野生大豆原生质体再生植株

1987年我们首次通过栽培大豆(Glycine max)的原生质体培养得到再生植株,最近又相继从栽培大豆的6个品种的原生质体分别获得再生植株,移栽于土中后均能正常开花结实。至于大豆野生种中的G.canescens和G.clandestina的原生质体培养已有再生植株或形成芽的报道。但在野生大豆G.soja的原生质体培养中仅见胚状结构的形成,迄今未获得再生植株。     

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。     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上, 用改进的PEG-高pH高钙法诱导草木樨状黄芪(Astragalus melilotoides)和木本霸王(Zygophyllum xanthoxylum)原生质体融合, 得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体, 使双亲原生质体及其同源融合产物均不能持续分裂而死亡, 融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系, 其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性, 结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

LpDH活性作为一种遗传标记在体细胞融合中的转移

用具有LpDH活性,但不能分化植株的烟草冠瘿瘤B 6 S 3为亲本的原生质体,和与之有相反特点的正常烟草xanthi品科叶肉原生质体间融合,由融合处理的原生质体形成了愈伤组织并再生了植株。对56株叶片的LpDH活性电脉分析表明,有75％植株含有不同程度的LpDH活性,即能合成章鱼碱。随植株发育成长,一些植株的LpDH活性有减弱或丢失现象。但叶片形态具有双亲部分特征,表明烟草冠瘿瘤的LpDHT活性标记可通过原生质体融合转移 到烟草xanthi细胞中。     

从胡萝卜与芹菜原生质体融合获得细胞杂种

试验以胡萝卜根和芹菜叶肉原生质体为亲本,用PEG高pH,高Ca~( )法诱导融合。异源融合率最高可达8％,一般在3％～5％之间。从150余棵融合再生植株中,经形态、同工酶谱、叶片中内含芳香物质等指标测试,鉴定出三棵(No 1,2,8)融合再生植株为芹莱和胡萝卜的细胞杂种。     

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。     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上,用改进的PEG-高pH高钙法诱导草木樨状黄（Astragalus melilotoides）和木本霸王（Zygophyllum xanthoxylum）原生质体融合,得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体,使双亲原生质体及其同源融合产物均不能持续分裂而死亡,融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系,其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性,结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

甘薯同一不亲和群内品种间体细胞杂种

利用PEG融合方法,融合甘薯(Ipomoea batatas)B不亲和群内品种‘koganesengan'和‘bitambi'的原生质体.将融合处理的原生质体进行培养,共获得45株再生植株.4株再生植株形态上表现出融合双亲的中间特性,其中2株染色体数为融合两亲之和(2n=12x(2n 2n)=180),另外2株分别为41～103和35～100,因细胞不同而不同.经RAPD分析,这4株再生植株分别具有双亲特异的DNA扩增带或双亲都不具有的新扩增带.鉴定这4株再生植株为杂交不亲和的B群内品种间体细胞杂种.     

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。     

甘薯同一不亲和群内品种间体细胞杂种

利用PEG融合方法,融合甘薯(Ipomoea batatas ) B不亲和群内品种‘koganesengan’和‘bitambi’的原生质体。将融合处理的原生质体进行培养,共获得45株再生植株。4株再生植株形态上表现出融合双亲的中间特性,其中2株染色体数为融合两亲之和（2n = 12x (2n + 2n) = 180）,另外2株分别为41～103和35～100,因细胞不同而不同。经RAPD分析,这4株再生植株分别具有双亲特异的DNA扩增带或双亲都不具有的新扩增带。鉴定这4株再生植株为杂交不亲和的B群内品种间体细胞杂种。     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

Construction of rice cybrid plants

Summary The mitochondrial genomes of rice cells were transferred to a fertile rice variety (N8) from a cytoplasmic male sterile variety (CMS) by asymmetric protoplast fusion based on metabolic complementation. Protoplasts derived from CMS were X-irradiated (125 krad) and electrofused with protoplasts which had been treated with iodoacetamide. Metabolic complementation, presumably between nuclear and cytoplasmic compartments, enabled fused protoplasts to form colonies at high efficiency. Restriction digest analysis of mitochondrial DNA (mtDNA) indicated that hybrid cells carried mtDNA derived from both parents. Of the plants regenerated from hybrid calli, 68% carried a diploid chromosome set (2n=24) and the rest of them carried 48 chromosomes. All of them expressed the aryl acylamidase I deficient phenotype encoded by the recessive allele of the fertile N8 parent. These results indicate that the novel somatic hybrid plants regenerated were cybrids, deriving their nucleus from the iodoacetamide treated parent and their mitochondria from both parents.     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

Production of a highly asymmetric somatic hybrid between rice and Zizania latifolia (Griseb): evidence for inter-genomic exchange

 A highly asymmetric and fertile somatic hybrid plant was obtained via protoplast fusion in an intergenric combination. Gamma-ray-irradiated Zizania latifolia (Griseb). Turcz. ex Stapf mesophyll protoplasts were electrofused with idoacetamide-inactivated rice protoplasts derived from a 2-month-old suspension cell culture. Two of the six putative hybrid calli regenerated plants. Cytological observation showed that the somatic chromosome numbers of both plants were the same as the rice parent (2n=24). Nevertheless, the hybrid nature and inter-genomic exchange events of one of the plants, i.e. SH6 (SH for somatic hybrid), were confirmed by Southern analysis using both total genomic DNA and moderate-copy, Z. latifolia-abundant DNA sequences as probes; in both cases, parental specific and/or new intergenomic recombinant hybridization fragments were detected. In both plant and seed morphology, the hybrid (SH6) was distinct from its rice parental cultivar, as well as from the wild donor species, Z. latifolia. Received: 15 August 1998 / Accepted: 30 September 1998     

Production and analysis of plants that are somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.)

In order to obtain plants that were somatic hybrids of barley (Hordeum vulgare L.) and carrot (Daucus carota L.), we fused protoplasts that had been isolated from 6-month-old suspension cultures of carrot cells with protoplasts isolated from barley mesophyll by electrofusion. After culture for 1 month at 25°C , the cells were cultured for 5 weeks at 4°C , and were then returned to 25°C for culture on a shoot-inducing medium. Three plants (nos. 1, 2 and 3) were regenerated from the cells. The morphology of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells had about 24 chromosomes, fewer than the sum of the numbers of parent chromosomes which was 32. Southern hybridization analysis with fragments of the rgp1 gene used as probe showed that the regenerated plants contained both barley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAs were also analyzed with several probes. The ctDNA of the regenerated plants yielded hybridization bands specific for both barley and carrot when one fragment of rice ctDNA was used as probe. Furthermore, the regenerated plants yielded a barley specific band and a novel band with another fragment of rice ct DNA as a probe. One of the regenerated plants (no. 1) yielded a novel pattern of hybridized bands of mt DNA (with an atp6 probe) that was not detected with either of the parents. These results indicated that the regenerated plants were somatic hybrids of barley and carrot and that recombination of both the chloroplast genomes and the mitochondrial genomes might have occurred. Received: 28 May 1996 / Accepted: 2 August 1996     

Asymmetric somatic hybridization between Triticum aestivum L. and Haynaldia villosa Schur

Suspension cell-derived protoplasts of wheat, inactivated with different concentrations (0-2.5mol/L) of IOA, were fused by PEG method with the Haynaldia villosa protoplasts which originated from the calli 4-5d after subculture and were irradiated with 60Co-γ ray. Cell colonies, calli or regenerated plants were obtained from different combinations of fusion. The calli and plants were verified to be hybrids by chromosome counting, isozyme analysis and morphological inspection.     

GFP expression as an indicator of somatic hybrids between transgenic Satsuma mandarin and calamondin at embryoid stage

Somatic hybridization was performed via electrofusion between embryogenic suspension-derived protoplasts of transgenic green fluorescent protein (GFP) Satsuma mandarin (Citrus unshiu Marc. cv. Guoqing No. 1) (G1) callus and mesophyll protoplasts of calamondin (Citrus microcarpa Bunge), and three embryoids expressing GFP under UV light were obtained after 60 days of culture. The three embryoids were considered not as diploid cybrids but true allotetraploid somatic hybrids, as it was based on: (1) citrus heterokaryons are generally more vigorous and have higher capacity for embryogenesis as compared with unfused and homo-fused embryogenic callus protoplasts; (2) the callus line of G1 Satsuma mandarin has lost the embryogenesis capacity; and (3) citrus diploid cybrids produced by symmetric fusion always possess nuclear genome of mesophyll parent, and calamondin without GFP gene was used as leaf parent in this study. Subsequent flow cytometry, simple sequence repeat and cleaved amplified polymorphic sequence analysis of one regenerated callus mass and three resulting plants validated this supposition, i.e., the callus was derived from transgenic G1 callus protoplasts, and the three plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from callus parent. The potential of transgenic GFP citrus callus as suspension parent in citrus somatic fusion to study the mechanism of cybrid formation, create new citrus cybrids, and transfer organelle-encoded agronomic traits was also discussed.     

Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana

Summary Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.Abbreviations PGM phosphoglucose mutase - PHI phosphohexose isomerase - MES 2[N-morpholino] ethane sulfonic acid - BH3 protoplast culture medium (Grosser and Chandler, 1987) - PEG polyethylene glycol - MT Murashige and Tucker (1969) basal medium - NAA 1-naphthaleneacetic acid - GA3 gibberellic acid - H+H and EME citrus embryogenic cell culture media (Grosser and Gmitter, 1990b) - B embryo germination medium - RMAN rooting medium Florida Agricultural Experiment Station Journal Series No. R-00298.     

Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

Production of asymmetric hybrids between Arabidopsis thaliana and Brassica napus utilizing an efficient protoplast culture system

Application of the protoplast culture method developed for Brassica protoplasts to protoplasts of Arabidopsis thaliana has increased the opportunities for interspecific hybridizations involving Arabidopsis. A more-efficient and much-simpler method was established compared to the earlier-reported protocol developed for A. thaliana protoplasts in which alginate beads were utilized. Mesophyll protoplasts of A. thaliana (ecotypes 'Landsberg erecta' and 'Wassilewskija') were cultured in the modified 8p liquid medium, which had been developed for Brassica protoplasts. For comparison, protoplasts were cultured in sodium alginate beads supplied with B5 medium according to the protocol for A. thaliana. The protoplasts divided with high frequencies in the 8p medium, and calli proliferated more rapidly than in the sodium alginate beads. High frequencies of shoot differentiation and regeneration were observed in calli of both ecotypes, from about 30% in the ecotype 'Wassilewskija' to about 60% for 'Landsberg erecta'. The more-rapidly the calli developed, the higher the regeneration frequencies were. Asymmetric hybrids between A. thaliana and Brassica napus were obtained by treating the protoplasts of A. thaliana with iodoacetamide (IOA) and B. napus protoplasts with UV-irradiation before fusion with polyethylene glycol (PEG). By using the culture procedure developed for Brassica protoplasts, calli developed and plants were regenerated. Although most of the plants regenerated after cell fusion were A. thaliana-like and were judged to be escapes from IOA treatment, more than ten plants showed hybrid features of both morphological and molecular characters. Among the hybrids that have flowered so far, both male-fertile and male-sterile plants have been obtained. Back-crossings to A. thaliana are now in progress as is morphological and molecular characterization of the plants.