Development of advanced technologies for germplasm conservation of tropical fruit species

Malaysia, Philippines, Thailand, Vietnam

Dr V. Ramanatha Rao
Phone: +60 3 89423891
Fax: +60 3 89487655
Email: v.rao@cgiar.org

• Griffith University, Australia
• Department of Agriculture, Thailand
• Institute of Agricultural Genetics, Vietnam
• University of the Philippines at Los Bans, Philippines
• Universiti Kebangsaan Malaysia, Malaysia

$746,480

01/01/2003 - 31/12/2005

Dr T K Lim

The Asia, Pacific and Oceania region has more than 400 tropical fruit species that are can provide income, nutrition, medicine, timber, fuel and livestock feed, but only a few have been exploited commercially. Tropical fruit species and their wild relatives play an important role in stabilising and sustaining ecosystems, particularly in Asia. Several native species are rapidly approaching extinction; thus, there is an urgent need to conserve this diversity. However, many species of tropical fruit are difficult or impossible to conserve by traditional methods such as seedbanks or field genebanks, and there are currently no efficient, appropriate methods for their long-term, sustainable conservation. New in-vitro technologies are therefore needed for conserving tropical fruit species. Such technologies were the subject of this project, which complemented an Asian Development Bank funded project, Conservation and use of tropical fruit species biodiversity in Asia, managed by the International Plant Genetic Resources Institute (IPGRI). The Asian Development Bank project involved 10 countries, including the Asian countries involved in this project.

This project aimed to conserve the genetic resources of selected tropical fruits and related species by developing new conservation methods and regeneration strategies, and disseminating these technologies to researchers and others within the Asia, Pacific and Oceania region.

The project involved five countries - Australia, Malaysia, Philippines, Thailand and Vietnam - and was managed by IPGRI through its regional office in Malaysia. Scientists in each of the participating countries focused on two or three species of major economic importance, such as Citrus species, litchi, longan, mango, papaya and persimmon. They also studied the wild relatives of these species.
As a first step, the project team developed cryopreservation methods for long-term conservation of the germplasm of the selected species. The method involved rapid freezing in liquid nitrogen, because this approach does not require conventional refrigeration equipment. A range of tissues, including shoot-tips, somatic embryos, zygotic embryos and pollen, were cryopreserved.
Project scientists also developed protocols for in-vitro micropropagation, slow growth and regeneration - techniques suitable for storage and distribution, and for providing material suitable for cryopreservation. For species that could not be cultured through these methods, somatic embryogenesis techniques were developed and implemented. Researchers and other users learned about the new techniques through joint meetings, training visits, workshops, IPGRI publications and various plant genetic resource networks.

In this project, conservation techniques have been developed for target tropical fruit species such as papaya, mango, Australian native fruits and several varieties of citrus, longan, litchi, Nephelium and persimmon. Development of conservation techniques included establishing a micropropagation system, optimizing cryopreservation protocols and investigating alternative conservation and regeneration strategies.

Papaya - Protocols for vitrification-based shoot tip cryopreservation were refined and successfully applied to a range of papaya genotypes and to Vasconcellea pubescens, a papaya wild relative. Factors that were optimized prior to liquid nitrogen (LN) exposure include age of culture, duration of overnight incubation and duration and temperature of exposure to the cryoprotectant. Post-LN factors that were tested and refined in this project included: duration of exposure to dark incubation; the effect of growth regulars in the culture medium on the rate of recovery of shoots; and rate of growth of plantlets in vitro. The effects of cryopreservation protocols on the growth of plants in vivo were also evaluated, including large-scale field trials. Papaya somatic embryos were recovered after cryopreservation and work on seed desiccation, germination and storage at a range of temperatures was carried out. It was shown that at any moisture content, seeds can be germinated with gibberelic acid (GA3) treatment or heat shock; the former was more effective. This also shows that papaya seeds may have dormancy right from the beginning. Papaya seeds were stored up to 12 months at a range of moisture contents and a range of temperatures, including cryostorage. Protocols for slow-growth of papaya in vitro were developed by modifying a medium previously developed for papaya micro-cutting. Plants were held under normal incubation conditions for 8 to 12 months before transfer.

Mango - Somatic embryogenesis was obtained and substantial progress was made with induction and maintenance of somatic embryos (SE) of mango suspension cultures and protocols for secondary embryogenesis. Successful cryopreservation of SE was obtained by pre-culturing embryo masses (EMs) in sucrose and Plant Vitrification Solution 2 (PVS2). Although 70% recovery was obtained, replication of these results is a major problem; more research is required to optimize the protocol.

Australian native fruits - Citrus australasica seeds demonstrated tolerance to desiccation and ultra-low temperatures, and had normal post-cryostorage morphology. Results on seed storage of C. inodora and C. garrawayi showed that these species have tolerance to desiccation and cryostorage, however reduced seedling vigour was observed. Seeds of both species survived cryopreservation with growth and acclimatization of plants post cryostorage.

A micro-propagation protocol was established for three Australian native Citrus species (C. australasica, C. inodora and C. garrawayi) that was suitable for mass multiplication and medium-term storage of this valuable germplasm. This regeneration protocol also allowed the investigation of shoot tip-based cryopreservation techniques. Encapsulation-dehydration protocols were applied to shoot tips of C. australasica. Survival of encapsulated shoot tips was minimal after desiccation to moisture contents suitable for cryopreservation. Shoot tips of C. australasica survived, however, and grew well with post cryopreservation using a standard vitrification protocol.

Somatic embryogenesis protocols were investigated for C. inodora, C. garrawayi and C. australasica using published methods and some media modifications. Embryogenesis was achieved in C. inodora and embryogenic tissue has been recovered from cryostorage using an encapsulation-dehydration protocol.

Davidsonia spp.
A micropropagation system was developed through the production of microcuttings in vitro. Protocols were developed for shoot and plantlet regeneration via organogenesis from a range of explants of D. pruriens and D. jerseyana. Preliminary experiments on vitrification and encapsulation-dehydration-based methods for cryopreservation were not successful. Work on organogenesis for D. johnsonii is ongoing. Genetic diversity studies have indicated that these three species are distinct.

Litchi and longan - Media for litchi and longan micropropagation were developed. Seed desiccation studies of litchi and longan identified optimum desiccation periods. Conservation of longan and litchi can be applied using encapsulation-dehydration technique of shoot tips.

Persimmon: A suitable media was identified for persimmon embryo culture and nodal cutting. Successful cryopreservation of embryonic axes (EA) was obtained through vitrification, but not for shoot tips. These results indicate that vitrification is not suitable for persimmon shoot tips.

Citrus
Optimum desiccation periods were identified for seeds of all the species studied. Protocols for adventitious root formation and regeneration of shoots for C. hystrix were developed. Regeneration via somatic (nucellar) embryogenesis was also developed for calamansi (X Citrofortunella macrocarpa) and mandarin (C. reticulata) using immature and mature seeds. For pummelo (C. grandis), callus was induced from juice vesicles and albedo tissues, but somatic embryogenesis and shoot regeneration was observed only in callus from albedo. A regeneration system via somatic embryogenesis was developed for lime using undeveloped ovules (immature seeds). Immature seeds of citron (C. medica), limon (C. limon) and native lime 'dalayap' (C. aurantifolia) showed varying degrees of callus and root formation. An effective slow-growth medium was identified for mandarin (C. reticulata).

Cryopreservation of desiccated seeds was studied for calamansi, mandarin, pummelo, native lime, limon and kubot (Philippine native Citrus sp.). The feasibility of low-temperature seed storage for short-term conservation of a few citrus species was investigated; storage of desiccated seeds of citron (C. medica) and kubot (Citrus sp.) could be used for medium-term conservation. For cryopreservation of embryogenic callus using encapsulation-dehydration technique, a suitable pretreatment and desiccation period were identified for C. reticulata and C. sinensis. For C. hystrix, the vitrification method was modified to obtain an acceptable level of survival.

Effects of desiccation and cryopreservation on lime, pummelo, calamansi, kubot, Tai Cat (mandarin type) and calamandarin (C. reticulata) were assessed using enzyme systems and no variants were observed. Random amplified polymorphic DNA (RAPD) primers were identified to study the genetic stability of Citrus regenerants (still to be tested).

Nephelium
Protocols for adventitious root formation and shoot regeneration were developed. Different steps in the vitrification procedure were studied and optimized. Further modification and study need to be carried out for the survival of shoot tips after cryopreservation. Slow-growth technique has shown potential for short-to-medium-term storage of germplasm. Suitable primers for RAPD have been identified for genetic stability studies on regenerants.