Development of a protocol for the rapid in vitro establishment of Eucalyptus clones

Thesis (MScAgric)--Stellenbosch University, 2020.ENGLISH ABSTRACT: The practice of cultivating Eucalyptus species using micropropagation has found favour in the development of successful forestry plantation programs due to its effectiveness in generating large numbers of juvenile plant stocks. Natural phytohormones and their synthetic analogues, called plant growth regulators (PGRs), are typically included in growth media during micropropagation. These compounds may be used to stimulate certain physiological processes in plants, allowing, for example, the in vitro manipulation of plant cell growth and differentiation, depending on the concentrations and combinations of specific PGRs in the medium. The development of micropropagation programs for Eucalyptus spp. are often clone-specific, necessitating optimisation for each clone. There is thus an inherent need for the development of a standardized protocol permitting the in vitro establishment and proliferation of multiple Eucalyptus clones. In the present study, the sensitivity and responsiveness of three Eucalyptus grandis × Eucalyptus nitens clones to various PGRs were investigated for the different in vitro growth stages. They include auxins such as indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-Naphthaleneacetic acid (NAA). Similarly, the cytokinins kinetin, 6-benzylaminopurine (BAP) and meta-topolin were tested. The effects of strigolactones on micropropagation were examined using the synthetic strigolactone analogue, GR24. Parameters such as explant multiplication, shoot elongation and rooting were examined for all clones. The explants were incubated on a reduced PGR maintenance medium, from which source material was derived for subsequent experimentation, enabling the growth stages to be investigated independently. The highest level of bud initiation and shoot growth was observed for all Eucalyptus clones when the growth medium was supplemented with 0.5 mg ℓ-1 of meta-topolin in combination with 0.1 mg ℓ-1 of IAA. Optimal elongation for all the clones was detected upon addition of meta-topolin at 0.05 mg ℓ-1 and IAA at 0.5 mg ℓ-1. The peak rooting response was obtained with 0.5 mg ℓ-1 of IBA for clones 2 and 3, whilst 0.029 mg ℓ-1 of GR24 in combination with 0.5 mg ℓ-1 of IAA elicited optimal rooting for clone 1. The most consistent rooting response across all clonal lines, however, was obtained with the GR24 treatment. Clone 2 was found to be the most responsive to in vitro stimulation by auxin and this observation was further probed via RT-qPCR. Expression of the genes encoding the auxin efflux and influx transporters PIN1 and AUX1, respectively, were analysed to assess whether clonal response could be linked to expression of a particular gene(s). Equal expression levels of PIN1:AUX1 were detected for clone 2. Clones 1 and 3, however, exhibited an expression profile whereby PIN1 transcript levels exceeded those of AUX1 when treated with 0.5 mg ℓ-1 of IAA. These expression profiles suggest that equivalent expression of PIN1:AUX1 correlated with greater responsiveness to exogenously supplied IAA for clone 2. The findings of this study thus suggest a novel approach for the rapid determination of in vitro responses of valuable Eucalyptus genotypes, by investigating culture growth stages and gene expression levels.AFRIKAANSE OPSOMMING: Die gewoonte om Eucalyptus-spesies deur mikrovoortplanting te kweek het guns gevind in die ontwikkeling van suksesvolle bosbou-plantasieprogramme vanweë die doeltreffendheid daarvan om menige jeugdige plantvoorrade te genereer. Natuurlike fitohormone asook hul sintetiese analoë, genaamd plantgroeireguleerders (PGRs), word tipies in groeimedia ingesluit tydens mikrovoortplanting. Hierdie samestellings mag gebruik word om sekere fisiologiese prosesse in plante te stimuleer wat, by voorbeeld, die in vitro-manipulering van plantselgroei en-differensiasie toelaat, afhangende van die konsentrasies en kombinasies van spesifieke PGRs in die medium. Die ontwikkeling van mikrovoortplantingsprogramme vir Eucalyptus spp. is dikwels kloonspesifiek, wat optimalisering vir elke kloon vereis. Daar bestaan dus ‘n inherente behoefte aan die ontwikkeling van ‘n gestandaardiseerde protokol wat die in vitro-vestiging en-vermenigvuldiging van veelvuldige Eucalyptus-klone toelaat. In die huidige studie was die sensitiwiteit en responsiwiteit van drie Eucalyptus grandis × Eucalyptus nitens klone teenoor verskeie PGRs ondersoek vir die verskillende in vitro-groeistadiums. Dit sluit ouksiene in soos indool-3-asynsuur (IAA), indool-3-bottersuur (IBA) en 1-Naftaleen-asynsuur (NAA). Desgelyks was die sitokiniene kinetien, 6-Bensielaminopurien (BAP) en meta-topolien getoets. Die uitwerkings van strigolaktone op mikrovoortplanting was ondersoek deur aanwending van die sintetiese strigolaktoon-analoog, GR24. Parameters soos eksplantvermenigvuldiging, skootverlenging en wortelskieting was vir alle klone ondersoek. Die eksplante was op ‘n verlaagde PGR-instandhoudingsmedium geïnkubeer, waarvan bronmateriaal afgelei is vir daaropvolgende eksperimentering wat die onafhanklike ondersoek van groeistadiums moontlik gemaak het. Die grootste vlak van knoppie-inisiëring en skoot-ontwikkeling was waargeneem vir alle Eucalyptus-klone toe die groeimedium aangevul is met 0.5 mg ℓ-1 meta-topolien in kombinasie met 0.1 mg ℓ-1 IAA. Optimale verlenging van al die klone was gewaar met toevoeging van meta-topolien by 0.05 mg ℓ-1 en IAA by 0.5 mg ℓ-1. Die piekwortelrespons was verkry met 0.5 mg ℓ-1 IBA vir klone 2 and 3, terwyl 0.029 mg ℓ-1 GR24 in kombinasie met 0.5 mg ℓ-1 IAA optimale wortelskieting vir kloon 1 ontlok het. Die mees konsekwente wortelskietrespons, oor alle klonale lyne, was egter met die GR24-behandeling behaal. Daar was gevind dat kloon 2 mees responsief is op in vitro-stimulasie deur ouksien en hierdie waarneming was verder ondersoek via RT-qPCR. Uitdrukking van die gene wat onderskeidelik die ouksien-uitvloei- en invloeivervoerders PIN1 en AUX1 kodeer was geanaliseer om te bepaal of klonale vatbaarheid gekoppel kan word aan die uitdrukking van ‘n spesifieke geen(e). Gelyke uitdrukkingsvlakke van PIN1:AUX1 was vir kloon 2 bespeur. Klone 1 en 3 het egter ‘n uitdrukkingsprofiel getoon waardeur PIN1-transkripvlakke dié van AUX1 oorskry het tydens behandeling met 0.5 mg ℓ-1 IAA. Hierdie uitdrukkingsprofiele suggereer dat ekwivalente uitdrukking van PIN1:AUX1 ooreenstem met groter responsiwiteit op eksogene verskafde IAA vir kloon 2. Die bevindinge van hierdie studie stel dus ‘n nuwe benadering voor vir die vinnige bepaling van in vitro-reaksies van waardevolle Eucalyptus-genotipes, deur die ondersoek van kultuur groeistadiums en geenuitdrukkingvlakke.Master