Author(s):Work by Dr Paul Lynch, Professor of Plant Biotechnology at the University of Derby, UK, with Dr Norzulaani Khalid University of Malaya in 2005

According to some sources there are approximately 250,000 higher plant species in the world today, but of these many thousands are endangered. Between 40-100 000 plants have been used by mankind over the years, but only approximately 7000 plant species are cultivated today, of which about 30 form the basis of the world’s agriculture. Of these, three crops - maize, wheat and rice - provide over half of humanities calorific and protein requirements. However their diversity is declining, and, for example over the last century the number of varieties of maize grown in America has fallen by over 90%. Such narrowing of the diversity of crop varieties makes staple food supply vulnerable to interruption by the effects of pests, diseases and global climate change. To maintain food security mankind must keep the diversity of plants and crop plants to allow for the breeding of plants for future needs.

Many plant species are traditionally and effectively conserved by conventional seed storage techniques, which involve drying seeds to approximately 5% moisture content and maintaining them at 18ºC (the temperature of a domestic freezer). Seeds that can be conserved this way are known as orthodox seeds. However, many plants, particularly tropical species, do not produce orthodox seeds; rather their seeds rapidly lose viability and cannot survive desiccation and/or cold storage. Such seeds are known as recalcitrant. For plants, which produce recalcitrant seeds or that are propagated vegetatively, e.g. banana and potato, traditional conservation involves maintenance in field collections. However, such collections remain vulnerable to loss due to the effects of extreme weather conditions, pests and diseases, and increasing maintenance costs. Plant biotechnology provides alternative approaches that can be used to safeguard the survival of such plant species.

The biotechnology techniques for plant conservation can be divided into three types:- 1. Short-term – This is basically micro propagation, where the plants are maintained under laboratory conditions in a state of active growth. However it is expensive to store plants in this way and they are vulnerable to microbial contamination, equipment failure and genetic change. 2. Medium-term – This involves modification of the culture conditions of the plants in tissue culture to slow the rate of growth, so that plants do not have to be transferred to fresh medium at the normal monthly intervals (subculture period). For example potatoes when grown on modified culture medium at only 4 ºC can be left for over a year between subcultures. However, plant material stored this way can be vulnerable to genetic changes. 3. Long-term – The only effective biotechnological approach for the long-term conservation of plant species is cryopreservation. This is the placing and holding of biological material at ultra-low temperatures (normally that of liquid nitrogen -196 ºC) in such a manner that the viability is maintained after thawing.

There are a variety of approaches to cryopreserving plant cells and tissues. However they all designed to avoid the formation of crystalline ice inside the cells that is extremely damaging. Rather, what cryobiologists are trying to achieve is the formation of the less damaging non-crystalline ice by a process known as vitrification. Essentially plant cryopreservation techniques provide means of protecting the cells/tissue to be frozen from chilling or desiccation damage while dehydrating them to a point were there is insufficient remaining water in the cells to allow the formation of crystalline ice. After storage in liquid nitrogen (the duration of which does not appear to affect the viability of the stored plants) tissue is rapidly thawed and transferred to appropriate growth conditions. Depending on the plant species and type of tissue frozen, re-growth can be seen in as little as seven days after thawing. After several weeks or months of growth, recovered plants can be returned to compost. The general consensus is that most plants recovered from cryopreservation are comparable to the original non-frozen ones.

There are now reports of hundreds of plant species that have been successfully cryopreserved and hence plant cryopreservation is currently moving into a new and exciting stage, where the technology is being tested in pilot scale gene banks. As part of a Higher Education Link Programme, supported by the British Council, Professor Lynch’s has worked with Dr Norzulaani Khalid of University of Malaya towards the transfer of cryopreservation technology to support the University’s banana biotechnology programme. Dr Norzulaani’s laboratory has developed embryogenic suspension cultures of Malaysian banana varieties such as mas, rashthali, jaribuaya and the disease resistant wild bananas that provide an improved method of micro propagating elite clones. This work will allow the provision of elite banana plants to a wide range of farmers and indeed as part of the British Council project has supplied plants to the poor farmers under the ‘Pusat Urus Zakat, Pulau Pinang’ and Synergy Farm.. The integration of cryopreservation will further improve the efficiency of this approach and provide a means of safeguarding these important cultures.

A controlled rate freezing approach is being taken for the cryopreservation of the embryogenic suspension cultures of banana from Dr Norzulaani’s laboratory, which has previously been successfully employed to cryopreserve similar cultures. This is essentially a classic cryopreservation approach which once optimised has the advantages of being technically not too demanding, suitable for the cryopreservation of large numbers of samples and has been successfully used to cryopreserve in gene banks of plant cell suspension cultures. To ensure the reproducibility of the controlled rate freezing approach the project is using a Planer controlled rate freezer. During visits to the University of Malaya in recent years Professor Lynch has taught a series of workshops and master classes on plant cryopreservation which have been attended by scientists and students from several Malaysian Universities, Research Institutes and companies and this is starting to result in the establishment of other new cryopreservation research towards the sustainable conservation of other examples of Malaysia’s rich plant biodiversity. Equipment for these workshops has been loaned by the Planer Products distributor in Malaysia.

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