callus, however, is not limited to differentiation towards plant regeneration. Thus, this study focused on callus production from an avocado explant that is true to type and less destructive to the parent plant – the leaf, which was sourced from both greenhouse plantlets and the in vitro shoots. In vitro avocado shoots were generated via three explanted materials: the embryonic axes, the seeds and the axillary buds. Shoots grown from embryonic axes had, significantly, the least number of leaves per shoot, and the smallest leaf size(length and breadth) while those obtained from the seeds had the highest of the observed parameters. However, for the purpose of this study, associated genetic variation with seed-derived shoots needed to be avoided as well as seasonal availability of the seeds. Hence, axillary buds were used for in vitro avocado shoot production. The greenhouse and the in vitro growth conditions triggered the expression of phenotypic plasticity in the avocado plants. One of the characteristics observed was the presence or absence of bundle sheath extensions –a criterion with which leaves are accordingly classified as heterobaric or homobaric and thus leaves from the greenhouse-and the in vitro-raised materials fall into these classifications, respectively. Another trait was that there were more chloroplasts (3 chloroplasts/100 μm2) in the greenhouse materials than their in vitro counterparts (1 chloroplasts/100 μm2), which implied that photosynthesis was more efficient in the former. Also, the in vitro leaves were thicker (171.19 ± 21.6 μm)with larger cells than greenhouse leaves (75.98 ± 8.6 μm). Histomorphological observation showed that in vitro materials were not as differentiated as the greenhouse materials. The immature state of differentiation of the in vitro plant did not support the development of its nodal explants towards organogenesis but favoured the induction of call us from its leaf explants. It showed that callus originated from all three leaf tissues (i.e. the vascular bundles, the mesophylls and the epidermal layers) in thein vitro materials while it was only induced from the vascular bundles and the epidermis of the greenhouse materials. It also showed that in the first two weeks of callus induction, leaf cells had increased in size and cell division had been induced –the processes that eventually led to callus formation. These processes were as a result of the synergistic relationship between 2,4-D and BAP in the induction medium. Leaf explants from in vitro shoots produced more callus (72.7 ± 10.35%) than explants from greenhouse plants (54.2 ± 15.40%). Seventy-nine different media were tested for optimal callus induction. The media were tested using four basal nutrient formulations: Murashige & Skoog (MS), Gamborg’s B5, Mango Medium for Somatic Embryos (MMSE) and B5+and three auxin types: 2,4-dichlorophenoxyacetic acid (2,4-D), picloram and naphthaleneacetic acid (NAA).The results showed that Gamborg’s B5 basal nutrient medium was more favourable (39.69 ± 14.95%) for callus induction than MS (21.43 ± 9.99%). All the MMSE-based induction media did not support callus formation while less than 10% of explants developed on B5+ on some of its associated induction media. The use of auxin as the only plant growth regulator (PGR) did not result in callus formation. However, the addition of the cytokinin,6-benzylaminopurine (BAP),to the auxin-enriched media resulted in some callus formation. In Gamborg’s B5 medium, PGRs within the range of 0.5 -2.0 mg/L 2,4-D and 0.2 –1.0 mg/L BAP were optimal for callus induction under dark incubation, of which 1.0 mg/L 2,4-D and 0.5 mg/L BAP was the most effective combination. The order of auxin effectiveness for inducing callus was 2,4-D˃ NAA ˃ picloram. Other factors that were monitored included light and dark incubation, explant size and explant surface in contact with the induction medium. The size of the leaf explant did not have any significant effect on callus formation (p = 0.380794). However, dark incubation significantly favoured more callus formation than light incubation (p=0.00124 and 0.00182 in B5 enriched with 2,4-D and picloram, respectively). Also, the adaxial surface making contact with the induction medium resulted in more callus formation (54%) than when the abaxial surface was in contact with the induction medium (22%).The callus formed, in this study, had a range of colour (white, brown cream and green), texture (fluffy, grainy, compact, soft and nodular) and origin (cut edges, end of vein and along the vein and lamina). There was a switch of preference between induction medium and developmental medium. While the combination of B5 and 2,4-D was optimal for callus induction, B5 was not suitable as a basal medium for further development. Also, phenolic compounds often accumulated in 2,4-D-derived callus which led to deleterious browning and subsequently the callus did not survive manipulation towards differentiation. On the other hand, MS, which was not preferred for callus induction, was more suited for the callus differentiating medium. Similarly, although NAA did not stimulate as much callus as 2,4-D, browning was less pronounced in the NAA-derived callus, the callus cells accumulated callose (one of the markers of embryogenic cells) in their cell walls. The NAA-derived callus subsequently showed some ‘greening’ responses at the differentiating phase before they died when subcultured. In most cases, subculturing callus to fresh medium led to loss of culture. Some groups of callus cells from the leaves of both the greenhouse-and the in vitro-derived materials developed to form proembryo-like structures, which gave an indication of possible further development towards organogenesis. Considering the paucity of published information on any in vitro procedures for the main cultivar ‘Edranol’ used in this study and for the use of avocado leaves as explants, the work reported here is fundamental. Thus, this study further enhanced our understanding of (i) the selective effectiveness of not fully-differentiated tissues in micropropagation procedures, (ii) the preferential effectiveness of the different basal nutrient media, B5 and MS, at either inducing callus or supporting further development of callus, (iii) the type of auxin used affecting the rate, type and quality of callus formed and (iv) avocado is a recalcitrant species, not only in its post-harvest seed behavior but also in its response to in vitro manipulations
A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy School of Animal, Plant and Environmental Sciences, Faculty of Science, University of the Witwatersrand, 2021