| Blue honeysuckle(Lonicera L.subsect.Caeruleae L.)is a small deciduous shrub of Lonicera Linn in Caprifoliaceae.It has many excellent agronomic traits,such as cold resistance,early maturity and wide adaptability to soil.Its fruit is rich in anthocyanins and other physiological active substances.The fruits of blue honeysuckle can be used for fresh food,processing or extraction of natural pigment.It is one of the small fruit trees with high nutritional value and economic value.More and more people pay attention to it.There are abundant wild resources of blue honeysuckle in China.In recent years,China has introduced many excellent varieties from abroad,gone out of the stage of relying solely on the collection and utilization of wild resources,and started large-scale artificial cultivation.Due to the destruction of wild resources by human collection and the impact of global climate change,the loss of germplasm resources of blue honeysuckle was aggravated.The conservation of plant germplasm resources is the basic work of development and utilization.Therefore,it is necessary to strengthen the conservation of blue honeysuckle germplasm resources.The cryopreservation method has the characteristics of relatively simple operation,less variation and long storage time.Nowadays,it has become an ideal method for the conservation of plant germplasm resources.In this study,the shoot tips of blue honeysuckle cultivar‘Огненныйопал’test-tube plantlets were used as materials,and the single factor experiment was carried out on the operation parameters of cold hardening,preculture,loading,vitrification solution dehydration,unloading and recovery culture in cryopreservation by using droplet vitrification method.Genotypes from five different varieties of L.caerulea were used to verify the method.The aim of this study was to establish a cryopreservation protocol for the shoot tips of blue honeysuckle in vitro,and to provide a new method for the long-term and effective conservation of blue honeysuckle germplasm resources,and lay a technical foundation for the cryopreservation of blue honeysuckle germplasm resources.The main results are as follows.Through the optimization of the parameters,the cryopreservation protocol of blue honeysuckle germplasm resources were established.The stem segments of 1.5 cm in length with terminal buds were cut from 4-week-old test tube plantlets,and cultured on subculture medium(MS+1.0 mg/L 6-BA+0.3 mg/L IBA+30 g/L sucrose+6.5 g/L agar,SCM)in 4℃and the dark for 15 days for cold hardening.2-3 mm long shoot tips of terminal buds were cultured on preculture medium(MS+0.5 mol/L sucrose+6.5 g/L agar)for 2 days(25℃±2℃,dark).The shoot tips were put into loading medium(MS+2.0 mol/L glycerol+0.4 mg/L sucrose)for 40 min(room temperature).After that,The shoot tips were treated with PVS2 for 40min(0℃).The shoot tip was then transferred to the droplet prepared with PVS2 on the aluminum foil(0℃)and quickly put into liquid nitrogen(LN)for 1 h.The material taken out from LN was quickly put into unloading solution(MS+1.2 mol/L sucrose)for 30 min(room temperature).Finally,the shoot tips were cultured on SCM for 5 days(25℃±2℃,dark)and then cultured under the condition of temperature 25℃±2℃,white fluorescent light intensity 50μmol/s·m~2,and photoperiod 16 h light/8 h dark.Five genotypes of L.caerulea(‘Огненныйопал’,‘Сибирячка’,‘Голубоеверетено’,L.caerulea subsp.pallasii,L.caerulea var.edulis)were cryopreserved.The average regeneration rate of shoot tip reached 44.7%after 30 days of recovery culture.Among them,the regeneration rate of‘Сибирячка’was the highest 55%,and that of L.caerulea var.edulis was the lowest36.7%.The cryopreservation protocol showed a good broad spectrum.To the best our knowledge,this is the first report on blue honeysuckle cryopreservation protocol by droplet vitrification. |
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