| Among the 25 species of Amelanchier,Amelanchier alnifolia(saskatoon)is one of the most economically valuable species in North America.This species has the characteristics of strong cold resistance and soil adaptability.The fruit of saskatoon has delicious flavor,rich bioactive substances and high nutritional value,which can be eaten or processed.It is one of the few species cultivated as fruit trees in this genus.Many excellent cultivars come from this species or have its genes.It has been successfully introduced and cultivated in China.It has a broad prospect in the development of fruit industry and ecological restoration.The safe conservation of crop germplasm resources is of great strategic significance.Cryopreservation is a safe and effective method for long-term conservation of germplasm resources.In this experiment,the shoot tips of saskatoon in vitro were used as materials to optimize the factors such as time duration of cold hardening,time duration of preculture,sucrose concentration in preculture medium,sucrose concentration in loading solution,time duration of loading,time duration of vitrification protective solution treatment,time duration of unloading and time duration of culture in the dark;11 different genotypes of saskatoon were used to verify the broad spectrum of the optimized parameters;The long-term storage stability of saskatoon shoot tips was verified by different storage time in liquid nitrogen(LN);ISSR Molecular markers were used to identify the genetic stability of regenerated plants after cryopreservation.The purpose of this study was to establish the cryopreservation protocol of shoot tip in vitro of saskatoon by droplet vitrification,and lay the technical foundation for cryopreservation of germplasm resources of Amelanchier.The main results are as follows.1.The optimized parameters and preservation protocol are as follows: Stem segments with terminal buds,about 0.5 cm long,were cut from 30-day-old tissue culture seedlings and cultured in SSM(MS + 1.0 mg / L 6-BA + 35 g / L sucrose + 7 g / L agar)for 2 wk at 4 in the dark.℃The apical shoot tips,about 2 mm long,with 4-5 leaf primordia,were quickly excised under stereomicroscope and cultured in PM(MS + 0.3 mol / L sucrose + 7 g / L agar)for 24 h(25℃22℃,dark).Shoot tips were treated in LS(MS + 2.0 mol / L glycerol + 0.4 mol / L sucrose)for20 min(room temperature),and then were treated with plant vitrification solution 2(PVS2)for40 min(0).℃PVS2 droplet(4.5 4L)was made on aluminum foil strip(2 cm×0.8 cm)(0℃),and the shoot tip was transferred into the droplet.The aluminum foil strip was quickly put into LN for 10 minutes,then transferred into a freezing tube(2 ml)and stored in LN for 1 hour.The aluminum foil strip with droplet of shoot tip was taken out from LN and quickly transferred into ULS(MS + 1.2 mol/l sucrose)for 20 min(room temperature).Shoot tips were cultured on SSM in dark for 3 days(25℃22℃),and then cultured under normal light and temperature.2.Eleven different genotypes of saskatoon were cryopreserved by this preservation protocol.The average regeneration rate was 50.92%.The highest was 68.33%(‘Thiessen’).The lowest was 43.33%(Smoky-1).The technology system has a good broad spectrum.The shoot tip regeneration rate of ‘Thyssen’ was not affected by LN storage for 90 days.The effect of this technique was stable.3.Fifteen pairs of ISSR primers were used for molecular marker detection,and no variation bands were found in the regenerated lines after cryopreservation.The heredity of their regenerated plants was stable. |
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