姓名班级学号实验日期科目植物生理学实验实验名称Effects of Hormones on Morphogenesis in Plant Tissue Culture 合作者指导教师成绩Effects of Hormones on Morphogenesis in Plant Tissue Culture IntroductionThe technology of plant tissue culture is based on the principle of totipotency which means the capacity to generate a whole plant from any cells or an explant.During the plant tissue culture,the hormones affect largely on morphogenesis,such as Auxin and Cytokinin.And the content and ratio of these hormones can make different results.NAA is the routine reagent of auxine and6-BA is the reagent of cytokinin.For the tissue culture,we prepare the Murashige and Skoog’s medium(MS medium).The MS medium contains hormones,macronutrients and micronutrients(detailed ingredients are in Table 2-1).The pH in the medium will be adjusted with1M NaOH.We should prepare10MS media with hormones.During the whole operation of tissue culture,six potential source of contamination are air,water, growth media,equipment,explant and people.We use the laminar flow hood to remove most of microbes from air flow.The water and the media have been autoclaved.The tools should be kept sterile in the70%alcohol and flamed by alcohol burner.And we sterilize the explants with Ca(ClO)2and70% ethanol and sterile water.We use the NAA to induce the callus to generate root.MaterialsFresh young leaves of tobacco(Nicotiana tabacum)MS medium:NH4NO3,KNO3,CaCl2,MgSO4,KH2PO4,MnSO4•H2O,ZnSO4•7H2O,KI,Na2MoO4•2H2O,CuSO4•5H2O,CoCl2•6H2O,Na2EDTA,FeSO4•7H2O,Nicotinic acid,Pyridoxine HCl(VB6), Thiamine HCl(VB1).Glycine,Mesoinositol.Agar,HgCl2,NAA,6-BA.2%Ca(ClO)2,sterile distilled water,70%ethanol,Beaker,Balance,10 Bottles,pH test strips,Laminar flow hood,Tween-80,Scissor,Forceps,Scalpels,Alcohol lamp. MethodPart1:Prepare10bottles of culture.1.Wash10bottles and dry them for a group.2.Our group’s task:Weigh50mg6-BA and mix with sterile distilled water to be50mL1mg/ml solutionin a bottle and mark the stock solution name,the date,and our group number.3.Other groups prepare the MS medium culture,agar,NAA and sugar.In Table2-1,MS=100ml A+1ml B+10ml C+5ml D+10ml E.Every3group prepare1L medium=MS+1mg/ml6-BA+0.2mg/L NAA+3%sugar+0.7%agar.Boil the solution with induction cooker to mix thoroughly.4.Adjust the pH of medium to5.8with1M NaOH or HCl.5.Repackage the1L medium to the bottles of3groups.10bottles of each group,30ml for each bottle.6.Sterilize the culture bottles by autoclaving at115℃for30min.Store them at room temperature. Part2:Operation of plant tissue culture.1.The space in the laminar flow hood has been sterilized by UV light for15min.We place all items inwell order to start working.2.Rinse4young leaves as explants in tap water for10-30min.The next operations are all aseptic.3.Take the leaves into70%ethanol for90s and gently agitate the explants.4.Pour out the ethanol and add some2%Ca(ClO)2with a drop of Tween-80,gently agitate for6min.When submerge for about6min,we found that the leaves are lightly wilting,so we stopped submerge without continuing to15-30min.5.Decant the disinfectant.Rinse with sterile distilled water4times.6.Transfer the leaves on a sterile metal plate,and cut out the edges of all leaves.Excise the leaves into5mm×5mm sized blocks and clamp the leave blocks into the medium bottles,1or2leaf blocks per bottle.The lower surface of leaf block should cover the medium.Because some leaves we used are lightly wilting,we choose several leaves of germfree tobacco bleedings to excise into blocks and cultivated them directly.7.Place the10bottles in the light growth chamber,where has light intensity of2000-3000lux for16h at25℃and8h-dark period at20℃.8.Observe them every week for6weeks,and record their growth.Part3:Callus differentiation or Rooting.1.Prepare the culture medium for callus differentiation(4bottles per people).The composition of themedium is as below:1/2MS+NAA0.5mg/L+2%sugar+0.7%agar(pH=5.8)2.Choose some good-looking callus from the5bottles.In the laminar flow hood,cut four pieces of stemswith leaves and insert them in the prepared agar bottles,flame the bottles to sterilize.And seal them to incubate at37℃for several weeks.3.Record the root growing situation every week.Results:pared with the leaves blocks in the bottle in zero week,09/10/2015(figure1),the callus in the3rdweek have some protuberances,which means the tissue have begun to differentiate(figure2).Since the 4th week,several young leaves came into being(figure3),and since the5th week,the young stems appeared(figure4).Finally,in the7th week,the callus had become bigger(figure5),while the callus ina bottle almost didn’t grew up since the3rd week(figure6).2.In the7th week,20/11/2015,we began to rooting cultivation(figure7),and in the4th week,18/12/2015,we observed that the parts we plant in the bottles had generated white slender roots(figure8). Discussion:1.During the tissue culture,the callus in a bottle just finished the preliminary differentiation,and since the3rd week,they almost didn’t continue to differentiate.We think there may be two potential reasons.The first reason is that the leaves blocks in this bottle is very weak,because we said there several leaves is wilted,which made them couldn’t furtherly differentiate.The second one is the bottle was contaminated by microbes that weaken the tissues.I think the first reason is most possible.2.During the rooting culture,the lower leaves became yellow and wilted(figure8),which maybe anormal apoptosis process,we think.Distribution:In the tissue culture operation,we separately prepare5bottles of leaves blocks.And in the rooting culture,I made my3bottles of samples while my partner made4bottles.Figures:Figure1.The leaves blocks(0week)Figure2.Callus tissues(3rd week)Figure3.Callus tissues(4th week)Figure4.Callus tissues(5th week)Figure5.Callus tissues(7th week)Figure6.Weak callus tissues(6th week)Figure 7.The young branch just planted (7thweek)Figure 8.The 3bottles after 4-week rooting culture.第4页Yellow and wiltedlower leavesWhite young roots.。
