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Conservation of Plant Germplasm

South Africa has a plethora of plant genetic resources. This richness is decreasing at an alarming rate. Plant in vitro technology offers a potential solution to both the long term storage of germplasm and mass production of species categorized as endangered.

The Research Team

Head:

    Professor David Mycock, School of Animal, Plant and Environmental Sciences, University of the Witwatersrand.

Collaborators:

    National
  1. Professor P. Berjak, School of Life and Environmental Sciences, University of Natal, Durban.
  2. Dr M.P. Watt, School of Life and Environmental Sciences, University of natal, Durban.
  3. Dr J.M. Farrant, Department of Botany, University of Cape Town.
  4. Dr V.M. Gray, School of Cell and Molecular Biology, University of the Witwatersrand.
    International
  1. Dr W.E. Finch-Savage, Horticultural Research International, Wellesbourne, UK.
  2. Dr C.W. Walters (Vertucci), National Seed Storage Laboratory, United States Department of Agriculture, Fort Collins, Colorado, USA.

Students:

    Ph.D Students:
  • Jonathan Groll
  • Candace Is cs
  • Phillipa Stewart
    M.Sc Students:
  • Mr K Naicker
  • Ms M Taylor
  • Kershree Padayachee

Introduction

Plant genetic resource conservation can be considered from two aspects. In situ conservation involves the establishment and/ or maintenance of natural reserves where species are allowed to remain in optimal ecosystems with the minimum of management. On the other hand, ex situ conservation involves the use of botanic gardens, field plantations, seed stores and gene banks. Within each category of conservation there are numerous approaches and associated problems. The research projects carried out in the department have particular relevance to certain aspects of ex situ conservation of plant germplasm, viz. seed stores and genebanks.

In terms of their storability seed-bearing plants fall into two categories, those that produce what are termed "orthodox" desiccation-tolerant seeds and those that have "recalcitrant", desiccation-sensitive propagules.

The ability of orthodox seeds to undergo maturation drying and withstand lowered temperatures has enabled humans to store this type of seed and, in fact seed storage can be considered as ancient as agriculture itself. However, storage for human needs often imposes the requirement of seed survival over periods which exceed the natural life span. It is consequently not surprising that the condition of orthodox seeds in storage will deteriorate more or less rapidly with resultant losses in vigour and viability. An additional factor, which is frequently dismissed as inconsequential, or is even completely neglected, is the r?of micro-organisms, in particular fungi, which are responsible for the spoilage or total destruction of stored seed.

Recalcitrant seeds, unlike their orthodox counterparts, do not undergo a period of maturation drying during their development, and as a consequence, they are shed from the parent plant at high water content. Since these seeds are sensitive to desiccation and generally also to chilling, they cannot be stored under the conditions that facilitate storage of orthodox seeds. A number of economically important tropical and sub-tropical crop species are characterised by having recalcitrant seeds e.g. Camellia sinensis (tea) and Hevea brasiliensis (rubber) amongst others. At present the only practical method of conservation of these seeds is by storage at ambient temperatures and relative humidities allowing maintenance of the seed water content at that characterising, or only a little below, the newly shed state. This approach which is termed "wet storage" is useful only in the short-term. Furthermore, the natural loss of viability of recalcitrant seeds, which is accompanied by leakage of biomolecules due to loss of membrane integrity, in conjunction with the relatively warm, humid storage conditions provides an excellent environment for fungal proliferation and this exacerbates deterioration. Because their seeds are short lived, and in order to maintain desirable cultivars crop species that produce recalcitrant seeds are vegetatively propagated.

Cassava (Manihot esculenta Crantz) is a woody perennial shrub cultivated for its starchy tuberous roots and is one of the main energy sources for nearly 500 million people of which 200 million are in Africa. As a source of human and animal nutrition cassava is not popular in South Africa, however, there is renewed interest in the cultivation of the crop as a source of commercial starch. Although this crop produces orthodox seeds, due to a high degree of heterozygosity, low fertility, poor seed set and sterile cultivated seed, the crop can only be propagated and conserved via vegetative means. Date palm (Phoenix dactylifera), which is assuming importance in the local agricultural industry, also falls into the same category as cassava in that desirable cultivars can only be propagated via vegetative methods.

In terms of germplasm conservation there are a number of problems associated with vegetative propagation e.g. annihilation of uniform stands by insects. As a number of these crops (orthodox and recalcitrant) are propagated in South Africa (see above), these problems are relevant to the local agricultural sector.

South Africa has a plethora of plant genetic resources, and in terms of economic, medicinal and scientific importance much of this indigenous diversity and richness is not realised. There are organisations at all levels of society from government down (the NRF included), that are aware of this wealth and who are attempting to understand, utilise and preserve this national heritage. Due to a myriad of factors (which are beyond the scope of this report) this richness is decreasing at an alarming rate. For species that are considered endangered a number of approaches can be taken and these strategies can vary depending on the level of remedial action. For instance, populations may be isolated and natural reproductive processes used to increase numbers (in situ conservation). Alternatively, plant numbers may be increased via human directed mass propagation and subsequent re-introduction into the natural environment (ex situ conservation). Superimposed on, and complimentary to, such approaches are the possibilities afforded by the preservation of propagules in seed stores and genebanks. Unfortunately for many South African species little is known about their eco-physiology, physiology (orthodox or recalcitrant) and storage requirements.

Plant in vitro technology offers a potential solution to both the long term conservation of these presently difficult to store germplasm categories and in the mass production of species that are categorised as endangered. In vitro technology has the distinct advantage that a variety of cells and organised tissues may be stored, e.g. single cells, meristems embryos (both zygotic and somatic). Principal amongst the in vitro conservation technologies is cryopreservation, that is the storage of plant tissues at temperatures below -80 oC and usually at liquid nitrogen temperature (-196 oC). Although other procedures, such as slow growth, can be considered, the optimisation and scientific understanding of the cryopreservation procedures has formed one of the major thrusts of my research. Obviously, such studies can only commence once the appropriate in vitro micropropagatory procedures for the species under consideration have been developed.

The procedures associated with plant tissue culture are basically dependent on two developmental processes, organogenesis and embryogenesis. Due to their economic importance, these procedures are fairly well documented for most crop species. However, the situation is generally not the same for South African indigenous species. We have therefore decided to develop the micropropagatory procedures (in particular somatic embryogenic procedures) for a number of endangered indigenous species and for two vegetatively propagated crops (cassava and date palm).

Consequently, we believe that our research has relevance to plant genetic resource conservation in the broadest sense, that is, the aims and objectives of the research are directed at the development and scientific understanding of long-term conservation methods for plants that are presently difficult to store by conventional methods. Two aspects are being considered: 1. Development of micropropagatory techniques for selected endangered South African indigenous plants and vegetatively propagated crop species. 2. Development of storage techniques for plant species (both indigenous and crops) which hitherto have been unstorable in the medium to long term.

Ultimate marriage between approaches (technologies) will enable the propagules produced by the micropropagatory techniques to be long-term stored. Similarly they may also have relevance to the conservation of species that produce recalcitrant seeds.

Development of micropropagatory procedures for selected indigenous and crop species

Tissue culture provides new methods for both the production and storage of plant germplasm, and this is particularly relevant to species that are vegetatively propagated or those that produce recalcitrant seeds (see earlier). The development of reproducible methods for the production of the in vitro tissues (particularly somatic embryos) of specific species has therefore been a priority. To this end a range of approaches (direct, indirect etc) have been tested to determine the optimum somatic embryogenic procedure for each species. A number of species have been studied viz. Clivia miniata (horticulturally important), Haworthia limifolia (endangered indigenous), Haworthia koelmaniorum (endangered indigenous), Mannihot esculenta (Cassava - vegetatively propagated), Phoenix dactylifera (date palm - vegetatively propagated), Siphonochilus aethiopicus (endangered indigenous) and Warburgia salutaris (endangered indigenous). The indigenous species selected were identified by the FRD s Indigenous Plant Programme as being of particular importance.

SOME RECENT PUBLICATIONS IN THIS FIELD

Naicker, K. and Mycock, D.J. (1997). Ultrastructural studies on the somatic embryoids of two Haworthia species. Proceedings of the Microscopy Society of Southern Africa, 27, 83.

Stewart, P. and Mycock, D.J. (1997). Somatic embryogenesis in Warburgia salutaris. Proceedings of the Microscopy Society of Southern Africa, 27, 84.

Taylor, M. and Mycock, D.J. (1997). The preparation of cassava somatic embryoids for TEM. Proceedings of the Microscopy Society of Southern Africa, 27, 85.

Groll, J., Gray, V.M. and Mycock, D.J. (1997). Friable embryogenic callus formation from thin cell layer explants of Cassava. African Journal of Root and Tuber Crops, 2, 154-158.

Mycock, D.J., Watt, M.P., Hannweg, K.F., Naicker, K., Makwarela, M. and Berjak, P. (1997). Micropropagation of two indigenous Haworthia spp. (H. limifolia and H. koelmaniorum). South African Journal of Botany, 63 (6), 345-350.

Development of cryostorage techniques

The development of cryopreservation methods requires the empirical determination, on a species basis, of the optimum conditions for cryostorage. Variables such as water content, the necessity for, and type and concentration of cryoprotectant, tissue developmental state, tissue desiccation tolerance/sensitivity and freezing and thawing rates have to be taken into consideration. Water content and size of the sample are possibly the principal factors in preparation for cryopreservation. Water content is important as it affects the amount of lethal ice crystal formation, and sample size regulates the rate of freezing and thawing. Generally the smaller, and within limits the drier the better. This however is not always possible. In terms of size and ease of physical manipulation embryos are ideal candidates for cryopreservation.

SOME RECENT PUBLICATIONS IN THIS FIELD

Is cs, C. and Mycock, D.J. (1997). Effects of early imbibition on the ultrastructure of isolated maize root meristems. Proceedings of the Microscopy Society of Southern Africa, 27, 81.

Mycock, D.J. (1998). Important variables in the development of cryostorage procedures for somatic embryoids. In: Progress in Seed Research: Proceedings of the second international conference on seed science and technology. (Eds) A.G. Taylor and X-L Huang, Communications services of the New York State Agricultural Experimental Station, Geneva, NY, 316-321.

Mycock, D.J. (1999). Addition of calcium and magnesium to a glycerol and sucrose cryoprotectant solution improves the quality of plant embryo recovery from cryostorage. Cryo-Letters, 20, 77-82.

Is cs, C. and Mycock, D.J. (1999). Ultrastructural effects of early imbibition and cryostorage on Zea mays L. root meristems. Cryo-Letters, 20, IN PRESS.

Berjak, P., Walker, M., Watt, M.P. and Mycock, D.J. (1999). Experimental parameters underlying failure or success in plant germplasm cryopreservation: A case study on zygotic axes of Quercus robur. L. Cryo-Letters, 20, IN PRESS.

 

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