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

Epigenetic mechanisms are highly dynamic events that modulate gene expression. As more accurate and powerful tools for epigenetic analysis become available for application in a broader range of plant species, analysis of the epigenetic landscape of plant cell cultures may turn out to be crucial for understanding variant phenotypes. In vitro plant cell and tissue culture methodologies are important for many ongoing plant propagation and breeding programmes as well as for cutting-edge research in several plant model species. Although it has long been known that in vitro conditions induce variation at several levels, most studies using such conditions rely on the assumption that in vitro cultured plant cells/tissues mostly conform genotypically and phenotypically. However, when large-scale clonal propagation is the aim, there has been a concern in confirming true-to-typeness using molecular markers for evaluating stability. While in most reports genetic variation has been found to occur at relatively modest frequencies, variation in DNA methylation patterns seems to be much more frequent and in some cases it has been directly implicated in phenotypic variation. Recent advances in the field of epigenetics have uncovered highly dynamic mechanisms of chromatin remodelling occurring during cell dedifferentiation and differentiation processes on which in vitro adventitious plant regeneration systems are based. Here, an overview of recent findings related to developmental switches occurring during in vitro culture is presented. Additionally, an update on the detection of epigenetic variation in plant cell cultures will be provided and discussed in the light of recent progress in the plant epigenetics field.

/pdf/an-epigenetic-view-of-plant-cells-cultured-in-vitro-gfrjwecxtc.pdf

An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond

Introduction. 1. Male Gametogenesis Development and Structure of Sperm D. Southworth, S.C. Russell. 2. Sperm and Generative Cell Isolation and Manipulation D. Southworth. 3. Pollen Germination and Pollen Tube Growth Tip Growth Mechanism in Sexual Plant Reproduction A. Moscatelli, M. Cresti. 4. Female Gametogenesis Ontogenesis of the Embryo Sac and Female Gametes S.D. Russell. 5. Embryo Sac Isolation and Manipulation D.D. Cass, J.D. Laurie. 6. In Vivo Fertilization T.B. Batygina, V.E. Vasilyeva. 7. In Vitro Fertilization E. Kranz. 8. Sexual Incompatibility F. Cruz-Garcia, B.A. MacClure. 9. Zygotic Embryogenesis Structural Aspects R. Czapik, R. Izmailow. 10. Zygotic Embryogenesis Hormonal Control of Embryo Development B. Fischer-Iglesias, G. Neuhaus. 11. Zygotic Embryogenesis: Developmental Genetics The Formation of an Embryo from a Fertilized Egg K. Schrick, T. Laux. 12. Somatic Embryogenesis T.A. Thorpe, C. Stasolla. 13. Synthetic Seeds of Asparagus officinalis L. K. Mamiya, et al. 14. Endosperm Development P.W. Becraft, et al. 15. Seed Maturation, Germination, and Dormancy A.B. Downie. 16. Gametophytic Apomixis A Successful Mutation of the Female Gametogenesis Y. Savidan. 17. Parthenocarpy State of the Art A. Spena, G.L. Rotino. 18. Androgenesis in Brassica A Model System to Study the Initiation of Plant Embryogenesis J.B.M. Custers, et al. 19. Androgenesis in Cereals S.K. Datta. 20. In Vitro Gynogenesis S.S. Bhojwani, T.D. Thomas. 21. Inheritance of Cytoplasmic Traits -- Embryological Perspectives T. Kuroiwa, et al. Subject Index.

Current trends in the embryology of angiosperms.

In vitro recalcitrance is the inability of plant cells, tissues and organs to respond to tissue culture manipulations. With respect to plant regeneration, recalcitrance can be a major limiting factor for the biotechnological exploitation of economically important plant species and it can also impair the wider application of in vitro conservation techniques. This first paper introduces a compilation of Symposium papers, collectively entitled “Do we understand in vitro plant recalcitrance?”, presented at the 1999 Congress of the Society for In Vitro Biology. The Symposium reviewed recalcitrance in the context of genetic predeterminism, molecular markers and gene expression patterns, whole and explant physiology, stress physiology, habituation, neoplastic progression and plant cancer. The symposium contributors present fundamental and applied investigative approaches which have the potential to enhance our current understanding of in vitro recalcitrance and to assist in overcoming the problems associated with nonresponsive plant cultures. This introductory paper presents the general concept of recalcitrance in relation to whole-plant and explant physiology and considers basic aspects of tissue culture manipulations in the context of recalcitrance problems.

Sepecial symposium: In vitro plant recalcitrance in vitro plant recalcitrance: An introduction

Micropropagation and accumulation of essential oils in wild sage (Salvia fruticosa Mill.)

The aim of this study was to micropropagate mature Quercus robur L. trees when material retaining physiologically juvenile characteristics (stump sprouts, epicormic shoots) is not available. Branch segments from 70–300 year-old trees were force-flushed and the flushed, partially rejuvenated or reinvigorated shoots were used as a source of explants for establishment of cultures. In vitro establishment and multiplication was achieved with seven of the eight selected trees. The proliferation capacity of cultures of vertically placed explants declined after several subcultures, but efficient shoot multiplication was achieved by culturing decapitated shoots placed horizontally on GD medium supplemented with 0.89 μM of 6-benzyladenine. Reculturing the same horizontal explant several times allowed both higher multiplication rates and a shorter subculture cycle (2 weeks). An initial dark period of 5 days generally improved rooting capacity, which ranged, depending on clone, from 15 to 46%.

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions.

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Salvia valentina Vahl and Salvia blancoana Webb & Heldr subsp. mariolensis Figuerola, two endemic species of Salvia from the Mediterranean coastal region of Spain, were successfully gegenerated in vitro from adult plants using two explant types (apical and nodal segments). Maximum shoot proliferation for both species was obtained with nodal explants: for S. blancoana on Murashige and Skoog medium supplemented with 6-γ-γ-dimethylallylaminopurine at 1 mg 1−1 (4.9 μM). and for S. valentina on the same medium with kinetin at 1–2 mg 1−1 (4.6–9.3 μM). The influence of apical dominance, and the explant viability in culture were found to be the main differences between the two species during the shoot multiplication phase. Rooting of shoots was low, specially for S. valentina. For both species, rooting was achieved in Murashige and Skoog medium without growth regulators. In general the addition of the auxins indole 3 acetic acid or indole-3-butyric acid did not improve the rooting or, in the case of naphthaleneacetic acid, had an inhibitory effect. In the best rooting media, rooting shoots increased in length. The rooted plantlets were acclimated to ex vitro conditions and a survival percentage > 70% was obtained under greenhouse conditions. This work was carried out as an ex situ conservation method for these Spanish endemic species.

In vitro propagation of two Spanish endemic species of Salvia through bud proliferation.

Salix tarraconensis Pau ex Font Quer, an endemic willow species from northeast Spain, was micropropagated with nodal segments. Shoot multiplication was obtained with different cytokinins, either on Murashige and Skoog medium or woody plant medium. Best results for shoot formation were obtained on Murashige and Skoog medium containing 4.9 μM of 6-γ-γ-dimethylallylaminopurine. Shoots showed strong apical dominance, and some cultures displayed apical necrosis. Benzyladenine gave the worst results; shoots displayed very slow growth, deformed leaves, and hyperhydrity. Good rooting of shoots was obtained with different auxins or without plant growth regulators on woody plant medium. The best results (90-100%) were obtained within 20 d. On rooting media with indole-3-butyric acid or indoleacetic acid, shoot elongation was good (35-40 mm length). Apical necrosis was observed in elongating shoots on rooting medium, but this disturbance favored axillary bud sprouting and formation of new shoots. Shoot length and quality of roots decreased gradually as the concentration of naphthaleneacetic acid increased. Plant survival was 90% 4 weeks after removal fromin vitro conditions.

https://www.jstor.org/stable/info/20064872

In vitro propagation of Salix tarraconensis Pau ex Font Quer, an endemic and threatened plant

Micropropagation of Limonium cavanillesii Erben, a threatened and endemic statice species from Valencia Community (Eastern Spain), was successfully achieved using inflorescence stem pieces as initial explants. Segments 20 mm long from basal parts of immature inflorescences and with axillary buds were cut, sterilised and established in vitro. Shoots obtained from indifferentiated buds were sectioned and then transferred to Murashige and Skoog (MS) medium with 2 mg l−1 kinetin to provide a plant stock.

J. B. Amo-Marco

Papers

Foreed flushing of branch segments as a method for obtaining reactive explants of mature Quercus robur trees for micropropagation

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

In vitro propagation of two Spanish endemic species of Salvia through bud proliferation.

In vitro propagation of Salix tarraconensis Pau ex Font Quer, an endemic and threatened plant

Micropropagation of Limonium cavanillesii Erben, a threatened statice, from inflorescence stems