“Role of the Centre for Pacific Crops and Tree’s in the sustainable conservation and the safe movement of material”

Valerie Saena Tuia, Logotonu Meleisea Wainaqabete, Amit Sukal, Sainimili Baculacula, Ulamila Lutu, Arshni Shandil, Unaisi Davete and Rohini Prasad

Centre for Pacific Crops and Trees, Secretariat of the Pacific Community, Suva, Fiji

Abstract: The Centre for Pacific Crops and Trees (CePaCT), a Pacific Genebank, based in Narere Fiji is an investment by the Secretariat of the Pacific Community established in response to the Heads of Agriculture and Forestry Meeting (HOAFS, 1996) recommendation to put in place policies to conserve, protect, and best utilize plant genetic resources in countries and through regional cooperation. CePaCT assists Pacific island countries (PICs) to sustainably conserve and utilize their plant genetic resources as well as sourcing improved crop diversity to address food and nutritional security and for improved resilience to climate change. The Centre uses in vitro technology to conserve collections of some of the Pacific’s important staple crops, and such as (Colocasia esculenta) and other edible aroids ( (Alocasia macrorrhizos), swamp taro (), (Xanthosoma sagittifolium)), banana (Musa sp), breadfruit (Artocarpus altilis), cassava (Manihot esculenta), sweet potato (Ipomoea batatas) and yam (Dioscorea sp). Other collections conserved by the Centre include kava (Piper methysticum), potato (Solanum tuberosum), vanilla (Vanilla fragrans) and some selected tree species (Pandanus tectorius and Santalum sp). CePaCT conserves a unique global collection of taro, largest in the world, comprising of over 1,100 taro accessions from the Pacific and Asia. CePaCT has a “state of the art” virus indexing facilities that virus test germplasm using internationally approved protocols in order to facilitate distribution of crop diversity (improved and traditional) to farmers. The Centre does virus indexing on aroids, banana, sweet potato and yam. Over 56,000 tissue culture plantlets from 14 crops have been distributed by the CePaCT from January 2001 to June 2012, to 46 countries including 24 PICs. In vitro technology is considered one of the safest and efficient methods available for the international movement of germplasm.

Key words: CePaCT, edible aroids, virus indexing, in vitro conservation, resilience, crop diversity

1. Background: The establishment of the Centre for Pacific Crops (CePaCT) by the Secretariat of the Pacific Community (SPC) is a result of all the past and ongoing efforts that started in early 1990 under a European Union project titled “The provision of tissue culture services to the Pacific region” that was based at the University of the South Pacific in . The importance of the tissue culture services was further strengthened when the taro industry of both was devastated by the outbreak of taro leaf blight (TLB) disease in 1993, has resulted in the loss of food security, export market, increased erosion of susceptible traditional varieties and increased vulnerability of other Pacific countries to TLB. The importance of sharing tolerant genetic diversity from outside of Samoa was also realized and taro varieties from , Palau and Federated State of Micronesia were brought in as tissue culture material to help restored food security. These TLB tolerant lines have formed the basis of new breeding programs to generate new diversity of taro in Samoa as a long term solution to TLB and sustainable food security. The Samoa TLB disaster has also prompted a 5 year AusAID Taro Genetic Resources: Conservation and Utilisation (TaroGen) that began in 1998 and coordinated by SPC. The aim was to re-collect all taro genetic resources of the Pacific, have them characterized, DNA fingerprinted for selection of a core collection and have it virus indexed for sharing by all partners. Capacity building on DNA fingerprinting and virus indexing was provided for regional staff and technology transfer was also part of the project. With funding provided by the AusAID, the Regional Germplasm Centre (RGC) was established, however the RGC has its name changed to CePaCT in 2007 recognizing the importance of trees in the food supply systems and other income generation activities. The initial SPC’s global taro collection comprises of taro established under this AusAID TaroGen regional project. Through importance of SPC networking and sharing of germplasm, the EU Taro Network for the South-east Asia and (TANSAO) enabled the Pacific countries to access new diversed genepool of Asian taro with tolerant traits to TLB. These new TLB lines progenies of Pacific and Asian parentage are widely distributed within the Pacific for evaluation. The importance of sharing and accessing new diversity outside the region, Pacific countries through SPC have agreed to place Annex 1 regional crop collections held in trust by SPC CePaCT into the Multilateral System (MLS) of the International Treaty for Plant Genetic Resources for Food and Agriculture (ITPGRFA). In June 2009, Hon Taua Kitiona Seuala, a Samoan Agricultural Minister then on behalf of Pacific Ministers of Agriculture and Forestry has signed the agreement at the 3rd Session of the Treaty Governing Body. All germplasm exchange and distribution from SPC have to be accompanied by the Standard Material Transfer Agreement provided under the ITPGRFA. The use of tissue culture technology by CePaCT involves the growing of crops under aseptic controlled conditions and is widely used for commercial propagation of many (Dodds and Lorin., 1982). There are multiple advantages of the in vitro system: [i] as a safe method for preservation and conservation of plants for future use, [ii] all year production of planting material, [iii] plant can be manipulated for increased multiplication rates, [iv] virus-indexed status of the crops allows for safe distribution without any quarantine risks, [v] sterility state of plant material provides for easier access to diversity from within and outside of the region and [vi] cost-effective method for distribution of plant material due to small size, easier and cheaper. The CePaCT continues to provide services to the region through sustainable conservation and utilization of their plant genetic resources as well as sourcing improved crop diversity to address food and nutritional security and for improved resilience to climate change.

2. Conservation: The Centre uses in vitro technology to conserve over 2,000 accessions of some of the Pacific’s important staple crops, and such as taro and other edible aroids (alocasia, swamp taro, xanthosoma), banana, breadfruit, cassava, sweet potato and yam. Other collections conserved by the Centre include kava (Piper methysticum), potato (Solanum tuberosum), vanilla (Vanilla fragrans) and some selected tree species (Pandanus tectorius and Santalum sp). SPC is a current recipient of a long term funding by the Global Crop Diversity Trust (the Trust) recognizing CePaCT asone of the leading Worlds Genebank Centre for conserving a unique in vitro global taro collection of over 1,100 accessions from the Pacific and Asia. SPC is the first non-CGIAR Centre to have received this funding from the Trust. At the request of the countries, the Centre also conserves important cultural collections such as the unique collection of yam (D.alata) from the Federated State of Micronesia, New Caledonia unique fei and maoli popoulu banana collection, taro and banana from French Polynesia as well as swamp taro from Kiribati, Federated State of Micronesia and Tuvalu. The Centre repatriates lost accessions from their traditional collections in the countries from its in vitro master collections for countries such as Cook Islands, Fiji, New Caledonia, Niue, Palau, PNG, Solomon Islands and Samoa. SPC collaborates with the University of the South Pacific (USP) on the conservation and safety duplication of the CePaCT Pacific collection as a backup to its main master collections conserved at CePaCT in Fiji. 3. Utilisation: The HOAFS 1996 recommendation (Taylor et al., 2003) encourages utilization and sharing of plant genetic resources, thus CePaCT should not be seen as just a museum of plants. CePaCT has been distributing over 56,000 tissue culture plantlets (36,922 tubes) from January 2001 to June 2012, of 14 crops to 46 countries including 24 Pacific island countries (PICS). Crops being distributed to PICs include aroids, banana, bele, breadfruit, cassava, cowpeas, potato, kava, sweet potato and yam. The following PICs that have received germplasm from SPC namely American Samoa, Cook Islands, Fiji, Federated State of Micronesia, Guam, Hawaii, Kiribati, Marshall Islands, New Caledonia, Nauru, Niue, Norfolk Islands, Northern Marianas, Palau, Papua , Pitcairn Islands, Samoa, Solomon Islands, French Polynesia, Tokelau, , Tuvalu, Vanuatu and Wallis and Futuna. For the first time SPC has distributed improved crop diversity (banana, cassava, sweet potato and taro) to Pitcairn Islands in May 2012 after several years of delays due to isolation, irregular boat travel schedules and New Zealand biosecurity regulations restricting transit of some of other improved material listed as risky bound for Pitcairn Islands. CePaCT works closely with countries on the evaluation of this germplasm using new evaluation forms developed and information of crops that performed well under local conditions is fed into the Pacific Genetic Resources Database (PacGen) developed by the Centre. The activities of the Centre have expanded dramatically over the years taken on board new projects encompassing food security, trade components and climate change adaptation. With funding by AusAid climate change project, CePaCT is actively working closely with the Pacific countries in evaluating its climate ready collection comprising of seven crops namely taro, banana, cassava, swamp taro, sweet potato, cassava and yam which have been indentified to have demonstrated tolerance to drought, salt, waterlogged, windy conditions and acid soils. The number of accessions will increase with time after evaluation information is received from the countries. 4. Sourcing of improved diversity The CePaCT continues to source new diversity from within and outside of the Pacific. The 1993 TLB scenario has proven Samoas taro genetic pool is very narrow and vulnerable. The new taro diversity is robust against TLB, an indication of broadened genetic base enhanced by integrated TLB tolerant genes contributed by Asian taro. It is also likely limited genetic pool also exists for other crops in the Pacific. Bringing diversity that exists outside of the Pacific is the long term solution to sustainable food security. In collaboration with International Genebank Centres, CePaCT sources improved crop diversity from the International Institute of Tropical Agriculture (IITA), Nigeria for yams, with Bioversity International, Belgium and Department of Employment, Economic Development and Innovation (DEEDI), Australia on banana, sweet potato is obtained from the Centre Internacional de la Papa (CIP), Peru and cassava acquired from the Centro Internacional de Agricultura Tropical (CIAT), Columbia. The accessions imported from these Genebanks are virus indexed material and SPC accessed them as in vitro cultures for further mass propagation and distribution. The PICs have limited resources and facilities to establish and virus test their germplasm, and this service is provided by SPC. CePaCT imports treated vegetative plant material from countries for establishment in the Biosecurity of Fiji approved screenhouse prior to establishment in tissue culture.

5. Virus Indexing The Centre houses a virus indexing laboratory which enables SPC to facilitate distribution and sharing of crop diversity both local and improved varieties to the Pacific and SPC partners. The improved diversity sourced from the region either sent as vegetative or as tissue cultures, is virus indexed by the Centre. The Centre can virus test for aroids, banana, sweet potato and yam using internationally approved protocols. There are several stages of the process, includes screening of the accessions at the in vitro stage, only those tested negative will be tested at the three and six month stage of crop growth in the post-entry quarantine (PEQ) screen house. This is a prolonged process and requires about 6-10 months until any accession can be declared clean and available for distribution, whilst those infected have to go through the cleaning process again until viruses are eliminated.

Three types of indexing technologies used involves the application of (1) molecular techniques, (2) serological and (3) symptomatology: molecular methods, namely polymerase chain reaction (PCR), Reverse transcriptase (RT) PCR, Immunocapture (IC)-PCR and Rolling Circular Amplification (RCA); serological techniques such as the double antibody sandwich enzyme- linked immunosorbent assay (DAS-ELISA), triple antibody sandwich ELIZA (TAS-ELISA) and nitrocellulose membranes ELISA (NCM-ELISA), and symptomatology using grafting techniques as used in sweet potato virus diagnostics.

The collection to date has 554 accessions of aroids, banana, potato, sweet potato, vanilla and yam already indexed to known crop viruses and available for distribution. The number of the collection will also increase with time once more accessions are tested negative to certain known crop viruses. For infected material, CePaCT has been investigating three methodologies for eliminating viruses as part of a Masters research, namely thermotherapy, electrotherapy and virus therapy (chemotherapy). The research has initially focused on yam viruses, in particular, Dioscorea badna virus (DBV) and virus (DAV).

6. Challenges in Distribution due to integrated badna viruses

The widespread presence of two major viruses in the Pacific especially the Dasheen Mosaic Virus (DsMV) and Taro bacilliform virus (TaBV), an integrated badna virus (Secretariat of the Pacific Community, 2004), have caused a major constraint in the distribution of accessions from CePaCT. The new elite Cycle-7 breeding lines of taro from Samoa have all been tested positive to TaBV. Based on Macanawai et al (2005), TaBV is transmissible and even through seed and pollen. Other badna viruses infect banana namely the Banana streak virus (BSV). The effect of badna viruses on plant growth is minimal and often a latent virus. This indeed can be a long term constraint if no effective methodologies for eliminating integrated viruses from plant material are available hence further research is urgently needed. CePaCT has looked at possible methods that could help improved badna work, such as the use of BSV antibodies (Phillips et al., 1999) and Dioscorea Badna Virus (DBV) antibodies for IC-PCR; Rolling Circular Amplification (RCA) using new primers designed to detect episomal viruses (non integrated sequence); and the use of internal primer controls NAD5 (Menzel et al., 2002) to check on integrity of nucleic acid extraction for example, RNA. Through network collaboration, CePaCT collaborates with virologists in IITA, Nigeria, Queensland University of Technology (QUT) and DEEDI, Australia, and DSMZ, Germany on modification of protocols used on the IC-PCR protocol that has been recommended to use with the DBV antibodies for detection.

The virus research on both taro and yam, is a time consuming process with a number of challenges facing with badna viruses. The presence of badna viruses continue to hinder accessibility of some of these unique varieties at CePaCT that have tolerant traits to drought and TLB disease important for adaptive capacity to climate change variability. Viruses do evolve with time, so as the new challenges on virus research, hence the need to develop more sensitive tests that are efficient and cost-effective in the long run. 7. Possible consideration Both DsMV and TaBV infect some of the known aroids and are widespread throughout Asia and the Pacific. The impact of the two viruses on yield and plant growth could be less than 20% as compared to Colocasia bobone disease virus (CBDV) confined to PNG and Solomon Islands (Secretariat of the Pacific Community., 2004). All of the new collection of TLB resistant taro varieties from Samoan (Cycle 7 breeding lines) and some of the aroids received from other PICs are infected by these widespread viruses. The same scenario occurred with D.esculenta and D.alata yams obtained from PICs are also infected with yam badna virus (DBV), but these accessions have tolerant traits to drought important for climate change adaptation. Similarly, some of the unique banana accessions such as the fei (soa’a), iholena and maoli popoulu types from the Pacific are infected with BSV. CePaCT is unable to distribute this new diversity, and therefore, farmers are denied the opportunity to access it. If this new diversity can’t be shared, countries prone to TLB and to other climatic extremes such as drought will become very vulnerable hence food security will be severly affected.Given that some of these viruses are widespread in the Pacific, countries might like to consider an import risk assessment (IRA) to be carried out on these common viruses (DsMV, TaBV, DBV, BSV) subject to country approval before accessing this new diversity from CePaCT if they are already present in their respective countries. 8. Conclusion In line with the mission of the Pacific Plant Protection Organisation, SPC CePaCT is actively distributing virus indexed germplasm within and outside of the Pacific as in vitro cultures. Not only CePaCT is internationally recognized as a Pacific Genebank, but a Centre that conserves the largest unique global collection of taro in the world. The establishment of the virus facility of the Centre using internationally approved diagnostic methodologies facilitates access of PICs to improved diversity at SPC. Countries also need to carry IRA on less harmful badna viruses as not to deny access to unique accessions at CePaCT important for food security. Using in vitro technology facilitates conservation and utilization of improved diversity through germplasm sharing important for sustainable food and nutritional security, as well as building resilience of communities to climate change through availability of climate ready crops that also have commercial potential.

References

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Dodds, John H. & Lorin W. Roberts, 1982. Experiments in Plant Tissue Culture Cambridge University Press, Cambridge London, New York, New Rochelle, Melbourne Sydney.

Mary Taylor, Valerie Tuia, Rajnesh Sant, Eliki Lesione, Raghani Prasad, Rohini Lata Prasad and Ana Vosaki., 2003. “Using in vitro techniques for the conservation and utilization of Colocasia esculenta var esculenta (taro) in a regional genebank”. Edited by Luigi Guarino, Mary Taylor and Tom Osborn in 3rd Taro Symposium, 21-23 May 2003, Tanoa International Hotel, Nadim Fiji Islands. Page 69-73.

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Secretariat of the Pacific Community., 2004. Development and Application of Virus Indexing Protocols for the International Movement of Taro Germplasm. Plant Biotchnology Program Science Research Centre, Queensland University of Technology, Australia and Regional Germplasm Centre, SPC, Suva Fiji. Report compiled by Graham Jackson., 2003

W.Menzel, W.Jelkmann, E.Maiss., 2002. “Detection of four apple viruses by multiplex RT-PCR assays with coamplification of plant mRNA as internal control”. Journal of Virological Methods (2002). Volume 99, Issue 1-2, Publisher Elsevier, Pages: 81-92