A study on the cryopreservation of almond buds by droplet-vitrification

Astract:【Objective】Almond (Amygdalus communis L.) is an important fruit tree in the world, and preservation of almond germplasm provides the basic materials for breeding of new varieties. This paper studied the cryopreservation of almond germplasm resources in order to provide a practical reference for preservation of almond germplasm resources in China.【Methods】Dormant buds of almond sterilized with 70% alcohol for 10 s and then with 0.1% Hg Cl2 for 7-8 minutes were used as the experimental materials, and an orthogonal design and single factor tests were used to evaluate the cryopreservation of dormant apical buds of almond by droplet vitrification. Factors including pre-culture sucrose concentration, pre-culture time, loading time and duration of exposure to PVS2 were tested. The dormant buds were suspended in 10 mL of liquid MS medium supplemented with 0, 0.1, 0.3, 0.5 or 0.7 mol · L-1 sucrose and pre-cultured in the dark at room temperature for 2 day. The dormant buds were excised and loaded for 0, 10, 20, 30 or 40 min at (24±2) ℃ in filteration-sterilized loading solution (LS) (2 mol·L-1 glycerol and 0.4 mol·L-1 sucrose dissolved in MS liquid medium) . After loading, the budswere treated for 0, 30, 60, 90, 120 or 150 min with filteration-sterilized PVS2. At the end of the dehydration period, explants were transferred into 5 μL droplets of vitrification solution attached to aluminum foil strips and rapidly immersed in LN for at least 24 h. For rewarming, the dormant buds were transferred to pre-heated (40 ℃) unloading solution (filteration-sterilized liquid MS medium with 1.2 mol·L-1 sucrose) for 30 s. After an equal volume of unloading solution (at room temperature) was added, the samples were incubated for a further 30 min. The buds were inoculated in MS liquid medium at different auxin concentrations, and the survival rate of almond was determined.【Results】Orthogonal test results showed that the effect of pre-culture time on the survival rate of dormant buds after cryopreservation was significant (P-1 sucrose for 2 days, treatment for 20 min with the loading solution (MS +2 mol · L-1 glycerol+0.4 mol · L-1 sucrose) , which was sucked out with a sterile plastic dropper. After loading, the dormant buds were treated with filteration-sterilized PVS2 (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol and 0.4 mol · L-1 sucrose in MS liquid medium) for 120 min under ice-water mixture at 0 ℃. Then 10 droplets of PVS2 each about 5 μl were placed on a sterile aluminum foil strip of 1 cm ´ 2.5 cm. A dormant bud was placed into each of the droplet. The droplets with a dormant bud were quickly dipped in liquid nitrogen, and placed into a freezing tube filled with liquid nitrogen, and sealed with a cover. These freezing tubes were placed in a nylon yarn bag and placed in a liquid nitrogen tank. After storage in liquid nitrogen for24 h, thawing was carried out at room temperature in a filteration-sterilized unloading solution (US) (1.2 mol · L-1 sucrose dissolved in MS liquid medium) for 30 min. The solution remaining on the surface of the dormant bud was removed with sterilized absorbent paper. After thawing and unloading of the samples processed by droplet vitrification, the buds were inoculated in MS liquid medium with 0.3 mol· L-1 sucrose and 7 g· L-1 agar and at a p H of 5.8, and incubated in darkness for 72 h. The explants were then transferred to MS liquid medium with 0.3 mol· L-1 sucrose, 0.3 mg· L-1 BAP, 0.5 mg· L-1 IAA, 0.3 mg· L-1 GA3 and 7 g· L-1 agar at a p H of 5.8 and cultured at a 16-h photoperiod and 28 ℃. After 30 days of culture, the survival rate of buds was 80.44%. The optimized program of cryopreservation used for the‘Shache 1'‘Shache 11'and‘Shashe 18'generated a survival rate of 87.41%, 85.44% and 83.02%, respectively.【Conclusion】The droplet vitrification method increased the survival rate of almond dormant buds after cryopreservation and proved to be viable for the long-term preservation of almond germplasm.