The droplet-vitrification cryopreservation protocol developed in collaboration with IITA (Ibadan, Nigeria) has been successfully tested on a total of 42 accessions of African and Asian yam, in IRD Montpellier and in Ibadan.
In collaboration with CIRAD Montpellier, a detailed histological study has been performed on yam shoot tips during their cryopreservation using the encapsulation-dehydration technique. We have shown for the first time, thanks to the combination of qualitative and quantitative analyses of shoot tip histological sections, that it was the sucrose dehydration step which was responsible for the main cellular plasmolysis, and not the subsequent physical dehydration step.
We performed a preliminary study on the impact of the successive steps of a cryopreservation protocol (droplet-vitrification) on gene expression. Except for two genes, whose expression level remained unchanged throughout the cryopreservation process, the expression of all the other 15 genes studied varied depending on the step of the cryopreservation protocol. On average, dissection was the step which induced the largest changes in expression level for the majority of the genes studied, followed by the loading treatment.
In collaboration with CIRAD Guadeloupe and Montpellier, we have shown for the first time that apices of in vitro plantlets could be cryopreserved using the droplet-vitrification technique. However, regrowth was lower, with the two clones tested, than that observed with the original encapsulation-dehydration technique.
Prunus and Rubus
In collaboration with INRA Bordeaux and Montpellier, we have shown that it was possible to cryopreserve shoot tips of in vitro plantlets of sour cherry (Prunus cerasus) using the vitrification technique. Cold treatment of in vitro mother-plants could be efficiently replaced with a preculture of shoot tips on sucrose-enriched medium, thereby significantly simplifying and shortening the protocol.
Similarly, in collaboration with the Fruit Research Institute of Cacak (Serbia), we have obtained growth recovery and multiplication of apices of P. cerasifera and Rubus fruticosus using droplet-vitrification.
Preliminary experiments performed in collaboration with CIRAD La Réunion have allowed obtaining limited survival after cryopreservation of vanilla shoot tips using droplet-vitrification. In the framework of an Open Science project funded by Agropolis Fondation, we have successfully experimented the V-cryoplate technique for cryopreservation of vanilla shoot tips.
In the framework of our collaboration with INRA Montpellier and Zagreb University (Croatia), we have shown for the first time that it was possible to use the droplet-vitrification technique for cryopreserving shoot tips of grape in vitro plantlets. We have underlined the critical importance of the physiological state of the buds employed for cryopreservation, by comparing the recovery of buds sampled at different levels on stems of in vitro plantlets, and that of buds sampled on microcuttings cultivated for various periods on media added with different growth regulators.
In collaboration with Cirad Montpellier, we have studied the possibility of using cryopreservation for elimination of grape viruses (a process called cryotherapy), using virus immunolocalisation techniques. The immunolocalisation protocol has been adapted to our material and experiments have been performed to compare shoot tips of virus-free and virus-infected cultivars, before and after liquid nitrogen exposure. Interestingky, we have noted that viruses can be eliminated even without liquid nitrogen exposure, by treatment with the highly concentrated vitrification solution employed.
We have shown for the first time that apices of in vitro plantlets of two endangered Mediterranean species (Lithodora rosmarinifolia and Limonium serotinum), provided by the University of Palermo (Italy), could withstand cryopreservation using droplet-vitrification, with modifications of the treatment conditions, compared with the original technique.
In collaboration with CEPROCOR (Argentina) research has been performed for the development of a cryopreservation protocols for the endangered Argentinian species Clinopodium odorum. High recovery has been achieved using the V cryo-plate technique and limited recovery with the D cryo-plate.
In collaboration with RDA (Korea) and Cirad Montpellier, we have established a cryopreservation protocol (droplet-vitrification) for hairy roots of this species, and performed a qualitative and quantitative histological study of the impact of the successive steps of the protocol on their structural integrity. We have shown that plasmolysis was mostly obtained during the sucrose preculture. However, when comparing sections performed at two different levels, we have shown that in the median part, plasmolysis was observed already during the sucrose pretreatment step, while in apical segments, plasmolysis was observed only after the loading step. These differences of reactivity can be related to differences in histological structure and in survival, the apical segments displaying higher survival compared to median sections.
Two cryopreservation protocols (droplet-vitrification and D-cryoplate) have been compared with date palm proembryogenic masses (PEMs) and the response of four date palm varieties to LN exposure is studied. When studying the effect of sucrose pretreatment on PEM survival, we have shown that sucrose pretreatment allows obtaining survival after LN exposure without treatment with a vitrification solution.