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。     

宜昌橙与伏令夏橙种间体细胞杂种

宜昌橙(Citrus ichangensis SwingIe)试管实生苗叶肉原生质体与伏令夏甜橙(C．Sin-ensis Osbeck)胚性悬浮细胞系原生质体,经聚乙二醇(PEG)诱导融合。再生出的胚状体为畸形,转移到生芽培养基中诱导产生丛芽。丛芽微嫁接在15天龄的枳壳砧上成为完整植株。幼叶染色体检查表明,两亲本均为二倍体2n=2x=18,融合后再生出的植株为四倍体2n=4x=36。过氧化物酶(POX)及谷草酰胺转氨酶(GOT)同工酶分析证明,这些四倍体均为体细胞杂种,它们同时含有双亲的酶带。杂种植株叶片形态更像甜橙。植株移八土壤后生长旺盛。     

电融合获得用于选择抗CTV砧木的酸橙与甜橙体细胞杂种植株

在已知参数条件下,通过电场诱导酸橙(Citrus aurantium L.)叶肉原生质体和沙漠蒂甜橙(C.sinensis Osbeck cv.Shamouti)的胚性愈伤组织原生质体融合,融合产物经培养再生出40棵植株.染色体检查表明所得到的植株具有36条染色体,为四倍体植株.再生植株具有翼叶,叶片厚,表现出多倍体的特征.采用2个10-碱基随机引物鉴别再生植株的杂种特性.在2个引物的扩增带型中,再生植株的随机扩增带图里出现了融合亲本的特征带.对再生植株染色体计数和RAPD分析的结果表明它们是酸橙和甜橙种间异源四倍体体细胞杂种植株.这些体细胞杂种植株的获得为选择具有酸橙优良性状、同时抗CTV的新型砧木提供了好的试材.     

电融合获得用于选择抗CTV砧木的酸橙与甜橙体细胞杂种植株

在已知参数条件下,通过电场诱导酸橙（Cit-rus aurantium L.）叶肉原生质体和沙漠蒂甜橙（C.sinensis Osbeck cv.Shamouti）的胚性愈伤组织原生质体融合,副合产物经培养再生出40棵植株。染色体检查表明所得到的植株具有36条染色体。为四倍体植株。再生植株具有翼叶,叶片厚,表现出多倍体的特征,采用2个10－碱基随机引物鉴别再生植株的杂种特性。在2个引物的扩增带型中,再生植株的随机扩增带图里出现了融合亲本的特征带。对再生植株染色体计数和RAPD分析的结果表明它们是酸橙和甜橙种间异源四倍体体细胞杂种植株。这些体细胞杂种植株的获得为选择具有酸橙优良性状、同时抗CTV的新型砧木提供了好的试材。     

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。     

柑桔种间原生质体融合植株再生及染色体数目变异的研究

为了获得具有抗病、优质丰产等优良性状的柑桔体细胞杂种,本研究应用当前推广良种朋娜脐橙胚性细胞原生质体和抗裂皮病、耐盐碱的红桔叶肉细胞原生质体作为亲本进行体细胞杂交研究。通过对原生质体分离,融合和培养过程中培养基调控等环节的研究,建立起原生质体融合及其后的胚状体再生系统,并从融合处理后的原生质体培养中获得了大最的胚状体,进而获得个别体细胞杂种植株。同时对融合后再生的胚状体染色体数目和同工酶分析,还揭示了在柑桔原生质体融合再生中,胚状体水平上存在淹广泛的遗传变异。其中有14.1%的胚状体为四倍体,近20%为超倍体的非整倍体,并讨论了变异发生及由胚状体再生植株困难的原因。     

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。     

RAPD为基础的柑橘体细胞杂种有性后代遗传研究

筛选31条谱带清晰、多态性强及重复性好的10 bp引物, 运用RAPD技术对以异源四倍体柑橘体细胞杂种(哈姆林甜橙(Citrus sinensis Osbeck)+粗柠檬(C. jambhiri Luss))为父本与二倍体单胚类型宜本杂4号(华农本地早橘(C. reticulataBlanco)×宜昌橙(C. ichangensis Swingle))杂交获得的杂种后代进行遗传分析. χ₂测验表明: 杂交父本与母本的特异标记总体上是随机传递给后代的, 父本即柑橘异源四倍体体细胞杂种融合双亲的单个特异标记也是随机传递给后代的. 通过组群平均数假设测验, 二倍体组群与三倍体组群间各类亲本标记的平均数不存在显著差异. 基于遗传距离的聚类结果显示: 杂种后代聚为3类: 第1类与融合亲本之一哈姆林甜橙聚为一类, 有15株杂种后代; 第2类与母本宜本杂4号聚为一类, 有16株杂种后代; 第3类与父本体细胞杂种(哈姆林甜橙+粗柠檬)聚为一类, 有61株杂种植株, 大多数杂种后代与父本的遗传关系更为密切.     

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。     

柑橘体细胞杂种有性后代的创造及杂种鉴定

以异源四倍体柑橘体细胞杂种[哈姆林甜橙(Citrus sinensis Osbeck) 粗柠檬(C.jambhiri Luss)]为父本与二倍体单胚类型宜本杂4号[华农本地早橘(C.reticulata Blanco)×宜昌橙(C.ichangen- sis Swingle)]杂交,采用胚抢救技术获得了110株有性后代植株,通过染色体计数及倍性分析仪鉴定,其中93株植株为3倍体,余下的17林植株为2倍体。RAPD分析表明:获得的有性后代植株均为杂种。     

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.     

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.     

GISH,AFLP and PCR-RFLP analysis of an intergeneric somatic hybrid combining Goutou sour orange and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Intergeneric somatic hybrids combining Goutou sour orange (Citrus aurantium L.) with trifoliate orange [Poncirus trifoliata (L.) Raf] were produced by electrofusion and their genetic inheritance analyzed by amplified fragment length polymorphism (AFLP), genomic in situ hybridization (GISH), and PCR-restriction fragment length polymorphism (PCR-RFLP). Sixteen mini-calluses were obtained after 20 days of culture; they all developed into embryoids on EME500 medium. Following several subcultures on shoot induction medium for a total culture period of 6 months, shoots regenerated. The plants grew vigorously with a well-developed root system and exhibited the trifoliate leaf character of P. trifoliata. Ploidy analysis verified that all of the regenerates were tetraploids (2n=4x=36) as expected. GISH analysis confirmed that 18 chromosomes came from trifoliate orange and the remaining 18 from Goutou sour orange, as with most symmetric somatic hybrid plants; moreover, chromosome translocations were also observed in one plant. AFLP analysis of 16 regenerates and their fusion parents indicated that all of the somatic hybrids except one were genetically uniform. Analysis of the somatic hybrid cytoplasmic genomes with universal primers revealed that their chloroplast DNA (cpDNA) banding patterns were identical to those of the mesophyll parent trifoliate orange, while their mitochondria (mt) genomes were of the callus parent sour orange. The potential of GISH in Citrus somatic hybrid analysis is discussed.The first two authors contributed equally to this paper.     

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.     

Regeneration and molecular characterization of intergeneric somatic hybrids between Citrus reticulata and Poncirus trifoliata

Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.     

Intertribal hexaploid somatic hybrid plants regeneration from electrofusion between diploids of Citrus sinensis and its sexually incompatible relative, Clausena lansium

 Chinese wampee [Clausena lansium (Lour.) Skeels], a sexually incompatible relative of citrus, is commercially cultivated in South China. In this study, embryogenic protoplasts of ‘Bonanza’ navel orange [Citrus sinensis (L.) Osbeck] were electrically fused with leaf protoplasts isolated from ‘Chicken Heart’ Chinese wampee. After 8 months of culture, fusion products regenerated into shoots. More than 70% of the shoots unexpectedly rooted well. Chromosome counting of several shoot- and root-tips revealed that their chromosome numbers were not 2n=4x=36 as expected, but 2n=6x=54, suggesting that chromosome doubling occurred rather than chromosome elimination in this intertribal fusion combination. RAPD analysis of embryoids and the leaves of unrooted and rooted shoots verified their hybridity. This is the first report of hexaploid somatic hybrid plant regeneration from fusion between diploids in Aurantioideae. Received: 20 June 1998 / Accepted: 18 August 1998     

Regeneration of Diploid Intergeneric Somatic Hybrid Plants Between Microcitrus a Electrofusion

Leaf-derived protoplasts of Rough lemon ( Citrus jambhiri Lush, 2n = 2x = 18) were electrofused with embryogenic suspension protoplasts of its relative, Microcitrus papuana 5wingle (2n = 2x = 18), with an intention of creating novel germplasm. Six plants were regenerated following protoplasts fusion. Cytological examination demonstrated that they were diploids with 18 chromosomes (2n = 2x = 18). RAPD (random amplified polymorphic DNA) analyses with six arbitrary 10-mer primers showed that the regenerated plants had identical band pattems to those of Rough lemon for primers OPA-07, OPAN-07, OPE-05 and OPA-08, whereas for the other two primers, OPA-04 and OPS-13, bands specific to M. papuana could be detected in the regenerated plants. Cytological and RAPD analysis revealed that the regenerated plants were diploid somatic hybrids between M. papuana and Rough lemon. The putative hybrids were morphologically similar to Rough lemon. This is the first report on production of diploid somatic hybrid plants between citrus with its related genus via symmetric fusion.     

Regeneration of <Emphasis Type="Italic">citrus sinensis</Emphasis> (+) <Emphasis Type="Italic">clausena lansium</Emphasis> intergeneric triploid and tetraploid somatic hybrids and their identification by molecular markers

Summary Somatic hybrid plants were regenerated via electrofusion between leaf-derived protoplasts of ‘Chicken heart’ sweet wampee (Clausena lansium) and embryogenic protoplasts of ‘Newhall’ navel orange (Citrus sinensis Osbeck). Most of the complete plantlets were formed via mini-grafting. Flow cytometry showed that most of the regenerants were tetraploids as expected, but unexpectedly three plantlets were triploids. Simple sequence repeat (SSR) analysis of seven randomly selected tetraploids and the three triploids showed that they had specific fragments from both fusion parents, thereby confirming their hybridity. Analysis of cytoplasmic genomes using universal primers revealed that their chloroplast DNA (cpDNA) band pattern was identical to the mesophyll parent, while their mitochondrial genomes were of the navel orange type. According to the SSR results, the triploids obtained in this study were most likely due to chromosome elimination of ‘Chicken heart’ sweet wampee prior to plant regeneration.