【作者】 由翠荣；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2009， 博士

【摘要】 本论文以仙客来（Cyclamen persicum Mill）已开花植株的新生叶片和叶柄为外植体,系统研究了仙客来体细胞胚的诱导条件并建立了完整的仙客来体细胞胚胎发生和发育体系;在此基础上,选择各发育期的体细胞胚为外植体,进一步建立了以球形胚为外植体的体细胞胚高频、高效诱导体系;同时以球形胚直接投放液体培养成功建立了胚性悬浮体系并筛选获得另一胚性丧失体系。以上述不同胚性培养体系为材料,对仙客来SERK基因（CpSERK）在体细胞胚性转化过程中的表达特性进行了初步的研究,获得的研究结果如下:以仙客来“葡萄酒红火焰纹”（Cyclamen cv“Wine Red Flame”）的新生叶片为外植体,成功诱导、筛选出胚性愈伤组织。所选择的愈伤组织诱导培养基为1/2MS附加2,4-D(2-4mg.L^-1)和2iP( 0.2-0.8mg.L^-1),胚性愈伤组织继代增殖培养基1/2MS+ 2 , 4-D2mg.L^-1+6-BA0.2mg.L^-1和1/2MS+2 , 4-D2mg.L^-1 +2iP0.2mg.L^-1。初培养所获得的松散愈伤组织经过3-4次的继代培养后,根据颜色、松散程度、水渍化程度、细胞的形态特征等指标进一步分类筛选。通过胚性能力的检测,证明并获得了两种不同形态的胚性愈伤组织,分别为棕红色松软愈伤组织和松脆愈伤组织,胚性愈伤组织固体继代一年以上仍能保持其胚性能力。将两种胚性愈伤组织进一步继代培养于不含激素的1/2MS培养基上,14d左右,体细胞胚开始在愈伤组织表面发生。松软愈伤组织的体细胞胚均以多发生的状态出现,一丛可达几十个甚至上百个。松脆愈伤组织的体细胞胚以单发生和多发生两种状态出现,即使多发生,体细胞胚发育的数目最多只有十几个。体细胞胚发生和发育具明显的不同步化,同一丛体胚中会出现球形胚、心形胚、鱼雷胚甚至成熟胚并存的现象,大多数体胚60-90d可发育成完整的小植株。连续7-8次继代培养的胚性愈伤体胚正常率为34.7%,随着继代次数的增加,胚性愈伤组织的胚性能力和正常体胚率明显下降,继代培养一年以上的胚性愈伤组织,其体胚正常率下降到7%以下。对两种胚性愈伤组织和松散的非胚性愈伤组织进行了活体细胞的比较观察,结果表明,胚性和非胚性愈伤组织中均含有不同大小的细胞和细胞团。两种胚性愈伤组织细胞中均含有胚性细胞和非胚性细胞,但数量上存在明显的差异。松软胚性愈伤组织多为含有数十个胚性细胞的胚性细胞团,而松脆胚性愈伤组织中的胚性细胞团胚性细胞数目只有几个或十几个,且在随机选取的相同视野中胚性细胞团的数量前者明显多于后者。通过连续的活体细胞追踪观察发现,胚性细胞团起源于胚性母细胞。一个胚性母细胞通过持续分裂形成包含有几个或十几个胚性细胞的胚性细胞团,而包含有几十个胚性细胞的胚性细胞团来源于成团存在的几个胚性母细胞。对体细胞胚胎发生和发育整个过程进行了细胞和组织学追踪,结合活体细胞和石蜡包埋切片技术显微观察结果表明,该培养体系的体细胞胚胎属于单细胞发生体系。体细胞胚的发生起源于胚性细胞团表面的单个胚性细胞,经历了二分体、四分体、八分体、多细胞原胚、球形胚、心形胚和鱼雷胚等时期,发育成完整植株。仙客来体细胞胚发育植株再生方式有两种:第一种为胚性细胞经多细胞原胚、球形胚、心形胚和鱼雷胚再生为小植株;第二种再生方式为球形胚不经过鱼雷胚而发育先形成球茎,然后是根或芽的形成,最后形成一完整植株。利用组织化学切片技术对体细胞胚发生和发育过程中淀粉的代谢进行了动态跟踪观察。结果显示在体细胞胚胎整个发生发育期内出现了4个淀粉粒积累的高峰期,分别为胚性细胞期、球形胚期、早期鱼雷胚期和体细胞胚再生为小植株的球茎和不定芽的发育期,淀粉的积累和消减与体细胞胚的发生、发育密切相关。为进一步研究高频率体胚体系,以再生胚胎为外植体,采用上述的胚性愈伤组织继代培养基1/2MS+2,4-D 2mg.L^-1 +6-BA 0.2 mg.L^-1和1/2MS+2,4-D 2 mg.L^-1 +2iP 0.2 mg.L^-1使其重新脱分化,初培养25-28d即成功诱导获得了胚性愈伤组织。在无激素的基本培养基上促使体细胞胚的发育,实验结果表明,除小球形胚外,以不同再生胚胎为外植体初培养愈伤组织诱导率均达95%以上,而且初代培养获得的愈伤组织就具备胚性能力。比较不同再生胚胎诱导获得的愈伤组织的胚性能力,表现为以大球形胚为外植体获得的胚性愈伤组织胚性能力最佳,以小球形胚和小无极球为外植体次之,无激素培养基培养30d每克愈伤组织获得体细胞胚数分别为734、423和342个,来源于单个胚胎外植体的体胚发生总数为118、80、69;同时,相同类型体细胞胚为外植体获得的愈伤组织的胚性能力个体间存在显著差异。以大球形胚为外植体,研究继代培养次数对胚性愈伤组织胚性能力的影响。1/2MS基本培养基培养50d,根据对体细胞胚的发生率、每克愈伤组织体胚发生总数、来源单个再生胚胎外植体的体胚发生总数以及正常体胚率四个指标综合考察,继代次数对体胚发生率无显著影响,但对愈伤组织的胚性能力和体胚正常率影响显著。胚性能力最强的愈伤组织为继代一次的愈伤,体细胞胚的发生率、每克愈伤组织体胚发生总数、来源单个再生胚胎外植体的体胚发生总数以及正常体胚率分别为100%,3304/g.FW,1296/ g.FW,8.54%。以大球形胚为外植体诱导获得的胚性愈伤组织为实验材料,以蔗糖浓度(0, 10, 30, 60, 90, 120g.L^-1)和不同的ABA浓度(0, 0.2, 0.5, 1.0, 2.0mg.L^-1)为指标,对体细胞胚的发育进行了进一步的条件优化。结果表明,1/2MS基本培养基中附加一定浓度的ABA可显著提高体细胞胚的正常率,但随着ABA浓度的增加,2.0mg.L^-1ABA明显抑制体细胞胚的发生,体胚发生数仅为292/ g.FW;培养基中附加0.5 mg.L^-1ABA,每克愈伤组织体细胞胚的发生数量为648,正常体胚率为12.65%;而未附加ABA的对照组,每克愈伤组织体细胞胚发生数为957,体胚的正常率仅有4.09%。体细胞胚的发生和发育均需要在基本培养中附加适量浓度的蔗糖,以体细胞胚的发生数量和正常体胚率两个指标综合考察,适宜范围为培养基附加30-60g.L^-1的蔗糖;其中60g.L^-1蔗糖胚性愈伤组织体胚的发生数量和正常体胚率均为最高,分别为1384/ g.FW,8.24%。蔗糖浓度低于10g.L^-1或高于120 g.L^-1均显著抑制体细胞胚的正常发育。以再生球形胚为外植体采用直接液体投放的方式进行悬浮培养建立胚性悬浮体系,结果表明,球形胚初代液体培养即可脱分化形成大量的胚性母细胞,2-3次继代胚性母细胞分裂形成胚性细胞团。悬浮培养体系的最佳接种量为5%PCV浓度,最适继代时间为14d;此胚性悬浮体系连续继代培养一年以上仍具有胚性能力,且获得的胚性细胞在无激素的液体和固体培养基中均能发育;液体培养基中体胚的发育、植株再生方式只有第二种方式,而固体发育时两种发育、再生方式兼备。悬浮培养一年半（36代）筛选获得的米黄色细胞团块经两种不同方式验证,已完全丧失胚性和体胚发生的能力,但可以稳定增殖。以上述获得的不同胚性体系为实验材料,研究仙客来体胚发生、发育相关基因的特异表达,利用Genbank搜索到的仙客来SERK基因cDNA序列,根据基因的保守序列利用Primer Premier 5.0软件设计了SERK基因的两对特异引物（SKF1/SKR1或SKF2/SKR2）,克隆获得两个SERK基因cDNA片段,分别命名为CpSERKa和CpSERKb;测序结果显示cDNA长度分别为464bp,编码154aa和768bp,编码255aa。CpSERKa和CpSERKb在核苷酸水平上有89.2%的相似性,而在蛋白质水平上相似性可达90.9%,是两个不同的SERK基因。获得的基因序列在GenBank比对搜索,结果显示CpSERKa与该物种的CpSERK3同源性最高,核苷酸水平相似性为98.5%,而蛋白质水平相似性为98.1%,推测可能为该基因的部分序列;而CpSERKb为与CpSERK3同源性很高的另一新的家族成员。CpSERKa和CpSERKb与拟南芥、胡萝卜、马铃薯和蜜柑等物种具有较高的同源性,在分子系统进化树中与AtSERK3邻近,位于同一分支,可能属于同一SERK基因家族。在不同的胚性体系中,CpSERKa和CpSERKb在具有胚性发生能力的组织和细胞中均有表达,而未获得或丧失胚性能力的组织中均不表达。对体细胞胚胎不同发育时期的表达结果表明,在多细胞原胚和球形胚期也有明显的表达; CpSERKa在鱼雷胚期仅表现出微弱的表达,而CpSERKb则没有表达产物产生。更多还原

【Abstract】 A protocol for somatic embryogenesis and development has been carried out,using young leaves and petioles from flowering plants as explants in Cyclamen persicum Mill. Another rapid and high-frequency somatic embryogenesis system has also been successfully achieved from globular embryo-explants. And an embryogenic suspension culture is further established by inoculating globular embryos into liquid medium. Meanwhile expression pattern of Cyclamen somatic embryogenesis receptor-like kinase （CpSERK） is investigated during acquisition of embryogenic competence of somatic cell from liquid and solid culture systems.The results show that embryogenic calli have been successfully induced and identified. The auxin 2,4-D (2-4mg.L^-1) and 2iP (0.2-0.8mg.L^-1) in half strength MS medium are important factors for the induction of somatic embryogenesis. The optimal sub-cultural media for embryogenic callus are supplemented with 1/2MS +2,4-D2mg.L^-1 + 6-BA0.2mg.L^-1 or 1/2MS+2,4-D2mg.L^-1 + 2iP0.2mg.L^-1. According to the color, friability, watery grades and morphological character et al., calli are classified and respectively proliferated after 3-4 sub-cultural periods on induction medium. Two types of embryogenic callus are verified and obtained, which one is white-brown and soft, and the other is similar to the former except for friability. Embryogenic competence of both calli can be retained for more than one year.Somatic embryos start to appear on the surface of both embryogenic calli on the same medium without growth regulators for about 14 days. Somatic embryos occur in clusters from soft embryogenic callus, and several scores of embryoids are observed in one cluster. However, somatic embryos from friable embryogenic callus occur in clusters in more cases, or occasionally in individual way, with a few or more than ten embryoids in one cluster. Somatic embryos developed asynchronously in the same cluster, containing different developmental stage of embryoids, such as globular embryos, heart-shaped embryos, torpedo-shaped embryos and even mature embryos. Majority of somatic embryos can be regenerated into whole plantlets for 60-90 days. Normal embryo frequency is 34.7% from embryogenic calli after 7-8 periods of subculture. Embryogenic competence and normal embryo frequency have decreased remarkably as subculture prolongs, being only below 7% of normal embryo frequency for more than one year.The observations of viable cells show that both embryogenic and non-embryogenic calli comprise different sizes and shapes of cells and cell aggregates. The number of embryogenic and non-embryogenic cells is obviously different in the two types of embryogenic calli. The soft embryogenic callus mostly comprises aggregates with more than several scores of embryogenic cells, but the friable embryogenic callus contains aggregates with several or more than ten embryogenic cells. And the number of embryogenic aggregates in the former callus is more than that of the latter callus in the same random scope. Embryogenic aggregates are derived from mother embryogenic cells by time-lapse track of viable cell. Embryogenic aggregates with several or more than ten cells are originated from an embryogenic mother cell by continuous division, but embryogenic aggregates with more than several scores of embryogenic cells could be from a few of mother embryogenic cells in groups.Histological and cytological analysis during somatic embryogenesis of cyclamen show that somatic embryo is originated from the single embryogenic cell, being developed into a whole plantlet through different stages, such as two-cell proembryo, four-cell proembryo, eight-cell proembryo, multicellular proembryo, globular, heart-shaped, torpedo-shaped embryo. The two pathways have been observed during cyclamen somatic embryo development and plant regeneration. The first pathway is that a whole plantlet is regenerated through different developmental stages, such as embryogenic cell, multicellular proembryos, globular embryo, heart-shaped embryo and torpedo-shaped embryo. The second pathway is that a tuber is firstly formed from globular embryo without torpedo-shaped embryo, then roots or buds appear, finally a whole plantlet is regenerated.Dynamic changes of starch metabolism are investigated during the development and regeneration of somatic embryos. Four accumulation peaks of starch grains respectively appear at the stage of embryogenic cells, globular embryos, the early torpedo-shaped embryos and the developing tuber of a plantlet, which may be closely related to somatic embryogenesis, development and plant regeneration for energy andsubstance. Embryogenic calli are successfully induced after 25-28 days from regenerated somatic embryos-explants on the above sub-cultural media(1/2MS+2,4-D 2mg.L^-1 +6-BA 0.2 mg.L^-1 and 1/2MS+2,4-D 2 mg.L^-1 +2ip 0.2 mg.L^-1). Primary callus percentages come to above 95%, except for small globular embryo-explants, using different types of somatic embryos regenerated from leaf-explants as explants. The initial calli have embryogenic capacity, which is optimal in embryogenic callus from large globular embryo-explants, secondly from small non-polar tuber-explants and small globular embryo-explants. The number of somatic embryos from the above three embryo-explants is respectively 734/g.FW, 423/g.FW and 342/g.FW and that of embryogenic calli is 118, 80 and 69 from single embryo-explants on free-growth-regulators medium for 30 days. Embryogenic competence shows significant difference among individuals from the same type of embryo-explants.Effects of sub-cultural periods on embryogenic competence are studied as large globular embryo explants. The results show that sub-cultural periods have no effects on embryogenesis percentage, but significant effects on embryogenic competence and normal somatic embryo frequency on the-free-growth-regulator medium for 50 days. Calli for the first sub-cultural period show the best embryogenic competence, and the somatic embryogenesis percentage, the total number of somatic embryos of per gram callus, the total number of somatic embryos from single embryo-explants and the frequency of normal somatic embryos are respectively 100%, 3304/g.FW, 1296/ g.FW and 8.54%.Effects of different concentration of sucrose (0, 10, 30, 60, 90, 120g.L^-1) and ABA(0, 0.2, 0.5, 1.0, 2.0mg.L^-1))have been evaluated on late development of somatic embryos. The frequency of normal somatic embryo has remarkably increased on the 1/2MS media supplemented with appropriate concentration of ABA. Higher concentration of ABA can clearly inhibit somatic embryogenesis, and the average number is only 292/g.FW on the medium with ABA 2.0 mg.L^-1. ABA (0.5 mg.L^-1) is the most effective for development of somatic embryos, which the average number is 648/g.FW and normal embryo frequency is 12.65%. The normal embryo frequency （4.09%）on the control medium was low as compared to the frequency of 9.92–12.65% on experimental medium supplemented with ABA. Appropriate concentration supplement of sucrose (30-60 g.L^-1) is also helpful to somatic embryogenesis and development. The total number of somatic embryos and the normal embryo frequency are optimal on the medium with 60 g.L^-1 sucrose, respectively 1384/ g.FW and 8.24%. Development of somatic embryos can be inhibited on the medium with less than 10g.L^-1 or more than 120 g.L^-1 of sucrose.Embryogenic suspension culture has been successfully established, using regenerated globular embryos as starting materials inoculated directly in liquid medium. Globular embryo-explants are dedifferentiated into embryogenic mother cells after initial suspension culture, from which embryogenic cell aggregates are derived for 2-3 periods of subculture. The optimal inoculated density is 5% PCV （packed cell volume） and the optimal period of subculture is 14 days in the suspension system. Somatic embryos have developed on solid or liquid media without growth regulators, and embryogenic competence in suspension culture can be retained for more than a year. The developmental pathway of somatic embryos belongs to the second pathway in liquid culture, but both developmental pathways occur on solid medium. It is further verified that yellow cells aggregates have completely lost embryogenic competence with better proliferation capacity.Specific expression of the related genes with somatic embryogenesis and development is investigated using the above different embryogenic systems as experimental materials. Two pairs of specific primers of SERK genes （SKF1/SKR1 或SKF2/SKR2）are designed using Primer Premier 5.0 software, according to the cDNA conserved sequences of cyclamen SERK genes which are BLAST searched against the GenBank database at the National Centre for Biotechnology Information （NCBI）. Two putative SERK gene fragments are cloned and named CpSERKa and CpSERKb, respectively. The 464bp fragment is CpSERKa, coding 154 amino acids and the 768bp fragment is CpSERKb, coding 255 amino acids. The two CpSERK fragments are distinct but similar at both nucleotide level （89.2% similarity） and protein level （90.9% similarity） within the overlapped segment. The results from GenBank BLAST search and multiple sequence comparison reveal that CpSERKa is most similar to the SERK homologue CpSERK3 in the same species, with the similarities of 98.5% at nucleotide levels and 98.1% at protein levels. And CpSERKa is the partial sequence of CpSERK3, but CpSERKb is the other member of the same family. The results are confirmed by the phylogenic analysis in which CpSERKa, CpSERKb and CpSERK3 are tightly grouped together adjacent to AtSERK3. CpSERKa and CpSERKb are also highly homologous to SERK genes in Daucus carota, Solanum tuberosum and some other species.Expression of both CpSERKa and CpSERKb has successfully detected in tissues and cells of embryogenic competence from different embryogenic systems, but not in non-embryogenic callus and cell aggregates of losing embryogenic competence. The results show that SERK genes are also expressed in multicellular proembryos and globular embryos. The slight expression of CpSERKa is detected, but CpSERKb is not expressed at the stage of torpedo-shaped embryos.更多还原