In vitro techniques have found increasing use in the conservation of threatened plants in recent years and this trend is likely to continue as more species face risk of extinction. The Micropropagation Unit at Royal Botanic Gardens, Kew, UK (RBG Kew) has an extensive collection of in vitro plants including many threatened species from throughout the world. The long history of the unit and the range of plants cultured have enabled considerable expertise to be amassed in identifying the problems and developing experimental strategies for propagation and conservation of threatened plants. While a large body of knowledge is available on the in vitro culture of plants, there are limited publications relating to threatened plant conservation. This review highlights the progress in in vitro culture and conservation of threatened plants in the past decade (1995–2005) and suggests future research directions. Works on non-threatened plants are also included wherever methods have applications in rare plant conservation. Recalcitrant plant materials collected from the wild or ex situ collections are difficult to grow in culture. Different methods of sterilization and other treatments to establish clean material for culture initiation are reviewed. Application of different culture methods for multiplication, and use of unconventional materials for rooting and transplantation are reviewed. As the available plant material for culture initiation is scarce and in many cases associated with inherent problems such as low viability and endogenous contamination, reliable protocols on multiplication, rooting, and storage methods are very important. In this context, photoautotrophic micropropagation has the potential for development as a routine method for the in vitro conservation of endangered plants. Long-term storage of material in culture is challenging and the potential applications of cryopreservation are significant in this area. Future conservation biotechnology research and its applications must be aimed at conserving highly threatened, mainly endemic, plants from conservation hotspots.

https://bioone.org/journals/In-Vitro-Cellular-and-Developmental-Biology---Plant/volume-42/issue-3/IVP2006769/CONSERVATION-iIN-VITRO-i-OF-THREATENED-PLANTS--PROGRESS-IN/10.1079/IVP2006769.pdf

Conservation in vitro of threatened plants - progress in the past decade

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Eucalypt domestication and breeding

Abscisic acid (ABA) plays a significant role in the regulation of many physiological processes of plants. It is often used in tissue culture systems to promote somatic embryogenesis and enhance somatic embryo quality by increasing desiccation tolerance and preventing precocious germination. ABA is also employed to induce somatic embryos to enter a quiescent state in plant tissue culture systems and during synthetic seed research. Application of exogenous ABA improves in vitro conservation and the adaptive response of plant cell and tissues to various environmental stresses. ABA can act as anti-transpirant during the acclimatization of tissue culture-raised plantlets and reduces relative water loss of leaves during the ex vitro transfer of plantlets even when non-functional stomata are present. This review focuses on the possible roles of ABA in plant tissue culture and recent developments in this area.

The role of abscisic acid in plant tissue culture: a review of recent progress

Synseed technology-a complete synthesis.

Several techniques have been developed for reinvigorating, rejuvenating or maintaining the juvenility of plants. None of these techniques is as effective as natural rejuvenation whereby the most mature plant parts, the reproductive organs, produce the most juvenile plant parts, the embryos, through gametogenesis and sexual reproduction. The most common criteria for identifying reinvigoration or rejuvenation are based on morphology, morphogenic capacity and the ability to produce cones or flowers. Doubts remain over whether true rejuvenation (reduced ontogenetic age) can be achieved by artificial methods such as serial vegetative propagation, or whether these methods merely provide reinvigoration (reduced physiological age) by temporarily removing environmental and physiological constraints to growth. Rejuvenation and reinvigoration are difficult to distinguish from each other but there is some evidence that artificial methods provide at least partial rejuvenation of plants. This review examines methods for delaying the maturation of juvenile plants and for reducing the ontogenetic or physiological age of more-mature plants. The review discusses theoretical and practical aspects of juvenility, rejuvenation and reinvigoration in clonal forestry, and identifies research areas in maturation that could lead to more-effective methods for establishing high-productivity plantations.

Maturation and related aspects in clonal forestry—part II: reinvigoration, rejuvenation and juvenility maintenance

Slow growth-storage, for up to 10 months, has been achieved for Eucalyptus grandis shoot cultures by either the addition of 10 mg l−1 abscisic acid to the growth medium or by the halving of nutrient supply (half MS) and removal of exogenous plant growth regulators. Reduction of light intensity or the addition of mannitol to the media were less effective in reducing growth rate. Isolated in vitro axillary buds encapsulated in calcium alginate and stored under low temperature and low light intensities survived for up to 3 months without loss in viability. Storage of such encapsulated fresh axillary buds at higher temperature resulted in a loss in viability. These methods have immediate applications to forestry breeding and clonal programs.

In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions.

Multiple shoots of two Eucalyptus grandis Hill ex Maid. x E. nitens (Deane & Maid.) Maid. clones (GN121 and GN107) generated from axillary buds were used for in vitro rooting studies. The highest rooting rates in clones GN121 (75%) and GN107 (65%) were achieved on modified 1/4-strength Murashige and Skoog (MS) (1962) medium (Ca 2+ and Mg 2+ levels as for 3/4-strength MS), 0.5 μM IBA, 0.4 μM biotin, 0.2 μM calcium pantothenate, 0.04 M sucrose and 0.4% (w/v) Gelrite ® . The optimal culture conditions were an initial 72-h dark incubation period followed by a 16-hour photope- riod at a photosynthetic photon flux density (PPFD) of 37 μmol·m -2 ·s -1 and 23 °C day/ 21 °C night for 7 days, after which the PPFD and temperature were increased to 66 μmol·m -2 ·s -1 and 27 °C day/21 °C night for 18 days. Plantlets were acclimatized with survival rates of 78% for GN121 and 58% for GN107 after 28 days. Chemical name used: indole-3-butyric acid (IBA).

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In Vitro Rooting and Subsequent Survival of Two Clones of a Cold-tolerant Eucalyptus grandis × E. nitens Hybrid

A simple method was established for the collection and short-term storage of shoot material from field-grown Eucalyptus grandis and E. grandis hybrids for micropropagation. Initial studies were undertaken with plants grown outside a greenhouse, which were neither fertilised nor treated with fungicides. The method was then tested and adapted for field-grown clones. It involved collecting 35–50 mm long stems with three nodes and no leaves, spraying them with 70% (v/v) ethanol and storing them in glass bottles containing moist sterile vermiculite for 48 h. Addition of 1 g l−1 calcium hypochlorite to the first culture medium (bud break) inhibited endogenous contamination. Multiplication yields after storage of field-grown explants were 160–264 shoots/100 explants, depending on clone. This offers an alternative, improved means for explant collection over present standard procedures of maintaining parent plants in hedges or transporting shoots to the micropropagation laboratory in buckets.

In vitro field collection techniques for Eucalyptus micropropagation

Success in Eucalyptus micropropagation varies with genotype. Although some protocols have proven suitable for suites of clones, many genotypes are recalcitrant to rooting. Their micropropagation is addressed empirically through the manipulation of auxins and cytokinins, which work antagonistically to produce roots and shoots, respectively. Rooting success of three genotypes with 0.1 mg/l indole-3-butyric acid (IBA) was initially recorded as 87, 45 and 41% for clones 1, 2 and 3, respectively. Further studies using the auxin signal transduction inhibitor n-chlorophenoxyisobutyric acid (PCIB) or the auxin conjugation inhibitor dihydroxyacetophenone (DHAP) indicated that the poor rooting response of clone 2 was not due to deficient auxin signal perception or auxin conjugation. Omitting kinetin during elongation, followed by auxin-free rooting, significantly increased root production in clone 2 (from 45 to 80.3%), but had no effect on clone 1. Gas chromatography-mass spectrometry (GC-MS) analysis of auxins and kinetin of shoots, prior to rooting, revealed a strong relationship (R2 = 0.943) between rootability and the shoot kinetin:auxin. Replacing kinetin with the less stable trans-zeatin significantly increased rooting of clone 2 (from 19 to 45%) and clone 3 (31 to 52%). It is suggested that root induction in poor-rooters is dependent on exogenous cytokinin depletion from in vitro shoots. Key words : Eucalyptus , phytohormone interaction, root quality.

/pdf/the-properties-and-interaction-of-auxins-and-cytokinins-4hj72w450u.pdf

M. Paula Watt

Papers

In vitro storage of Eucalyptus grandis germplasm under minimal growth conditions.

In Vitro Rooting and Subsequent Survival of Two Clones of a Cold-tolerant Eucalyptus grandis × E. nitens Hybrid

In vitro field collection techniques for Eucalyptus micropropagation

The properties and interaction of auxins and cytokinins influence rooting of shoot cultures of Eucalyptus