番木瓜茎尖的玻璃化法超低温保存及其植株再生

 研究了番木瓜( Carica papaya L. ) 茎尖玻璃化法超低温保存的一些影响因素。结果表明: 无菌试管苗在MS + BA 0.5 mg/L + NAA 0.1 mg/L + GA₃ 1.0 mg/L 的培养基上生长较好。番木瓜茎尖超低温保存较佳体系是: 3～5 cm的茎尖在含有5 %二甲基亚砜(DMSO) 培养基上预培养3 d , 解剖镜下剥取含1～2 个叶原基的茎尖(1.5～2.5 mm长) , 先在室温下于60 %的玻璃化溶液(PVS2) 中装载40～50 min , 再用PVS2于0 ℃下处理30 min , 换1 次PVS2 溶液后迅速投入液氮。保存24 h 后, 在40 ℃水浴中化冻, 用1.2 mol/L 的蔗糖培养液洗涤两次, 转入继代培养基上再培养, 成活率和再生率分别达53.7 %和52.6 %。再生植株生根后可移栽成活。     

香蕉茎尖的玻璃化法超低温保存及其植株再生

 以香蕉(Musa spp. ) 为试材, 对其离体培养茎尖玻璃化法超低温保存影响因素进行研究。结果表明, 不定芽在MS + 3.0～5.0 mg/L 6-BA + 0.1 mg/L NAA的培养基上分化较好。香蕉茎尖超低温保存较佳体系是: 2.0～3.0 cm的茎尖在含0.4 mol/L蔗糖培养基上预培养2 d, 剥取带1～2个叶原基的茎尖(长1.0～1.5 mm) , 室温(25℃) 下装载液(MS + 2 mol/L甘油+ 0.4 mol/L蔗糖) 装载20～30 min, 然后用玻璃化溶液( PVS2 ) 于0℃下处理40 min, 换1次PVS2后迅速投入液氮。保存至少1 h后, 在40℃水浴中化冻90 s, 用1.2 mol/L蔗糖培养液洗涤2次, 每次10 min, 然后转入含0.3 mol /L蔗糖的MS培养基上,暗培养10～15 h后转移到含0.5 mg/L 62BA的MS培养基中, 暗培养1周后转移到正常光下, 3个香蕉品种(巴西蕉、广东香蕉2 号、广东粉蕉1 号) 的成活率分别为75.9%、40.0%和69.6% , 再生率分别为63.4%、35.0%和63.4%。再生植株生长和分化正常, 生根后可移栽成活。     

大蒜茎尖玻璃化法超低温保存技术研究

 用山东‘苍山大蒜’进行了茎尖玻璃化法超低温保存技术的研究。5～8 mm大蒜茎尖在MS +0.7 mol/L蔗糖的固体培养基上预培养7 d, 切取3.0～3.5 mm的茎尖, 在20℃下经60% PVS2 处理60 min,再于0℃下用PVS2 处理5～60 min后, 换适量新鲜PVS2 , 浸入液氮。保存2 d或1个月后取出, 在37℃水浴中解冻2 min, 用MS + 1.2 mol/L蔗糖液体培养基洗涤2次, 每次10 min, 经过恢复培养, 茎尖成活率最高可达到100%。     

桃离体茎尖的超低温保存及植株再生

 以简单玻璃化法为基本方法, 研究了影响桃离体茎尖超低温保存后存活率的因子———低温驯化时间、蔗糖预培养时间、玻璃化液处理时间及化冻后植株再生条件; 建立了较为适宜的超低温保存技术程序———选择继代培养30 d的试管材料, 5℃低温驯化3～4周, 在含017 mol/L蔗糖的固体培养基预培养2 d, 再经玻璃化液PVS3处理100 min后浸入液氮, 化冻后茎尖存活率可达60%以上。     

树莓茎尖玻璃化法超低温保存及植株恢复培养研究

对离体树莓茎尖的玻璃化法超低温保存进行了研究。结果表明,离体树莓茎尖超低温保存程序为在含0.8mol/L蔗糖的固体MS培养基预培养4天,低温驯化21天,再经玻璃化液PVS2处理60分钟后浸入液态氮。保存结束,茎尖在40℃水浴5分钟解冻,存活率达80%,植株再生率达75%。     

玻璃化法超低温保存猕猴桃离体茎尖及其植株再生

 以中华猕猴桃矮型种质为离体培养材料, 建立试管无性系, 对其离体茎尖的玻璃化法超低温保存技术进行研究, 探讨猕猴桃种质长期保存的适宜途径。结果表明, 含1～2个叶原基(1.5～2.5 mm) 的茎尖, 在5%二甲基亚砜(DMSO) + 5%蔗糖+MS培养基上预培养4 d后, 于室温下用2 mol/L甘油+ 0.4 mol/L蔗糖预处理30 min, 再在0℃下用玻璃化液(PVS2 ) 脱水处理40 min并迅速投入液氮中, 保存24 h后接种至继代培养基上再培养, 成活率和再生率分别为56.7%和51.6%。再生植株生根后可移栽成活。     

马铃薯(Solanum tuberosum L.)茎尖超低温保存的研究

以克新8号为试验材料,通过优化超低温保存马铃薯茎尖中影响成活及再生的几个关键因素,成功建立了基于玻璃化法的马铃薯茎尖超低温保存体系。茎尖在MS+0.3M蔗糖的培养基上预培养1天后,经2M甘油+0.4M蔗糖的加载液室温加载30 min,浸入到PVS2溶液中(00C)处理60 min,将处理后的茎尖转至含有0.05 mg/L GA3的MS后培养基上培养,成活率和再生率达到94.8%和78.1%。并将建立的玻璃化法应用到其它4个品种,平均再生率为65.5%,研究结果可为中国马铃薯种质资源的超低温保存提供技术支持。     

食用百合种质的玻璃化法超低温保存技术初探

以离体茎尖为试材 ,采用玻璃化法 ,对食用百合离体超低温保存技术进行了初步研究。结果表明 ,用2～ 3mm茎尖 ,在MS +0 .5mol·L-1蔗糖浓度的培养基上预培养 1～ 2d ,室温下植物玻璃化溶液 (PVS2 )处理 2 0min ,换入新鲜的PVS2 ,迅速投入液氮中 ,2d后取出 ,在 4 0℃水浴中解冻 2min ,再在 2 5℃水浴中解冻 10min ,用1.2mol·L-1蔗糖液体培养基洗涤 2 0min ,接种在 6 -BA 0 .5mg·L-1+NAA 0 .1mg·L-1+GA3 0 .3mg·L-1+蔗糖 30 g·L-1+琼脂 7g·L-1的MS培养基上 ,2 5℃弱光培养 1周后转为正常光下培养 ,2周后再生率达到 5 2 .6 %。     

“童子一号”草莓玻璃化法超低温保存技术研究

以玻璃化法进行"童子一号"草莓离体茎尖的超低温保存研究,探讨了低温锻炼时间、预培养时间、预处理时间、PVS2处理时间等对存活率的影响。结果表明:将4℃条件下低温锻炼30d的健壮草莓苗茎尖,接种在固体预培养培养基中预培养4d,预处理30min后,吸出预处理液,加入PVS2,0℃处理80min后,更换新鲜的PVS2,并迅速投入液氮60min后,置于40℃快速化冻1min;室温洗涤3次并取出茎尖,接种到再生培养基恢复培养;成活率最高可达43.00%,且再生植株分化正常。     

李离体茎尖的超低温保存

 以简单玻璃化法为基本方法,研究了影响李离体茎尖超低温保存的因子—继代培养时间、低温驯化时间、PVS3处理时间以及化冻后茎尖存活检测;建立了较为简单的李离体茎尖超低温保存技术程序：即选择继代培养120 d的试管材料,经过5 ℃低温驯化21 d,0.7 mol·L^-1蔗糖预培养1 d,PVS3处理100 min,浸入液氮。化冻后茎尖存活率可达60 %以上。     

银条茎尖玻璃化法超低温保存及其植株再生

 为长期稳定保存银条种质, 建立了玻璃化法超低温保存其茎尖的技术体系。选择继代4次的 银条无菌壮苗, 5 ℃低温驯化14 d; 剥取5 mm的茎尖, 在含有0.5 mol·L ^{- 1}蔗糖的MS (无Ca^{2 +} ) 液体培养基预培养1 d; 25 ℃条件下经改良MS + 2PVS₂装载20 min; 在- 20 ℃, 95%乙醇浴中以PVS₂脱水处理4h; 更换新鲜的PVS₃后投入液氮, 保存24 h后取出冷冻管在40 ℃水浴中化冻1 min; 以含有1.2 mol·L ^{- 1}蔗糖的改良MS (无Ca^{2 +} ) 溶液洗涤2 次, 每次10 min; 冻存后的茎尖相对存活率超过70%。将冻存后的茎尖转接到再生培养基上, 暗培养20 d后转入正常光照条件下培养, 存活率达63.7%; 继续培养得到正常分化和生长的再生植株, 生根后可移栽成活。     

通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究

以草莓品种明宝为材料,取茎尖做初代培养,继代扩繁5次后,取2mm左右的茎尖,利用玻璃化超低温技术对SMYEV进行脱除,并利用结合内标的多重RT-PCR技术对再生植株进行病毒检测。结果表明,在病毒脱除过程中,预培养蔗糖浓度为0.5mol/L,处理3d;装载处理为25℃,60min;玻璃化处理为0℃,120min;液氮处理60min后,进行40℃水浴2min,草莓茎尖的存活率为76%,而草莓轻型黄边病毒的脱除率为95%。只有通过液氮处理才可以脱除该病毒,液氮处理前的玻璃化处理脱毒率为0。利用超低温脱毒法可以简便有效地脱除SMYEV。     

马铃薯茎尖小滴玻璃化法超低温保存及其再生植株的遗传稳定性

以马铃薯试管苗为试材,对其茎尖小滴玻璃化法超低温保存的影响因素进行了研究,并对再生植株进行了遗传稳定性检测。结果表明,马铃薯茎尖依次在含有0.3 mol · L-1和0.5 mol · L-1蔗糖的液体MS培养基中预培养各1 d后,在0 ℃下PVS2处理30 min,转到铝箔条上PVS2小滴上（约15 μL）,将粘有茎尖的铝箔条在液氮里蘸一下,然后直接装入盛满液氮的冷冻管中,投入液氮至少保持1 h。室温下用含有1.2 mol · L-1蔗糖的MS液体培养基解冻并洗涤30 min后,接种到MS + 0.5 mg · L-1 Zeatin + 0.1 mg · L-1 NAA＋1.0 mg · L-1 GA3恢复培养基上,存活率和再生率最高达79.91%和62.52%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。     

Cryopreservation of carnation (Dianthus caryophyllus L.) shoot tips by encapsulation-vitrification

Shoot tips excised from in vitro cultured plants of Dianthus caryophyllus L. (cv. Pallas, cv. Pink Candy and cv. Wanessa) were successfully cryopreserved using an encapsulation-vitrification method. Shoot tips (2–3 mm in length) were encapsulated in sodium alginate, precultured on liquid Murashige and Skoog (1962) medium supplemented with various sucrose concentrations (0.25, 0.5, 0.75, 1.0 M) for 24 h or 48 h and dehydrated with the vitrification solution PVS2 (up to 4 h) at 24 °C or 0 °C prior to direct immersion in liquid nitrogen (−196 °C). A maximum of shoot regeneration from cryopreserved shoot tips was obtained with the following combinations: preculture in 0.5 M sucrose and 180 min dehydration treatment at 0 °C for cv. Pallas (60% shoot formation), or preculture in 0.75 M and 200 min dehydration at the same temperature for cv. Pink Candy (66.6% shoot formation) and cv. Wanessa (73% shoot formation).     

Cryopreservation of Malus cultivars: Comparison of two droplet protocols

Two droplet procedures, droplet-vitrification (PVS2) and droplet (DMSO) were applied for cryopreservation of in vitro cultured apple (Malus domestica Borkh., cvs. Florina, Idared, Colmar and Rebra) plants. The highest post-thaw regrowth rates (70% for cv. Florina, 66% for cv. Idared, 63% for cv. Colmar and 60% for cv. Rebra) were achieved after using the droplet-vitrification (PVS2) protocol. The excised shoot tips (2–3 mm in length) were precultured in 0.5 M sucrose enriched media for 24 h. Subsequently they were transferred in PVS2 vitrification solution for 30 or 40 min (depending on cultivar) at 24 °C and then immersed in liquid nitrogen. Rewarming was performed in liquid MS medium at 24 °C. Plants regenerated from cryopreserved shoot tips did not display any sign of morphological alteration or abnormalities in growth in comparison with control plants.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.