In Vitro Conservation Of Citrus Germplasm

It is the need for the sustainable development to keep the biodiversity. Plant genetic resource plays an important role in the sustainable development of agriculture. Citrus is one of the most important fruit trees in the world and has important economic values. China is the major origin place of citrus. During the natural evolvement and culture, many types or cultivars with important or potential values have been formed. It is strategic for the world citrus industry sustainable development to keep those citrus types and cultivars. Planting in the open field is the traditional method for the conservation of citrus germplasm. Citrus germplasms conservation in China is imperi led because of diseases and insect pests, natural disasters and shortage of fund. Many citrus germplasms are facing loss or have been lost. In order to settle those problems, we began to study citrus germplasm conservation in vitro. The major results are as follows:(1) Embryoids of 21 cultivars or types of citrus such as cara cara and so on, and embryonic calli of 10 cultivars have been obtained through culture in vitro of undeveloped ovules of 23 cultivars, as provided materials for the conservation in KJtmThe medium for citrus embryoids forming plantlets has been optimized. An effective and broad-used recipe for regeneration has been established. The optimal medium is: MT + BA 2. 0 mg /L + IBA 0.2 mg/L + GA30.5 mg/L + glucose 30g/L.(2) Approaches for in vitro culture of mature citrus stems were conducted. A high efficient method based on the principle of the resistance of fungi spores to disinfectant becoming weak after germination to obtain disinfected mature explants has been developed. The explants were kept for 2 d in culture room after the primary disinfection and were disinfected for the second time. Then the materials were cut into stems of about 0. 5 cm long with one bud and inoculated in the medium for germination. Four citrus cultivars had been tested and the average contamination rate and success rate were 21. 07% and 71. 01% respectively. The medium for primary culture of mature citrus had been optimized, and the best medium was MT + glucose 30 g/L + BA 2. 0 mg/L + IBA 0. 1 mg/L + GA3 0. 5 + CH 500 mg/L. Getting rid of the oldtissue gradually during subculture could avoid forming of callus.(3) We have studied the conservation of citrus plantlets at normal temperature (25 2 and with a 14 h photoperiod provided by daylight-type fluorescent lamps) and low temperature (5 1 with a 16 h/d photoperiod, 11 mol/m2 s) conditions. The result showed that the sub-culture times of Cara cara navel orange could be prolonged to 13 months and 2 years respectively and the materials conserved at low temperature were easy to be contaminated when cotton plugs were used to seal the tubes.(4) Cryopreservation of citrus protoplasts has been studied. Results showed that the best treatment time of PVS was 3 min and bovine serum albumin added in PVS was favorable to cryopreservation of protoplasts. Protoplasts derived from different cultivars embryogenic calli have different survival rate after cryopreservation. The survival rates of protoplasts of different ploidy citrus sinensis (L.) cv. Valencia varied, the diploid cells was the highest and the hexaploid the lowest. Protoplasts after cryopreservation could divide in BH3 medium and could form colonies.(5) We studied and improved the basic conditions for citrus shoot-tips cryopreservation by vitrifiction. The role of additive GSH (reduced form of glutathione) in cryopreservation of citrus shoot-tips using the vitrification procedure was investigated for the first time. The results showed that GSH was useful in cryopreservation citrus shoot-tips. The average survival and regeneration rate after cryopreservation of 12 cultivars were 84.05% and 77.93% respectively.(6) Mature shoot-tips of four citrus cultivars have been cryopreserved and the average survival and regeneration rate were 56. 93% and 46. 93% respectively. Mature explants of somatic hybrid of Citrus reticulata +Ponci...