苜蓿与百脉根原生质体培养及体细胞杂交的研究
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摘要
紫花苜蓿被誉为“牧草之王”，其营养价值位列各种牧草之首。然而，青饲时易造成家畜臌胀病成为限制其营养价值发挥与利用的重要原因。百脉根又称“瘠地苜蓿”，由于富含缩合单宁，单独饲喂其鲜草或直接放牧不会引起臌胀病发生。目前，利用体细胞杂交技术将百脉根细胞中控制单宁合成的基因转入紫花苜蓿成为改良苜蓿品质、创造新品种的重要方法和途径。但以适宜西北内陆地区栽种和利用的苜蓿品种作为原生质体分离材料并开展体细胞杂交的研究较少。因此，本论文拟通过对杂花苜蓿、紫花苜蓿和百脉根等3种豆科牧草不同品种进行体细胞培养、原生质体培养及清水紫花苜蓿和里奥百脉根体细胞杂交的研究，建立和优化3种豆科牧草原生质体分离和培养体系及紫花苜蓿和百脉根体细胞杂交技术体系。取得的主要结果如下：
1、苜蓿原生质体分离和培养
1）以MS固体培养基为基本培养基，适于甘农1号杂花苜蓿和甘农4号紫花苜蓿愈伤组织诱导的最佳外植体均为下胚轴，最佳激素浓度与配比及诱导率分别为3.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，96.7%；2.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，100%；适于阿尔刚金紫花苜蓿愈伤组织诱导的最适激素浓度和配比为2.0mg·L~(-1)2,4-D+0.5mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，下胚轴和子叶对出愈率的影响不显著（p＞0.05），诱导率分别为100%，90%。试验证明，诱导出的苜蓿愈伤组织经过一段时间的继代调整，均可酶解分离出大量活力较高的原生质体。
2）以4个苜蓿品种的胚性愈伤组织为酶解材料，甘农1号、甘农4号和清水紫花苜蓿的最佳酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶，最适酶解时间分别为12h、14h和10h。阿尔冈金的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶+0.3%离析酶+0.3%崩溃酶，最适酶解时间为12h。适宜4个苜蓿品种原生质体酶解的最佳甘露醇浓度分别为：甘农1号0.75mol·L~(-1)，甘农4号0.65mol·L~(-1)，阿尔冈金0.6mol·L~(-1)，清水紫花苜蓿0.55~0.6mol·L~(-1)。最佳愈伤组织继代培养时间均为第12d。适宜4个苜蓿品种愈伤组织酶解的最佳预处理措施分别为，甘农1号0.55mol·L~(-1)蔗糖或CPW溶液中处理1h，甘农4号预低温处理1h或0.55mol·L~(-1)蔗糖溶液中处理1h，阿尔冈金和清水紫花苜蓿均为0.55mol·L~(-1)CPW溶液中处理1h。4个苜蓿品种原生质体最高产量可达2.48×10~6个·g~(-1)（甘农4号），最大活力值为93.1%（清水紫花苜蓿）。
3）采用液体浅层培养法和固液双层培养法，4个苜蓿品种在含KM8P培养液的培养基上培养第2~3d，均可观察到原生质体再生细胞的第1次及多次分裂，第7~14d形成肉眼可见的微愈伤组织，转入含0.5~3mg·L~(-1)2,4-D，0.5~1mg·L~(-1)NAA、0~0.5mg·L~(-1)6-BA和0~0.5mg·L~(-1)KT的固体培养基增殖与继代，最终均获得了原生质体再生的愈伤组织。固液双层培养法较液体浅层培养法形成再生细胞团的时间较早，操作简便且不易污染，更有利于苜蓿原生质体早期的分裂和再生。
2、百脉根原生质体分离和培养
1）以MS固体培养基为基本培养基，适于里奥愈伤组织诱导的最佳外植体为下胚轴，最佳激素浓度和组合为：1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT，诱导率为92.2%；在仅含0.5~3.0mg·L~(-1)KT的MS基本培养基上下胚轴和子叶诱导1个月左右均可直接生成胚状体和小苗，经2~3次继代发育成完整植株。
2）适于里奥无菌苗子叶和胚性愈伤组织原生质体分离的最适酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶，最佳酶解时间分别为6h及12h，最适甘露醇浓度均为0.55mol·L~(-1)。里奥愈伤组织分离原生质的最佳继代培养时间为第8d；0.55mol·L~(-1)CPW溶液中预质壁分离1h或预暗培养1d均可显著（p＜0.05）增加其原生质体的产量和活力。子叶和愈伤组织原生质体最高产量和活力分别可达4.13×106个·g~(-1)，65%和3.8×106个·g~(-1)，67.4%。
3）采用液体浅层培养法，由子叶和胚性愈伤组织分离的原生质体在KM8P+1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT+13.0%（0.7mol·L~(-1)）甘露醇的培养基上可持续分裂并形成肉眼可见的微愈伤组织，当愈伤组织长到直径0.5mm左右时，转入含相同激素浓度的MS固体培养基上增殖和继代。大量的体细胞胚胎和再生小植株在含MS+2.0mg·L~(-1)KT+0.7%琼脂+12.0%蔗糖的培养基中发生。从原生质体第一次分裂至再生植株仅需3~4个月。再生植株移栽成活率达95%。3、苜蓿和百脉根的体细胞杂交
1）3~10mmol·L~(-1)的IOA和40~70μg·m L~(-1)的R-6G分别处理10min，可使清水紫花苜蓿和里奥百脉根原生质体的活力及植板率明显下降，各处理浓度与原生质体活力和植板率之间均存在极显著的负相关（p＜0.01）。培养第7d，所有处理均可见再生的小细胞团，培养30~40d，2种抑制剂临界浓度下原生质体均丧失形成愈伤组织的能力。适宜2种豆科牧草的抑制剂及临界浓度分别是：清水紫花苜蓿3mmol·L~(-1)IOA或40μg·m L~(-1)R-6G；百脉根5mmol·L~(-1)IOA或50μg·m L~(-1)R-6G。
2）FDA、罗丹明B、罗丹明6G和吖啶橙均可使苜蓿原生质体清晰染色，可用于细胞融合时亲本原生质体的标识。其中，FDA染色最为清晰，是检测原生质体活力的理想染料。吖啶橙和DAPI可对细胞核进行特异性染色，可用于融合时细胞核的观察和计数，前者还可用于检测融合细胞的活力，为进一步研究摸索融合条件和融合细胞的培养提供理论依据。
3）在已摸索出的最佳酶解和培养条件下，用3mmol·L~(-1)IOA和50μg·mL~(-1)R-6G分别处理清水紫花苜蓿和里奥百脉根愈伤组织来源的原生质体，利用PEG高Ca高pH值法诱导二者融合，最佳PEG浓度为35%，异源融合率为3.1%。利用固液培养法，杂种融合细胞在培养第4d出现第一次细胞分裂，2周后形成肉眼可见的微愈伤组织。待愈伤组织直径达0.5cm左右，转入MS固体培养基。经3~4次继代，最终获得杂种愈伤组织。
Alfalfa (Medicago sativa L.) is praised as‘the king of the forage’, its nutritionalvalue listed first among all kinds of forages. However, Livestock bloat diseasebecome the important limit reason for utilization and extension of its nutritional valuewhen green feeding with alfalfa. Birdsfoot trefoil (Lotus corniculatus Linn.) is praisedas ‘the alfalfa of infertile regions’, and it cann’t cause the tympanites when onlyfeeding its fresh grass or directly grazing in birdsfoot trefoil field because it is rich indense tannin. Now, it is an important ways and means to transfer the controllingtannins synthetic gene by means of somatic hybridization technology for improvingalfalfa quality and creating new alfalfa germplasm. But only a little investigation wasreported on the protoplasts isolation and somatic hybridization using the materials ofcultivated alfalfa varieties adapted northwest china. Thus, this paper wanted toestablish and optimize the experimental system of protoplast isolation and culture ofthree legume forages via somatic cell culture, protoplast culture，and to obtain thesomatic cell hybridization system via the protoplast fusion between M. sativa L.and L.corniculatus Linn. The main results of this research were as follows:
1、Protoplast isolation and culture of alfalfa
1）For callus induction of ‘Gannong No.1’and ‘Gannong No.4’, the optimumexplant on MS basic medium both was hypocotyl, and the best hormoneconcentration and combination were3.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA with the rate of induction of96.7%and2.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA with the rate of induction of100%,respectively. There wasn’t significant influence (p＞0.05) of differentexplants on callus induction from‘Algonquin’and2.0mg·L~(-1)2,4-D+0.5mg·L~(-1)NAA+0.5mg·L~(-1)6-BA were propitious both for hypocotyl andcotyledon with the rate of induction100%and90%, respectively.
2）The optimum enzyme solution combination of protoplasts isolation for‘Gannong No.1’,‘Gannong No.4’and ‘Qingshui’were2%celluloseonozuka R~(-1)0+0.5%pectinase Y-23+0.3%Driselase with the optimumenzymolysis time of12,14and10h, respectively. For ‘Algonquin’, the bestenzyme solution combination and enzymolysis time were2%cellulose onozuka R~(-1)0+0.5%pectinase Y-23+0.3%hemicellulose+0.3%macerozyme+0.3%driselase with12h enzyme digestion. The optimumenzyme osmoticum pressure for4varieties was:0.75mol·L~(-1)for ‘GannongNo.1’,0.65mol·L~(-1)for Gannong No.4’,0.6mol·L~(-1)for ‘Algonquin’ and0.55~0.6mol·L~(-1)for‘Qingshui’. The optimum callus subculture time for4varieties was12d. The best pretreatment methods for four varieties calli were:preplasmolyzing with0.55mol·L~(-1)sucrose or CPW solutions for1h for‘Gannong No.1’, preculture in4℃in1d or with0.55mol·L~(-1)sucrosesolutions for1h for‘Gannong No.4’, preplasmolyzing with0.55mol·L~(-1)CPW solutions for1h both for‘Algonquin’ and ‘Qingshui’. The highestyield and viability of four alfalfa varieties were2.48×106·g~(-1)(‘GannongNo.4’) and93.1%(‘Qingshui’).
3）The first divisions of four alfalfa varieties occurred in2~3days of culture andmini calli could be seen after7~14days with the liquid thin layer andsolid-liquid double layers culture methods both containing the KM8P medium.The regenerated calli from protoplasts of4cultivars were finally obtained bypropagation and subculture on the MS medium with0.5~3mg·L~(-1)2,4-D，0.5~1mg·L~(-1)NAA,0~0.5mg·L~(-1)6-BA and0~0.5mg·L~(-1)KT. Thesolid-liquid double layers culture method is advantageous than the liquid thinlayer because of the earlier cell division, more simple operate and not easy topollution during protoplast culture period.
2、Protoplast isolation and culture of birdsfoot trefoil
1）For callus induction of‘Leon’, the optimum explant on MS basic mediumwas hypocotyl, and the best hormone concentration and combination was1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT with the rate of induction of92.2%. Thehypocotyls and cotyledons of L. corniculatus produced numerous somaticembryos and plantlets on MS medium only containing0.5~3.0mg·L~(-1)KTafter about1month in the culture. And after subcultured2~3times, theentirely plants were obtained.
2）The best enzyme combination for both cotyledons and calli were2%celluloseonozuka R~(-1)0+0.5%pectinase Y-23+0.3%hemicellulose with0.55mol·L~(-1)mannitol. The optimum enzymolysis time was6h for cotyledons and12h for calli. Results indicated that both yield and viability of calli protoplastssignificantly increase (p＜0.05) through pre-culture for8d, preplasmolyzingwith0.55mol·L~(-1)CPW solutions for1h, or under darkness for1d. The highestyield and viability of protoplasts for cotyledons and calli were4.13×10~6·g~(-1)，65%and3.8×106·g~(-1)，67.4%, respectively.
3）The protoplast isolating from cotyledons and calli could constantly divide andform mini visible calli on KM8P medium with1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT+13.0%（0.7mol·L~(-1)） mannital. Numerous somatic embryos andregenerated plantlets appeared on MS medium with2.0mg·L~(-1)KT+0.7%agar+12.0%sucrose by the propagation and subculture of calli. Whenregenerated mini calli grew to0.5cm, the protoplast culture medium wasneeded to change into MS solid medium.Only3~4months was needed toobtain the regenerated plantlets from the first divisions of protoplasts. Thesurvival rate of regenerated plant from protoplast arrived at95%.
3、Somatic hybridization of M. sativa L.‘Qingshui’and L. corniculatus Linn.‘Leon’
1）The viability and plate efficiency of protoplasts from‘Qingshui’and ‘Leon’were obviously reduced under affecting of3~10mmol·L~(-1)IOA and40~70μg·m L~(-1)Rhodamine6G for10min. Correlation analysis revealed thatthe viability and plate efficiency of2species and effects of IOA and R-6Gwith different concentrations had significantly negative correlation（p＜0.01）.All protoplasts regenerated small aggregated cells cluster under the influenceof IOA and R-6G after culture for7days. The development of theseprotoplasts stopped and could not form calli because affecting of IOA andR-6G with a critical concentration after culture for30to40days. Theinhibitor and critical concentrations suitable for3forages were:3mmol·L~(-1)IOA or40μg·m L~(-1)R-6G for M. sativa L.‘Qingshui’and5mmol·L~(-1)IOA or50μg·m L~(-1)R-6G for L. corniculatus Linn.‘Leon’.
2）FDA, Rhodamine B, R-6G and Acridine Orange could dye and markprotoplasts of alfalfa clearly, which could identify parents protoplasts fromfusion clusters. FDA was the most optimal dye for testing the livability ofprotoplasts. Acridine Orange and DAPI can dye specific on nucleus of protoplasts, so that to count and survey nucleus in cell fusion. The former canalso test the viability of fusion cell and provide the theory basis by identifyingthe heterocaryon and testing their viability for exploring the cell fusioncondition and culturing the fusion cell.
3）Under the optimum enzymolysis and culture conditions, the protoplasts of‘Qingshui’ treated with3mmol·L~(-1)IOA and the protoplast of ‘Leon’ treatedwith50μg·m L~(-1)R-6G before somatic hybridization. The best fusioncondition with PEG was35%for the hybrid cell and the heterologous fusionrate was3.1%. The first division of hybrid cells occurred in4d and cellclusters could be seen after2weeks with solid-liquid double culture method.When hybrid cells grow to0.5cm, the protoplast culture medium was neededto change into MS solid medium. The hybrid calli was obtrained aftersubculture for1~2times.
1、苜蓿原生质体分离和培养
1）以MS固体培养基为基本培养基，适于甘农1号杂花苜蓿和甘农4号紫花苜蓿愈伤组织诱导的最佳外植体均为下胚轴，最佳激素浓度与配比及诱导率分别为3.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，96.7%；2.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，100%；适于阿尔刚金紫花苜蓿愈伤组织诱导的最适激素浓度和配比为2.0mg·L~(-1)2,4-D+0.5mg·L~(-1)NAA+0.5mg·L~(-1)6-BA，下胚轴和子叶对出愈率的影响不显著（p＞0.05），诱导率分别为100%，90%。试验证明，诱导出的苜蓿愈伤组织经过一段时间的继代调整，均可酶解分离出大量活力较高的原生质体。
2）以4个苜蓿品种的胚性愈伤组织为酶解材料，甘农1号、甘农4号和清水紫花苜蓿的最佳酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%崩溃酶，最适酶解时间分别为12h、14h和10h。阿尔冈金的最佳酶液组合为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶+0.3%离析酶+0.3%崩溃酶，最适酶解时间为12h。适宜4个苜蓿品种原生质体酶解的最佳甘露醇浓度分别为：甘农1号0.75mol·L~(-1)，甘农4号0.65mol·L~(-1)，阿尔冈金0.6mol·L~(-1)，清水紫花苜蓿0.55~0.6mol·L~(-1)。最佳愈伤组织继代培养时间均为第12d。适宜4个苜蓿品种愈伤组织酶解的最佳预处理措施分别为，甘农1号0.55mol·L~(-1)蔗糖或CPW溶液中处理1h，甘农4号预低温处理1h或0.55mol·L~(-1)蔗糖溶液中处理1h，阿尔冈金和清水紫花苜蓿均为0.55mol·L~(-1)CPW溶液中处理1h。4个苜蓿品种原生质体最高产量可达2.48×10~6个·g~(-1)（甘农4号），最大活力值为93.1%（清水紫花苜蓿）。
3）采用液体浅层培养法和固液双层培养法，4个苜蓿品种在含KM8P培养液的培养基上培养第2~3d，均可观察到原生质体再生细胞的第1次及多次分裂，第7~14d形成肉眼可见的微愈伤组织，转入含0.5~3mg·L~(-1)2,4-D，0.5~1mg·L~(-1)NAA、0~0.5mg·L~(-1)6-BA和0~0.5mg·L~(-1)KT的固体培养基增殖与继代，最终均获得了原生质体再生的愈伤组织。固液双层培养法较液体浅层培养法形成再生细胞团的时间较早，操作简便且不易污染，更有利于苜蓿原生质体早期的分裂和再生。
2、百脉根原生质体分离和培养
1）以MS固体培养基为基本培养基，适于里奥愈伤组织诱导的最佳外植体为下胚轴，最佳激素浓度和组合为：1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT，诱导率为92.2%；在仅含0.5~3.0mg·L~(-1)KT的MS基本培养基上下胚轴和子叶诱导1个月左右均可直接生成胚状体和小苗，经2~3次继代发育成完整植株。
2）适于里奥无菌苗子叶和胚性愈伤组织原生质体分离的最适酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶，最佳酶解时间分别为6h及12h，最适甘露醇浓度均为0.55mol·L~(-1)。里奥愈伤组织分离原生质的最佳继代培养时间为第8d；0.55mol·L~(-1)CPW溶液中预质壁分离1h或预暗培养1d均可显著（p＜0.05）增加其原生质体的产量和活力。子叶和愈伤组织原生质体最高产量和活力分别可达4.13×106个·g~(-1)，65%和3.8×106个·g~(-1)，67.4%。
3）采用液体浅层培养法，由子叶和胚性愈伤组织分离的原生质体在KM8P+1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT+13.0%（0.7mol·L~(-1)）甘露醇的培养基上可持续分裂并形成肉眼可见的微愈伤组织，当愈伤组织长到直径0.5mm左右时，转入含相同激素浓度的MS固体培养基上增殖和继代。大量的体细胞胚胎和再生小植株在含MS+2.0mg·L~(-1)KT+0.7%琼脂+12.0%蔗糖的培养基中发生。从原生质体第一次分裂至再生植株仅需3~4个月。再生植株移栽成活率达95%。3、苜蓿和百脉根的体细胞杂交
1）3~10mmol·L~(-1)的IOA和40~70μg·m L~(-1)的R-6G分别处理10min，可使清水紫花苜蓿和里奥百脉根原生质体的活力及植板率明显下降，各处理浓度与原生质体活力和植板率之间均存在极显著的负相关（p＜0.01）。培养第7d，所有处理均可见再生的小细胞团，培养30~40d，2种抑制剂临界浓度下原生质体均丧失形成愈伤组织的能力。适宜2种豆科牧草的抑制剂及临界浓度分别是：清水紫花苜蓿3mmol·L~(-1)IOA或40μg·m L~(-1)R-6G；百脉根5mmol·L~(-1)IOA或50μg·m L~(-1)R-6G。
2）FDA、罗丹明B、罗丹明6G和吖啶橙均可使苜蓿原生质体清晰染色，可用于细胞融合时亲本原生质体的标识。其中，FDA染色最为清晰，是检测原生质体活力的理想染料。吖啶橙和DAPI可对细胞核进行特异性染色，可用于融合时细胞核的观察和计数，前者还可用于检测融合细胞的活力，为进一步研究摸索融合条件和融合细胞的培养提供理论依据。
3）在已摸索出的最佳酶解和培养条件下，用3mmol·L~(-1)IOA和50μg·mL~(-1)R-6G分别处理清水紫花苜蓿和里奥百脉根愈伤组织来源的原生质体，利用PEG高Ca高pH值法诱导二者融合，最佳PEG浓度为35%，异源融合率为3.1%。利用固液培养法，杂种融合细胞在培养第4d出现第一次细胞分裂，2周后形成肉眼可见的微愈伤组织。待愈伤组织直径达0.5cm左右，转入MS固体培养基。经3~4次继代，最终获得杂种愈伤组织。
Alfalfa (Medicago sativa L.) is praised as‘the king of the forage’, its nutritionalvalue listed first among all kinds of forages. However, Livestock bloat diseasebecome the important limit reason for utilization and extension of its nutritional valuewhen green feeding with alfalfa. Birdsfoot trefoil (Lotus corniculatus Linn.) is praisedas ‘the alfalfa of infertile regions’, and it cann’t cause the tympanites when onlyfeeding its fresh grass or directly grazing in birdsfoot trefoil field because it is rich indense tannin. Now, it is an important ways and means to transfer the controllingtannins synthetic gene by means of somatic hybridization technology for improvingalfalfa quality and creating new alfalfa germplasm. But only a little investigation wasreported on the protoplasts isolation and somatic hybridization using the materials ofcultivated alfalfa varieties adapted northwest china. Thus, this paper wanted toestablish and optimize the experimental system of protoplast isolation and culture ofthree legume forages via somatic cell culture, protoplast culture，and to obtain thesomatic cell hybridization system via the protoplast fusion between M. sativa L.and L.corniculatus Linn. The main results of this research were as follows:
1、Protoplast isolation and culture of alfalfa
1）For callus induction of ‘Gannong No.1’and ‘Gannong No.4’, the optimumexplant on MS basic medium both was hypocotyl, and the best hormoneconcentration and combination were3.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA with the rate of induction of96.7%and2.0mg·L~(-1)2,4-D+1.0mg·L~(-1)NAA+0.5mg·L~(-1)6-BA with the rate of induction of100%,respectively. There wasn’t significant influence (p＞0.05) of differentexplants on callus induction from‘Algonquin’and2.0mg·L~(-1)2,4-D+0.5mg·L~(-1)NAA+0.5mg·L~(-1)6-BA were propitious both for hypocotyl andcotyledon with the rate of induction100%and90%, respectively.
2）The optimum enzyme solution combination of protoplasts isolation for‘Gannong No.1’,‘Gannong No.4’and ‘Qingshui’were2%celluloseonozuka R~(-1)0+0.5%pectinase Y-23+0.3%Driselase with the optimumenzymolysis time of12,14and10h, respectively. For ‘Algonquin’, the bestenzyme solution combination and enzymolysis time were2%cellulose onozuka R~(-1)0+0.5%pectinase Y-23+0.3%hemicellulose+0.3%macerozyme+0.3%driselase with12h enzyme digestion. The optimumenzyme osmoticum pressure for4varieties was:0.75mol·L~(-1)for ‘GannongNo.1’,0.65mol·L~(-1)for Gannong No.4’,0.6mol·L~(-1)for ‘Algonquin’ and0.55~0.6mol·L~(-1)for‘Qingshui’. The optimum callus subculture time for4varieties was12d. The best pretreatment methods for four varieties calli were:preplasmolyzing with0.55mol·L~(-1)sucrose or CPW solutions for1h for‘Gannong No.1’, preculture in4℃in1d or with0.55mol·L~(-1)sucrosesolutions for1h for‘Gannong No.4’, preplasmolyzing with0.55mol·L~(-1)CPW solutions for1h both for‘Algonquin’ and ‘Qingshui’. The highestyield and viability of four alfalfa varieties were2.48×106·g~(-1)(‘GannongNo.4’) and93.1%(‘Qingshui’).
3）The first divisions of four alfalfa varieties occurred in2~3days of culture andmini calli could be seen after7~14days with the liquid thin layer andsolid-liquid double layers culture methods both containing the KM8P medium.The regenerated calli from protoplasts of4cultivars were finally obtained bypropagation and subculture on the MS medium with0.5~3mg·L~(-1)2,4-D，0.5~1mg·L~(-1)NAA,0~0.5mg·L~(-1)6-BA and0~0.5mg·L~(-1)KT. Thesolid-liquid double layers culture method is advantageous than the liquid thinlayer because of the earlier cell division, more simple operate and not easy topollution during protoplast culture period.
2、Protoplast isolation and culture of birdsfoot trefoil
1）For callus induction of‘Leon’, the optimum explant on MS basic mediumwas hypocotyl, and the best hormone concentration and combination was1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT with the rate of induction of92.2%. Thehypocotyls and cotyledons of L. corniculatus produced numerous somaticembryos and plantlets on MS medium only containing0.5~3.0mg·L~(-1)KTafter about1month in the culture. And after subcultured2~3times, theentirely plants were obtained.
2）The best enzyme combination for both cotyledons and calli were2%celluloseonozuka R~(-1)0+0.5%pectinase Y-23+0.3%hemicellulose with0.55mol·L~(-1)mannitol. The optimum enzymolysis time was6h for cotyledons and12h for calli. Results indicated that both yield and viability of calli protoplastssignificantly increase (p＜0.05) through pre-culture for8d, preplasmolyzingwith0.55mol·L~(-1)CPW solutions for1h, or under darkness for1d. The highestyield and viability of protoplasts for cotyledons and calli were4.13×10~6·g~(-1)，65%and3.8×106·g~(-1)，67.4%, respectively.
3）The protoplast isolating from cotyledons and calli could constantly divide andform mini visible calli on KM8P medium with1.0mg·L~(-1)2,4-D+2.0mg·L~(-1)KT+13.0%（0.7mol·L~(-1)） mannital. Numerous somatic embryos andregenerated plantlets appeared on MS medium with2.0mg·L~(-1)KT+0.7%agar+12.0%sucrose by the propagation and subculture of calli. Whenregenerated mini calli grew to0.5cm, the protoplast culture medium wasneeded to change into MS solid medium.Only3~4months was needed toobtain the regenerated plantlets from the first divisions of protoplasts. Thesurvival rate of regenerated plant from protoplast arrived at95%.
3、Somatic hybridization of M. sativa L.‘Qingshui’and L. corniculatus Linn.‘Leon’
1）The viability and plate efficiency of protoplasts from‘Qingshui’and ‘Leon’were obviously reduced under affecting of3~10mmol·L~(-1)IOA and40~70μg·m L~(-1)Rhodamine6G for10min. Correlation analysis revealed thatthe viability and plate efficiency of2species and effects of IOA and R-6Gwith different concentrations had significantly negative correlation（p＜0.01）.All protoplasts regenerated small aggregated cells cluster under the influenceof IOA and R-6G after culture for7days. The development of theseprotoplasts stopped and could not form calli because affecting of IOA andR-6G with a critical concentration after culture for30to40days. Theinhibitor and critical concentrations suitable for3forages were:3mmol·L~(-1)IOA or40μg·m L~(-1)R-6G for M. sativa L.‘Qingshui’and5mmol·L~(-1)IOA or50μg·m L~(-1)R-6G for L. corniculatus Linn.‘Leon’.
2）FDA, Rhodamine B, R-6G and Acridine Orange could dye and markprotoplasts of alfalfa clearly, which could identify parents protoplasts fromfusion clusters. FDA was the most optimal dye for testing the livability ofprotoplasts. Acridine Orange and DAPI can dye specific on nucleus of protoplasts, so that to count and survey nucleus in cell fusion. The former canalso test the viability of fusion cell and provide the theory basis by identifyingthe heterocaryon and testing their viability for exploring the cell fusioncondition and culturing the fusion cell.
3）Under the optimum enzymolysis and culture conditions, the protoplasts of‘Qingshui’ treated with3mmol·L~(-1)IOA and the protoplast of ‘Leon’ treatedwith50μg·m L~(-1)R-6G before somatic hybridization. The best fusioncondition with PEG was35%for the hybrid cell and the heterologous fusionrate was3.1%. The first division of hybrid cells occurred in4d and cellclusters could be seen after2weeks with solid-liquid double culture method.When hybrid cells grow to0.5cm, the protoplast culture medium was neededto change into MS solid medium. The hybrid calli was obtrained aftersubculture for1~2times.
引文
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