This review describes the current status of development of methods for cryopreservation (at -196ºC) of plants germplasm in Argentina. Arachis pintoi, a forage legume, has been maintained as seeds using vitrification method. Additionally, apical meristems, shoot tips, and somatic embryos have been cryopreserved using encapsulation-dehydration. Zygotic embryos, encapsulated and dehydrated, have permitted the cryopreservation of seven species of the genus Ilex. Various explants (apical meristems, uninodal segments, buds and somatic embryos) of Melia azedarach have been cryopreserved using the encapsulation-dehydration method. Protocols based in encapsulation-dehydration have also been developed for shoot tips of Citrus sinensis, seeds and protocorms of Oncidum bifolium and anthers of Oryza sativa. Vitrification protocols have been developed forcryopreservation of shoot tips of Solanum tuberosum and seeds of Toona ciliata.Fil: Mroginski, Luis Amado. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Rey de Badaró, Hebe Yolanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentin

Mroginski, Luis Amado

Rey de Badaró, Hebe Yolanda

English

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

270Adv. Hort. Sci., 2007  21(4): 270-273Received for publication 25 May 2007.Accepted for publication 21August 2007.Cryopreservation of plant germplasm in ArgentinaL. Mroginski, H. ReyIBONE (UNNE-CONICET), Sargento Cabral 2131, Corrientes (3400), Argentina.Key words: Arachis, Citrus, cryopreservation, Ilex, Melia, Oncidium, Oryza, Solanum.Abstract: This review describes the current status of development of methods for cryopreservation (at -196ºC)of plants germplasm in Argentina. Arachis pintoi, a forage legume, has been maintained as seeds using vitrifica-tion method. Additionally, apical meristems, shoot tips, and somatic embryos have been cryopreserved usingencapsulation-dehydration. Zygotic embryos, encapsulated and dehydrated, have permitted the cryopreserva-tion of seven species of the genus Ilex. Various explants (apical meristems, uninodal segments, buds and somaticembryos) of Melia azedarach have been cryopreserved using the encapsulation-dehydration method. Protocolsbased in encapsulation-dehydration have also been developed for shoot tips of Citrus sinensis, seeds and proto-corms of Oncidum bifolium and anthers of Oryza sativa. Vitrification protocols have been developed forcryopre-servation of shoot tips of Solanum tuberosum and seeds of Toona ciliata.1. IntroductionArgentina is an agricultural country with differentclimates. Many of its economically important plantspecies (such as corn, wheat, beans, cucumbers) have -from a germplasm preservation point of view - ortho-dox seeds and are maintained in conventional seed-banks (mostly belonging to the National Institute ofAgriculture) distributed in 10 places of Argentina.However, seeds of other plant species (such as, pea-nuts, cotton, tea, “yerba mate”, oranges, various plantsused for timber), being either recalcitrant or subortho-dox, are highly sensitive to desiccation and cannot bestored at low temperature. Many plant crops (such aspotatoes, strawberry, cassava, sweet potato) have alsobeen maintained by vegetative propagation. Geneticresources of these crops are actually maintained eitherin situ or ex situ as plants in the field; losses of plantmaterial may occur due to diseases or accidents. Addi-tionally, a great deal of labor and time is required forthe maintenance of these field collections. Cryopreser-vation is an alternative strategy for efficient and econo-mical long-term conservation of these plant species.Research on cryopreservation of plant species inArgentina was initiated in 2001. At present, no collec-tion is maintained under cryopreservation, but at leasttwo institutions (IBONE, and the Facultad de CienciasAgrarias, UNMP-INTA) are making efforts to developprotocols in order to establish long-term banks forsome species of economical or ecological importance.The present paper provides an overview of the currentstatus of the development of plant germplasm cryopre-servation in Argentina.2. Arachis pintoi (“pinto peanut”)Arachis pintoi, a novel and important forage legumein tropical and subtropical areas of the world, has twocytotypes, one diploid, 2n=2x=20 chromosomes, andone, triploid with 2n=3x=30 chromosomes. The formerproduces relatively few seeds and they show high lossof viability after a few months of storage. The triploidcytotype does not produce seeds and propagation byvegetative procedures is obligatory. Thus, it is neces-sary to maintain collections of plants in the field forconservation of its germplasm.Table 1 summarizes the results obtained at IBONEwith cryopreservation of different plant material ofArachis pintoi. Seeds of the diploid cytotype wereeasily maintained at -196ºC using vitrification method.As much as 90% of them germinated after rewarming(Rey and Mroginski, 2007).Different in vitro cultured explants can also beemployed for germplasm preservation of both diploidand triploid cytotypes, with survival percentages ran-ging from 17 to 60%, depending upon the explants. Themethod employed was encapsulation-dehydration.Encapsulation was carried out by the classical proce-dure of sodium alginate-calcium chloride. The enca-271psulated explants were pregrown for three days inliquid medium with progressively increasing sucroseconcentration (0.5, 0.75 and 1 M of sucrose for 24 hreach). The beads with explants were dehydrated withsilicagel for 5 hr to 25% moisture content and immer-sed in liquid nitrogen followed by rapid cooling.Between rewarming and reculture of the explants itwas necessary to culture them for two days in liquidmedium with 1 M sucrose for 24 hr followed by 0.75M sucrose for 24 hr. It is interesting to note that usingthis method, it was not possible to employ uninodalsegments. Preculture-desiccation was also successfullyused for freezing somatic embryos (Rey, 2004).3. Ilex speciesThe genus Ilex (Aquifoliaceae), which includesabout 220 species, inhabits temperate and tropicalregions of South America. The most interesting amongthem, in terms of economic importance, is I. paragua -riensis because of its value for making a stimulatorybeverage called “mate”. Seeds of most of the Ilex spe-cies contain rudimentary embryos that remain at theimmature heart-shaped stage for a long time after thefruits reach maturity. An additional problem for seedconservation of these species is the fact that they arehighly sensitive to desiccation and cannot be stored atlow temperature. In other words, the seeds are recalci-trant and are therefore not suitable for long-term con-servation using conventional seed storage methods.Thus, germplasm of Ilex spp. is maintained in the fieldas ex situ genebanks.Two strategies for in vitro cryopreservation of Ilexspecies were tested, including cryopreservation of fruitor isolated embryos followed by embryo culture (Fig.1). In the first case, it was only possible to regenerateplants from five out of eight species tested (Table 2)when the embryo were isolated from fruits that werecryopreserved using slow freezing (1°C min- 1 till -4 0 ° C )with a cryoprotective solution containing 50% sucroseand 50% glycerol and stored in liquid nitrogen (Mro-ginski et al., 2006). However, survival was low (3-23%), yet percentages increased (Table 3) whenembryos were cryopreserved by using the encapsula-tion-dehydration and rapid cooling (Mroginski et al.,2007).SeedsApical meristemsShoot tipsUninodal segmentsSomatic embryosMaterial cryopreserved Methodvitrificationencapsulation-dehydrationencapsulation-dehydrationencapsulation-dehydrationencapsulation-dehydrationpreculture-desiccationrapidrapidrapidrapidrapidrapid90175702724---206003014Results (% survival)Diploid TriploidCoolingTable 1 - Cryopreservation of germplasm of Arachis pintoiFig. 1 - Strategies for cryopreservation of germplasm of Ilex spp.Ilex brasiliensisI. brevicuspisI. dumosaI. intergerrimaI. microdontaI. paraguariensisI. pseudoboxusI. theezansPlant spp. Rapid cooling Slow cooling0000000030130310230Table 2 - In vitro germination (%) of zygotic embryos from cryopre-served fruits of Ilex species (modified from Mroginski et al.,2006)Ilex brasiliensisI. brevicuspisI. intergerrimaI. dumosaI. paraguariensisI. pseudoboxusI. taubertianaI. theezansPlant spp. Embryos formingplants (%)710403057331039Table 3 - Effect of cryopreservation (rapid cooling) by encapsula-tion/dehydration of Ilex paraguariensis r u d i m e n t a r yembryos on their in vitro germination after 60 days in cul-ture on 1/4 MS, 3% sucrose and 0.1 mg/l Zeatin2724. Melia azedarach (Paradise tree)The paradise tree, native to the Asiatic Middle East,is an important forest tree in Argentina with excellentadaptability to a wide range of soil and climate condi-tions. Table 4 summarizes the results obtained atIBONE using cryopreservation of different plant mate-rial of Melia azedarach. Various explants were enca-psulated (Fig. 2 A), pretreated in liquid medium withsucrose concentration increased daily (0.5 M/0.75M/1.0 M), dehydrated with silica gel to 21-26% moi-sture content, then frozen in liquid nitrogen using arapid cooling or slow cooling method (Scocchi et al.,2004 b; Scocchi, 2005; Scocchi et al., 2007). The cryo-preserved explants, when rewarming and recultured invitro, regenerated shoots (Fig. 2 B), and whole plants(Fig. 2 C) which were successfully transplanted to pots(Fig. 2 D) and to the field (Fig. 2 E).5. Other plant speciesTable 5 summarizes the results obtained  with diffe-rent plants, such as Citrus sinensis, when shoot tipswere cryopreserved using the encapsulation-dehydra-tion method (Dolce et al., 2004) or seeds and proto-corms of the orchid Oncidium bifolium, preserved bythe same procedure (Flachsland et al., 2006). Likewise,seeds of Toona ciliata (Scocchi et al., 2004 a) andshoot-tips of Solanum tuberosum were stored at -196ºCthrough a vitrification protocol (Digilio, 2004). It isinteresting to note that with rice (Oryza sativa) theobjective of the work was to solve one of the limita-tions of the anther-culture technique, which is the shortperiod of time during which flower explants are availa-ble (Marassi et al., 2006).6. ConclusionsCryopreservation has been considered an ideal alter-native for germplasm conservation of plant species ofeconomical importance for A rgentina and for nativeendangered plant species. However, reduced financialsupport has characterized the small projects which startedin 2001, yet, significant progress has been made withplant species of economic interest such as Arachis pintoi,C i t rus sinensis, Melia azedarach a n d Ilex paraguariensis.U n d o u b t e d l y, action needs to be taken to increase thecryopreservation research in A rg e n t i n a .AcknowledgementsL. Mroginski and H. Rey are members of CONICETof Argen-tina. The authors wish to thank the ANPCyT and CONICET fortheir financial support.ReferencesDIGILIO A., 2004 - Crioconservación de variedades nativas deSolanum tuberosum spp. andigenum Juz.& Bukasov. - M.S.Thesis, Universidad Nacional de Mar del Plata, Argentina.DOLCE N., SCOCCHI A.M., MROGINSKI L.A.,  2004 - C r i o c o n -s e rvación de ápices de Citrus sinensis. - FACENA, 20: 37-45.FLACHSLAND E., TERADA G., SCOCCHI A.M., REY H.Y.,MROGINSKI L.A., ENGELMANN F., 2006 - Cryopreserva -Apical meristemsUninodal segmentsNon-dormant budsDormants budsSomatic embryosPlant material Rapid cooling Slow cooling4323454006057705236Explants forming shoots (%)Table 4 - Cryopreservation of germplasm of Melia azedarach using theencapsulation-dehydration methodFig. 2 - Cryopreservation of germplasm of Melia azedarach. A) Enca-psulated meristems; B) Shoot regeneration of encapsulatedmeristems; C) Plant regeneration; D) Regenerated plants tran-sferred to pots; E) Regenerated plants growing in field condi-tions.Citrus sinensisOncidium bifoliumOryza sativaSolanum tuberosumToona ciliataPlant species MaterialcryopreservedShoot tipsSeedsProtocormsAnthersShoot-tipsSeedsE-D.E-D.E-D.E-D.VV6511152-1135Dolce et al., 2004Flachsland et al., 2006Flachsland et al., 2006Marassi et al., 2006Digilio, 2004Scocchi et al., 2004 aResults(% survival)ReferencesMethod (z) Coolingslow (y)rapidrapidslowrapidrapidTable 5 - Summary of results obtained with cryopreservation of other plant spp. in Argentina(z) E-D= encapsulation dehydration; V= vitrification.(y) Slow cooling (at 1ºC min -1 to -30ºC and then immersion in liquid nitrogen).273tion of seeds and in vitro-cultured protocorms of Oncidiumbifolium Sims. (Orchidaceae) by encapsulation-dehydration. -CryoLetters, 27(4): 235-242.MARASSI M.A., SCOCCHI A., GONZALEZ A.M., 2006 - Plantregeneration from rice anthers cryopreserved by an encapsula -tion/dehydration technique. - In Vitro Cell. Dev. Biol. Plant.,42: 31-36.MROGINSKI L.A., SANSBERRO P.A., SCOCCHI A.M., LUNAC., REY H., 2006 - Effect of fruit cryopreservation on in vitrogermination of zygotic embryos of several species of Ilex. -Acta Horticulturae, 725: 417-420.MROGINSKI L.A., SANSBERRO P.A., SCOCCHI A.M., LUNAC., REY H.Y., 2007 - A cryopreservation protocol for immatu -re zygotic embryos of species of Ilex (Aquifoliaceae). - Biocell.(In press).REY H.Y., 2004 - Conservación in vitro de germoplasma de Ara-chis Pintoi (Leguminosae). - Ph.D. Thesis, Universidad Nacio-nal del Nordeste, Argentina.REY H.Y., MROGINSKI L.A., 2007 - Cryopreservation of Ara-chis pintoi (Leguminosae) seeds by vitrification . - Seed Scien-ce & Tecnology (In press).SCOCCHI A., 2005 - Conservación in vitro de germoplasma deparaíso (Melia azedarach). - Ph.D. Thesis, Universidad Nacio-nal del Nordeste, Argentina.SCOCCHI A.M., DIERINGER E., MROGINSKI E., MROGIN-SKI L.A., 2004 a - Conservación de semillas de cedro austra -liano (Toona ciliata). - Plant Genetic Resources Newsletter,137: 1-4.SCOCCHI A.M., FALOCI M., MEDINA R., OLMOS S., MRO-GINSKI L.A., 2004 b - Plant recovery of cryopreserved apicalmeristem-tips of Melia azedarach L. using encapsulation/dehy -dration and assessment of their genetic stability. - Euphytica,135: 29-38.SCOCCHI A.M., VILAS.K., MROGINSKI L.A., ENGELMANNF., 2007 - Cryopreservation of somatic embryos of paradisetree (Melia azedarach). - CryoLetters (In press).

Cryopreservation of plant germplasm in Argentina

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Cryopreservation of plant germplasm in Argentina

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