Development of a seed cryobank to preserve the citrus genetic resources of INRAE-Cirad germplasm: a complementary strategy to the tree field conservation
Abstract
In genebanks, fruit trees are often maintained as grafted scions on rootstocks in
field or greenhouse settings and are therefore exposed to various risks of decay. To
guarantee a secure long-term conservation, cryopreservation in liquid nitrogen is
currently developed for many plant species. The success of seed freezing depends on
their ability to tolerate cold and desiccation. Citrus seeds are described as semiorthodox because they are partially tolerant to desiccation. Seeds of many citrus are
polyembryonic and thus offer the possibility of clonal propagation by seedling. A seed
cryopreservation program for polyembryonic cultivars of the INRAE-Cirad citrus
collection has been initiated. We present here the protocol and the adaptations for
cultivars sensitive to desiccation. The control of the water content of the seeds is
ensured by incubation in atmospheres with controlled relative humidity to eliminate
intracellular free water lethal to the cells during freezing. The temperature and
duration of incubation during this desiccation step are factors that have a role in
maintaining germination capacity. Genetics also has a strong influence on the success
of cryopreservation operations. Citrus related to citron (Citrus medica) such as lemons
and limes are much more tolerant of low water content than cultivars related to
mandarin (C. reticulata) such as sweet and sour oranges. Seeds of Fortunella and
Poncirus trifoliata and some of their hybrids are unsuitable for cryopreservation
because they are too sensitive to moisture content decrease. Apex cryopreservation
could be an alternative for these non-orthodox citrus trees. However, regeneration by
sowing of frozen seeds is simpler than regeneration of apexes that need to be
micrografted to develop a plant. Although improvements to the protocol are still
needed to increase germination rates of some varieties, we estimate that we are able to
cryopreserve in seed form about half of the citrus collection (~500 accessions).