欧石楠体细胞胚发生过程中的DNA甲基化

利用甲基化敏感扩增多态性（MSAP）方法,对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对,共扩增得到506条带,统计显示,大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31．42％、27．86％和29．05％,3种试材发生甲基化变异的有175条带,变异率为34．58％。体细胞胚诱导形成胚性愈伤组织过程中,甲基化水平降低,而在再生苗中有所恢复,与大田苗接近。在外侧胞嘧啶甲基化水平上,胚性愈伤组织的甲基化水平有所增加,且在再生苗中可部分维持。另外,在175条变异带中,再生苗恢复到大田苗DNA甲基化模式的有62条,占总变异条带的35．43％,而与胚性愈伤组织维持相同DNA甲基化模式的有59条,占33．71％。回收部分甲基化变异条带,最终得到8条有效的基因组DNA序列。BLASTnI：对分析表明,在欧石楠基因组中,包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA基化修饰现象。     

高温胁迫对不同热敏型辣椒同工酶及DNA甲基化的影响

分别以耐热型晚熟紫色辣椒‘7036’和热敏型早熟绿色辣椒‘9050’为材料,研究了40℃高温胁迫对其POD同工酶以及DNA甲基化表达的影响。结果显示:(1)在POD同工酶表达方面,未经高温胁迫的辣椒‘7036’比其它处理多出1条带,而同工酶活性在2种辣椒中呈现不同的变化趋势。(2)随着高温胁迫时间延长,辣椒‘7036’DNA无甲基化和全甲基化比率增加,半甲基化和总甲基化比率降低,而辣椒‘9050’只有高温处理7d(T_7)的甲基化水平变化与之类似,但半甲基化和全甲基化比率的绝对值远低于辣椒‘7036’。(3)高温处理7d的DNA甲基化模式中,辣椒‘7036’去甲基化C型条带比率较高,辣椒‘9050’以超甲基化B型条带为主。实验表明,高温胁迫下紫色辣椒‘7036’的POD同工酶活性恢复,半甲基化水平大幅下降和全甲基化水平快速上升以及去甲基化等变化可能与其耐高温胁迫特性相关。     

不同因素对草莓玻璃化试管苗恢复的影响

旨在探索草莓玻璃化试管苗恢复措施。以‘丰香’草莓玻璃化试管苗为材料,采用正交设计的方法,在继代培养基中添加不同浓度组合的活性炭、聚乙烯醇和钙离子(氯化钙),研究不同组合对草莓玻璃化试管苗恢复的影响,同时比较恢复后的正常试管苗与原来的玻璃化苗及正常苗的生理生化指标。结果表明,正交设计的9种处理间草莓玻璃化苗的恢复率差异显著,恢复率最高的是处理9(1 g/L活性炭+2 g/L聚乙烯醇+166 mg/L钙离子),恢复率为89.07%,其次是处理4(0.5 g/L活性炭+166mg/L钙离子),81.05%、处理8(1 g/L活性炭+1 g/L聚乙烯醇),73.87%。方差分析表明,活性炭、聚乙烯醇、钙离子浓度对草莓玻璃化苗恢复的影响都极为显著,影响大小依次是钙离子>活性炭>聚乙烯醇。恢复苗的各项生理生化指标与玻璃化苗差异显著,与正常苗差异不显著。结合各处理对玻璃化苗的恢复率、生理生化指标及增殖系数,综合考虑认为使草莓玻璃化试管苗恢复的最佳处理为处理9。     

紫斑牡丹与牡丹种间杂交后代的DNA分子证据

据推测紫斑牡丹Paeoniarockii (S .G .HawetL .A .Lauener)T .HongetJ .J.Li是直接或间接参与中国牡丹品种群起源的野生种之一 ,杂交是栽培牡丹品种的重要培育途径。但尚未见到DNA分子方面相关证据的报道。本研究以紫斑牡丹作母本 (♀ ) ,分别以 3个牡丹品种‘海棠争润’ (P suffruticosa‘HaiTangZhengRong’)、‘胭脂红’(P suffruticosa‘YanZhiHong’)和‘盛丹炉’ (P suffruticosa‘ShengDanLu’)作父本(♂ ) ,进行人工杂交 ,获得了杂交后代。利用DNAISSR (Inter simplesequencerepeats,简单重复序列间隔区 )标记技术构建的亲子代DNA指纹图谱显示 ,在杂交后代中检测到了分别来自双亲的特征带 ,因而在DNA水平上证实了花瓣基部带紫斑的栽培牡丹品种杂交起源的可能性     

5-azaC对小麦幼苗生长及盐胁迫后DNA甲基化变异的影响

DNA甲基化是调节植物生长发育,调控逆境基因表达的表观遗传机制之一。该研究采用不同浓度的DNA甲基化抑制剂5-azaC处理耐盐性不同的春小麦种子,分析其对种子萌发及盐胁迫后叶片基因组DNA甲基化的影响,探究DNA甲基化与小麦耐盐性之间的相关性。结果表明:(1)5-azaC显著抑制幼苗根长伸长,降低根系鲜重和干重。(2)甲基化敏感扩增多态性(MSAP)分析发现,单独盐胁迫后甲基化水平上升, 5-azaC预处理材料经盐胁迫后甲基化水平呈下降趋势。(3)盐胁迫后基因组同时发生DNA去甲基化和DNA甲基化。敏盐品种‘新春6号’DNA去甲基化比率上升,DNA甲基化增加的比率下降;耐盐品种‘新春11号’DNA去甲基化比率和DNA甲基化增加的比率均上升,但去甲基化比率大于DNA甲基化增加的比率,说明盐胁迫引起的基因组DNA去甲基化为主,5-azaC预处理提高了盐胁迫下DNA去甲基化的比率。(4)DNA甲基化修饰位点序列分析发现,在核糖体亚基蛋白、蛋白激酶和转座子序列均存在DNA甲基化修饰现象,说明存在多种代谢途径共同参与了盐胁迫调控。     

植物试管繁殖的成本与效益浅析

近30年来,植物细胞和组织培养技术有了飞跃的发展。1960年Morel用兰花茎尖离体培养获得脱病毒植株后,国内外相继建立了兰花工业。在兰花试管快繁高效益的刺激下,观赏植物、果树和园艺植物农林植物药用及工业原料植物的试管快繁和脱病毒技术的研究和应用取得很大的进展。国内外先后建立了试管苗产业,进行了规模化和商业化生产,这是植物组织培养应用最大、最有成效的一个方面［’,’‘,”,”,‘’＋‘’］。现在世界上几乎所有地区均在开展植物组织培养的研究与应用,大部分是研究植株的再生及快速繁殖“”””,’‘’“,‘”据报…     

‘章姬’草莓茎段愈伤组织诱导及高频植株再生体系的建立

该研究以‘章姬’脱毒苗带节匍匐茎段为外植体,以MS为基本培养基,进行单因素预实验,选取适合‘章姬’草莓生长的植物激素种类和质量浓度范围,进而通过L_9(3~4)正交实验研究不同植物激素种类及其质量浓度对愈伤组织诱导、丛芽发生及植株再生的影响。结果表明:采用的3种基本培养基中,B_5和1/2MS对抑制培养过程中的褐化现象明显优于MS,而附加20 g·L~(-1)Na_2S_2O_3在保证材料存活的前提下,大大降低了褐化率;去除褐化的材料在MS+0.1 mg·L~(-1)6-BA+0.05 mg·L~(-1)2,4-D+0.1 mg·L~(-1)NAA中可同步进行愈伤组织诱导和丛芽发生;丛芽增殖培养基为MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA,30 d后繁殖系数可达12.86;试管苗生根则在1/2MS+1.0 g·L~(-1)AC中进行,35 d后可获得生长健壮的再生植株,生根率92.50%。再生苗移栽成活率在95%以上。该研究建立了‘章姬’草莓体外高效快速繁殖体系,对短期内为‘章姬’草莓栽培提供大量种性稳定、质量优良的种苗具有重要意义,同时为其他草莓品种的体外快繁提供了技术参考。     

草莓高频离体再生体系的研究

以6个草莓品种为试材,研究了影响草莓不定芽再生的各种因素,建立离体叶片高效再生系统。结果表明,外植体基因型、激素种类及配比、叶龄等是影响草莓再生的主要因子,其中‘鬼露甘’叶片最佳芽诱导培养基为MS 2.0 mg/L 6-BA 0.1 mg/L IBA,‘嫜姬’叶片愈伤组织的诱导以MS 3 mg/L 6-BA 0.2 mg/L 2,4-D较好,而且1周左右的暗培养可以防止外植体的褐化。芽伸长的最适培养基为MS 0.5 mg/L 6-BA 0.5 mg/L IBA,生根的最适培养基为MS 0.2 mg/L IBA,试管苗移栽后成活率为87%。     

牡丹试管苗玻璃化的研究

以MS为基本培养基,研究品种、琼脂、6-BA、大量元素、蔗糖、光照等因素对牡丹试管苗玻璃化的影响。结果显示：不同品种的牡丹试管苗在组织培养的过程中,出现不同程度的玻璃化,增加培养基中琼脂浓度、降低细胞分裂素6-BA的浓度、降低大量元素的浓度和增加光照强度或采用自然光照均可以有效地降低试管苗玻璃化。     

多效唑对草莓种质离体保存的影响

取离体培养形成的草莓单芽接种到1／2MS附加不同浓度多效唑和0．5mg／L6—BA的培养基上,研究了多效唑对草莓试管苗生长及其保存的影响。结果表明,多效唑对草莓试管苗芽的分化有明显的促进作用,对草莓试管苗的伸长具明显的抑制作用。当多效唑浓度为0．04mg／L,苗高为对照(多效唑浓度为0mg／L)的29％～63％,当多效唑浓度较高(0．2mg／L)时,延迟了试管苗的发根,且抑制根的伸长。在继代培养中,多效唑抑制芽的分化,同时抑制苗的生长。在本试验中,不同浓度的多效唑对草莓试管苗保存成活率差异不十分明显,但野生草莓品种在离体保存中多效唑浓度不宜高,以0．1mg／L为好。     

Epigenetic instability in genetically stable micropropagated plants of <Emphasis Type="Italic">Gardenia jasminoides</Emphasis> Ellis

Gardenia jasminoides Ellis is an evergreen tropical plant and favorite to gardeners throughout the world. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides Ellis, the efficiency remained low. In addition, no information is available on the genetic and epigenetic fidelity of the micropropagated plants. Here, we report on a simplified protocol for high efficient micropropagation of G. jasminoides Ellis cv. “Kinberly” based on enhanced branching of shoot-tips as explants. The protocol consisted of sequential use of three media, namely, bud-induction, elongation and root-induction. By using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism (MSAP), we analyzed the genetic and DNA methylation pattern stability of 23 morphologically normal plants randomly taken from a sub-population (>100) of micropropagated plants originated from a single shoot-tip. We found that of >1,000 scored AFLP bands across the 23 micropropagated plants, no incident of genetic variation was detected. In contrast, of 750 scored MSAP bands, moderate but clear alteration in several DNA methylation patterns occurred in the majority of the 23 micropropagated plants. The changed methylation patterns involved both CG and CHG sites representing either hyper- or hypo-methylation, which occurred without altering the total methylation levels partly due to concomitant hyper- and hypo-methylation alterations. Our results indicated that epigenetic instability in the form of DNA methylation patterns can be susceptible to the in vitro micropropagation process for G. jasminoides Ellis, and needs to be taken into account in the process of large-scale commercial propagation of this plant.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.     

Morphological and molecular analysis of genetic stability in micropropagated Fragaria×Ananassa cv. pocahontas

Summary Micropropagated strawberry plants (Fragaria×ananassa L.) grown on 5 μM and 15 μM BA medium or cold-stored were grown in the field to examine morphological variation. Except for plant height, morphological characteristics did not differ for field-grown plants micropropagated on 5 μM and 15 μM BA medium. Cold-stored plants were less vigorous, both vegetatively and reproductively, than BA-treated plants. Random amplified polymorphic DNA (RAPD) markers were used to determine if cold storage or supraoptimal levels of N⁶-benzyladenine (BA) in the culture medium caused genetic changes leading to somaclonal variation. No mutations were observed in 246 loci amplified by the 29 primers tested. Possible changes in methylation patterns of ribosomal DNA genes of strawberries were also examined. Changes in methylation patterns were observed in only one DNA sample from plants grown on 15 μM BA medium and in one of the cold-stored plants. Length polymorphism was observed in two samples from plantlets derived from one explant. The low levels of RAPD variation and methylation observed, and the apparently epigenetic changes in morphological characteristics in plants used in this study, indicated that mutations had not occurred. Part of a thesis submitted by M. B. K. in partial fulfillment of the requirements for the MS degree. The use of trade names in this publication does not imply endorsement by the U.S. Department of Agriculture or Oregon State University.     

Epigenetics in plant tissue culture

Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at the molecular level, viz., changes in DNA methylation and alterations of histone methylation or acetylation. Various studies have been carried out with animals, and with plant cells or tissues that have grown in tissue culture but only little work has been done with shoots generated by axillary branching. We present various molecular methods that are being used to measure epigenetic variation. In micropropagated plants mostly differences in DNA methylation have been examined. Epigenetic changes are thought to underlie various well-known tissue-culture phenomena including rejuvenation, habituation, and morphological changes such as flower abnormalities, bushiness, and tumorous outgrowths in, among others, oil palm, gerbera, Zantedeschia and rhododendron.     

Consistent and Heritable Alterations of DNA Methylation Are Induced by Tissue Culture in Maize

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.     

Effect of in vitro shoot multiplication and somatic embryogenesis on 5-methylcytosine content in DNA of Myrtus communis L.

Reversed-phase HPLC analysis of 2-deoxynucleosides was performedto study the amount of 5-methylcytosine in genornic DNA of Myrtuscommunis L. About 11% cytosines were found to be methylated inDNA of field growing shoots. This amount showed no variation after theestablishment of shoots in vitro or their subsequentrooting and acclimatisation to ex vitro conditions.Therefore, adult elite plants can be micropropagated and transferred to thefield without global DNA methylation changes. Likewise, no trend in5-methylcytosine content was introduced by increasing the number of subculturesin either adult- or seedling-originated shoot stocks. On the other hand, nodifference was found in DNA methylation after plant regeneration fromembryogenic calli. The significance of a tissue culture model system with noglobal hypo- or hypermethylation is discussed.     

Genetic analysis of enhanced-axillary-branching-derived Eucalyptus tereticornis Smith and E. camaldulensis Dehn. plants

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. Received: 8 October 1996 / Revision received: 22 July 1997 / Accepted: 30 July 1997