脐橙与柠檬种间细胞电融合再生杂种植株

‘纽荷尔’脐橙（Ｃｉｔｒｕｓｓｉｎｅｎｓｉｓ（Ｌ．）Ｏｓｂｅｃｋ）胚性细胞悬浮原生质体与‘尤力克’柠檬（Ｃｉｔｒｕｓｌｉｍｏｎ（Ｌ．）Ｂｕｒｍ．ｆ）叶肉原生质体经电场诱导融合,培养８个月,首批再生了１棵植株,形态学观察,染色体计数及ＲＡＰＤ分析证明异源四倍体体细胞杂种,该杂种的获得为三倍体无籽柠檬品种培育提供了杂交亲本。     

柑橘细胞电融合参数选择及种间体细胞杂种植株再生

以“Ｐａｇｅ”橘柚（ＣｉｔｒｕｓｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｐａｒａｄｉｓｉＭａｃｆ）胚性悬浮系原生质体和粗柠檬（Ｃ．ｊａｍｂｈｉｒｉＬｕｓｈ）叶肉原生质体为融合试材,摸索了适合柑橘的电融合参数。研究表明,在ＡＣ（交变电场）１２５Ｖ／ｃｍ、ＤＣ（直流脉冲）１２５０Ｖ／ｃｍ、ＡＣ作用时间６０ｓ、ＤＣ脉冲时间５０μｓ、ＤＣ脉冲个数为３、ＤＣ脉冲间隔为０．５ｓ时融合率较高,双核异核体率可以达到１５％以上。利用不同类型原生质体比重的不同,融合后通过控制离心时间,可以收集到更高比例的双核异核体。融合产物经培养,３～４个月再生小植株。染色体计数结合形态学观察表明,再生的１５０株小植株中７８％为四倍体,其余为二倍体叶肉亲本再生类型。再生植株经过氧化物酶同工酶及ＲＡＰＤ分析,表明得到了体细胞杂种,还证明获得了１株叶肉亲本同源四倍体再生类型。     

原生质体电融合再生柑橘属间体细胞杂种

电场诱导酸橙(CitrusaurantitumL.)叶肉原生质体与澳洲指橘(MicrocitruspapuanaSwingle)悬浮系原生质体融合,融合产物经培养再生出具有三种叶片形态的植株,绝大部分与酸橙叶片完全相似(即叶肉亲本型植株);有2棵植株叶片大且厚;有1棵植株出现二叶和三叶。细胞学检查表明这些植株均为二倍体。采用RAPD标记对前两种植株进行杂种特性分析,共选用4个10-mer随机引物进行扩增。在引物OPAA-17的扩增产物中,再生植株的带型与酸橙一致;在引物OPA-08的扩增产物中,再生植株的带型与酸橙或澳洲指橘相似;而在引物OPA-04和OPA-07的扩增产物中,再生植株的带型出现三种情况,4个引物的扩增结果证明所分析的植株均是体细胞杂种。综合染色体检查和RAPD标记的结果,表明获得了酸橙与澳洲指橘属间二倍体体细胞杂种植株。     

电融合法产生骆驼刺与鹰嘴紫云英属间体细胞杂种

采用电融合法获得了骆驼刺和鹰嘴紫云英属间体细胞杂种。骆驼刺原生质体来自发根农杆菌A4转化系细胞,并经碘乙酰胺处理;鹰嘴紫云英原生质体从甲硫氨酸抗性系细胞分离。双亲及同源融合产物均不能在无激素的筛选培养基上持续分裂,融合后的杂种细胞由于双亲生理互补效应可恢复持续分裂能力而被筛选出来。本实验优化了电融合参数,如直流脉冲、交流脉冲和脉冲次数。融合产物经培养获得的杂种细胞系经形态学、染色体数目检查、生化及随机扩增多态性DNA分析,鉴定出10个杂种克隆,并从3个杂种克隆再生了小植株。     

原生质体融合获得柑桔种间体细胞杂种

粗柠檬(Citrus jambhiri Lush)叶肉原生质体与哈姆林甜橙(C.sinensis L.Osbeck)胚性悬浮细胞系原生质体经PEG诱导融合,培养7天时原生质体恢复分裂。再生的胚状体在含有GA_3的培养基中萌发出茎芽。茎芽经生根诱导成为完整植株。对首批再生的5棵植株进行染色体检查,结果表明,全为四倍体,2n=4x=36。淀粉胶电泳分析过氧化物酶同工酶,结果显示这5棵植株为体细咆杂种。粗柠檬和哈姆林甜橙在该位点上均为同质结合,基因型分别是MM和FF。体细胞杂种含有双亲的酶带,基因型为MMFF。杂种植株生长旺盛,根系发达,叶片及植株形态介于双亲之间。本文对其作为砧木品种的可能性等问题作了讨论。     

澳洲指橘与柑橘属间原生质体电融合再生二倍体体细胞杂种

电场诱导粗柠檬（CitrusjambhiriLush,2n＝2x＝18）叶肉原生质体与澳洲指橘（MicrocitruspapuanaSwingle,2n＝2x＝18）悬浮系原生质体融合,融合产物培养后再生出丛芽,经试管嫁接得到完整植株。再生植株的细胞学检查表明它们具有18条染色体,为二倍体;植株的叶片形态与叶肉亲本（粗柠檬）一样;用6个10-mer随机引物分析再生植株的杂种特性：在4个引物（OPA－07、OPAN－07、OPE－05和OPA－08）的扩增带型图中,再生植株的带型与粗柠檬完全一样,澳洲指橘的特征带未在植株中出现;在引物OPS-13和引物OPA－04的扩增带型图中,再生植株都具有澳洲指橘的特征带。细胞学和RAPD分析的结果表明,通过对称融合得到了澳洲指橘与粗柠檬的属间二倍体体细胞杂种植株。这是柑橘属间对称融合再生二倍体叶肉亲本类型植株的首例报道。     

RAPD分析─鉴定柑桔体细胞杂种的快速方法

本文利用改进的DNA提取方法,从Volkamer柠檬(Citrus volkameriana Ten. and Pasq.)和酸橙(C. aurantium L.)及其原生质体杂种植株的叶片中抽提总DNA,进行RAPD(Random Amplified Polymorphic DNA)分析。结果表明: 在随机选取的15种引物中,有10种可单独或与其它引物一道鉴定这一组合的体细胞杂种。与形态学性状观察、同工酶及ONA杂交分析等方法比较,RAPD分析是一种可在试管苗期即可直接、准确、快速鉴定柑桔体细胞杂种的方法。     

原生质体电融合再生柑橘属间体细胞杂种

Leaf derived protoplasts of Sour orange (Citrus aurantium L.) were fused electrically with embryogenic protoplasts of Microcitrus papuana Swingle. Plants were regenerated from the fusion products, which were characteristic with three types of leaf morphology. Most of the plants were identical to Sour orange (namely, Leaf-parent-type plant) and two plants had large and thick leaves whereas one plant had bifoliate and trifoliate leaves. Chromosome examination showed that these plants were diploid with 18 chromosomes (2n = 2x = 18). RAPD analysis was employed to verify the hybrid characteristics of the plants in the first two types. Four 10-mer arbitrary primers with polymorphism were chosen. Band pattern of the plants was similar with the leaf parent (Sour orange) for the primer OPAA-17. Band pattern of the plants was similar with either Sour orange or M. papuana for OPA-08. As for OPA-07 and OPA-04 three kinds of band profiles were detected. Results of RAPD marker, together with chromosome determination, indicated that all of the analyzed plants were intergeneric diploid somatic hybrids between Sour orange and M. papuana.     

柑橘与黄皮属间体细胞杂种植株的异常倍性变异

黄皮属（Ｃｌａｕｓｅｎａ）是芸香科柑橘亚科黄皮族植物,有３０余个种,其中１１个种原产于中国。黄皮在我国广东、广西、福建等省区有大面积商业栽培,其中鸡心黄皮是最著名的品种［１］。本研究以柑橘属优良脐橙品种“朋娜”为胚性愈伤组织亲本,以黄皮属品种鸡心黄皮...     

柑橘与枳属间体细胞杂种再生及其对脚腐病抗性的评价

Page橘柚 (CitrusreticulataBlanco×C .paradisiMacf.)胚性细胞悬浮系原生质体与枳 (Poncirustrifoliata (L .)Raf.)叶肉原生质体经电场诱导融合 ,4～ 5个月再生 15 0余棵小植株。再生植株根系发达 ,叶片具三叶特征。随机检查 2 0余株再生苗根尖染色体数目 ,表明都为四倍体 (2n =4x =36 )。随机取 7株进行RAPD分析 ,表明被检测植株都为杂种。用引起脚腐病的寄生疫霉菌 (PhytophthoraparasiticaDastar)毒素接种体细胞杂种及双亲叶片 ,结果表明 ,Page橘柚中度感病 ,枳高抗 ,体细胞杂种为抗病类型。     

Intergeneric Somatic Hybrid Plants Between Citrus and Poncirus trifoliata and Evaluation of Their Root Rot Resistance

Protoplasts of Page tangelo (Citrus reticulata Blanco×C. paradisi Macf.) cell suspension culture were electrically fused with mesophyll protoplasts isolated from trifoliate orange (Poncirus trifoliata (L.) Raf.). More than 150 plantlets regenerated after 4-5 months of culture. The regenerated plants were trifoliate with well developed root systems. Root-tip chromosome counting of more than 20 randomly selected plants revealed that they were all tetraploids (2n=4x=36). RAPD analysis of 7 randomly selected plants verified their hybridity. Inoculation of citrus Phytophthora parasitica Dastar toxin on leaves of somatic hybrids and both parental genotypes showed that Page tangelo was moderately susceptible, and trifoliate orange was highly resistant while the somatic hybrids were resistant. The potential of this somatic hybrid as rootstock is also discussed.     

柑桔原生质体融合再生叶肉亲本型植株的遗传分析

叶肉亲本（粗柠檬）型植株叶形指数、气孔特征与亲本粗柠檬无异,与同组合体细胞杂种差异显著。染色体计数为二倍体（２ｎ＝２ｘ＝１８）。过氧化物酶（ＰＯＸ）、多酚氧化酶（ＰＰＯ）、谷草转氨酶（ＧＯＴ）同工酶图谱与粗柠檬一致。ＲＡＰＤ分析表明,在具多态性的５４个随机引物中,大多数引物（５２个）上的图谱与粗柠檬相同。但ＯＰＷ－１２上,叶肉亲本型植株含有哈姆林甜检的特征谱带。ＯＰＶ－０４扩增产物显示,叶肉亲本型     

Optimization of Electrofusion Parameters and Interspecific Somatic Hybrid Regeneration in Citrus

This study was primarily attempted to optimize the electrofusion parameters using protoplasts isolated from cell suspension cultures of "Page" tangelo ( Citrus reticulata Blanco x C. paradisi Macf) and mesophyll protoplasts of rough lemon ( Citrus jambhiri Lush) as fusion partners. It was shown that the binuclear heterokaryons frequency reached 15% with the following parameters: alternate current (AC) 125 V/cm, AC time 60 s, direct current (DC) 1 250 V/cm, DC pulse width 50 μs, DC pulse interval O. 5 s, No. of DC pulse 3. Considering the fact that different types of protoplasts have different specific weights, higher frequency of the binuclear heterokaryons was obtained by controlling the centrifugation time after fusion. The fusion products regenerated into plantlets after 3 to 4 months of culture. Chromosome counting of the root tips and morphological observation of the regenerants verified that 78% were tetraploids and the rest were diploids with the leaf morphology of mesophyll parent. Peroxidase (POX) isozyme and RAPD analysis indicated that interspecific somatic hybrids were obtained and an autotetraploid plant of mesophyll parent type was also verified.     

柑桔种间体细胞杂种的花粉特性及贮藏研究

对粗柠檬与“哈姆林”甜橙种间体细胞杂种的花粉特性(形态、花粉量、生活力等)进行了观察、检测,并于-20℃的低温条件下开展了贮藏试验。结果表明：花粉离体萌发的适宜培养基为0．8％琼脂 16％蔗糖 10mg／kg硼酸,萌发率为15．31％,其生活力介于两亲本之间,可作为杂交亲本进一步被利用。贮藏试验结果显示：以真空、干燥(CaCl2或硅胶作干燥剂)贮藏花粉的效果较好。授粉试验证明,贮藏花粉与新鲜花粉的座果率差异不显著,说明低温、真空和干燥是贮藏粗柠檬与“哈姆林”甜橙种间体细胞杂种花粉的较适条件。     

青花菜与白菜间体细胞杂种获得与遗传特性鉴定

为获得芸薹属白菜Brassica campestris与青花菜Brassica oleracea var. botrytis的种间体细胞杂交体,以青花菜和白菜的子叶与下胚轴为材料,分离制备原生质体,用40％聚乙二醇 (Polyethylene glycol,PEG) 进行原生质体融合。融合细胞在以0.3 mol/L 蔗糖、0.3 mol/L葡萄糖为渗透稳定剂,附加0.2 mg/L 2,4-D+0.5 mg/L 6-苄氨基嘌呤 (6-BA) +0.1 mg/L 1-萘乙酸 (NAA) +0.1 mg/L激动素 (Kinetin,Kin) 的改良K8p培养基中液体浅层培养。将包埋于0.1％琼脂糖的8~10个细胞期的细胞在添加0.3 mol/L蔗糖和2 mg/L 6-BA+2 mg/L玉米素 (Zeatin,ZEA) +1 mg/L NAA+0.5 mg/L Kin的Kao培养基中诱导愈伤组织。愈伤组织转到MS+5 mg/L ZEA+2 mg/L IAA诱导不定芽。将长1～2 cm的不定芽转到1/2 MS+0.2 mg/L NAA诱导生根。将生根的植株转移到花盆,并对其杂种性质进行形态学、细胞学和分子生物学鉴定。结果表明,融合细胞培养2～7 d后发生第1次分裂,培养35 d后植板率为0.66％,不定芽再生率达3.7％。形态学观察显示,绝大多数再生植株的叶面积较大,株型和叶型为两种杂交亲本的中间型。染色体计数结果显示,再生植株染色体数目为2n=38。流式细胞仪测定DNA含量显示,再生植株DNA含量是亲本之和。随机扩增多态性DNA (Random amplified polymorphic DNA,RAPD) 和基因组原位杂交 (Genomic in situ hybridization,GISH) 分析结果证明再生植株具有双亲基因组。体细胞杂种花粉育性比较低,杂交、回交后其育性逐渐获得恢复。     

Variation of Intergeneric Somatic Hybrids Between Citrus sinensis cv. Valencia and Fortunella crassifolia cv. Meiwa

Intergeneric somatic hybrid plants derived from protoplast fusion between Citrus sinensis (L.) Osbeck cv. Valencia and Fortunella crassifolia Swingle cv. Meiwa have been grown in the fields for 6 years. The plants grew less vigorously with uneven canopy size and some shoots frequently died. Chromosome count showed that the plants, containing non-tetraploid cells besides amphidiploid, were chimeras. Most somatic hybrid plants exhibited a new band of EST isozyme. RAPD analysis verified that several plants lacked some of the parents' markers. The results indicated that the intergeneric somatic hybrids were genetically unstable.     

甜橙与酸橙体细胞杂种核质组成鉴定

采用流式细胞术(flow cytometry,FCM)、简单重复序列(simple sequence repeat,SSR)和酶切扩增多型性序列(cleaved amplifiedpolymorphic sequence,CAPS)等技术分析酸橙(Citrus aurantium L.)叶肉原生质体和甜橙(C.sinenis Osbeck cv.Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种.FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体.用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本.在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种.通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样.结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本.讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用.     

甜橙与酸橙体细胞杂种核质组成鉴定(英文)

采用流式细胞术(flow cytometry, FCM)、简单重复序列(simple sequence repeat, SSR)和酶切扩增多型性序列(cleaved amplified polymorphic sequence, CAPS)等技术分析酸橙(Citrus aurantium L. )叶肉原生质体和甜橙(C. sinenis Osbeck cv. Shamouti)胚性愈伤组织原生质体电融合再生的体细胞杂种。FCM研究结果表明,所有的体细胞杂种植株荧光强度是二倍体对照的2倍,说明所分析的植株为四倍体。用SSR和CAPS分析了体细胞杂种的核质遗传组成,在试验的4对SSR引物中,有2对能区分开融合亲本。在2对引物中,体细胞杂种植株包含双亲的全部特异带,表明它们为异核杂种。通用引物扩增结合限制性内切酶酶切能鉴别融合亲本,在具有多型性的引物/酶组合中,所有体细胞杂种的线粒体和叶绿体DNA带型与胚性亲本(甜橙)完全一样。结果表明体细胞杂种核基因组来自双亲,而胞质基因组来自悬浮系亲本。讨论了所用技术的特点、柑橘四倍体体细胞杂种核质遗传规律及本组合体细胞杂种的应用。