AUTHENTICITY. I declare that this thesis is my own work, except for those sections explicitly

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3 AUTHENTICITY I declare that this thesis is my own work, except for those sections explicitly acknowledged, and to my knowledge the main content of the thesis has not been previously submitted for a degree at any other university. Rajnesh R. Prasad Sant DATE: 27/09/2001

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5 ABSTRACT From this study it was discovered that the hormone-free clonal propagation system developed by Thinh (1997) for Colocasia esculenta var. antiquorum, based on enhanced axillary branching (multiple shoot formation) through liquid TDZ medium shake culture and subsequent carry over effect on hormone-free medium, did not work with the tropical taro variety, Tausala ni Samoa, of Colocasia esculenta var. esculenta type. After two four-week cycles on liquid TDZ shake medium cultures, no enhancement in proliferation rates was noted and no carry over effect was observed when these plants were transferred to hormone-free media. The vitrification method of cryopreservation was experimented with cultivars of the tropical taro (Colocasia esculenta var. esculenta) and the technique was shown to have potential for the cryopreservation of taro from Pacific Island countries. Out of the eight taro cultivars experimented with, three, namely E399, CPUK and TNS, were successfully cryopreserved with average recovery rates of 20, 29 and 29%, respectively. The optimum vitrification protocol for the cultivars E399 and CPUK was; using shoot-tip donor plants cultured on solid MS in large jars for three months as sources of shoot-tips, which consisted of the apical dome surrounded by two leaf primodia; preculturing these shoottips overnight (16hr) on 0.3 M sucrose medium; loading with liquid MS supplemented with 2 M glyceroi M sucrose for 20 min at 25 C, dehydrating with PVS2 for 12 min at 25 C followed by rapid immersion in LN. Thawing was done by shaking the shoot-tips rapidly for 90 sec in waterbath at 40 C, followed by rehydration in liquid MS medium supplemented with 1.2 M sucrose for 15 min. The shoot-tips were then plated on a layer of filter paper on MS medium supplemented with 0.3 M sucrose and left overnight in the dark. Next day, they were

6 transferred onto MS medium supplemented with 0.1 M sucrose and maintained in the dark for three days, then transferred to dim light (10 u.molm"v) for one week before exposure to normal culture conditions. Piantlets were produced after about two months. For cultivar TNS, the optimum vitrification protocol was preconditioning explants on solid MS supplemented with 90 g/1 sucrose for seven weeks prior to dissecting and cryopreserving the shoot-tips without any preculture, The vitrification procedure was same as described above. During this study, it was found that the vitrification protocol has to be optimized for each individual taro cultivar.

7 DEDICATION This thesis is dedicated to my parents, Ram Prasad and Jai Wati, who had the vision and courage to rise above their experiences.

8 AKNOWLEDGEMENTS I would like to acknowledge my supervisors, Dr. Mary Taylor, the Tissue Culture Specialist of Regional Germplasm Center at the Secretariat of the Pacific Community and Professor Anand Tyagi, Head of the Department of Biology at The University of the South Pacific, whose efforts have made this project a success. Dr. Taylor's relentless and enterprising efforts as a supervisor in the still infant field of cryopreservation of tropical species deserve a special mention. The help and support given to me by the RGC personnel, Samila, Eliki, Raghani, Rohini and Kiran was invaluable. My special thanks to Samila for inaugurating me in the 'art and craft' of plant tissue culture. I wish to extend my gratitude to staff of SPC and technical staff of USP, whose names 1 do not mention out of the fear of inadvertently missing out some, for their help and support. I am indebted to two people, without the advice, suggestions and guidance of whom this project may not have succeeded. Firstly, Dr. Barbara Reed of National Clonal Germplasm Repository, Oregon, USA, through whom I got my introduction to the world of cryopreservation. She had been my mentor throughout my research. Secondly, Dr. Nguyen Tien Thinh of Nuclear Research Institute, Dalat City, Vietnam, who showed me the finer details of the vitrification method he developed for taro. It was only after his expert insight and suggestions that I achieved positive results.

9 My wife, Nischal Lata, deserves special recognition for her understanding and courage for the trials she endured as a newly wed wife of a research student and her help with the proofreading of my thesis. Last, but not the least, I would like to thank my sponsors, AusAID, who under the TaroGen project provided the funds with which this work was carried out.

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17 LIST OF APPENDICES Appendix 1: Recent successful cryopreservation of shoot apices by vitrification 89 Appendix 2: Bacteriological 523 Medium (Viss) 91

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21 CHAPTER INTRODUCTION 1.1 Description of Taro (Colocasia esculenta (L.) Schott Aracea) Taro is a common name of a species of the Colocasia genus that belongs to the Araceae family. It is a herbaceous plant one or two metres tall with peltate shaped leaves attached to one metre stout petioles that clasp around the base. The underground cylindrical corms are 30cm in length and 15cm in diameter with short internodes and a superficial and fibrous root system (Purseglove, 1972; Strauss, 1983). The yellow unisexual flowers are borne on a stout peduncle that is shorter than the petiole with about 20cm spathe covering the spadix. The spadix has male flowers on the upper portion and a mixture of female and sterile flowers on the lower part (Purseglove, 1972; Wilson, 1990). Only some taro cultivars flower naturally. However, flowering can be achieved through artificial induction (Katsura et al, 1986; Miyazaki et at, 1986; Wilson, 1990). The nutritive composition of corms is as follows: water 63% - 85%, carbohydrates 13% - 29%, protein 1.4% - 3.0%, fat 0.2% - 0.4%, fiber 0.6% - 1.2% and ash 0.6% - 1.3%. Vitamins B and C are present in appreciable amounts in the corm. The leaves contain 87.2% water, 6.0% carbohydrates, 3.0% protein, 0.8% fat 1.4% fiber, 1.6% ash, and they are excellent source of vitamin C (Purseglove, 1972).

22 1.2 Taxonomy Taro (Colocasia esculenta (L.) Schott) belongs to the monocotyledonous Araceae along with two other important root crops, Alocasia and Xanthosoma. There are confusions in the taxonomy of different Colocasia cultivars with edible tubers. The general agreement now, as first suggested by Purseglove (1972) and Pluckett (1983), is that there is one species, Colocasia esculenta (L.) Schott, with two botanical varieties. The first variety is Colocasia esculenta var. esculenta, commonly known as taro, dasheen or cocoyam. The second variety is Colocasia esculenta var. antiquorum, commonly known as taro or eddoe. The important differences between the two cultivars include; antiquorum has small central corm with many side cormels or tubers while esculenta has a large central corm. In antiquorum, the sterile spadix is longer than male section and is three times or more than that of esculenta. Antiquorum is hardier of the two - withstands lower rainfalls, colder climates and lighter poorer soils, and has longer storage periods (Purseglove, 1972). Throughout this study, taro will be used to refer to.colocasia esculenta var. esculenta. 1.3 Distribution and Use Taro is commercially cultivated throughout the humid and semi-humid tropics, as well as some warm regions of temperate areas with irrigation or rainy conditions. Worldwide, taro ranks fourteenth among staple vegetable crops with about 9.2 million tonnes produced globally from 1.8 million hectares with an average yield of 5.1 t/ha (FAO, 1996). Regions with active taro cultivation include South-East Asia, Pacific Islands (Papua New Guinea [PNG] inclusive) the South and Central China, Japan, India, West Indies and West and North Africa.

23 Almost all parts of taro plants are useful, either palatable or otherwise. However, as previously stated, the tuber is the most nutritive and considered most delicious. The conns are roasted, baked or boiled. The young leaves, including laminas and petioles, are used as vegetables. Blanched young shoots can also be eaten like asparagus. Some common dishes include the Hawaiian 'Poi', 'Che Mon Sap' of Viet Nam, and 'Callaloo' soup in Trinidad (Purseglove, 1972; Thinh, 1997). Taro corn) puree is used as a low allergic and easily digested baby food. Taro corm confections and leaves are also commercially processed in some countries (Nip, 1989; Tuia, 1997). It is also used as a traditional medicine. Root extracts are used to treat rheumatism and acne, while leaf extracts help clot blood, neutralize snake poison and act as a purgative (Winarno, 1990). Corms from wild plants and inferior cultivars as well as cooked or fermented silage from waste leaves and corms are used as animal feeds. ( 1.4 Importance of Taro in Pacific Taro cultivation has been practiced in the Pacific Island countries (PICs) for centuries. It is thought to have reached the Polynesian islands about two thousand years ago from the Indo- Malayan region. Lebot et al. (2000) have suggested that there are two distinct gene pools in South Asia and Melanesia and these probably reflect natural differentiation of the species on each side of the Wallace line. Over generations, taro has found its way into the rituals and fabric of local custom and culture in the Pacific and forms an important part of traditional ceremonies and auspicious occasions. It is also a staple food in the Pacific region and almost all parts, including the corm, leaves, stem and flowers are prepared as various delicacies. Taro is of great economic importance in the Pacific region. It is an important, and in some cases the major, export of a number of Pacific Island countries (Jackson, 1994; Taylor, 2001).

24 There are believed to be about two thousand taro cultivars in the Pacific region (Hunter et al., 1998). A report from an Australian Center for International Agricultural Research (ACIAR) funded project presented at the taro genetic conservation strategy workshop in Suva, Fiji (5" 1-7 th September 2001) revealed the significant taro diversity in the region, especially in PNG. Concerted efforts are required to conserve this diversity as significant genetic erosion has occurred due to agricultural intensification, pests, diseases, natural disasters, and civil unrests. Taro diversity is important for long-term food security as highlighted in the case of taro leaf blight (TLB) disease outbreak in Samoa in The introduction of exotic TLB tolerant and resistant varieties from Palau and the Philippines was used to combat the disease. 1.5 Conservation of Taro Germplasm The options for taro germplasm conservation can be categorized in two main areas, in situ, and ex situ conservation In situ conservation In situ conservation is the conservation of ecosystems and natural habitats and the maintenance and recovery of viable populations of species in their natural surroundings and, in the case of domesticated or cultivated species, in the surroundings where they have developed their distinctive properties. It encompasses genetic reserves, on-farm and home garden conservation (Maxted et al, 1997; Engelmann, 2000). In situ conservation of cultivated species is primarily concerned with the on-farm maintenance of traditional crop varieties (or land races) and with forage and agroforestry species (Taylor, 2001). Farmers have been practicing on-farm conservation for centuries, through the selection and maintenance of those varieties best suited to the local ecological conditions and social and

25 cultural requirements. However, it is a strategy about which very little is known. It is only in relatively recent years that studies have been conducted in attempts to determine the scientific basis of on-farm conservation. In addition to this, crops grown in the field situation are vulnerable to pest and/or disease attack, and to climatic extremes. It is also a system that is at the mercy of farmers' desire and ability to maintain the various cultivars (Taylor, 2001). A preliminary study was carried out within the Taro Genetic Resources Conservation and Utilization Project (TaroGen) that showed the impact of market pressure on the varieties chosen by the farmers for cultivation. Economically good varieties attracted more interest from fanners' than traditional ones, leading to abandoning of traditional varieties. This influence of new varieties on the impact of traditional varieties highlights the need to generate more knowledge and information about on-farm conservation. f Ex situ conservation Ex situ conservation can be in the form of biosphere reserves, botanical gardens, field genebanks, seeds and in vitro storage which includes slow growth and cryopreservation (Maxted et al., 1997; Engelmann, 2000; Taylor, 2001). The first two forms are not suitable for taro germplasm conservation Seeds Most taro plants do not flower naturally hence storage of taro germplasm as seeds has not been a feasible option. However, the technology to make taro flower through gibberellic acid induction (Miyazaki et al., 1986; Katsura et al, 1986; Wilson, 1990) is now available and the possibility to store taro germplasm as seed is being investigated under the TaroGen project.

26 Although seeds could be used to conserve genes, they are not an option for maintaining genetically true-to-type germplasm. 1,5,2.2 Field Genebanks According to a Consultative Group on International Agricultural Research (CGIAR) report (1989), field genebanks are more aptly called field collections, as they tend to be working collections. As a short to medium term activity, field collections are effective as working collections. They can be a useful system for conserving farmer-preferred varieties. In addition to this, they also facilitate screening, evaluation, breeding and distribution. Field genebanks face setbacks in that maintenance is labour-intensive and expensive due to demands for heavy machinery such as tractors and harvesters, vehicles for transport of material to and from field, labour, fuel, repairs, fertilizers and chemicals, buildings for storage, insurance and so on (Jerret and Florkwoski, 1990). The cost and maintenance of field genebanks as opposed to in vitro storage (slow growth) is being investigated under the TaroGen project. Other issues that are particularly pertinent to the Pacific situation include climatic extremes such as hurricanes, flooding and drought, limited resources, high staff turnover, and land ownership disputes. Another drawback is the loss of plants through insects, pests, pathogens, and diseases as in the Solomons in 1975 and the annihilating outbreak of TLB in Samoa in 1993 (Jackson, 1994). Individual plants may also be lost through human errors. The regional countries do not have the economy to facilitate collection and genetic conservation of taro in field genebanks. Losses of taro genetic resources have already occurred

27 in some countries due to natural disasters (flooding and cyclones in Vanuatu), diseases (TLB in Samoa) and ethnic conflicts (Solomon Islands) In vitro storage In vitro storage of taro involves micropropagating shoot tips and maintaining them under slow growth conditions in tissue culture laboratories. The conditions that can be used to induce slow growth include: lowered incubation temperatures, raised osmolarity of the culture medium and decreased oxygen concentration in the culture containers. Some studies have showed that taro can be stored under slow growth conditions (high osmolarity and low temperature) for over a year (Staritsky et at, 1986; Zandvoort, 1987; Bessembinder et al., 1993; Taylor, 1998). In vitro storage offers the following advantages: ( explants free from pathogens and safe from natural disasters relatively smaller space required to house cultures better (visual) monitoring of cultures high propagation potential of cultures slow growth conservation in in vitro is apt for short to medium term storage for accessions that are being distributed on regular basis The drawbacks can be: high establishment costs of a tissue culture lab and recurrent funding needed for maintenance with very precise storage conditions, frequent monitoring, costly chemicals, and specialized staff. Labour needs and costs for the running of an in vitro active genebank can also be high, especially if operating on a large scale. Jerret and Horkowski (1990)

28 have estimated that two to three technical staff are necessary for initiating, maintaining, and subculturing a collection of 1000 accessions cultures can be lost through bacterial and/or fungal contaminations, human errors and power failures in the absence of backup facilities genetic instability could compromise genetic conservation. Genetic instability due to somaclonal variation may have genotypic and phenotypic consequences such as changes in chromosome number and structure, loss of secondary product production, and characteristical changes such as disease resistance or plant height (Withers, 1987; Taylor, 1988; Ashmore, 1997; Thinh, 1997). To date there have been no studies to assess the genetic stability of taro under slow growth storage. For the PICs, the lack of technical staff and the high costs of establishing and maintaining tissue culture labs make in vitro conservation not a viable option Crvopreservation In light of the various challenges and shortcomings experienced with both genetic reserves and field genebanks, cryopreservation is the safest and most cost-effective option for the long-term conservation of Plant Genetic Resources (PGR) at present (Withers and Engels, 1990; Maxted et al., 1997; Engelmann, 2000). Cryopreservation has the following advantages over other PGR conservation methods: large number of accessions can be stored in a small space (for example: a 25L Dewar could store germplasm for 8000 individual plants) for unlimited time the collection is housed in the safe and secure confines of a laboratory and this obliterates the external threats which other ex situ and in situ methods are susceptible to ensures protection against loses through culture contaminations

29 maintains genetic fidelity of the germplasm collection. However, the following requirements have to be met in setting up cryopreservation facilities for any crop: well developed tissue culture technology for the crop round-the-clock electricity supply a reliable supply of liquid nitrogen modern, well-maintained laboratory facilities appropriate training for staff (university courses, regional workshops, sponsorship for study and travel) (Ashmore, 2001). 1.6 TaroGen Project (' As mentioned before, an outbreak of TLB (Phytophthora colocasiae) in the Samoan Islands in 1993 had a devastating impact on the taro industry as well as subsistence fanners. Farmers were forced to abandon the crop, which had severe economic implications. It also resulted in the loss of genetic resources. Collaborated effort of concerned authorities, interested parties and international donors to address the problem resulted in the TaroGen project in The project is funded by Australian Aid for International Development (AusAID) and implemented by the Secretariat of the Pacific Community (SPC). It aims to collect, improve and conserve the regional taro varieties. According to the project design document of TaroGen (1997), the aim of the conservation component of the project is to overcome the difficulties that all Pacific Island countries face in maintaining field collections of taro which are costly and subject to loss from natural disasters, pest attack and neglect due to fluctuating financial

30 support. To facilitate the regional countries' conservation efforts, a Regional Germplasm Centre (RGC) has been established at SPC, Suva within the project. The RGC was established in response to the realization that increased collaboration among the countries of the region is crucial in PGR conservation in the Pacific (Sajise, 2001). As part of the TaroGen project, taro conservation strategies were to be investigated. These included on-farm conservation, in vitro slow growth storage and cryopreservation. Cryopreservation was considered necessary, as one of the outputs of the project would be a collection of approximately two thousand taro accessions collected from PICs. This is valuable material accumulated because of its genetic diversity. The only safe and effective method for conserving this taro germplasm on a long-term basis would be cryopreservation. The purpose of this study is to investigate the applicability of cryopreservation for storage of regional taro germplasm. The vitrification protocol developed by Thinh (1997) on Japanese taro would be assessed for its competency with taro from PICs. 10

31 CHAPTER 2: LITERATURE REVIEW 2.0 CRYOPRESERVATION 2.1 Introduction Cryopreservation is storage at ultra low temperatures such as that of liquid nitrogen (-196 C) where all cellular divisions and metabolic processes cease and plant material has unlimited span of storage without alteration or modification. In 1956, Sakai carried out the first successful experiment on storage of plant tissue by freezing with liquid nitrogen on mulberry twigs (Sakai, 1995). In 1968, Quatrano showed cultured cells of flax pretreated with dimethylsulphoxide (DMSO) to withstand freezing to -50 C. Several new cryopreservation techniques have been developed in. recent years for application to a larger range of tissues and organs, in myriad infrastructural situations (Engelmann, 1997a; Takagi, 2000). 2.2 Freezing Implications The principal challenge faced during cryopreservation is avoiding the irreversible damage caused, to tissue by the crystallisation of water contained by all organic matter (cells, callus, shoot tips, embryos). During freezing, tee can form either outside the cells (extracellular freezing) or inside (intracellular freezing). Cryopreservation involves dehydrating plant material prior to liquid nitrogen (LN) cooling to avoid water crystallisation damage. However, plant cells can survive cooling only to a minimum critical temperature, which is species dependant, and can recover only after a 40-50% critical minimum volume reduction (Merymann et at, 1977; Sakai, 1995). Excess shrinkage and excessive dehydration can cause

32 cell injuries in various ways. Endocytotic vesicles may form on cell plasmolysis through membrane infolding and fusion. These vesicles could cause cells to burst on rehydration. Other forms of injury could be a dysfunctional cell membrane, coagulated, precipitated and subsequently toxic cell contents, and protoplasmic ph alterations. Cellular proteins can also get denatured at low temperatures (Levitt, 1980; Gordon-Kamn and Steponkus, 1984a,b,c; Singh and Miller 1985; Steponkus and Lynch, 1989; Steponkus et at, 1993; Pearce, 2001). In light of above, the dehydration process should be optimised to dehydrate plant material enough to cryopreserve, whilst avoiding excess shrinkage and excessive dehydration injuries. 2.3 Dehydration and Vitrification Cryopreservation employs two mechanisms for the treatment of intracellular water. The first mechanism exploits freeze-induced dehydration, where slow cooling causes cell exterior and medium to cool and form ice. The supercooled, but still liquid interior, responds by shedding water to equilibrate built up internal aqueous vapour pressure (Engelmann, 1997a; Ishikawa et at, 2000). The second mechanism, termed vitrification, involves the transition of water directly from the liquid phase into an amorphous phase or glass, whilst avoiding the formation of intraprotoplasmic ice crystals (IIC). Rapid thawing prevents IIC formation due to devitrification. Vitrification (glass formation) occurs readily in highly concentrated and viscous solutions that become solid without forming crystals upon rapid cooling (Burke et at, 1976; Fahy et at, 1984; Steponkus, 1985; Sakai et at, 1990; Engelmann, 1997a). Effective vitrification solutions, called cryoprotectants, consist of high molecular weight compounds or osmoticums such as 5-10% w/v or v/v levels of DMSO, sucrose, glycerol, abscisic acid (ABA) or proline (Kartha, 1985; Benson, 1999). Treating plant material (cells in suspension, calluses, embryos 12

33 and apices) firstly with cryoprotectants and then rapidly cooling causes the vitrification of the cells. This avoids mechanical damage caused by the formation of HC. The glass formed has lower vapour pressure than extracellular ice and therefore, further dehydration of cells and excessive cell shrinkage is avoided. The glass also prevents the buildup of cell solutes and thus prevents solute toxicity and ph alterations. The highly viscous glass stops all chemical reactions requiring molecular diffusion. These factors render the plant material dormant and stable for unlimited length of time (Fahy el al., 1984; Sakai et ah, 1990; Fujikawa & Jitsuyama, 2000). However, very high concentrations of cryoprotectants (CP) are toxic to plant materials. Hence the concentration, combination and length of exposure to CP has to be optimized for different species and type of plant material to be stored. 2.2 Cryopreservation Strategics ' According to Sakai (1993), all the cryopreservation protocols developed over the years fall into four general categories based on the dehydration treatments before LN immersion. All strategies aim to overcome cell damage resulting from intracellular crystallization and freeze dehydration. This is achieved either by glass formation intracellularly (classical methods) or both intra and extracellularly (complete vitrification method). The four general categories are: Conventional slow prefreezing method This method relies on dehydration of cells by the formation of extracellular ice on slow cooling so that the cytosol gets concentrated enough to vitrify on LN cooling. The explants are treated with cryoprotectants (at 0 C for 2-3 hrs) then subjected to slow cooling at rates ranging 13

34 from 0.1"C /min to 10 C /min depending on species and type of explant using a programmable freezer. An ice inoculation step at temperatures of -7 C to -10 C facilitates extracellular crystallization. The explants are plunged in LN after further slow freezing to -35 C to -40 C, where any unfrozen extracellular solution and cytosol are sufficiently concentrated to vitrify. Although an important and efficient protocol, its drawbacks are in the expensive equipment and long procedural time required (Thinh, 1997). Furthermore, the equipment used in this method requires high maintenance and therefore is not really viable for low-tech laboratories Simple freezing method This method involves exposing material to concentrated cryoprotectants for extracellular dehydration instead of slow freezing. The explants are then subjected to prefreezing temperatures (-30 to -40 C) in an ordinary freezer for a length of time dependant on species (e.g. 1 hr) before rapid cooling in LN. Although this method eliminates the use of expensive freezing equipment and ice inoculation step, it is not suitable for use with multicellular structures such as apical meristems or shoot tips (Sakai et al., 1991; Nishizawa et at, 1992) Complete vitrification method ' > Complete vitrification is when, both, extracellular and intracellular glasses form upon cooling in LN. This is achieved by sufficiently dehydrating cells, tissues and organs with concentrated vitrification solutions (PVS) at 0 C or 25 C prior to plunging in LN. PVS not only dehydrates cells, but also penetrates into cellular interspaces inducing freeze tolerance. However, the osmotic and chemical toxicity of PVS to plant tissues necessitates optimizing exposure. This can be achieved either by gradually increasing the concentration of the additive cryoprotectant 14

35 solutions (i.e. 10-»20->40-»60->80-M00%) or loading the plant material with less concentrated cryoprotective solutions prior to PVS exposure. The latter is referred to as twostep vitrification method (Takagi, 2000). The steps for complete vitrification method can be generalized as follows: 1. preparation and selection of appropriate samples 2. preculturing dissected shoot-tips with osmoticum (e.g M sucrose) 3. treatment with loading solutions 4. dehydration by exposure to PVS at 0 C or 25 C 5. rapid immersion in LN 6. rapid rewarming (40 C) 7. rehydration with unloading solution (e.g. 1.2M sucrose) 8. conditioning apices under favourable conditions for recovery (Takagi, 2000). The complete vitrification method has been successfully applied to a range of explant types. These include protoplasts (Langis and Steponkus, 1991), cell suspensions (Huang et al, 1995), nucellar cells (Sakai et al, 1990), somatic embryos (Uragami, 1989), shoot tips and meristems (Yamada et al, 1991; Niino et al, 1992b,c; Reed, 1992; Matsumoto et al., 1994; Matsumoto et al, 1998; Towill and Jarret> 1996), axillary buds (Takagi et al, 1994) and bud clusters (Kohmura et al, 1992). This method has the advantages of being simple, quick and easy to carry out and no expensive or sophisticated equipment is required Air drying method It involves concentrating cellular liquids to vitrifiable levels through evaporation with sterile air from a laminar airflow cabinet (LAF) or silicagel. Optimal water content for successful 15

36 vitrification is 20-30%. This could be achieved by drying periods of two to ten hours, depending on the plant species. Plants are conditioned to withstand such levels of dehydration by low temperature hardening or preculturing with ABA or high sucrose concentrations (Dereuddre et al, 1990; Uragami et al., 1990; Plessis et al, 1991; Dumet et al, 1993a; Sakai, 1993). Air drying of explants may be direct or after being first encapsulated in alginate beads as in the recent technique of encapsulation-dehydration (ED) (Takagi, 2000). The steps for encapsulation-dehydration method can be generalized as follows: 1. preparation and selection of appropriate samples 2. encapsulation of dissected shoot apices in alginate beads 3. preculturing beads with osmoticum (e.g. sucrose, sorbitol, glycerol) 4. desiccation in (LAF) or with silica gel 5. rapid immersion in LN I 6. rapid rewarming (40 C) 7. conditioning apices under favourable conditions for recovery (Takagi, 2000). Apart from the incipient success of Fabre and Dereuddre (1990) with ED of Solatium shoot tips, other successes with cryopreservation are cell suspensions (Bachiri et al., 1995), somatic embryos (Dereuddre et al., 1991; Hatanaka et al., 1994) and shoot tips (Plessis et al, 1991; Niino and Sakai, 1992a; Suzuki et al, 1994). Air drying and the modified ED method also have the advantages of being devoid of expensive appliances and easy to conduct. However, the disadvantages are that they can be time consuming and explants exhibit a slower post LN regrowth (Thinh et al., 2000). 16

37 2.3 Technical Issues for Cryopreservation of Shoot Apices Introduction Shoot apices, meristems or shoot tips, are plant organs with an ordered structure and less differentiated cells. This enables vigorous recovery into plantlets without callus formation or genetic artifacts (Ashwood-Smith, 1985; Kartha, 1985; Sakai, 1993; Thinh, 1997). Kartha et al. (1980) and Bajaj (1983) found plants regenerated from cryopreserved apices of strawberry and cassava to be normal. Oil palms regenerated from cryopreserved somatic embryos also had normal vegetative and floral development (Engelmann, 1991). A comparative study on the effects of slow growth and cryogenic storage on the stability of plants revealed that while cryopreserved Panax ginseng and Catharanthus roseus maintained normal secondary metabolite production, slow growth reduced it markedly (Mannonen et al., 1990). A study on potato plants by Harding (1991) showed plants regenerated from cryopreserved apices to be normal, whereas those stored on mannitol supplemented medium for six months under slow growth conditions depicted modifications in the Restriction Fragment Length Polymorphism (RFLP) pattern. Other studies on evaluation of plants rejuvenated from cryopreserved material also show that plant material can be safely stored under LN without change. Cote et al. (2000) studied the infield behaviour bananas {Musa AA sp.) obtained after regeneration of cryopreserved embryogenic cell suspension and found no difference at the agronomic level between plants produced from cryopreserved embryogenic cell suspensions and control plants. Genetic examination of Picea glauca engelmanni complex for somaclonal variation resulting from cryopreservation by Cyr et al. (1994) detected no genetic variation. Similarly, randomly amplified polymorphic DNA (RAPD) fingerprint evaluation of white spruce [Picea glauca 17

38 (Moench) Voss.] trees regenerated from embryogenic clones cryopreserved for 3 and 4 years, suggested primary genetic stability (DeVerno et al., 1999). The new vitrification methods have been optimized to successfully cryopreserve shoot apices with good recovery. To date a number of shoot apices of vegetatively propagated tropical monocots (VPTM) have been successfully cryopreserved (Appendix 1). Factors pertaining to successful cryopreservation of shoot apices include: Efficient in vitro culture system The most common source of apices for cryopreservation is in vitro maintained shoot cultures. The advantages of in vitro cultures are that duplication of adequate numbers of plant material can be achieved quickly, the material is pests and pathogen free, and the right conditions can be maintained to induce the optimum physiological conditions essential for cryo survival. An adequate tissue culture system is also necessary for effective preculturing of dissected apices and vigorous recovery of cryopreserved shoot apices without intermediate callus formation. In vitro culture system may be needed to be optimised for cryo success as what may be effective for conventional micropropagation may not be best for cryo success. Culture conditions such as light, solid or liquid media have been found to play crucial roles for cryo success (Thinh, 2001). -._ Selection of size and development stage of shoot apices Shoot tips, comprised of the apical dome with a few leaf primodia, are generally considered to be the best propagules for cryopreservation of clones (Towill, 1996; Takagi, 2000). The optimum type and shape of shoot tip to be used is species dependent. The best type of shoot tips for the VPTM, banana, Cymbidium, Cymbopogon, pineapple and taro, were found to be 18

39 partly covered ones mm in size (Thinh, 1997; Thinh et al, 2000). These ST consisted of the apical dome partly covered by the second leaf primodia. Escobar et al. (1997) found that cryopreserving smaller cassava shoot tips (1-2 mm and apical dome partly covered by 2-3 leaf primodia) markedly increased the recovery rate. Another essential factor for cryopreservation success is the optimal developmental stage at which the explants are used for freezing. Explants from rapidly growing cultures are recommended as actively dividing cells have characteristics, such as under developed vacuolar system and dense cytoplasm, which render them more cryopreservation tolerance (Kartha and Engelmann, 1994; Withers, 1985; Engelmann, 1997a). It has also been found that increasing the duration for which mother plants are maintained on standard medium without subculture also increases the recovery rate after cryopreservation (Thinh, 1997; Yongjie et al, 1999). f Factors affecting post-thaw survival of shoot apices in the vitrification method Thinh et al. (2000) categorised the various factors affecting VPTM, namely banana, Cymbidium, Cymbopogon, pineapple, and taro cryopreservation success as follows; in descending order of importance: Explant structure The best explants for cryopreservation are meristems with partially covered apical domes. Such explants are also referred to as shoot-tips (ST). Taro shoot structure, like most vegetatively propagated monocots, consists of apical meristems very well covered by the outer leaf petioles, which are tubular shaped and have interfolded, thick leaf bases. Two consecutive leaf petioles of an apex are buffered by air spaces. Thinh (1997) postulated that such apex organization could prevent vitrification solutions from penetrating to apical dome cells. Hence 19

40 the best shoot-tip structure was found to consist of the apical dome covered by two leaf primordia Loading treatment Cryoprotectants (section 2.3) can be a source of cellular injury. However, their toxic effects can be minimized by pretreatment (referred to as loading treatment in this study) with sugars, sugar alcohols, and amino acids introduced via solid or liquid medium (Nag and Street, 1975; Reid and Walker-Simmons, 1990; Luo and Reed, 1997). Pretreatment chemicals benefit plant cells and tissue by rendering reduced cell size and the cytoplasm to vacuole ratio, enhanced ability to take up cryoprotectants, resistance to dehydration injury through cell wall and/or cell membrane modifications, stabilized membrane bilayers and enzymes and prevention of toxic levels of compounds accumulating in membranes during dehydration and freezing by acting in a colligative manner (Heber et at, 1971; Volger and Heber 1975; Withers and King 1979; Steponkus, 1984; Crowe et al, 1990; Dumet et at, 1993b; Luo and Reed, 1997). Thinh et al. (2000) found that loading treatment enhanced markedly, both tolerance to PVS2 dehydration (see below) and post-thaw survival of meristems of all the species tested. This is corroborated by other studies of different species such as rye protoplasts (Langis and Steponkus, 1991), meristems of wasabi and lily (Matsumoto et al, 1994,1995a) and meristems and callus of currant (Luo and Reed, 1997). Thinh (1997) found that exposing taro ST to a loading solution consisting of 2M glycerol + 0.4M sucrose in liquid Murashige and Skoog (1962) (MS) for 20 minutes gave the highest recovery rates. 20

41 PVS2 dehydration Only sufficiently dehydrated plant material can survive LN cooling. One of the most effective and universally used solutions for dehydrating plant material is Plant Vitrification Solution 2 (PVS2) developed by Sakai et al, It consists of 30% glycerol + 15% ethylene glycol + 15% dimethylsulphoxide + 0.4M sucrose in liquid MS. However, PVS2 can be harmful to plant material due to high osmotic pressure and chemical toxicity. Hence, exposure time to PVS2 has to be optimized for different species, and in some cases cultivars (Thinh, 2001). It has been found that PVS2 sensitive plants can be adequately dehydrated by incubating at 0 C instead of the normal 25 C. Thinh (1997) optimized taro ST exposure to PVS2 as lomin at 25 C Preculture with high sucrose concentrations ( In the studies by Thinh et al. (2000) previously stated, it was found that preculturing the dissected ST on MS medium supplemented with 0.3M sucrose enhanced the post-thaw survival of ST of all the plant species, with banana as the only exception. Several theories have been put forward to explain the positive effects of preculturing. One suggestion is that the high osmotic pressure resulting from the high level of sucrose in a culture medium can trigger certain responses in plant cells, such as the accumulation of ABA and/or proline. The presence of these substances can protect against freezing damage. Mohapatra et al. (1998) report that ABA induces the synthesis of new proteins that confer cold tolerance to plants. Studies have found that sucrose and proline stabilize membrane biiayers and enzymes during desiccation and freezing (Steponkus, 1984; Crowe et al., 1990; Dumet et al, 1993b) The preculture medium also facilitates in holding dissected ST awaiting cryopreservation. However, the preculturing stage can be eliminated when using preconditioned plants (section ). 21

42 Sucrose preconditioning While cold-hardening (4-5 C) of ST donor plants has enhanced the cryopreservation success of temperate species, it is not an alternative with temperate species (Bajaj, 1985; Thinh, 1997; Thinh et at., 2000). However, preconditioning the shoot-tip donor plants on medium supplemented with high sucrose concentrations has enhanced cryopreservation success of tropical species. Such plants undergo physical and physiological changes that assist in withstanding cryo treatments. The plants have stunted growth, with thicker leaf blades, shorter petioles and more rigid tissues. This change enables better dissection of the tiny ST with little damage. The ST become morphologically uniform and have reduced water content with a build up of stress-responsive solutes (soluble sugars and free proline). This mediates more efficient vitrification of cytosol on immersion in LN (Thinh, 1997;Thinh et ah, 2000). 22

43 CHAPTER STUDY ON IN VITRO CULTURE OF TARO USING SHAKE CULTURE WITH LIQUID 0.5 TDZ MEDIUM 3.1 Introduction In vitro plants are the preferred sources of explants for cryogenic work. They have advantages over field and screen house materials in being less loaded with pathogens (bacteria and fungus), more physiologically homogenous and are already adapted to in vitro culture conditions. Taro has been micropropagated via callus or protocorm-like bodies induced by combinations of cytokinins (kinetin or BAP) and auxins (2,4-D or NAA or IAA) or even incorporations of taro extracts in the culture media (Mapes and Cable, 1972; Jackson et al., 1977; Irawati and Webb, 1983; Oosawa et al., 1984; Tim et al., 1990; Sabapathy and Nair, 1992,1995). These systems could compromise the genetic stability of the germplasm collection through artifacts such as somaclonal variations (Scowcroft, 1984). Nagata (1995), Clemente et al. (1994), Thinh (1997) developed systems for multiplication through enhancement of axillary branching or reproduction from axillary buds. In addition to the multiplication systems mentioned above, a three-stage cycle (0.5 tng/1 TDZ mg/1 - BAP mg/1 TDZ) system was developed in the laboratory at The University of the South Pacific, Samoa by the Pacific Regional Agriculture Programme (PRAP) tissue culture project (1997). This system was used to bulk up the required number of taro plants in this study (section 4.1.1). This system has the drawbacks of being lengthy, costly due to high prices of TDZ and BAP and the use of such relatively high concentrations of growth hormones could compromise cryo success (Reed, 2001:pers. comm.; Thinh, 2001;pers. comtn.) 23

44 Thinh (1997) established a non-hormone system for clonal production of taro plants (Colocasia esculenta var. antiquorum). Shoot-tips maintained on liquid shake culture supplemented with mg/1 TDZ had an average production rate of 4.3 offshoots after five subcultures. The offshoots continued to proliferate at a similar rate when transferred to hormone free medium. Cormel slices from these plants also produced three to four offshoots when placed on solid MS medium. The applicability of this protocol to a tropical taro {Colocasia esculenta var. esculenta) cultivar was investigated. 3.2 Materials and Method Taro plant material In vitro plants of the Fijian cultivar, Tausala ni Samoa, maintained on hormone free solid MS were obtained from the RGC. Two centimetre explants including the corm base and an adjoining length of stem were excised from these plants and used for the experiment Cycle One Twenty explants were inoculated on 0.5 mg/1 TDZ in liquid MS medium in McCartney bottles. (hereafter referred to as tubes). Ten tubes were placed on the shaker at 80 rpm while the other ten were left standing on the shelf under normal culture conditions (section 4.2). Observations were recorded on weekly basis Cycle Two After the fourth week, the tubes for both treatments in cycle two were divided into three categories depending on the number of offshoots per explant: 24

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47 3.4 Discussion The hormone-free clortal propagation system developed by Thinh (1997) for Colocasia esculenta var. antiquorum, based on enhanced axillary branching (multiple shoot formation) through liquid TDZ medium shake culture and subsequent carry over effect on hormone-free medium, did not work with the tropical taro variety Tausala ni Samoa of Colocasia esculenta var. esculenta type. After two four-week cycles on liquid TDZ shake medium cultures, no enhancement in proliferation rates was noted and no carry over effect was observed when these plants were transferred to hormone-free media. In contrast, the three-stage system being used in this study produced an average of four to six offshoots from the variety (Tausala ni Samoa) during routine bulking up. Furthermore, when these plants were transferred to hormone free medium, some carry over effect of TDZ was noted as these plants produced offshoots at similar rates in the first subculture on hormonefree medium. However, these effects diminished on subsequent subcultures on hormone-free medium. The different response of this variety of taro (Tausala ni Samoa of esculenta type) to liquid TDZ shake medium could be a genotypic response as the study carried out by Thinh (1997), used antiquorum type of taro cultivated in Japan. In tissue culture studies with these two types of taro, var. antiquorum has always been shown to be more responsive in tissue culture (Jackson et ah, 1977; Arditti and Strauss, 1979; Irawati and Webb, 1983; Tim et al., 1990). As var. antiquorum has a small central conn with pronounced lateral branching, it is possible that little manipulation of the tissue culture system is required to encourage further branching, compared to the var. esculenta, which produces a central corm with very few lateral branches. A comparative investigation of responses of different cultivars of var. esculenta to shake and non-shake cultures would be an interesting study. 27

48 Finally, it was interesting to note that there was no difference in proliferation whether cultures in liquid medium were shaken or left to stand. This implies that aeration is not crucial with cultures of taro in liquid medium. 28

49 CHAPTER METHODOLOGY 4.1 Materials and Method 4.1,1 Plant material In vitro stock plants of taro (Colocasia esculenta var. esculentd) were obtained from the Secretariat of the Pacific Community (SPC) Regional Germplasm Centre (RGC), Suva. The regional taro collection is comprised of taro varieties from several different PICs. A sample of varieties was selected to be representative of the diversity in the region by choosing varieties from all countries contributing to the regional collection. The plants were exposed to the following micropropagation cycle to bulk up sufficient material for experimentation: four weeks on MS solid medium thidiazuron (TDZ), followed by three weeks on solid MS benzylaminopurine (BAP), then for three weeks on solid MS TDZ. When sufficient material had been generated, the accessions were cultured in full strength liquid MS for one month to lessen any carry-over effects of the plant growth hormones. From these plants, 2 cm explants, including some basal corm and an adjoining length of leaf cluster bases, were excised and conditioned on the vaiious media stated in section Initially, the following cultivars were bulked up for experimentation: 29

50

51 4.2 In vitro Culture Conditions The basic medium used to micropropagate material for this study was that developed by Murashige and Skoog (1962) for tobacco tissues and always contained 30 g/1 sucrose, unless stated otherwise. The ph of the culture medium was adjusted to 5.8 ± 0.1 with 0.1 M KOH and/or HC1 solutions. The gelling agent used for solid medium was 7.5 g/1 Agar Type A (Sigma Chemical Co., Germany). The medium was sterilized by autoclaving at 121 C at 103 kpa for 15 min. Two types of micropropagation culture vessels were used: McCartney bottles containing 10 ml medium, referred to as Small Jars (SJ), and 100 ml Mayonnaise bottles containing 15 ml medium, referred to as Large Jars (LJ). SJ were initially used for micropropagating all in vitro plants. Later, LJ were used for cultures in an attempt to improve the quality of ST donor mother plants. Culture vessels Were incubated at 25 C under light intensity of 50 umolm'v 1 (cool white fluorescent lamps) and a photoperiod of 16 hr. All tissue culture consumables were obtained from Biolab Scientific Ltd. - New Zealand. 4.3 Procedure for Bacterial Screening The in vitro plants were inoculated on half strength liquid MS at ph 6.9 for one month to encourage bacterial and fungal growth (Reed et at, 1995; Tanprasert and Reed, 1997a,b). All the explants that failed to produce contaminants in that medium were tested on bacteriological 523 medium (Appendix 2). The bases of the explants were streaked on the 523 medium plates and the explants were cultured on solid MS. The plates were monitored for bacterial growth for up to a month (Viss et ah, 1991; Reed and Buckley, 1999). The explants depicting bacterial growth were discarded. 31

52 4.4 Vitrification of VPTM The steps for the vitrification procedure are outlined in general below. During this study the techniques used were modified for many of the steps in the vitrification procedure. The initial method was modified to optimize results by reducing handling damage, and to ensure that the times for each treatment were accurate in their duration. Both methods are described below under Initial Method and Modified Method General procedures Conditioning The explants are conditioned with different sucrose concentrations before being cryopreserved. The conditioning consists of either preculture or preconditioning: Preculture In vitro plants are cultured on solid MS. Shoot-tips (ST) are dissected from these plants and precultyred on solid MS medium supplemented with 0.3 M sucrose in 100 x 15 mm Petri dishes and left overnight (16 hi") in the dark at 25 ± 0.5 C before being cryopreserved. [This treatment will be referred to as overnight preculture (ONP) treatment throughout this study]. Preconditioning Preconditioning involves culture of the mother plants on high sucrose medium. ST were dissected from these plants and directly cryopreserved without preculture. 32

53 Shoot-tip dissection ST dissected from in vitro plants conditioned in the different ways described above are mm in size and consist of the apical dome surround by two leaf primodia. ST dissection is done under the binocular-dissecting microscope placed in the LAF using scalpel blades Loading treatment This involves loading the ST for 20 min at 25 C with a loading solution (LS) that consists of 2 M glycerol M sucrose prepared in liquid MS at ph Dehydration with PVS2 I* This involves dehydrating the ST with the plant vitrification solution 2 (PVS2) (Sakai et al., 1990) which consists of 30% w/v glycerol + 15% w/v ethylene glycol (EG) + 15% w/v DMSO M sucrose prepared in liquid MS at ph Immersion in liquid nitrogen The ST are immersed very quickly in LN Thawing The ST are removed very quickly from the LN and warmed at 40 C in a water bath. 33

54 Unloading This involves rehydrating the ST by incubating for 15 min at 25 C in liquid MS M sucrose solution, referred to as unloading solution (ULS) Recovery The ST are inoculated on recovery medium and incubated under favourable conditions for regrowth into plantlets Initial Method Conditioning f Same as in section Shoot-tip dissection ST dissection was done under the binocular dissecting microscope (Olympus) placed in the LAF cabinet (BTR Environmental Pty Ltd.), using size 12 scalpel blades (Paragon, England) Loading treatment A 1 ml polyvinyl cryovial (Nalgene) was placed in the LAF cabinet and filled with 0.8 ml of LS. Seven ST were collected at the tip of a scalpel blade with a needle and dipped into the cryovial of LS. After a few minutes the sides of the cryovial was tapped lightly to sink any floating ST. The ST were immersed in the LS for 20 min at 25 C. 34

55 Dehydration with PVS2 After 20 min, the LS was sucked out of the cryovial with a sterile Pasteur pipette. The ST were then washed three times with PVS2 solution. PVS2 solution was dispensed into the cryovials using the Pasteur pipette with enough force to swirl the ST. The ST were left in the last wash of PVS2 for 10, 12, 15 and 30 min Immersion in liquid nitrogen PVS2 was sucked out of the cryovial with the sterile Pasteur pipette and a fresh sample was filled in. The cryovial was attached to a cryocane, which was put in a cryocan and plunged quickly into LN contained in the Dewar flask. The ST were left overnight in LN. The cryocane with the cryovial containing the ST was removed from the LN and plunged quickly into waterbath at 40 C and kept in for 1 min. Then it was transferred to 25 C water for 1.5 min. The cryovial was then taken inside the LAF cabinet for unloading the ST. PVS2 was sucked out of the cryovial with the sterile Pasteur pipette and the ST were washed three times with ULS. The ULS was dispensed into the cryovials using the Pasteur pipette with enough force to swirl the ST. The ST were left in the last wash of ULS for 15 min. 35

56 4.4.2,8 Recovery After 15 inin, the ST were sucked up into the Pasteur pipette and dropped onto a sterile filter paper placed in a Petri dish. Using a needle, the ST were transferred onto solid MS supplemented with 0.1 M sucrose. The ST were maintained in dim light for a week then transferred to normal culture conditions as stated in section 4.2. If ST were green after twenty days, they were recorded as surviving the cryopreservatioii process. A recovery scoring was only recorded if the ST were still green and had started to grow into plantlets after four to six weeks Modified Mfithod Conditioning ( Same as in section Shoot-tip dissection Same as in section Loading treatment Seven ST were wrapped in a tissue paper prior to loading. The wrapping procedure was as follows: (Figure 4.1) a sterile 2x2 cm tissue paper (TP) was spread in a Petri dish and wetted with a few drops ofls 36

57 using scalpel blade, and looking under the microscope, the ST were carefully put onto the TP. All ST were placed in the center of the TP. 1 st Fold: two sharp pointed forceps, held in each hand, were used to hold two corners of the TP and fold the TP in half - trapping the ST in the middle. 2 nd Fold: the same two corners of the TP, together with the corners of the layer underneath it, were held again and folded back. 3 rd Fold: another two corners, at right angles one to the ones held previously, of the folded TP were held and folded perpendicular to the first two folds but reaching only half way across. 4 lh Fold: the opposite two corners were then folded over on the remaining unfolded half. The TP with the wrapped ST was immersed in 15 ml of LS contained in a 40 mm sterile glass Petri dish for 20 min at 25 C. Proline pretreatment (PPT) (' As an alternative investigation, trials were carried out where the ST of cultivar E399 were soaked in 5% proline solution for 2 hours prior to loading with LS. PVS2 exposure times of 10, 12, 15, 30 and 40 min at 25 C were investigated with PPT Dehydration with PVS2 The TP was lifted from the LS with a pair of sterile forceps, and blotted dry on two layers of sterile filter paper in a Petri dish. The TP was then immersed in 5 ml of PVS2 contained in a 15 ml Petri dish for the times stated in section

58 Immersion in liquid nitrogen When 50 sec of PVS2 exposure time was left, the TP with the ST was transferred to a 1 ml polyvinyl cryotube containing 0.8 ml fresh PVS2. The cryotube was attached to a cryocane and plunged quickly into LN contained in a wide mouth flash exactly at the end of the PVS2 exposure time. The ST were held in the LN for at least an hour Thawing The cryocane with the cryovial containing the ST was transferred very quickly from the LN to a waterbath at 40 C and shaken vigorously for 90 sec. The cryovial was transferred to the LAF cabinet for unloading the ST. The TP was removed from the cryovial with a pair of sterile forceps, blotted dry on two layers of sterile filter paper in a Petri dish and immersed in 5 ml of PVS2 contained in a 15 ml Petri dish. After 10 min, the TP was unwrapped so that the ST could float in the ULS for the next 5 At the end of 15 min, using the scalpel blade the ST were lifted from the ULS onto a sterile filter paper placed on solid MS supplemented with 0.3 M sucrose. The plated ST were left overnight in the dark at 25 C. The following day, they were inoculated on solid MS M sucrose plates. The cultures were maintained in the dark for three days and then transferred to 38

59 dim light for one week before exposure to normal culture conditions as stated in section 4.2. Survival and recovery were scored as in section Experimental Procedures Using the above procedures, the following variables were investigated: PVS2 dehydration times of 12, 15, 30 min at 25 C and prolonged PVS2 exposure at 0 C (on ice) for cultivar E399 at 40, 60 and 90 min PPT trials for cultivar E399 at PVS2 exposure times of 10, 12, 15, 30 and 40 min at 25 C Overnight preculture on 0.3 M sucrose medium Preconditioning on 60, 90 and 120 g/1 sucrose medium for 4 and 7 weeks (NOTE: 60, 90 and 120 g/1 sucrose media are referred to as 60S, 90S and 120S, respectively, throughout this study) ( Age of ST donor plants; 1, 2, 3 and 4 months for ONP trials and 4 and 7 weeks for high sucrose preconditioning trials Size of culture containers; post-ln recoveries of ST dissected from mother plants grown in small jars and large jars were evaluated 39

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63 5.2.2 Post-LN recovery rates for all trials conducted for all culture conditions at PVS2 exposure time of 12 tnin As presented in section 5.1 above, the best results were attained with PVS2 exposure time of 12 min. Other results for this treatment were 17% average recovery for two months old explants of cultivar CPUK with 60S preconditioning. The recovery rate for an individual trial under these conditions was 50% with the same cultivar (Table 5.6). Using 90S preconditioning, 7% average recovery was obtained for cultivar CPUK using seven weeks old explants. No success was achieved with cultivar E399 for 60S preconditioning with 12 min PVS2 exposure time. However, 7% average recovery for seven weeks old explants with 90S preconditioning was obtained with this cultivar. 12% and 7% average recoveries for three and four months old plants respectively, was achieved with cultivar TNS for ONP treatment. Under the same conditions, the recovery rate for individual trials were 71% for three months old plants and 14 and 29% for two months old plants. For TNS cultivar, average recoveries of 10% each for one and two months old explants, was obtained with 60S preconditioning. Under the same conditions, the recovery rates for individual trials were 29% for one month old explants and 14 and 43% for two months old explants Post-LN recovery rates for all trials conducted for all culture conditions at PVS2 exposure times of 15 and 30 min v As shown by the results presented in Tables 5.4 and 5.5, PVS2 exposure times of 15 and 30 min are not favourable for post-ln recovery with the taro cultivars investigated. The only success achieved for both times under all conditions was 4% average recovery for two months old plants of cultivar E399 with ONP treatment. However, high survival rates for ONP trials for these two cultivars (20 and 30% for two of the three E399 trials and 20, 30 and 40% for the three TNS trials) were obtained with PVS2 exposure time of 30 min. Ten percent 43

64 survival was also obtained for two of the three 120S trials for cultivar TNS with PVS2 exposure time of 30 min (Table 5.5). In all of these survival cases, one outer leaf of the shoottip stayed green and had some expansion but turned brown and died after 4 to 6 weeks Effects of prolonged PVS2 on post-ln recovery rates All trials conducted for prolonged PVS2 exposure at 0 C (on ice) for cultivar E399 at 40, 60 and 90 min were unsuccessful (section 4.5). 5.3 Effects of Prolonged PVS2 on Post-LN Recovery Rates All proline pretreatment trials conducted for cultivar E399 were unsuccessful (section f ). As shown in Table 5.6, it appears that eultivar E399 is the most tolerant to PVS2 as it had recoveries for all 10, 12 and 15 min exposure times. 5.4 Effects of Different Preconditioning on Post-LN Recovery Rates Effects of overnight preculture on 0.3 M sucrose medium on post-ln recovery rates No post-ln recoveries were obtained for cultivars CIMG, CIRA, N13, N14 and P3 using ONP treatment. As shown in Tables 5.3 and 5.6, cultivars CPUK, E399 and TNS could be successfully cryopreserved through overnight preculture on 0.3 M sucrose medium. For cultivars CPUK and E399, the best recovery rates were obtained with this treatment (section 5.1). As shown in Table 5.2, low average recovery rates of 12% for three months old plants and 7% four months old plants were obtained for cultivar TNS with ONP treatment. 44

65 However, a high recovery rate of 71% for an individual trial was obtained for this cultivar (Table 5.6). ONP treatment trials for ST of cultivar E399 dissected from plants preconditioned using 60S conditions were unsuccessful (Table 5.2). 5.4,2 Effects of preconditioning on high sucrose concentrations on post-ln recovery rates Effects of preconditioning on 60 g/l sucrose medium on post-ln recoverv rates The only success obtained for cultivar E399 with preconditioning treatments was a low recovery rate of 10% for 60S preconditioning with PVS2 exposure time of 10 min (Table 5.1). No successful recoveries were obtained for cultivar CPUK for 60S preconditioning with PVS2 exposure time of 10 min (Table 5.1). For this cultivar, an average recovery rate of 17% for PVS2 exposure time of 12 min was achieved with this treatment (Table 5.2). The recovery rate for an individual trial under these conditions was 50% (Table 5.6). The data in Tables 5.1, 5.2 and 5.6 show that cultivar TNS achieved the highest recoveries of all cultivars with 60S preconditioning treatment. An average recovery rate of 14% was obtained with PVS2 exposure time of 10 min (Table 5.1) whereas an individual trial under the same conditions had a recovery rate of 43% (Table 5.6). As shown in Table 5.2, a low average recovery rate of 10% with PVS2 exposure time of 12 min was obtained for cultivar TNS. The recovery rates for individual trials werfr 14, 29 and 43%. Generally, it can be said that preconditioning on 60 g/l sucrose medium was not very effective in enhancing post-ln recovery rates Effects of preconditioning on 90 g/l sucrose medium on post-ln recoverv rates As can be seen from Table 5.2, no post-ln recovery was achieved for cultivar CIMG using 90S preconditioning. This was also true for cultivar CPUK using 90S preconditioning with 45

66 PVS2 exposure time of 10 min (Table 5.1). However, a low 7% average recovery rate with 12 min PVS2 exposure using 90S preconditioning was obtained for this cultivar (Table 5.2). Using 90S preconditioning, the trials for cultivar E399 were unsuccessful with 10 min PVS2 exposure time (Table 5.1), while low average recovery rates of 6% were obtained for both 12 min and 15 min PVS2 exposure times (Tables 5.2 and 5.4). While no success was obtained for cultivar TNS with 90S preconditioning for 10 min and 15 min PVS2 exposure times (Tables 5.2 and 5.4), the optimum average recovery rate (29%) for all 90S preconditioning trials was obtained with 12 min PVS2 exposure. The recovery rates for individual trials were 17, 17, 29 and 100% (Table 5.6). All successes for all cultivars stated above using 90S preconditioning were for explants preconditioned for seven weeks. An interesting observation was that three cryopreserved shoot-tips had one axillary bud each appearing from their bases. One of such shoot-tips was from a CPUK trial while other two were from two separate TNS trials. The new buds were alive and growing for two weeks for the CPUK and one of the TNS trials, then turned brown and died. The new bud in the other TNS trial survived to grow into a plantlet Effects of preconditioning on 120 g/1 sucrose medium on post-ln recovery rates For cultivar E389, an average recovery rate of 3% was achieved using 120S preconditioning and a PVS2 exposure time of 10 min (Table 5.1). However, this cultivar had endogenous bacterial contamination, hence this result may not represent its true success potential for cryopreservation. A low average recovery rate of 3% was also obtained for cultivar TNS with 120S preconditioning for PVS2 exposure time of 10 min (Table 5.1). 46

67 5.5 Effects of Age of ST Donor Mother Plants on Post-LN Recovery Rates From the evaluation of the post-ln recovery rates of ST dissected from mother plants grown for different lengths of time without subculture, it was discovered that using older mother plants as sources of ST is more conducive to post-ln viability. As stated in section 5.1, the optimum age of ST donor mother plants for cultivars CPUK and E399 was three months with ONP treatment. However, leaving the mother plants too long without subculture conferred a decline in post-ln recoveries as no recoveries were achieved for either of cultivars CPUK and E399 with four months old plants (Table 5.2). For cultivar TNS, seven weeks old plants were best with 90S preconditioning treatment. 5.6 General Observations/Results ' All plants grown in large jars for one to four months on hormone free solid MS were on average 17 to 20 cm tall, had 2 to 4 leaves that were 2.5 to 5 cm long and 1.5 to 3.5 cm wide and produced 12 to 25 roots that ranged from 11 to 25 cm in length (Figure 5.1). These plants produced very few offshoots (0 to 4). In contrast, plants grown in small jars were of roughly half the size and dimensions of those from large jars (Figure 5.1). These characteristics correlated with the quality of ST dissected from plants grown in different jars. The ST dissected from plants grown in large jars were larger, less succulent, more rigid, more homogenous and easier to dissect than their counterparts grown in small jars. Different growth was observed for different cultivars when subjected to medium with high sucrose concentrations. While cultivar TNS did not grow at all on 90S medium and had retarded growth on 60S medium, the cultivars CPUK and E399 grew almost normally on 47

68 60S medium and had retarded growth on 90S medium (Figure 5.2). In addition to this, all the cultivars listed in Table 4.1 had no growth on 120S medium. For the cultivar TNS, the successes from 90S culture conditions (Table 5.6) came from explants that had no growth at all. The outer two to three leaves of the 2 cm explants cultured on the 90S medium turned brown and the explant had a dead appearance. However, on dissecting the inner ST was healthy, turgid and very easy to dissect. ONP and 120S trials were conducted on the taro cultivars listed in Table 4.1. All trials were unsuccessful except a 120S trial for cultivar E389 (Table 5.1). While the ST cryopreserved from these plants died on the recovery medium, the bacteria flourished. All trials conducted for ST donor plants grown in small jars and/or liquid MS were unsuccessful,,except the cultivar E389 success stated above. During the post-ln recovery process, the surviving shoots-tips could be identified two to four days after thawing from LN. The survivors would swell and start turning green. Four to six weeks later they would form the first leaves and begin rooting. About two months after thawing, they would form small plantlets (Figure 5.3).

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