In vitro propagation of Corymbia torelliana × C. citriodora (Myrtaceae) via cytokinin-free node culture

Abstract

Hybrids between Corymbia torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora subsp. variegata (F.Muell.) A.R.Bean & M.W.McDonald are used extensively to establish forestry plantations in subtropical Australia. Methods were developed for in vitro seed germination, shoot multiplication and plantlet formation that could be used to establish in vitro and ex vitro clone banks of juvenile Corymbia hybrids. Effects of sodium hypochlorite concentration and exposure time on seed contamination and germination, and effects of cytokinin and auxin concentrations on shoot multiplication and subsequent rooting, were assessed. A two-step surface sterilisation procedure, involving 70% ethanol followed by 1% sodium hypochlorite, provided almost no contamination and at least 88% germination. A novel method of cytokinin-free node culture proved most effective for in vitro propagation. Lateral bud break of primary shoots was difficult to induce by using cytokinin, but primary shoots rooted prolifically, elongated rapidly and produced multiple nodes in the absence of exogenous cytokinin. Further multiplication was obtained either by elongating lateral shoots of nodal explants in cytokinin-free medium or by inducing organogenic callus and axillary shoot proliferation with 2.2 µm benzyladenine. Plantlets were produced using an in vitro soil-less method that provided extensive rooting in sterile propagation mixture. These methods provide a means for simultaneous laboratory storage and field-testing of clones before selection and multiplication of desired genotypes.

Tissue Culture of Corymbia and Eucalyptus

“…Cytokinins are sometimes supplemented with an auxin, usually 0.05-5.4 µM NAA. Auxins can promote eucalypt rooting and shoot elongation in cytokinin-free proliferation media [45,46]. However, cytokinins prevent adventitious rooting and so it is unclear why auxin rooting hormones are added to shoot proliferation media that contain cytokinins.…”

“…For example, raising the NaOCl concentration progressively from 134 to 402 to 670 mM reduced seed germination of C. torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson × C. citriodora from 88% to 74% to 64%, respectively, and it reduced the percentage of plated seeds with shoots of sufficient length (>5 mm) for subculture from 78% to 65% to 52%, respectively [45].…”

“…Shoot proliferation is performed in the light, usually on semi-solid medium containing 58.4 mM or 87.6 mM sucrose (Table A1). Sucrose at 87.6 mM has been used in proliferation media for Corymbia species and hybrids even when 58.4 mM sucrose was used in the establishment medium [45,46,64,69,87]. A lowered sucrose level of 43.8 mM has been used during elongation [92] or both proliferation and elongation [93] of E. benthamii × E. dunnii shoots.…”

“…Seeds are also an appropriate explant source for producing a genetically diverse collection of plants for conservation, revegetation, fodder production, or horticulture. Seeds are often the simplest explants to initiate into tissue culture because they are easy to decontaminate and because the juvenility of young seedlings makes them conducive to callogenesis or rapid shoot proliferation [35,45,54,55]. The use of shoot tips, nodes, or axillary buds as explants allows true-to-type propagation of selected trees, but these explants can be difficult to decontaminate, especially for eucalypts that are covered in hairs.…”

“…The gibberellins, GA 3 at 0.29-0.58 µM or GA 4 at 0.5 or 1 µM, have been added for a single passage to promote elongation of E. benthamii × E. dunnii and E. impensa Brooker & Hopper shoots, respectively, prior to root induction [70]. Shoot culture often provides lower plant production rates than alternative methods that include a callus phase, but the repeated use of intact organs is thought to minimize the risk of releasing or inducing somaclonal variation [35,45,46]. Genetic variation has been reported after shoot culture of E. camaldulensis and E. tereticornis clones, although much of this variation was attributed to mislabelling during the tissue culture process [16,109].…”