榅桲的组织培养快繁及耐盐性研究
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摘要
本研究以新疆苹果型榅桲为试材,研究了榅桲的茎段及子叶组织培养快繁途径,分别采用榅桲组培苗和种子对其耐盐性作了初步探讨。组培快繁试验中分别研究了不同灭菌剂、灭菌时间、培养基、激素组合等对榅桲外植体灭菌、初代培养、继代培养和生根培养的影响。耐盐性研究中,探讨了不同盐种类、不同盐浓度及不同盐胁迫时间对榅桲组培苗生长性状、生理指标的影响。同时也研究了上述盐胁迫因素对榅桲种子萌发情况、酶活性和离子分布的影响。分析结果表明:
1)用榅桲茎段进行组培快繁研究中,0.1%升汞溶液消毒5min,灭菌效果最好;初代培养基以MS+6-BA 0.6mg/L效果较好,萌芽率和增殖倍数均较高,分别为93.3%和4.7;继代培养基以MS+6-BA0.6 mg/L+IBA0.2mg/L和MS+6-BA0.6mg/L+IBA0.4mg/L组合交替使用更有利于组培苗的增殖和壮苗;生根培养基以1/2MS(I)+NAA1.0mg/L生根率最高,达50%,平均根数为7-10根。短时间(7d)暗培养对诱导榅桲苗生根有利,随着暗培养天数的增加叶焦边现象逐渐加重,甚至出现顶端枯死现象;6-BA对诱导榅桲组培苗生根无益且易出现畸形叶。
2)在榅桲成熟胚子叶不定植株再生研究中,1/4MS(I)+6-BA5mg/L+NAA0.5mg/L组合诱导成熟胚子叶芽分化效果最好,芽分化率可达77.5%;继代培养基以MS+6-BA 0.6mg/L+IBA0.2mg/L效果较好,增殖倍数约达4倍;生根培养基以速蘸IBA50mg/L 3S后接入活性炭培养基中生根率最高为75%,根数较多,但根生长缓慢,30d平均根长还不到1cm。移栽炼苗采用培养室培养钵炼苗法,以锯末为介质,15d左右长出新根,30d后成活率为78%。
3)组培苗耐盐性研究中,萌芽率、增殖倍数、平均株高均随着盐浓度的增大而降低,膜透性和盐害率随着盐浓度的增大而升高。三种保护酶SOD、CAT、POD活性除POD活性随着Na2SO4浓度的增加降低外,其它均随着盐浓度的增大呈先上升后下降趋势。NaCl胁迫下叶绿素a、b和总叶绿素含量的变化趋势相同,均呈下降-升高-下降趋势,但始终在空白处理时最高,最大盐浓度时最低。Na2SO4胁迫下叶绿素a和总叶绿素含量的变化趋势相同,呈下降趋势,叶绿素b与之不同,呈先升高后下降趋势。类胡萝卜素的含量变化没有规律性。认为,萌芽率、增殖倍数、平均株高、膜透性、盐害率、SOD、CAT、POD活性均可作为鉴定榅桲耐盐性评价指标,叶绿素可作为抗盐评价的参考指标。两种盐胁迫下,榅桲组培苗更能耐受NaCl胁迫,NaCl和Na2SO4分别在浓度为0.6%和0.8%时开始对榅桲组培苗造成严重伤害,在0.8%和1.0%时至死率超过50%。
4)榅桲种子耐盐性研究中,随着盐浓度的增大,发芽率和成苗率均呈下降趋势。NaCl胁迫时,SOD、CAT活性均随着盐浓度的增大呈先上升后下降趋势,POD活性呈上升趋势。Na2SO4胁迫时,随着盐浓度的增大POD、CAT活性呈先上升后下降趋势,SOD活性呈降低趋势。所以认为,不同种类盐胁迫下,榅桲种子的耐盐机制不同。随着盐浓度的增大,芽苗和培养基中的Na+均呈上升趋势,种皮中的Na+呈先下降后上升趋势。K+在各组织中的变化没有规律性。NaCl胁迫下,随着盐浓度的增加,芽苗和种皮中Na+/K+呈升高-降低趋势,但始终高于空白处理中Na+/K+的值,培养基中Na+/K+呈升高-降低-升高趋势。Na2SO4胁迫时芽苗和种皮中Na+/K+呈升高-降低-升高趋势,培养基中Na+/K+呈升高趋势。综合分析,发芽率、成苗率、SOD、CAT、POD、榅桲芽苗中Na+含量可作为榅桲种子耐盐性评价指标。K+、Na+/K+变化较复杂,能否作为榅桲种子耐盐性鉴定的指标还有待于研究。Na2SO4胁迫对榅桲种子发芽的影响比NaCl胁迫大。以盐胁迫浓度对榅桲种子发芽率和成苗率为标准来看,Na2SO4和NaCl浓度分别为100mmol和150mmol时严重影响榅桲种子发芽。
In this research , Cydonia oblonga Mill of apple model in XinJiang was used to study the reproduction approaches of tender-stem and cotyledon .Then used the tissue culture plants and seeds to have a primary discuss on the salt-tolerance of quince .In the experiment of reproduction , studed the effect on setting axenic explants,primary culture ,succeed culture and rooting culture of different disinfectant、sterilization time、culture medium、hormone combinations,etc.The study on salt-tolerance ,we discussed the effect on regeneration plant’s growth character, Physiological index of different salt , concentration and time .we also had studied on the effect on seed buding,enzyme and ion of salt-stress.The results showed that:
1)Using the tender-stem of Cydonia oblonga Mill to regeneration, HgCl2 0.1% for 5 min is the best for sterilization; The better Primary medium is MS+6-BA 0.6mg/L,its germination rate and multiplication multiple are all higher, they are 93.3% and 4.7; Using MS+6-BA0.6 mg/L +IBA0.2 mg/L and MS+6-BA0.6mg/L+IBA0.4 mg/L by turns is better for multiplication and strong seedlings; Rooting medium chose 1/2MS(I)+NAA1. 0 mg/L can achieve the highest 50% rootage rate and 7-10 average root number; Short dark treatment(7d) is benefit for rooting but as the time longer the leaf-burnt became more serious and even lead to dead of stem tip;(6)6-BA is not benefit for rooting and even lead to abnormal leaves.
2)Using the cotyledon of Cydonia oblonga Mill to regeneration, 1/4MS(I)+6-BA5mg/L+NAA0.5mg/L is best for planted regeneration ; regeneration rate is 77.5%;MS+6-BA 0.6mg/L+IBA0.2mg/L is better for multiplication , it can be about 4 times more ; active carbon and dipped IBA50mg/L 3S is benefit for rooting ,rootage rate is 75% and the number is more ,but the growing of roots is slowly ,shorter then 1cm after 30d .In the culture room and used earthen bowls to transplant young plants ,sawdust is medium ,new roots would come out in 15d and 78% could be alive after 30d .
3)In the research of regeneration plant salt-tolerance, the burgeon ratio ,multiplication multiple ,average tallness were all reduced along with the increasing degree salt-stress , MP and impair rate is increased . SOD、CAT、POD were all showed up-down except POD was showed down when Na2SO4 added .Under NaCl stress Chla ,Chlb and ChlT had the same movement ,showed down-up-down ,and highest at blankness lowest at most harvest salt concentration . Under Na2SO4 stress, Chla and chlT were all falled , Chlb was rised then falled . Chlx.c had no regular changed. We considered, burgeon ratio ratio, multiplication multiple,MP,impair rate SOD,CAT,POD can be the index of salt-tolerance estimation Cydonia oblonga Mill salt-tolerance estimation and Chl can be the reference index . Under the two salt stress, Cydonia oblonga Mill regeneration plants could be even more able to bear NaCl,and was injured hardly at 0.6% and 0.8% of NaCl and Na2SO4, 50% more died at 0.8% and 1.0% .
4) In the research of Cydonia oblonga Mill seed salt-tolerance, the bud ratio and the seeding rate were all decreased . Under NaCl stress, SOD、CAT were showed down along with the increasing degree salt-stress when POD was showed up . Under Na2SO4stress, POD、CAT were showed up-down when SOD showed down .So we think , Cydonia oblonga Mill seed had different mechanism for different kinds of salt . Na+ in young plant and medium are increased along with the increasing degree salt-stress when Na+ in seed capsule was showed down-up . K+ in all organises had no ruled change . Na+/K+ in young plant and seed capsule were showed up-down and higher than the Na+/K+ of blank . Na+/K+ in medium showed up-down-up . Under Na2SO4 stress, Na+/K+ in young plant and seed capsule were showed up-down-up,when Na+/K+ in medium showed up . For all , bud ratio , seeding rate, SOD, CAT, POD, Na+ in young plant can be the index of Cydonia oblonga Mill seed salt-tolerance estimation . The change of K+ and Na+/K+ is complex ,whether can be the index of Cydonia oblonga Mill seed salt-tolerance estimation need futhermore research . Na2SO4 is more disadvantaged to Cydonia oblonga Mill buding, and 100mmol /L and150mmol/L of Na2SO4 and NaCl can have serious effect on Cydonia oblonga Mill buding .
1)用榅桲茎段进行组培快繁研究中,0.1%升汞溶液消毒5min,灭菌效果最好;初代培养基以MS+6-BA 0.6mg/L效果较好,萌芽率和增殖倍数均较高,分别为93.3%和4.7;继代培养基以MS+6-BA0.6 mg/L+IBA0.2mg/L和MS+6-BA0.6mg/L+IBA0.4mg/L组合交替使用更有利于组培苗的增殖和壮苗;生根培养基以1/2MS(I)+NAA1.0mg/L生根率最高,达50%,平均根数为7-10根。短时间(7d)暗培养对诱导榅桲苗生根有利,随着暗培养天数的增加叶焦边现象逐渐加重,甚至出现顶端枯死现象;6-BA对诱导榅桲组培苗生根无益且易出现畸形叶。
2)在榅桲成熟胚子叶不定植株再生研究中,1/4MS(I)+6-BA5mg/L+NAA0.5mg/L组合诱导成熟胚子叶芽分化效果最好,芽分化率可达77.5%;继代培养基以MS+6-BA 0.6mg/L+IBA0.2mg/L效果较好,增殖倍数约达4倍;生根培养基以速蘸IBA50mg/L 3S后接入活性炭培养基中生根率最高为75%,根数较多,但根生长缓慢,30d平均根长还不到1cm。移栽炼苗采用培养室培养钵炼苗法,以锯末为介质,15d左右长出新根,30d后成活率为78%。
3)组培苗耐盐性研究中,萌芽率、增殖倍数、平均株高均随着盐浓度的增大而降低,膜透性和盐害率随着盐浓度的增大而升高。三种保护酶SOD、CAT、POD活性除POD活性随着Na2SO4浓度的增加降低外,其它均随着盐浓度的增大呈先上升后下降趋势。NaCl胁迫下叶绿素a、b和总叶绿素含量的变化趋势相同,均呈下降-升高-下降趋势,但始终在空白处理时最高,最大盐浓度时最低。Na2SO4胁迫下叶绿素a和总叶绿素含量的变化趋势相同,呈下降趋势,叶绿素b与之不同,呈先升高后下降趋势。类胡萝卜素的含量变化没有规律性。认为,萌芽率、增殖倍数、平均株高、膜透性、盐害率、SOD、CAT、POD活性均可作为鉴定榅桲耐盐性评价指标,叶绿素可作为抗盐评价的参考指标。两种盐胁迫下,榅桲组培苗更能耐受NaCl胁迫,NaCl和Na2SO4分别在浓度为0.6%和0.8%时开始对榅桲组培苗造成严重伤害,在0.8%和1.0%时至死率超过50%。
4)榅桲种子耐盐性研究中,随着盐浓度的增大,发芽率和成苗率均呈下降趋势。NaCl胁迫时,SOD、CAT活性均随着盐浓度的增大呈先上升后下降趋势,POD活性呈上升趋势。Na2SO4胁迫时,随着盐浓度的增大POD、CAT活性呈先上升后下降趋势,SOD活性呈降低趋势。所以认为,不同种类盐胁迫下,榅桲种子的耐盐机制不同。随着盐浓度的增大,芽苗和培养基中的Na+均呈上升趋势,种皮中的Na+呈先下降后上升趋势。K+在各组织中的变化没有规律性。NaCl胁迫下,随着盐浓度的增加,芽苗和种皮中Na+/K+呈升高-降低趋势,但始终高于空白处理中Na+/K+的值,培养基中Na+/K+呈升高-降低-升高趋势。Na2SO4胁迫时芽苗和种皮中Na+/K+呈升高-降低-升高趋势,培养基中Na+/K+呈升高趋势。综合分析,发芽率、成苗率、SOD、CAT、POD、榅桲芽苗中Na+含量可作为榅桲种子耐盐性评价指标。K+、Na+/K+变化较复杂,能否作为榅桲种子耐盐性鉴定的指标还有待于研究。Na2SO4胁迫对榅桲种子发芽的影响比NaCl胁迫大。以盐胁迫浓度对榅桲种子发芽率和成苗率为标准来看,Na2SO4和NaCl浓度分别为100mmol和150mmol时严重影响榅桲种子发芽。
In this research , Cydonia oblonga Mill of apple model in XinJiang was used to study the reproduction approaches of tender-stem and cotyledon .Then used the tissue culture plants and seeds to have a primary discuss on the salt-tolerance of quince .In the experiment of reproduction , studed the effect on setting axenic explants,primary culture ,succeed culture and rooting culture of different disinfectant、sterilization time、culture medium、hormone combinations,etc.The study on salt-tolerance ,we discussed the effect on regeneration plant’s growth character, Physiological index of different salt , concentration and time .we also had studied on the effect on seed buding,enzyme and ion of salt-stress.The results showed that:
1)Using the tender-stem of Cydonia oblonga Mill to regeneration, HgCl2 0.1% for 5 min is the best for sterilization; The better Primary medium is MS+6-BA 0.6mg/L,its germination rate and multiplication multiple are all higher, they are 93.3% and 4.7; Using MS+6-BA0.6 mg/L +IBA0.2 mg/L and MS+6-BA0.6mg/L+IBA0.4 mg/L by turns is better for multiplication and strong seedlings; Rooting medium chose 1/2MS(I)+NAA1. 0 mg/L can achieve the highest 50% rootage rate and 7-10 average root number; Short dark treatment(7d) is benefit for rooting but as the time longer the leaf-burnt became more serious and even lead to dead of stem tip;(6)6-BA is not benefit for rooting and even lead to abnormal leaves.
2)Using the cotyledon of Cydonia oblonga Mill to regeneration, 1/4MS(I)+6-BA5mg/L+NAA0.5mg/L is best for planted regeneration ; regeneration rate is 77.5%;MS+6-BA 0.6mg/L+IBA0.2mg/L is better for multiplication , it can be about 4 times more ; active carbon and dipped IBA50mg/L 3S is benefit for rooting ,rootage rate is 75% and the number is more ,but the growing of roots is slowly ,shorter then 1cm after 30d .In the culture room and used earthen bowls to transplant young plants ,sawdust is medium ,new roots would come out in 15d and 78% could be alive after 30d .
3)In the research of regeneration plant salt-tolerance, the burgeon ratio ,multiplication multiple ,average tallness were all reduced along with the increasing degree salt-stress , MP and impair rate is increased . SOD、CAT、POD were all showed up-down except POD was showed down when Na2SO4 added .Under NaCl stress Chla ,Chlb and ChlT had the same movement ,showed down-up-down ,and highest at blankness lowest at most harvest salt concentration . Under Na2SO4 stress, Chla and chlT were all falled , Chlb was rised then falled . Chlx.c had no regular changed. We considered, burgeon ratio ratio, multiplication multiple,MP,impair rate SOD,CAT,POD can be the index of salt-tolerance estimation Cydonia oblonga Mill salt-tolerance estimation and Chl can be the reference index . Under the two salt stress, Cydonia oblonga Mill regeneration plants could be even more able to bear NaCl,and was injured hardly at 0.6% and 0.8% of NaCl and Na2SO4, 50% more died at 0.8% and 1.0% .
4) In the research of Cydonia oblonga Mill seed salt-tolerance, the bud ratio and the seeding rate were all decreased . Under NaCl stress, SOD、CAT were showed down along with the increasing degree salt-stress when POD was showed up . Under Na2SO4stress, POD、CAT were showed up-down when SOD showed down .So we think , Cydonia oblonga Mill seed had different mechanism for different kinds of salt . Na+ in young plant and medium are increased along with the increasing degree salt-stress when Na+ in seed capsule was showed down-up . K+ in all organises had no ruled change . Na+/K+ in young plant and seed capsule were showed up-down and higher than the Na+/K+ of blank . Na+/K+ in medium showed up-down-up . Under Na2SO4 stress, Na+/K+ in young plant and seed capsule were showed up-down-up,when Na+/K+ in medium showed up . For all , bud ratio , seeding rate, SOD, CAT, POD, Na+ in young plant can be the index of Cydonia oblonga Mill seed salt-tolerance estimation . The change of K+ and Na+/K+ is complex ,whether can be the index of Cydonia oblonga Mill seed salt-tolerance estimation need futhermore research . Na2SO4 is more disadvantaged to Cydonia oblonga Mill buding, and 100mmol /L and150mmol/L of Na2SO4 and NaCl can have serious effect on Cydonia oblonga Mill buding .
引文
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