Progresses in cryopreservation of Pyrus spp and evaluation of genetic stability of the recovered shoots
Caboni, Emilia; Condello, E; Meneghini, M; Palombi, M.A; Frattarelli, A; Damiano, Damiano (2008)
Caboni, Emilia
Condello, E
Meneghini, M
Palombi, M.A
Frattarelli, A
Damiano, Damiano
Julkaisusarja
Agrifood Research Working papersMTT:n selvityksiä
Numero
153
Sivut
s. 36
MTT
2008
Tiivistelmä
Encapsulation-dehydration and vitrification methods were applied to shoot tips (2-4 mm in length) of Pyrus communis (Cv William, Decana and Rootstock FAR 40) and Pyrus pyraster to established a suitable protocol of cryopreservation for application in Pyrus germplasm collection of the CRA - Fruit Tree Research Center in Rome. The protocol, previously established for Pyrus pyraster with recovery of 60% after liquid nitrogen immersion and consisting in dehydration of Na-alginate beads for 2 days in 0.75M sucrose and desiccation to 20% moisture content (fresh weight basis), is being tested on Pyrus communis genotypes optimising the procedures of pre-treatment, of dehydration (Glucose or sucrose at various concentration and with time of application ranging from 1 to 5 days) and of dessication in silica (8 to 20 hours of application) according to the genotype. The highest recovery (26%) has been so far obtained in William with a dehydration in sucrose 1M for 3 days and a silica desiccation of 10 hours. Other factors, such as cold and/or high sugar concentration pre-treatments, are now under evaluation to optimise the protocol. The cryopreservation of the pear genotypes by the vitrification method was also considerated. PVS composition and time of application of the vitrification solution were tested and preliminary results will be discussed. Genetic stability of the shoots of Pyrus pyraster, recovered after cryopreservation by encapsulation-dehydration, was also evaluated. Fifteen single bud derived lines were used for genetic analyses by RAPDs and SSRs. In RAPD analysis, among a total of 24 ten-mer primers used to amplify all the genotypes, 15 showed reproducible and well resolved bands. These primers produced a total of 66 fragments ranging from about 500 to 2500 base pairs size. SSR marker amplification was performed using 19 pair-primers which produced 57 reproducible fragments. Microsatellite fragments ranged from 60 to 600 base pairs. Both markers did not reveal any polymorphism between cryopreserved shoots and the original genotype. These results cannot represent the final proof that no somaclonal variation occurres cryopreserving wild pear by encapsulation-dehydration method, however, RAPDs and SSRs were shown to be more efficient than other markers in determine somaclonal variation in tissue culture and their combination gives more robust results than when they are used singularly, allowing us to be more confident in the stability of the cryopreserved material and in the possibility of a wider application of the encapsulation-dehydration method for long term conservation of Pyrus genotypes of the germoplasm collection.
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