Chen, Aiping

Luo, Yupeng

Yan, Run

Zhang, Bo

English

University of Kentucky

University of Kentucky UKnowledge International Grassland Congress Proceedings 21st International Grassland Congress / 8th International Rangeland Congress Effects of Different Enzyme Combination and Dissociation‐Time on the Protoplast Isolation of Alfalfa Aiping Chen Xinjiang Agricultural University, China Yupeng Luo Xinjiang Agricultural University, China Run Yan Xinjiang Agricultural University, China Bo Zhang Xinjiang Agricultural University, China Follow this and additional works at: https://uknowledge.uky.edu/igc  Part of the Plant Sciences Commons, and the Soil Science Commons This document is available at https://uknowledge.uky.edu/igc/21/1-4/21 The 21st International Grassland Congress / 8th International Rangeland Congress took place in Hohhot, China from June 29 through July 5, 2008. Proceedings edited by Organizing Committee of 2008 IGC/IRC Conference Published by Guangdong People's Publishing House This Event is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in International Grassland Congress Proceedings by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. 瞯 ]170　 瞯 　 Multifunctional Grasslands in a Changing World 　 Volume Ⅰ 　Grasslands/Rangelands Resources and Ecology ——— Ecology of Grasslands/RangelandsEffects of different enzyme combination and dissociation‐time on the protoplast isolation ofalfalfaChen A i‐p ing , L uo Y u‐peng , Y an Run ,Zhang BoKey L aboratory o f G rassland Resource and Ecology o f X inj iang , College o f Pratacultural Engineering , X inj iangA gricultural University ,Urumqi , 830052 ,China . E‐mail :klcap＠ １２６ .comKey words : alfalfa ,the kinds of enzyme ,concentration of enzyme ,dissociation‐time ,protoplastIntroduction At present , as donor of protoplast dissociation is different to alfalfa , some factors have influence on protoplastdissociation , such as the kinds of enzyme , the concentration and dissociation‐time . Using hypocotyl of alfalfa as testedmaterials , by setting different the kinds of enzyme , the concentration and combination of enzyme , dissociation‐time in thisstudy in order to provide theory and practical guidance for extension of alfalfa biotechnology .Materials and methods According to the routine methods for inducement cultivating asepsis seedling . After asepsis seedling wascultivated １０d , the hypocotyls were chopped .Usually １ .０ g chopped hypocotyls in a CPW‐１３ solution ( Frearson et al . , １９７３) were incubated in the dark at ２５ ℃ for １h thensubmitted to １０ ml filter‐sterilized enzyme solution Ⅰ .The same method was treated in the enzyme solution Ⅱ , Ⅲ , Ⅳ as theenzyme mixtures Ⅰ . Chopped hypocotyls were shaken at ６０ rpm for ６h in the dark .Usually １ .０ g chopped hypocotyls in a CPW‐１３ solution were incubated in the dark at ２５ ℃ for １h then submitted to １０ mlenzyme solution Ⅳ ,Chopped hypocotyls were shaken at ６０ rpm ,２５ ℃ for ４h ,６h , ８h and １０h in the dark .The mixture was passed through a nylon sieve (３８ .５ μm pore sizes) and １５ ml of CPW９M solution was added . The protoplastswere collected by centrifugation for ５ min at ６０ rpm and resuspended in the washing solution . The washing treatment was donetwice . Protoplasts , free of debris were carefully removed from the interface of the solutions , and protoplasts were rinsed twicewith １５ ml of KM８p medium( ( Kao and Michayluk , １９７５ ) A small sample of protoplasts in the washing solution was stainedwith ０ .０１％ ( w / v ) phenosafranine .The yield of protoplasts was determined with a Malassez counting chamber .Results and discussion Even conspecific plant , initiative materials are different , there are different to concentration of enzymeand dissociation‐time . Chopped hypocotyls were treated in four enzyme solutions for ６h , it was better the condition ofprotoplast dissociation in enzyme solution Ⅱ in this experimentation ;In the same condition of protoplast dissociation ,protoplastdissociation production gradual increase along with protraction of dissociation‐time ,but dissociation‐time is overlong will resultin sharp increase of cell debris ,it will affect protoplast culture ,and prior dissociative protoplast is able to occur fracture . It wasbetter dissociation‐time that the hypocotyls were treated for ６h , at the same time cell debris was less .Table 1 The corcertration and combintin o f ensyme .Name Ceihilose( ％ )Pectinose( ％ )macerorymeR‐１０ ( ％ )enryme soltion Ⅰ １ :０ *.８ ０ Denryme soltion Ⅱ １ :０ *.８ ０  .５enryme soltion Ⅲ ２ :０ *.８ ０ Denryme soltion Ⅳ ２ :０ *.８ ０  .５Table 2 The e f f ects o f di f f erent enz yme combintion o fal f al f a hy pocotyk cop rotop last dissociation f or on .Name yield of vinble pro toplast( １０６ / ml) cellbreabageenryme soltion Ⅰ ０ 换.２６ large amountenryme soltion Ⅱ ０ 换.９５ smallenryme soltion Ⅲ ０ 换.３１ quterareenryme soltion Ⅳ ０ 换.４０ large amountTable 3 The e f f ects o f di f f erent enz yme dissociation‐time o f al f al f a hy pow ty ls onp rotop last dissociz tion in enryme solitionⅡ .dissocio tion‐time( h) yield( １ × １０６ g － １ ) visbility ( ％ ) yield of vinble protoplast ( １ × １０６ g － １ ) cell brealage４  ０ 趑.２２ ７９２０ ０ 揪.３３ quterare６  ０ 趑.９６ ８６９０ １ 揪.０９ small８  １ 趑.５６ ８１１０ ０ 揪.７４ small１０ 2１ 趑.０１ ９０２０  ０ 揪.５６ large amountReferenceWang Hai‐bo , Yu Yong‐xiong .( ２００６ ) . The Research of Protoplast Dissociate Conditions of Alfalfa [ J] . Chinese Journal o fG rassland , ２８(５) :３３‐３７ .

Effects of Different Enzyme Combination and Dissociation‐Time on the Protoplast Isolation of Alfalfa

https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=2349&amp;context=igc

Effects of Different Enzyme Combination and Dissociation‐Time on the Protoplast Isolation of Alfalfa

Abstract

Similar works

Full text

Available Versions

University of Kentucky