Studies On Protoplast Culture And Somatic Hybridization Between Alfalfa And Birdsfoot Trefoil

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 alfalfa1)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^-12,4-D+1.0mg·L^-1NAA+0.5mg·L^-16-BA with the rate of induction of96.7%and2.0mg·L^-12,4-D+1.0mg·L^-1NAA+0.5mg·L^-16-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^-12,4-D+0.5mg·L^-1NAA+0.5mg·L^-16-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^-10+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^-10+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^-1for 'GannongNo.1',0.65mol·L^-1for Gannong No.4',0.6mol·L^-1for 'Algonquin' and0.55⁰.6mol·L^-1for'Qingshui'. The optimum callus subculture time for4varieties was12d. The best pretreatment methods for four varieties calli were:preplasmolyzing with0.55mol·L^-1sucrose or CPW solutions for1h for'Gannong No.1', preculture in4℃in1d or with0.55mol·L^-1sucrosesolutions for1h for'Gannong No.4', preplasmolyzing with0.55mol·L^-1CPW 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 in2³days of culture andmini calli could be seen after7¹4days 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 with0.5³mg·L^-12,4-D,0.5¹mg·L^-1NAA,0⁰.5mg·L^-16-BA and0⁰.5mg·L^-1KT. 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 trefoil1)For callus induction of'Leon', the optimum explant on MS basic mediumwas hypocotyl, and the best hormone concentration and combination was1.0mg·L^-12,4-D+2.0mg·L^-1KT with the rate of induction of92.2%. Thehypocotyls and cotyledons of L. corniculatus produced numerous somaticembryos and plantlets on MS medium only containing0.5³.0mg·L^-1KTafter about1month in the culture. And after subcultured2³times, theentirely plants were obtained.2)The best enzyme combination for both cotyledons and calli were2%celluloseonozuka R^-10+0.5%pectinase Y-23+0.3%hemicellulose with0.55mol·L^-1mannitol. 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^-1CPW solutions for1h, or under darkness for1d. The highestyield and viability of protoplasts for cotyledons and calli were4.13×10⁶·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^-12,4-D+2.0mg·L^-1KT+13.0%(0.7mol·L^-1) mannital. Numerous somatic embryos andregenerated plantlets appeared on MS medium with2.0mg·L^-1KT+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.Only3⁴months 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 of3¹0mmol·L^-1IOA and40⁷0μg·m L^-1Rhodamine6G 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^-1IOA or40μg·m L^-1R-6G for M. sativa L.'Qingshui'and5mmol·L^-1IOA or50μg·m L^-1R-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^-1IOA and the protoplast of 'Leon' treatedwith50μg·m L^-1R-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 for1²times.