Tissue Culture of Corymbia and Eucalyptus

Eucalypts are among the world’s most widely planted trees, but the productivity of eucalypt plantations is limited by their often-low amenability to true-to-type propagation from cuttings. An alternative approach to cutting propagation is tissue culture, which can be used to micropropagate valuable genotypes rapidly while simultaneously preserving germplasm in vitro. This review describes the use of tissue culture methods such as shoot culture, organogenesis, and somatic embryogenesis for micropropagating eucalypts. This review also discusses the use of cool storage, encapsulation, and cryopreservation methods for preserving eucalypt germplasm and delaying tissue maturation under minimal-growth conditions.

The productivity of eucalypt plantations has been limited by low amenability to clonal propagation from cuttings.Some species from high rainfall or riparian habitats, such as flooded gum (E.grandis), river red gum (E.camaldulensis), and rainbow gum (E.deglupta Blume), have long been considered amenable to cutting propagation [11][12][13][14][15][16].Nonetheless, efficient commercial-scale propagation of these species has relied on the development of intensively managed 'mini-cuttings' or 'micro-cuttings' systems for maintaining stock plants and producing cuttings in the nursery (Figure 1a).The difference between these two systems is that nursery stock plants in the mini-cuttings system are raised from small and often serially-propagated rooted cuttings, whereas nursery stock plants in the micro-cuttings system are raised initially in tissue culture [16][17][18][19][20][21][22][23][24][25][26].The species and hybrids that are propagated in these systems (e.g., many millions of E. grandis and E. grandis × E. urophylla plants per annum) are generally suited to high rainfall sites in the tropics or subtropics.Hardwood plantations are increasingly being established on drier and colder sites where land is more readily available and less expensive.These sites require other eucalypt species such as spotted gum (C.citriodora), Gympie messmate (E.cloeziana F.Muell.), southern blue gum (E.globulus), or shining leaf gum (E.nitens) that are more drought-or cold-tolerant, but which are also much more difficult to propagate from cuttings [14,[27][28][29][30][31][32][33][34].One of the great challenges in hardwood forestry is to develop efficient methods for the clonal propagation of eucalypts, particularly for those species that are difficult to propagate from cuttings.One of the most promising approaches is tissue culture, which can be used to micropropagate valuable genotypes rapidly whilst simultaneously preserving germplasm in vitro (Figure 1b,c) [35][36][37][38][39][40][41][42].

Establishment of Aseptic Cultures
Culture initiation is the first and often the limiting phase during in vitro propagation of trees because primary explants are typically non-aseptic and are, therefore, the main source of inoculum for microbial contamination in vitro.The initial explants for eucalypt tissue culture are typically seeds, shoot tips, nodes, or axillary buds (Table A1).Seeds do not provide true-to-type clonal propagation from a selected mother tree, but they can be used as the starting point for producing multiple clones from selected families in a vegetative family forestry program.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 proliferation capacity of shoot tips, nodes, or axillary buds and the subsequent growth of their plantlets may also be influenced strongly by the position of the tree from which the explant was harvested.Maturation effects such as reduced rooting capacity, shorter internode length, and decreased stem growth [54,55,[59][60][61][62] can become evident from very early stages (i.e., from relatively low explant positions) during the development of eucalypt trees [44,[63][64][65][66][67].
Surface sterilisation of the initial explant is required for contaminant-free initiation into a tissue culture medium.However, non-aseptic shoot cultures of E. benthamii Maiden & Cambage have been maintained successfully by incorporating an active chlorine source into all stages of the tissue culture to suppress microbial growth [68].Surface sterilisation of eucalypt explants typically involves rinsing in non-sterilised water or detergent solution, soaking in 70% ethanol for 30-60 s, immersing in a sterilant such as mercuric chloride (HgCl 2 ), sodium hypochlorite (NaOCl), or calcium hypochlorite (Ca(OCl) 2 ) with constant agitation (Table A1), and then rinsing in sterile distilled water.Other surface sterilants such as hydrogen peroxide (H 2 O 2 ), combinations of H 2 O 2 and ethanol, and alkyldimethylbenzalkonium chloride have been used occasionally for decontaminating eucalypt explants [47,[69][70][71][72][73][74][75][76].A drop of detergent or wetting agent such as Tween 20 ® is often added to the solution to improve contact between the sterilant and the explant surface, which is often hairy in the case of eucalypt shoots or leaves.The use of chlorine-based sterilants such as NaOCl or Ca(OCl) 2 is strongly recommended over the use of HgCl 2 because of the high mammalian toxicity and long-term environmental persistence of HgCl 2 [35,77,78].Eucalypt explants are generally treated with NaOCl at concentrations of 67-1340 mM for 1-30 min (Table A1).However, there can be a fine balance between applying sufficient sterilant to prevent microbial contamination and applying so much sterilant that it reduces seed germination or shoot growth.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].
An optimal balance of medium components including mineral salts, vitamins, organic supplements, and hormones contributes to the success of tissue culture for eucalypts and other plants.However, eucalypt explants are often placed initially onto simple culture media with minimal additives.Half-or full-strength Murashige and Skoog (MS) basal salts or media [79] are commonly used during culture establishment, typically with the addition of no organic additives other than 58.4 mM (2%) or 87.6 mM (3%) sucrose (Table A1).Establishment media sometimes include additives such as myo-inositol, thiamine, biotin, or calcium pantothenate, with or without hormones such as benzyladenine (BA), naphthalene acetic acid (NAA), or kinetin (Table A1).Reduced levels of mineral nutrients have also been used in some establishment media.For example, reduced levels of NH 4 NO 3 and KNO 3 have been used in MS-based establishment media for C. citriodora nodes [80] and greatly reduced CaCl 2 levels have been used during in vitro germination of E. dunnii, E. globulus, and E. saligna seeds [81][82][83][84].
Eucalypt initiation and shoot proliferation are usually performed on semi-solid media that incorporate gelling agents such as 6-8 g L −1 agar, 1.5-4.0g L −1 Gelrite, or 1.5-2.5 g L −1 Phytagel, which are adjusted to pH between 5.6 and 6.0 (Table A1).However, liquid media have been used to establish E. × phylacis L.A.S.Johnson & K.D.Hill nodes and shoot tips into culture [71].Paper supports over liquid MS salts have been used during establishment of axillary shoot tips of C. citriodora trees into culture [69], while seeds of C. maculata (Hook.)K.D.Hill & L.A.S.Johnson, E. sideroxylon A.Cunn.ex Woolls, and E. urophylla have been germinated on sterile moistened filter paper prior to transfer to semi-solid media for shoot culture or callogenesis [85][86][87].Anti-browning agents such as polyvinylpyrrolidone (PVP), ascorbate, and activated charcoal are sometimes added to the establishment medium to improve eucalypt explant survival [16,[88][89][90][91][92][93][94][95].Antibiotics can also be used during establishment to reduce bacterial or fungal contamination.Caution is warranted in the use of antibiotics because they may have only a bacteriostatic or fungistatic effect, with contaminants emerging at later and more-costly stages of the tissue culture process [96].Establishment of eucalypt explants, including the germination of seeds, is usually performed in the light, although cultures are sometimes established in darkness if the primary explant is being used to induce callus.

Shoot Culture
Shoot culture relies on the capacity to promote the outgrowth of existing axillary and accessory buds that occur at the base of each leaf axil.Eucalypt shoots have mostly been proliferated on full-strength, or sometimes half-strength, MS media or MS salts (Table A1).Media that use MS salts are usually supplemented with organic additives, including some that are constituents of MS medium (e.g., 500-555 µM myo-inositol, 1-6.25 µM thiamine-HCl, 4.06 µM nicotinic acid, 2.43 µM pyridoxine-HCl, and/or 26.64 µM glycine) and some that are not (e.g., 0.4 µM biotin and/or 0.2 µM calcium pantothenate).Other proliferation media for eucalypts have included woody plant medium (WPM) [94,97], JADS media [98][99][100], DKW medium [101], mixtures of MS and de Fossard nutrients [102], and MS basal salts with either White vitamins or B5 vitamins [85,87,90,103].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.The sucrose level has been dropped from 58.4 mM in the proliferation phase to 29.2 mM to promote elongation of E. grandis × E. urophylla shoots prior to transfer to root induction media [104].
Mineral nutrient levels have sometimes been adjusted in MS-based media for eucalypt shoot proliferation.The concentrations of KNO 3 and NH 4 NO 3 have been reduced by half in full-strength MS media for shoot culture of C. citriodora [80].Shoots of C. torelliana × C. torelliana exhibit micronutrient deficiencies in half-strength MS medium, but shoot proliferation and shoot length are not increased by using full-strength MS medium or by doubling the micronutrient levels in half-strength MS medium [46].A greatly decreased KNO 3 concentration of 1.88 mM, but increased MgSO 4 •7H 2 O concentration of 3.76 mM, has been used during the establishment and proliferation of E. dunnii, E. grandis × E. camaldulensis, E. grandis × E. urophylla, and E. urophylla × E. grandis shoots [105].Calcium chloride concentrations are often reduced to one-sixth of their full-strength MS levels, including for proliferation of E. globulus and E. saligna shoots [82,83].
Eucalypts have multiple buds within each leaf axil [106].Outgrowth of these buds is promoted by cytokinins such as 0.44-6.66µM BA and, occasionally, 0.23-9.29 µM kinetin that are added to the proliferation medium (Table A1).Cytokinins are sometimes supplemented with an auxin, usually 0.05-5.4µM 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.Cytokinin levels are often reduced during long-term maintenance of cultures or for a single passage prior to root induction [80,92,93,107,108].The gibberellins, GA 3 at 0.29-0.58µM 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].

Organogenesis
Organogenesis involves the formation of adventitious buds in tissues that would not have otherwise formed buds.The production of adventitious eucalypt shoots usually seems to occur through an intervening callus phase (i.e., by indirect organogenesis) [35], although the anatomical origin and development of the new shoots is often not investigated thoroughly.Callus can be induced from eucalypt hypocotyls, cotyledons, nodes, internodes, shoot apices, leaves, immature flowers, and stamens (Table A1).Explants from the base of the seedling such as the hypocotyls and cotyledons are typically the most responsive because these organs contain juvenile cells that have undergone minimal ageing [44,54,55,67].However, eucalypt shoots regenerating from the hypocotyls are often easier to proliferate subsequently than shoots regenerating from the cotyledons.
Adventitious eucalypt shoots usually appear to form via an intervening callus phase, but some media formulations and explant types may favour direct organogenesis.For example, nodular regenerating structures form on the hypocotyls of E. globulus zygotic embryos plated onto MS medium containing 16.2 µM NAA [47].These resemble somatic embryos, but microscopic examination demonstrates that they are formed via an organogenic, rather than an embryogenic, developmental pathway.These authors [47] suggested that the numerous reports of eucalypt somatic embryogenesis using similar protocols may, in fact, have described organogenesis, and that histological examination of numerous serial sections is required to confirm an embryogenic pathway.Similarly, some reports of eucalypt organogenesis from nodes must be treated with caution because eucalypt leaf axils contain multiple accessory buds, which may be released from dormancy by the same growth regulators, especially cytokinins, that induce callus formation.Callus overgrowth may conceal these growing buds, and so shoot formation could be occurring by either (or both) axillary shoot proliferation or adventitious shoot regeneration.The use of callus to regenerate shoots can provide very rapid plant production, but it also has the potential to release or induce somaclonal variation [35,45].There are only two reports of possible somaclonal variation arising during organogenesis of eucalypts.Haploid and triploid variants have been identified following callogenesis and organogenesis of E. urophylla [130] and amplified fragment length polymorphism analysis has identified genetic variation within clones, without apparent phenotypic variation, following callogenesis and organogenesis of E. globulus [131].

Somatic Embryogenesis
Somatic embryogenesis involves the formation of bipolar structures (i.e., with both a shoot meristem and a root meristem), typically along a morphological and physiological pathway that resembles the development of zygotic embryos.Histological examination is required to ultimately confirm the developmental pathway of embryo-like structures, although this has not been attempted in many reports of eucalypt embryogenesis [47].In practice, the morphology of the proliferative tissue might not be important provided that the tissue can be converted easily into either somatic emblings or plantlets.
Eucalypt somatic embryogenesis is initiated on semi-solid MS-based media typically with 87.6 mM or, sometimes, 58.4 mM sucrose (Table A1).However, B5 medium with 146 mM sucrose has been used for initiating somatic embryos on C. citriodora cotyledons [132], and N7 medium [118] has been used for callogenesis and somatic embryo formation from E. urophylla hypocotyls [86].MS nutrient levels are generally not adjusted for somatic embryogenesis although half-strength MS medium with one-sixth CaCl 2 has been used for E. dunnii [81] and MS medium with half-strength KNO 3 and NH 4 NO 3 has been used for E. microtheca [121].Embryogenic callus is often, though not always, induced in darkness.

Adventitious Root Formation
Germination of the bipolar structures formed during somatic embryogenesis requires media that stimulate growth from the existing root and shoot meristems.In contrast, the unipolar structures formed during shoot culture or organogenesis usually must be converted into plantlets by inducing adventitious roots at the base of the shoot.Root induction on eucalypt shoots is typically performed on semi-solid media similar to those used during the shoot establishment and proliferation phases (Table A1).However, the sucrose concentration is sometimes reduced from 87.6 mM to 58.4 mM or from 58.4 mM to 43.8 mM, and root induction is often performed in darkness.Glucose at 88 or 176 mM has been used, instead of sucrose, during root induction on E. globulus and E. saligna shoots [139].Activated charcoal at 83.3-833 mM is often incorporated into the root induction media, including for E. camaldulensis, E. globulus, E. grandis, E. grandis × E. urophylla, E. regnans F.Muell., and E. saligna [14,[82][83][84]88,[139][140][141][142][143][144].Activated charcoal may act by adsorbing inhibitory compounds, decreasing phenolic oxidation, altering medium pH, or reducing irradiance at the base of the shoot [145,146].
Levels of mineral nutrients are often reduced during the root induction phase for eucalypt shoots.Mineral adjustments have included the use of MS salts with 2.74 mM NaH 2 PO 4 , or a reduction in MS-medium strength to 1/10, for C. citriodora shoots [69,147].They have also included the use of MS medium with half-strength macronutrients, or half-strength nitrates, for E. camaldulensis shoots [116,143], or the use of MS macronutrients with half-strength micronutrients for E. grandis × E. urophylla shoots [148].More often, MS medium is simply reduced to half-strength during root induction, including for shoots of C. citriodora × C. torelliana, C. ptychocarpa (F.Muell.)K.D.Hill & L.A.S.Johnson,C.torelliana × C. citriodora, E. camaldulensis, E. camaldulensis × E. tereticornis, and E. grandis × E. urophylla [88,103,107,120,149,150].Half-strength MS medium with 1/10 KNO 3 and 2.5× MgSO 4 has been used for E. grandis shoots [104], and half-strength MS medium with full-strength vitamins, 2.66 µM riboflavin, and 0.93 µM β-carotene has been used recently for E. grandis × E. urophylla shoots [114].MS medium has also been reduced to quarter-strength during root induction, including for E. grandis, E. grandis × E. nitens, and E. grandis × E. urophylla shoots [124,[151][152][153]. MS nutrients at quarter-strength, but with half-or three-quarters-strength CaCl 2 and MgSO 4 , have been used for root induction on E. grandis × E. nitens and E. grandis × E. urophylla shoots [154,155].MS macronutrients at quarter-strength but with one-eighth-strength nitrogen sources and full-strength micronutrients have been used for E. marginata Donn ex Sm. root induction [156].
Eucalypt shoots are frequently transferred to auxin-free media after a short period on root induction medium (Table A1).This allows root and shoot elongation, which can be inhibited by long periods of exposure to exogenous auxin [165,166].Alternatively, shoots can be transferred to potting medium immediately after auxin treatment, bypassing one of the culture passages typically associated with rooting and ex-flasking.For example, C. torelliana × C. citriodora shoots can be transferred after 3-7 days on IBA-containing medium to tubes containing sterile potting mix, with the tubes placed in sterile 1-L plastic containers that are covered initially with another container to create a humid sealed volume of 2 L [41,45,46,64,120].Shoots of E. benthamii × E. dunnii, E. grandis × E. camaldulensis, E. grandis × E. tereticornis, and E. grandis × E. urophylla have been transferred directly ex vitro after auxin treatment [92,167].Shoots of E. benthamii × E. dunnii and E. grandis × E. camaldulensis have also been transferred directly ex vitro without an auxin treatment, as have E. cloeziana and E. dunnii shoots [92,96,99,104,119].
The process of ex-flasking shoots and acclimatising them to nursery conditions is one of the limiting steps in the micropropagation of many plants.One of the innovations in tissue culture that has been expected to improve ex-flasking capacity of eucalypt shoots has been the use of temporary immersion systems that provide repeated cycles of shoot wetting and drying [95,161,[168][169][170][171].These systems have the potential to increase nutrient and hormone uptake by repeatedly refreshing the medium in contact with the shoot surface during the wetting cycles while also conditioning the shoot for ex vitro conditions during the drying cycles (e.g., by promoting cuticle formation [170][171][172]).Temporary immersion has increased proliferation more than 2-fold and increased nursery survival from 5% to 67% for shoots of E. grandis × E. nitens [168], and increased proliferation more than 5-fold for shoots of E. grandis × E. urophylla [171].Temporary immersion and continuous immersion systems have both provided high rooting (100%) and nursery survival (76%) with E. camaldulensis shoots [161].Another technique that supports the acclimatisation capacity of eucalypt shoots is photoautotrophic culture, in which shoots are maintained under conditions of high CO 2 concentration, but low sugar concentration, to promote photosynthetic carbon fixation and transpiration [173][174][175][176][177]. Photoautotrophic culture has provided excellent nursery survival with shoots of E. camaldulensis (86-96%) and E. urophylla × E. grandis (100%) [175][176][177].

In Vitro Preservation
One of the advantages of tissue culture is the capacity to preserve germplasm in vitro for long periods without the large investments in land, labour, water, fertiliser, and pesticide that would be required for plantation-or nursery-based germplasm archives [42,[51][52][53]178,179].In vitro storage can also delay the maturation of valuable clones, especially if their shoots or callus are stored under minimal-growth or nil-growth conditions [39,40,42,[180][181][182]. Plantation trees generally display higher adventitious rooting capacity, stem growth, internode length, and developmental commitment to vegetative growth when they are propagated from juvenile, rather than mature, explants or cuttings [54,55,61,62,183,184].However, many eucalypt species progress through some of these juvenile-to-mature phase transitions at a very young age and low canopy height [11,39,40,44,63,64,67,185].This may be the one of the reasons why seeds (or in vitro seedlings) have been the initial explant source in 54% of the eucalypt tissue-culture techniques in which an explant source has been stated (Table A1).Propagation of selected adult trees often relies on the ability to obtain juvenile tissue at the base of the tree by inducing coppice shoots or epicormic shoots [54,55].Shoot tips, nodes, or axillary buds from nursery stock plants or adult trees have been the initial explant source in 46% of the eucalypt tissue-culture techniques in which an explant source has been stated (Table A1).This includes 24% of the techniques that used explants from nursery stock plants, 18% that used explants from the canopy of adult trees, and 4% that used explants from coppice shoots or epicormic shoots at the base of adult trees (Table A1).Coppice and basal epicormic shoots may be more juvenile than upper-canopy shoots but they are not as juvenile as seedling explants.Some plantation growers subculture difficult-to-root clones in vitro (e.g., for 10-12 passages) to rejuvenate their stock plant material prior to use in the nursery.Other plantation growers have moved away from employing clonal forestry programs that clonally propagate selected adult individuals to employing vegetative family forestry programs that propagate multiple clones from selected seedling families.Tissue culture techniques such as cool storage, synthetic seed preservation, and cryopreservation can preserve juvenile tissue in vitro with little or no growth.These techniques, therefore, have great potential to improve nursery efficiency and tree productivity in forestry plantation programs.Nonetheless, there are few reports of eucalypt germplasm storage under growth-limiting conditions.
Cool storage of shoots has been attempted for E. grandis and C. torelliana × C. citriodora.Storage at 10 • C and reduced irradiance (4 µmol m −2 s −1 ) allowed the preservation of E. grandis shoots on full-strength MS medium for 6 months, although shoots did not survive to 8 months [178].However, E. grandis shoots could be stored for 10 months at 24-28 • C on half-strength MS medium, or on full-strength MS medium with 37.8 µM abscisic acid (ABA) [178].Shoots of C. torelliana × C. citriodora have been stored on half-strength MS medium for 12 months at 14 • C and reduced irradiance (10 µmol m −2 s −1 ) [40].These shoots were subsequently ex-flasked and their performance as nursery stock plants compared with plants of the same clones that had been stored for the same 12-month period either ex vitro in the nursery or in vitro at 25 • C. Cool storage at 14 • C delayed clonal maturation, with adventitious rooting and total root mass of many clones being higher after cool storage than after ex vitro nursery storage [40].Adventitious rooting was sometimes also higher after cool storage at 14 • C than after storage at 25 • C [40], providing empirical evidence that minimal-growth storage can delay germplasm maturation and improve subsequent plant growth.
Synthetic seed preservation has also been attempted for E. grandis and C. torelliana × C. citriodora.Plant germplasm can generally be preserved, as synthetic seeds, by encapsulating small explant such as shoot tips, nodes, or axillary buds in calcium alginate [53,[186][187][188][189][190][191].Encapsulation can limit the size of the shoots, especially when the synthetic seeds are preserved under minimal-growth conditions of low temperature, reduced irradiance, or decreased nutrient supply [56,178,[192][193][194][195][196].Almost 50% of encapsulated axillary buds of E. grandis have been preserved successfully for 6 months at 10 • C and 4 µmol m −2 s −1 irradiance when the synthetic seeds, containing full-strength MS medium, were stored in jars containing a small volume of sterile distilled water [178].Between 76% and 100% of encapsulated shoot tips or nodes of C. torelliana × C. citriodora have been preserved successfully for 12 months at 14 • C in darkness when the synthetic seeds, containing highly-diluted MS medium, were stored in Petri dishes containing either agar alone, agar with 29.2 mM sucrose, or MS medium with 29.2 nM sucrose [56].The most effective storage substrate, MS medium with 29.2 mM sucrose, provided 92-100% regrowth capacity [56].This high regrowth capacity after 12 months of storage means that synthetic seed techniques can provide major commercial advantages in managing the workflow requirements for propagule production in commercial laboratories.Synthetic seeds can be constructed and stored throughout the year and then retrieved in one large batch, without requiring a peak labour commitment in the weeks prior to despatch.Storage of synthetic seeds beyond 6 months or 12 months has not been tested for either E. grandis or C. torelliana × C. citriodora, respectively.Further research is warranted to determine whether synthetic seeds could be stored for much longer than 1 year.If this were the case, alginate encapsulation would provide an extremely convenient, low-cost, and space-effective means to preserve germplasm.
Cryopreservation has been attempted for E. globulus, E. grandis, E. grandis × E. camaldulensis, E. grandis × E. urophylla, E. gunnii, and E. gunnii × E. dalrympleana Maiden.Cryopreservation has proven challenging because of the desiccation sensitivity of eucalypt buds [52,197].However, axillary buds of E. grandis × E. camaldulensis have been cryopreserved successfully, with 49% regrowth, by placing encapsulated explants on semi-solid MS media with progressively increasing sucrose and glycerol concentrations (each 0.4, 0.7 then 1.0 M), drying them in empty Petri dishes to a moisture content ≤25% before freezing, and re-growing them on media with progressively decreasing sucrose and glycerol concentrations [198].E. gunnii has also been cryopreserved successfully, with 62-73% regrowth, by transferring encapsulated shoot tips into liquid media with progressively increasing sucrose concentrations (0.3, 0.5, 0.75 then 1.0 M), drying them over silica gel before freezing, and re-growing them on MS medium with BA, NAA, and 87.6 µM sucrose [199].The same technique provided 43% and 13% regrowth from alginate-encapsulated shoot tips of E. gunnii × E. dalrympleana and E. globulus, respectively [199].Shoot-tip and axillary-bud cryopreservation has proven challenging for some eucalypts, but it has a major advantage over cool storage and synthetic seed preservation in potentially being able to store plant germplasm for many years without the need for periodic subcultures for recovery and re-storage.Cryopreservation has been used very successfully to store embryogenic callus of other tree species [36,48], but there are no reports of embryogenic-callus cryopreservation for eucalypt species.

Conclusions
Tissue culture provides a means to rapidly propagate selected eucalypt trees, or their progeny, in a clonal forestry or vegetative family forestry program.Eucalypt tissue cultures are usually initiated from shoot tips, nodes, axillary buds, or seeds, typically after surface sterilisation using detergent, aqueous ethanol solution, and sterilants such as NaOCl or Ca(OCl) 2 .Eucalypt plants can be multiplied through: (1) shoot culture, by proliferating shoots from existing axillary and accessory buds in the leaf axils; (2) organogenesis, by inducing adventitious buds, often through an intervening callus phase; or (3) somatic embryogenesis, by forming bipolar structures with both a shoot and root meristem, often following formation of an embryogenic callus.Eucalypt tissue culture is often performed on semi-solid MS-based media, although a wide range of media formulations and support systems have been employed.Shoots arising from shoot culture or organogenic culture are converted into plantlets using an auxin such as IBA to induce adventitious roots, although some eucalypts form adventitious roots spontaneously in the absence of exogenous auxin.Ex-flasking capacity can be improved by techniques such as temporary immersion and photoautotrophic culture that pre-acclimatise shoots for transfer to nursery conditions.There are few reports of eucalypt germplasm conservation in vitro despite the multitude of techniques for eucalypt plantlet or embling production.Nonetheless, cool storage, synthetic seed storage, and cryopreservation have all been successful, albeit following attempts with only a few eucalypt species.These preservation techniques for eucalypt germplasm have been under-utilised, given that in vitro preservation can delay or prevent the maturation of juvenile clones prior to their mass-production for hardwood plantations.The development of efficient clonal-propagation methods for eucalypts has been one of the great challenges in hardwood forestry.Micropropagation and in vitro preservation are now contributing to provide the best-possible hardwood trees for the global plantation estate.Single nodes and shoot tips from the only wild tree 63 mM alkyl-dimethyl-benzalkonium chloride for 10 min and 9.2 mM HgCl 2 for 30 s Liquid 1 2 MS medium with 100 µM Na.EDTA, 1 µM thiamine HCl, 2.5 µM pyridoxine HCl, 4 µM nicotinic acid, 500 µM myo-inositol, 100 mg L −1 MES buffer, 0.01% potassium citrate:citrate (10:1), 416.5 mM activated charcoal, and 1 µM zeatin, followed by semi-solid medium with the same organic compounds and 0.5 µM zeatin Nodular callogenesis on 1  2 MS medium with 100 µM Na.EDTA, 1 µM thiamine HCl, 2.5 µM pyridoxine HCl, 4 µM nicotinic acid, 500 µM myo-inositol, 100 mg L −1 MES buffer, and 5 µM TDZ, then shoot regeneration and development on the same medium but with 1 µM GA 4 , then with 0.5 µM zeatin and 1 µM GA 4 , then with 0.1 µM BA and 1 µM zeatin, then with 0.5 µM zeatin and 2 µM IAA, then with either 0.5 µM zeatin and 0.5 µM GA 4 or 1 µM zeatin and 0.5 µM IAA

Table A1 .
Culture techniques, explants, and media used in the micropropagation of eucalypts.Media are aseptic and semi-solid unless stated otherwise.
1 2 MS basal salts with 58.4 mM sucrose MS medium with 87.6 mM sucrose, then proliferation on MS medium with 4.4 µM BA and 87.6 mM sucrose 1 2 MS medium with 19.6 µM IBA and 58.4 mM sucrose for 7 days, then sterile vermiculite and perlite 8 g L −1 agar pH 5.8