Main Menu
Development of advanced technologies for germplasm conservation of tropical fruit species
Project ID
CP/2000/002
Inactive project countries
Malaysia
Commissioned Organisation
Bioversity International, Malaysia
Project Leader
Dr V. Ramanatha Rao
v.rao@cgiar.org
Phone:
+60 3 89423891
Fax:
+60 3 89487655
Project Budget
$746,480.00
Start Date
01/01/2003
Finish Date
31/12/2005
ACIAR Research Program Manager
Dr T K Lim
Related publications
Overview Objectives
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.
Project Background and Objectives
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.
Progress Reports (Year 1, 2, 3 etc)
Year 1:
Year 1 (01/01/2003-31/12/2003)
The outputs expected for all three objectives in year 1 were fully achieved. The five partner countries are working on identified priority tropical fruits to develop new techniques to conserve germplasm. The initial stages of the project involved developing micropropagation systems for the identified crops (mango, papaya, Australian native fruits, Nephelium, citrus, persimmon, litchi and longan). Successful tissue culture systems were established to provide plant material for cryopreservation work.
Cryopreservation techniques such as encapsulation dehydration (ED), vitrification, new encapsulation-dehydration (NED) and slow freezing were attempted to conserve different plant materials. Developing-country partners were trained in using these techniques through a course organised at Griffith University, Australia in September, 2003. At the end of the course, each trainee developed a re-entry plan in line with project activities, to be implemented upon return to their institutions.
Initial work on cryopreservation of papaya, citrus, Australian native fruits (citrus and Davidson plum) showed promising results. Work on mango, persimmon, litchi and longan has progressed well in establishing micropropagation systems.
Alternative conservation strategies such as slow growth as well as storage of seed in liquid nitrogen were attempted. Positive results were obtained for papaya and citrus. Established micropropagation techniques were shared among partner countries working on the same crop to adapt to the different species that they are working on.
Year 2:
Cryopreservation studies for the identified crops (mango, papaya, Australian native fruits, Nephelium, citrus, persimmon, litchi and longan) were performed using materials propagated in Year 1. Preliminary cryopreservation studies were already reported for some crops like papaya, citrus and Australian native fruits in Year 1.
Somatic embryogenesis have been developed for Citrofortunella microcarpa, Citrus reticulata, C. aurantifolia, C. sinensis and C. suhuiensis and multiple shoot formation was obtained for Citrofortunella microcarpa, C. reticulata and C. hystrix. Protocols have been refined for micropropagation and shoot and plantlet regeneration via organogenesis of some Australian native fruits. Micropropagation and plantlet regeneration from in vitro nodal cuttings of three Australian Citrus species (Citrus inodora, C. garrawayi and C. australasica) has been achieved. Adventitious shoots were obtained for both litchi and longan. Persimmon callus was formed and used for cryo-assays. Adventitious buds were formed from embryonic shoots of persimmon.
Genetic fidelity testing for tissue cultured and non-tissue cultured plants of Citrofortunella macrocarpa, C. reticulata, C. aurantifolia and C. grandis were evaluated using 11 enzyme systems and no differences was found in banding patterns between tissue cultured and non-tissue cultured plants.
In developing cryopreservation techniques, seed desiccation sensitivity was tested for Citrus reticulata, C. sinensis, C. medica, Citrofortunella macrocarpa, C. aurantifolia, C. hystrix and C. nobilis as well as for longan seeds and excised embryonic axes of persimmon. Experiments with desiccation of C. australasica seeds showed tolerance to ultra low temperatures and had normal morphology post-cryopreservation. In another study, the optimum duration was found for C reticulata and C. nobilis.
Encapsulation dehydration (ED) and vitrification methods were tried for several Citrus varieties with successful recovery. The results suggested that ED may not be a suitable technique for cryopreservation of somatic embryos. Cryopreservation of embryonic axes of C. grandis using ED method was performed and the results provided initial data on the potential of cryopreservation of C. grandis embryonic axes. Promising results were obtained for C. hystrix as well using the ED method. Cryopreservation of mature Citrofortunella macrocarpa seeds using desiccation proved to be a very simple method of preserving its germplasm. Embryogenic callus of Som-Keaw-Wan (C. reticulata) Som-Shokun (C reticulata), Som Chengh (C. sinensis) were cryopreserved using new ED. Results showed that pretreatment affected the percentage of moisture content after desiccation in various time. The results of this experiment showed that new ED with pretreatment solution was better than conventional ED. For C reticulata and C. nobilis, the pregrowth duration had significant effect on survival of its cryopreserved embryonic axes.
Papaya shoot tip cryopreservation protocol was improved to be applicable to a wide range of genotypes by optimization factors influencing vitrification. Eight different genotypes were selected to compare the old and refined protocol and all were successfully cryopreserved with recovery rates varying from 36% to 60%.
Preliminary tests for cryopreservation of Nephelium were carried out. Progress on developing a suitable cryopreservation method for this difficult species has been made and the results obtained so far has provided guidance on the approach for further work.
For Australian native species, shoot tips of Citrus australasica were successfully recovered after encapsulation and desiccation and Murcott tissue was cryopreserved using a slow freezing approach.
Fine suspension cultures of mangoes were cryopreserved and viable embryos were recovered post-cryopreservation. Recent experiments have successfully replaced the toxic DMSO in the vitrification stage.
Slow growth protocol was developed for mandarin by testing varying concentration of mannitol and sorbitol. Using MS medium without growth regulator and supplemented with sucrose as carbon source slowed down growth of mandarin shoots. Slow growth protocols papaya plants in vitro were refined and cryopreservation of shoot tips and seeds achieved by using modified medium for the papaya micro-cutting system. Plants can be held for 8 to 12 months before transfer. The use of fructose in lieu of sucrose slows the growth considerably and allows incubation at 25C. This is essential, as tropical species cannot be incubated at low temperatures due to tissue damage. Considerable progress has been made with experiments on papaya seed desiccation, germination and storage at different temperatures. It was found that desiccation below 15% severely reduces germination percentage and that seed at any moisture content can be germinated with gibberellic acid or heat shock treatments. Seed that were stored for 1 month had germination percentages as high as 80%.
The project's Second Annual Meeting was organized as planned in Hanoi, Vietnam from 8-11 December, 2004 and country coordinators had the opportunity to review and discuss their work together. Techniques and information were shared among the country coordinators.
Year 3:
See Final Report (25/02/06)
Project Outcomes
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.
Location
There are no project locations defined for this project.
