枇杷花药胚胎发生发育、再生植株的RAPD分析及EMS诱变研究
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摘要
花药培养对植物育种具有重要的意义,是获得单倍体及纯合体植株最有效的方式之一。本研究以‘大五星'枇杷为试材,优化了枇杷花药胚胎发生及植株再生体系。在此基础上,对枇杷花药胚胎的发生、发育过程进行了观察;研究了枇杷花药胚胎发育过程中几种主要的代谢物质动态变化;对花药胚胎再生植株的遗传稳定性进行了RAPD分析;对花药胚胎进行了EMS离体诱变研究。主要的研究结果如下:
(1)‘大五星'枇杷花药,在一定条件下能够通过胚胎发生途径再生花药植株,本试验中采用的技术体系较为稳定。
(2)适当控制接种密度,枇杷花药在培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA 0.02mg/L,附加蔗糖70 g/L和琼脂6.5 g/L上,愈伤组织的形成率为68.14%,其形态多以脆嫩、较致密的浅黄色愈伤组织为主。诱导出的花药愈伤组织接近花药大小时,尽快剥离花药壁后接种到胚胎诱导培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA0.02mg/L,附加蔗糖30 g/L和琼脂6.5 g/L上,愈伤组织再分化形成胚胎的比率为58.33%。
(3)枇杷花药胚胎的增殖能力较强,能够满足获得大量次生胚的需要。在固体增殖培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA 0.02mg/L,附加蔗糖30 g/L和琼脂6.5 g/L上能获得5.39的平均增殖率,而在相同配方的液体震荡培养中能获得26.10的平均增殖率。液体培养在枇杷花药胚胎的增殖中具有一定的优势,能在短期内获得数量较多、发育时期相对一致的花药胚胎,且畸形胚的发生率很低。
(4)‘大五星'枇杷花药胚胎的萌发率和转化率较低,干燥处理对提高枇杷花药胚胎的萌发和成苗的作用重大。未经干燥处理的花药胚胎难以萌发,干燥处理后的花药萌发率为23.38%,其中正常萌发的胚胎仅为29.23%,正常萌发的胚胎最终能够形成完整植株比率为68.42%。
(5)对试管苗的染色体观察发现:部分植株为单倍体,部分植株为二倍体,其中单倍体占了较大比例。单倍体的获得证实了至少部分胚胎来自于花粉,而其中的二倍体则可能是单倍体的自然加倍,也可能是胚胎本身来自于花药壁愈伤组织。
(6)解剖镜下观察和石蜡切片观察都显示枇杷花药胚胎的形态发育包括球形胚、心形胚、鱼雷胚和子叶胚,同时还观察到部分子叶畸形的胚胎。石蜡切片显示:枇杷花药胚胎次生胚的起源有三种情况:直接起源于子叶胚表皮细胞;起源于子叶胚表皮下层细胞;起源于胚性愈伤组织细胞。当次生胚发生以直接途径进行时,次生胚起源于子叶胚表皮细胞和靠近表皮细胞的下层细胞,观察发现:其中多数是起源于靠近表皮细胞的下层细胞。当次生胚以愈伤组织间接途径进行增殖时,子叶胚愈伤组织化形成胚性愈伤组织,次生胚多起源于愈伤组织的内部细胞。胚性细胞均经历同样的从单细胞原胚到多细胞原胚的分化。
(7)培养方式对次生胚发生有一定的影响。固体培养时次生胚的增殖途径包括起源于表皮细胞的直接途径和起源于愈伤组织内部细胞的间接途径。液体震荡培养时,次生胚的增殖途径主要以起源于表皮和靠近表皮细胞的下层细胞的直接途径。固体培养基中的出现畸形胚的比率较高,而液体培养基中几乎没有畸形胚的出现。
(8)随着枇杷花药胚发育的进行,胚的体积不断增加,鲜重也随之增大,其中以鱼雷胚到子叶胚增加最多。在枇杷花药胚的整个发育过程中,干物质含量总体上表现为随着发育的进行逐渐上升。本试验研究发现:可溶性蛋白质、可溶性糖类、核酸三类物质与枇杷花药胚的发育与成熟密切相关。在枇杷花药胚胎发育的转折时期,都有可溶性蛋白质,可溶性糖类和核酸的变化。枇杷花药胚发育过程中蛋白质的合成动态,大致呈“S”形。枇杷花药胚在从胚性愈伤组织到球形胚阶段的早期胚时期一直保持较高的可溶性蛋白质水平。可溶性蛋白质的两次积累高峰分别出现在早期胚时期和子叶胚时期。在枇杷花药胚发育的不同时期及其成熟培养的过程中,碱性蛋白含量总体上表现为高于酸性蛋白质,且碱性蛋白的变化趋势和可溶性总蛋白变化相似。枇杷花药胚发育的过程中可溶性糖和果糖含量的变化动态相似,两次积累高峰分别出现在胚性愈伤组织时期和分化胚时期。在枇杷花药胚发育的过程中,DNA变化动态与总核酸变化动态相似,分别在胚性愈伤组织和分化胚出现两次积累高峰,DNA合成高峰先于RNA合成高峰出现。在胚性愈伤组织和子叶胚中,RNA的含量都很低,而DNA合成十分活跃。
(9) RAPD分析显示,在所采用的4个引物中多数植株的谱带高度一致,少数植株的谱带在不同引物中存在不同程度的差异,谱带缺失较为明显。通过对枇杷花药胚胎再生植株的RAPD分析初步表明:枇杷花药胚胎再生植株总体上遗传较为稳定,同时也有一定数量的变异发生,而关于变异的来源则有待进一步的研究。
(10)浓度为0.1%-0.7%的EMS处理0.5h、1h、2h后均能得到不同比例的存活胚胎,而0.9%相当于或接近EMS对枇杷花药胚胎的致死浓度。0.3%的EMS处理0.5h获得的存活率是所有EMS处理中存活率最高的,仅略低于对照,且与对照差异不显著。EMS处理后的材料产生新胚比率都显著低于对照。其中0.3%和0.5%的EMS处理后的材料都能获得较高的产生新胚的比率。各处理上产生的新胚均能继续增殖,并且增殖能力较对照有所提高。特别是0.7%的EMS处理的材料通过较其它处理更长时间的培养能产生新胚,新胚的增殖能力明显强于其它各处理。
(11) EMS诱发了枇杷花药胚胎有丝分裂异常的出现,随着EMS浓度的升高,畸变率逐渐上升。染色体畸变类型中以发生不均等分裂和染色体桥居多,其次为多极分裂和双核。而且一些细胞出现了染色体数目的变化,表现为染色体数目加倍的趋势。由此可见,EMS处理可能对枇杷花药胚胎染色体数目有一定的影响。
(12) EMS处理后的次生胚维持与对照相近的萌发能力。多数处理萌发率较对照略高,正常萌发率和对照差异不大,萌发情况和未经处理的枇杷花药胚胎之间没有明显的区别。EMS处理后的枇杷花药次生胚经萌发后,染色体数目变化较大。EMS导致了萌发的胚根中单倍体减少,二倍体增多,还产生了一定比例的3倍体(2n=3x=51)、4倍体(2n=4x=68)及非整倍体(2n+1、2n-1)。出现多倍体和非整倍体比例最高的是0.3%和0.5%的EMS处理2h,为16.67%。综合分析枇杷花药胚胎EMS诱变采用的最佳浓度为0.5%,处理时间为2h。
Anther culture has special significance for the genetic improvement of plants and has been proved as one of the best effective ways of obtaining haploid and homozygotic plants.In the present study,investigations were carried out of the anther culture and plant regeneration system,the embryogenesis and embryo development process,several primary physiological and chemical changes during the development of anther-derived embryos,RAPD analysis of regenerated plants and EMS in vitro mutation of anther-derived embryos in loquat(Eriobotrya japonica Lindl.cv.'Dawuxing').The main results are as follows.
(1) A stable and repeatable anther culture and plant regeneration system of loquat (Eriobotrya japonica Lindl.cv.'Dawuxing') was improved in this study.
(2) The callus induction rate was 68.14%on MS medium supplemented with 0.05mg·L~(-1)ZT,0.02mg·L~(-1)NAA,0.02mg·L~(-1)IBA,70 g·L~(-1) sucrose and 6.5g·L~(-1) agar.The rate of embryogenesis of anther callus was 58.33%on MS medium supplemented with 0.05mg·L~(-1) ZT,0.02mg·L~(-1) NAA,0.02mg·L~(-1) IBA,30 g·L~(-1)sucrose and 6.5g·L~(-1)agar.
(3) The multiplication ability of anther-derived embryos was strong.When cultured on MS solid medium supplemented with 0.05mg·L~(-1)ZT,0.02mg·L~(-1)NAA,0.02mg·L~(-1)IBA, 30 g·L~(-1) sucrose and 6.5g·L~(-1)agar,each primary embryo could produce 5.39 new embryos on the average.When cultured in MS liquid medium with the same supplements,the primary embryos averaged 26.10 new embryo formation.Compared with solid culture, the liquid culture helped produce abundant anther-derived embryos with comparatively identical development stages in short time and with few abnormal embryos.
(4) Though the germination rate and conversion rate of anther-derived embryos were low,the desiccation of anther-derived embryos was effective for improving the germination rate and conversion rate:the anther-derived embryos hardly germinated without desiccation,and after desiccation,the germination rate of anther-derived embryos was 23.38%,in which 29.23%of the embryos germinated normally and 68.42% of normally germinated embryos at last formed plantlets.
(5) Chromosome observation showed that most of plantlets were haploids,the others were diploids.The acquisition of haploids indicated that some of these embryos originated from microspores.
(6) Both microscope and histological observations showed that the anther-derived embryos of loquat developed through the typical globular,heart,torpedo and cotyledon stages.But some abnormal embryos were also observed.Histological observations showed that the origin of secondary embryogenesis of loquat anther had two ways:one was from epidermal cells and under epidermal cells and the other was from embryonic calluses.
(7) Culture methods had some effects on secondary embryogenesis.When adopting solid culture,the origin of secondary embryogenesis was from both epidermal cells and under epidermal cells of embryonic calluses.When adopting liquid shake culture,the origin of secondary embryogenesis was mostly from epidermal cells and under epidermal cells.The rate of abnormal embryos was quite high in solid culture medium,whereas in liquid culture medium abnormal embryos were seldom observed.
(8) In the whole developmental stages of anther-derived embryos,the water contents were high,and the dry mass contents were low.As a whole,the dry mass contents increased gradually with the anther-derived embryos development.Soluble sugars, soluble protein and nucleic acid were closely related to the development and maturation of loquat anther-derived embryos.In the important turning point of the development of loquat anther-derived embryos,soluble sugars,soluble protein and nucleic acid changed significantly in metabolism.During development of loquat anthers embryos,the dynamics of protein synthesis were roughly in "S" shape.The anther-derived embryos contained high soluble proteins from embryogenic calluses to globular embryos.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage. Alkaline protein contents were higher than acidic protein contents,and alkaline proteins contents and total soluble protein contents had the same change trend during the development and maturation of anther-derived embryo.Soluble sugar contents and fructose contents had the same change trend.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage.At early stage,soluble protein accumulation was due to the sugar transformation.After cotyledon stage,proteins were transformed to sugars in order to prepare energy for the anther-derived embryos' further development,maturation,germination and plant regeneration.RNA contents of anther-derived embryos were low at embryogenic callus stage and cotyledon stage. However,DNA were actively synthesized at the two stages.The DNA synthesis peak appeared earlier than that of RNA during the development of'anther-derived embryos.At the early development stage,the ratio of RNA to DNA ascended,which showed that the transcription and translation function were boosted up,and protein and ribosome accumulated gradually.But the ratio of RNA to DNA diminished gradually with the maturation of anther-derived embryos.
(9) The RAPD analysis with four primers showed that the bands of some regenerated plants were coincident,but of a few regenerated plants the bands were inconsistent obviously.RAPD analysis primarily showed that the anther-derived embryo regenerated plants had comparative hereditary stability.At the same time,mutation variations occurred in some of the regenerated plants.
(10) When EMS concentration ranged 0.1%-0.7%and the exposure time of treatment was 0.5h,1h and 2h,respectively,loquat anther-derived embryos could retain different survival rates.But 0.9%was approximately the deadly concentration of EMS to anther-derived embryos.Maximal survival rate(91.56%) was obtained in the treatment of 0.3%EMS for 0.5h.After EMS treatments,the percentage of primary embryos producing new embryos was remarkably lower than CK.The new embryos produced on all treatments could maintain good multiplication ability like CK.The anther-derived embryos needed more time to produce new embryos when EMS concentration was 0.7%. And these new embryos had stronger multiplication ability than others.
(11) EMS induced abnormal mitosis in anther-derived embryo cells and embryonic cells.The rate of abnormal mitosis of these cells increased with the EMS concentration. The abnormal mitosis included chromosome multipolar,dikaryon,chromosome bridge, chromosome multi-bridge,chromosome laggards,and chromosome unequal division.In some cells the chromosome number changed.
(12) The secondary embryos after EMS treatments had the approximately same germination rate and conversion rate as CK.Chromosome observation revealed that the chromosome number changed in some of the radicels germinated from anther-derived embryos with EMS treatment.After EMS treatment the haploid(n=17) proportion decreased,diploid proportion increased,and there were also certain frequencies of polyploids(2n=3x=51,2n=4x=68) and aneuploids(2n+1,2n-1).The maximal proportion (16.67%) of polyploids and aneuploids were obtained in the treatment of 0.3%and 0.5% EMS for 2h.
(1)‘大五星'枇杷花药,在一定条件下能够通过胚胎发生途径再生花药植株,本试验中采用的技术体系较为稳定。
(2)适当控制接种密度,枇杷花药在培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA 0.02mg/L,附加蔗糖70 g/L和琼脂6.5 g/L上,愈伤组织的形成率为68.14%,其形态多以脆嫩、较致密的浅黄色愈伤组织为主。诱导出的花药愈伤组织接近花药大小时,尽快剥离花药壁后接种到胚胎诱导培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA0.02mg/L,附加蔗糖30 g/L和琼脂6.5 g/L上,愈伤组织再分化形成胚胎的比率为58.33%。
(3)枇杷花药胚胎的增殖能力较强,能够满足获得大量次生胚的需要。在固体增殖培养基:MS+ZT0.05mg/L+NAA 0.02mg/L+IBA 0.02mg/L,附加蔗糖30 g/L和琼脂6.5 g/L上能获得5.39的平均增殖率,而在相同配方的液体震荡培养中能获得26.10的平均增殖率。液体培养在枇杷花药胚胎的增殖中具有一定的优势,能在短期内获得数量较多、发育时期相对一致的花药胚胎,且畸形胚的发生率很低。
(4)‘大五星'枇杷花药胚胎的萌发率和转化率较低,干燥处理对提高枇杷花药胚胎的萌发和成苗的作用重大。未经干燥处理的花药胚胎难以萌发,干燥处理后的花药萌发率为23.38%,其中正常萌发的胚胎仅为29.23%,正常萌发的胚胎最终能够形成完整植株比率为68.42%。
(5)对试管苗的染色体观察发现:部分植株为单倍体,部分植株为二倍体,其中单倍体占了较大比例。单倍体的获得证实了至少部分胚胎来自于花粉,而其中的二倍体则可能是单倍体的自然加倍,也可能是胚胎本身来自于花药壁愈伤组织。
(6)解剖镜下观察和石蜡切片观察都显示枇杷花药胚胎的形态发育包括球形胚、心形胚、鱼雷胚和子叶胚,同时还观察到部分子叶畸形的胚胎。石蜡切片显示:枇杷花药胚胎次生胚的起源有三种情况:直接起源于子叶胚表皮细胞;起源于子叶胚表皮下层细胞;起源于胚性愈伤组织细胞。当次生胚发生以直接途径进行时,次生胚起源于子叶胚表皮细胞和靠近表皮细胞的下层细胞,观察发现:其中多数是起源于靠近表皮细胞的下层细胞。当次生胚以愈伤组织间接途径进行增殖时,子叶胚愈伤组织化形成胚性愈伤组织,次生胚多起源于愈伤组织的内部细胞。胚性细胞均经历同样的从单细胞原胚到多细胞原胚的分化。
(7)培养方式对次生胚发生有一定的影响。固体培养时次生胚的增殖途径包括起源于表皮细胞的直接途径和起源于愈伤组织内部细胞的间接途径。液体震荡培养时,次生胚的增殖途径主要以起源于表皮和靠近表皮细胞的下层细胞的直接途径。固体培养基中的出现畸形胚的比率较高,而液体培养基中几乎没有畸形胚的出现。
(8)随着枇杷花药胚发育的进行,胚的体积不断增加,鲜重也随之增大,其中以鱼雷胚到子叶胚增加最多。在枇杷花药胚的整个发育过程中,干物质含量总体上表现为随着发育的进行逐渐上升。本试验研究发现:可溶性蛋白质、可溶性糖类、核酸三类物质与枇杷花药胚的发育与成熟密切相关。在枇杷花药胚胎发育的转折时期,都有可溶性蛋白质,可溶性糖类和核酸的变化。枇杷花药胚发育过程中蛋白质的合成动态,大致呈“S”形。枇杷花药胚在从胚性愈伤组织到球形胚阶段的早期胚时期一直保持较高的可溶性蛋白质水平。可溶性蛋白质的两次积累高峰分别出现在早期胚时期和子叶胚时期。在枇杷花药胚发育的不同时期及其成熟培养的过程中,碱性蛋白含量总体上表现为高于酸性蛋白质,且碱性蛋白的变化趋势和可溶性总蛋白变化相似。枇杷花药胚发育的过程中可溶性糖和果糖含量的变化动态相似,两次积累高峰分别出现在胚性愈伤组织时期和分化胚时期。在枇杷花药胚发育的过程中,DNA变化动态与总核酸变化动态相似,分别在胚性愈伤组织和分化胚出现两次积累高峰,DNA合成高峰先于RNA合成高峰出现。在胚性愈伤组织和子叶胚中,RNA的含量都很低,而DNA合成十分活跃。
(9) RAPD分析显示,在所采用的4个引物中多数植株的谱带高度一致,少数植株的谱带在不同引物中存在不同程度的差异,谱带缺失较为明显。通过对枇杷花药胚胎再生植株的RAPD分析初步表明:枇杷花药胚胎再生植株总体上遗传较为稳定,同时也有一定数量的变异发生,而关于变异的来源则有待进一步的研究。
(10)浓度为0.1%-0.7%的EMS处理0.5h、1h、2h后均能得到不同比例的存活胚胎,而0.9%相当于或接近EMS对枇杷花药胚胎的致死浓度。0.3%的EMS处理0.5h获得的存活率是所有EMS处理中存活率最高的,仅略低于对照,且与对照差异不显著。EMS处理后的材料产生新胚比率都显著低于对照。其中0.3%和0.5%的EMS处理后的材料都能获得较高的产生新胚的比率。各处理上产生的新胚均能继续增殖,并且增殖能力较对照有所提高。特别是0.7%的EMS处理的材料通过较其它处理更长时间的培养能产生新胚,新胚的增殖能力明显强于其它各处理。
(11) EMS诱发了枇杷花药胚胎有丝分裂异常的出现,随着EMS浓度的升高,畸变率逐渐上升。染色体畸变类型中以发生不均等分裂和染色体桥居多,其次为多极分裂和双核。而且一些细胞出现了染色体数目的变化,表现为染色体数目加倍的趋势。由此可见,EMS处理可能对枇杷花药胚胎染色体数目有一定的影响。
(12) EMS处理后的次生胚维持与对照相近的萌发能力。多数处理萌发率较对照略高,正常萌发率和对照差异不大,萌发情况和未经处理的枇杷花药胚胎之间没有明显的区别。EMS处理后的枇杷花药次生胚经萌发后,染色体数目变化较大。EMS导致了萌发的胚根中单倍体减少,二倍体增多,还产生了一定比例的3倍体(2n=3x=51)、4倍体(2n=4x=68)及非整倍体(2n+1、2n-1)。出现多倍体和非整倍体比例最高的是0.3%和0.5%的EMS处理2h,为16.67%。综合分析枇杷花药胚胎EMS诱变采用的最佳浓度为0.5%,处理时间为2h。
Anther culture has special significance for the genetic improvement of plants and has been proved as one of the best effective ways of obtaining haploid and homozygotic plants.In the present study,investigations were carried out of the anther culture and plant regeneration system,the embryogenesis and embryo development process,several primary physiological and chemical changes during the development of anther-derived embryos,RAPD analysis of regenerated plants and EMS in vitro mutation of anther-derived embryos in loquat(Eriobotrya japonica Lindl.cv.'Dawuxing').The main results are as follows.
(1) A stable and repeatable anther culture and plant regeneration system of loquat (Eriobotrya japonica Lindl.cv.'Dawuxing') was improved in this study.
(2) The callus induction rate was 68.14%on MS medium supplemented with 0.05mg·L~(-1)ZT,0.02mg·L~(-1)NAA,0.02mg·L~(-1)IBA,70 g·L~(-1) sucrose and 6.5g·L~(-1) agar.The rate of embryogenesis of anther callus was 58.33%on MS medium supplemented with 0.05mg·L~(-1) ZT,0.02mg·L~(-1) NAA,0.02mg·L~(-1) IBA,30 g·L~(-1)sucrose and 6.5g·L~(-1)agar.
(3) The multiplication ability of anther-derived embryos was strong.When cultured on MS solid medium supplemented with 0.05mg·L~(-1)ZT,0.02mg·L~(-1)NAA,0.02mg·L~(-1)IBA, 30 g·L~(-1) sucrose and 6.5g·L~(-1)agar,each primary embryo could produce 5.39 new embryos on the average.When cultured in MS liquid medium with the same supplements,the primary embryos averaged 26.10 new embryo formation.Compared with solid culture, the liquid culture helped produce abundant anther-derived embryos with comparatively identical development stages in short time and with few abnormal embryos.
(4) Though the germination rate and conversion rate of anther-derived embryos were low,the desiccation of anther-derived embryos was effective for improving the germination rate and conversion rate:the anther-derived embryos hardly germinated without desiccation,and after desiccation,the germination rate of anther-derived embryos was 23.38%,in which 29.23%of the embryos germinated normally and 68.42% of normally germinated embryos at last formed plantlets.
(5) Chromosome observation showed that most of plantlets were haploids,the others were diploids.The acquisition of haploids indicated that some of these embryos originated from microspores.
(6) Both microscope and histological observations showed that the anther-derived embryos of loquat developed through the typical globular,heart,torpedo and cotyledon stages.But some abnormal embryos were also observed.Histological observations showed that the origin of secondary embryogenesis of loquat anther had two ways:one was from epidermal cells and under epidermal cells and the other was from embryonic calluses.
(7) Culture methods had some effects on secondary embryogenesis.When adopting solid culture,the origin of secondary embryogenesis was from both epidermal cells and under epidermal cells of embryonic calluses.When adopting liquid shake culture,the origin of secondary embryogenesis was mostly from epidermal cells and under epidermal cells.The rate of abnormal embryos was quite high in solid culture medium,whereas in liquid culture medium abnormal embryos were seldom observed.
(8) In the whole developmental stages of anther-derived embryos,the water contents were high,and the dry mass contents were low.As a whole,the dry mass contents increased gradually with the anther-derived embryos development.Soluble sugars, soluble protein and nucleic acid were closely related to the development and maturation of loquat anther-derived embryos.In the important turning point of the development of loquat anther-derived embryos,soluble sugars,soluble protein and nucleic acid changed significantly in metabolism.During development of loquat anthers embryos,the dynamics of protein synthesis were roughly in "S" shape.The anther-derived embryos contained high soluble proteins from embryogenic calluses to globular embryos.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage. Alkaline protein contents were higher than acidic protein contents,and alkaline proteins contents and total soluble protein contents had the same change trend during the development and maturation of anther-derived embryo.Soluble sugar contents and fructose contents had the same change trend.The two accumulation peaks appeared at the embryogenic callus stage and differentiation stage.At early stage,soluble protein accumulation was due to the sugar transformation.After cotyledon stage,proteins were transformed to sugars in order to prepare energy for the anther-derived embryos' further development,maturation,germination and plant regeneration.RNA contents of anther-derived embryos were low at embryogenic callus stage and cotyledon stage. However,DNA were actively synthesized at the two stages.The DNA synthesis peak appeared earlier than that of RNA during the development of'anther-derived embryos.At the early development stage,the ratio of RNA to DNA ascended,which showed that the transcription and translation function were boosted up,and protein and ribosome accumulated gradually.But the ratio of RNA to DNA diminished gradually with the maturation of anther-derived embryos.
(9) The RAPD analysis with four primers showed that the bands of some regenerated plants were coincident,but of a few regenerated plants the bands were inconsistent obviously.RAPD analysis primarily showed that the anther-derived embryo regenerated plants had comparative hereditary stability.At the same time,mutation variations occurred in some of the regenerated plants.
(10) When EMS concentration ranged 0.1%-0.7%and the exposure time of treatment was 0.5h,1h and 2h,respectively,loquat anther-derived embryos could retain different survival rates.But 0.9%was approximately the deadly concentration of EMS to anther-derived embryos.Maximal survival rate(91.56%) was obtained in the treatment of 0.3%EMS for 0.5h.After EMS treatments,the percentage of primary embryos producing new embryos was remarkably lower than CK.The new embryos produced on all treatments could maintain good multiplication ability like CK.The anther-derived embryos needed more time to produce new embryos when EMS concentration was 0.7%. And these new embryos had stronger multiplication ability than others.
(11) EMS induced abnormal mitosis in anther-derived embryo cells and embryonic cells.The rate of abnormal mitosis of these cells increased with the EMS concentration. The abnormal mitosis included chromosome multipolar,dikaryon,chromosome bridge, chromosome multi-bridge,chromosome laggards,and chromosome unequal division.In some cells the chromosome number changed.
(12) The secondary embryos after EMS treatments had the approximately same germination rate and conversion rate as CK.Chromosome observation revealed that the chromosome number changed in some of the radicels germinated from anther-derived embryos with EMS treatment.After EMS treatment the haploid(n=17) proportion decreased,diploid proportion increased,and there were also certain frequencies of polyploids(2n=3x=51,2n=4x=68) and aneuploids(2n+1,2n-1).The maximal proportion (16.67%) of polyploids and aneuploids were obtained in the treatment of 0.3%and 0.5% EMS for 2h.
引文
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