Somatic hybridization between Solanum tuberosum and species of the S. nigrum complex: Selection of vigorously growing and flowering plants

Fusion experiments were performed between diploid (2n = 2x = 24) or tetraploid (2n = 4x = 48) potato genotypes and four species of the Solanum nigrum complex, namely S. nigrum (2n = 6x = 72), S. villosum (2n = 4x = 48), S. chenopodioides (2n = 2x = 24) or S. americanum (2n = 2x = 24 and 2n = 6x = 72). All five accessions of the S. nigrum-species were successfully hybridized with at least one of the potato genotypes. Somatic combining abilities were influenced by the ploidy level as well as the genotype of the parental species. The use of kanamycin or hygromycin resistance as cell-selectable markersystem had no influence on somatic combining ability, but such markers can be useful to improve efficient selection of somatic hybrids in sufficient numbers. At least 20% of the hybrids of each successful combination performed well in vitro. However, only 60 genotypes out of 761 somatic hybrids were vigorous as well as flowering in the greenhouse. Analysis of the DNA content of somatic hybrids could be used as a criterion for the indirect selection in vitro of hybrids that were vigorous in the greenhouse. Flowering somatic hybrids of S. nigrum (+) 2x potato and S. americanum (+) 4x potato were selected with the aim of introgression of resistance traits after recurrent backcrossing with cultivated potato. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potential of Nicotiana glauca (Grah.) as a source of resistance to black root rot Thielaviopsis basicola (Berk. and Broome) Ferr. in tobacco improvement

Nicotiana glauca, a wild relative of Nicotiana tabacum, is an attractive potential source of black root rot (Thielaviopsis basicola)‐resistant germplasm. Moreover, it shows a resistance or tolerance to PVY, TEV, anthracnose and powdery mildew. In this study its potential as a source of resistance to black root rot caused by Th. basicola was investigated. Nicotiana glauca GG (2n = 2x = 24) was crossed as male with two N. tabacum TT (2n = 4x = 48) flue‐cured cultivars: ‘BY103’ and ‘K 326’, both of which are susceptible to black root rot. Amphihaploid F₁ TG (2n = 3x = 36), amphidiploid TTGG (2n = 6x = 72) and sesquidiploid TTG (2n = 5x = 60) hybrids were obtained. The resultant sesquidiploid hybrids were used as maternal components in backcrossing to N. tabacum and a segregating post‐sesquidiploid TTg (2n = 5x = 52–54) offspring was obtained. Amphihaploids exhibited a level of resistance to black root rot characteristic of N. glauca. The expression of resistance varied in the sesquidiploid generation, possibly reflecting cytological instabilities in that progeny. A wide variation in response to black root rot was found for post‐sesquidiploids a clear reflection of extensive chromosome segregation in that hybrid generation.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

四倍体大白菜的选育

利用二倍体和四倍体大白菜,通过正反交,连续6年共做52个组合,从1985年的水仙花(四倍体)×后36高(二倍体)和1987年的BP058(二倍体)×水仙花(四倍体)两个组合得到了少量杂交种,经系谱选择并在人工气候室加代,提高孕性,均于第四代基本稳定。1989年将这两个杂交种分别定名为翠宝和翠绿。它们具有多倍体植物叶厚、叶面有泡状突起、蕾大、花大、气孔也大的特点,经染色体镜检,染色体数2n=4x=40。这两个品种生长速度快,生育期80天左右,包球紧实,抗病性强,品质优良,适口性好,耐贮藏运输,亩产7000～7500kg。     

普通小麦与Elymus rectisetus异附加系的分子细胞遗传学鉴定

Elymus rectisetus (Nees in Lehm) A. Löve et Connor是目前小麦族中发现的唯一的无融合生殖种,为了鉴定和标记从普通小麦与E. rectisetus BC₂F₂衍生后代中选育的2n=44株系1026A1、1057A1和1035A2的外源染色体,应用细胞学、基因组原位杂交和RAPD方法进行了研究。经细胞学鉴定,3个株系花粉母细胞减数分裂中期Ⅰ（PMC MⅠ）染色体构型均为2n=22Ⅱ,与普通小麦Fukuhokomugi杂交F₁的PMC MⅠ染色体构型均为2n =21Ⅱ+1Ⅰ,两两杂交F1的PMC MⅠ染色体构型均为2n=21Ⅱ+2Ⅰ,表明它们是分别附加了1对互不相同外源染色体的普通小麦-E. rectisetus二体异附加系。标记E. rectisetus品系1050的基因组DNA为探针DNA,对3个异附加系进行原位杂交,分别鉴定出附加的1对E. rectisetus染色体。应用13个引物对2个亲本和3个异附加系进行RAPD分析,获得了可分别用于检测1026A1和1057A1中所附加的E. rectisetus染色体遗传物质的分子标记OPB-14_900bp、OPE-09_750bp和OPB-14_1000bp。     

Assessment of genetic relationships among Cucumis spp. by SSR and RAPD marker analysis

The first successful production of a sterile interspecific hybrid obtained from a cross between Cucumis hystrix Chakr. (2n = 2x = 24) and Cucumis sativus var. sativus L. (2n =2x = 14), and its subsequent fertility restoration through chromosome doubling provide an effective means for investigating genetic relationships among Cucumis spp. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to investigate relationships among C. s. var. sativus L., C. s. var. hardwickii (R.) Alef., C hystrix, C. hytivus Chen & Kirkbride (the amphidiploid species from chromosome doubling of the C. sativus x C. hystrix interspecific hybrid, 2n = 38), C. melo (2n =2x = 24) and C. metuliferus Meyer and Naudin (2n =2x= 24). A total of 109 SSR bands and 398 RAPD primed sites were used to calculate Jaccard's distance coefficients for cluster analysis using a unweighted pair‐group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using SSR and RAPD markers were highly concordant, such that the correlation between SSR and RAPD genetic distance (GD) estimates was r = 0.94. SSR and RAPD analysis of 22 accessions allowed for their grouping into two distinct groups designated as CS and CM. While group CS consisted of 11 C. sativus genotypes, and the C. hytivus and C. hystrix accessions, group CM included six C. melo genotypes and C. metuliferus. The GD values between C. hystrix and C. sativus ascribed by SSR and RAPD matrices were 0.59 and 0.57, respectively. These GDs were smaller than those detected between C. hystrix and C melo (0.87 and 0.70 derived from SSR and RAPD markers, respectively).     

稻属种间杂种(Oryza sativa×O.latifolia)再生植株的形态与染色体变化

供试材料 种间杂种[O.sativa(AA,2n=24)×O.latifolia(CCDD,2n=48)]F_1的幼穗,杂种基因型是异源三倍体(ACD)3n=36。利用幼穗诱导愈伤组织与再生植株,经过多次继代培养后,再生植株分为2大类型:(1)完全不结实株,形状与 F_1杂种相似,染色体数2n=36。(2)结有少量种子的植株,性状介于双亲之间,染色体数2n=24。我们的结果表明:幼穗     

Assessment of genetic and phenotypic variation among intraspecific somatic hybrids of potato, Solanum tuberosum L.

Intraspecific somatic hybrids have been produced by protoplast fusion in eight combinations involving 10 dihaploids (2n= 2x= 24) in an attempt to provide new material for potato breeding. Cytological analysis revealed extensive variation in chromosome number, such as aneuploid, aneusomatic and mixoploid hybrids. Most of the hybrids represented the expected chromosome number of 48; however, the frequency of aneuploids reached 50% in some combinations. Some hybrids carried structurally rearranged chromosomes and exhibited a high frequency of aberrant anaphases. Isozyme and random amplified polymorphic DNA (RAPD) patterns of the hybrids from the same fusion combination were uniform. In the field, somatic hybrids showed wide phenotypic variation in 20 morphological characters. There was a significant correlation between certain leaf characters and the ploidy level, which may be used to distinguish the tetraploid hybrids from hexaploids and octoploids. Tuber yield and flowering intensity were highest in tetraploid hybrids (2n= 4x= 48). Eighteen of the 73 hybrids reached higher yields than the standard variety ‘Adretta’. Floral development and fertility were restored in hybrids derived from fusions between non-flowering or sterile dihaploids.     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

马铃薯2x-2x,4x-2x和2x-4x杂交产生的四倍体杂种块茎产量和特性的优势表现及遗传分析

利用能产生2n配子的二倍体杂种与四倍体普通栽培种(S.tuberosum L.)和四倍体新型栽培种(Neo—tuberosum)进行了2x-2x,4x-2x和2x-4x间的杂交,获得了18个杂交组合的4x杂种家系,将这些家系与其10个4x亲本品种(品系)及对照品种在同一地点进行了连续两年的比较试验。结果表明:4x杂种家系单株平均块茎产量比亲本两年分别高16％和19％;其中最好的6个杂种家系比较好的4个品种(品系)分别高20％和43％;杂种产量的优势主要表现在单株块茎数目的显著增多,但在块茎商品率方面则与4x亲本差异不显著,而且杂种块茎的表现还不如4x亲本及对照品种,这是由于具2n配子特性的2x亲本这些性状较差所致。试验结果还表明杂种的遗传背景愈复杂其杂种优势也愈强,进一步说明马铃薯的产量与遗传异质性(多样性)的强弱可能有明显关系,利用2n配子特性可获得较高的遗传异质性。     

Alteration of the genomic composition of Solanum nigrum (+) potato backcross derivatives by somatic hybridization: selection of fusion hybrids by DNA measurements and GISH

Fusion experiments were performed with a first (BC₁‐6738) and a second (BC₂‐9017) generation backcross hybrid of 6x Solarium nigrum (+) 2x potato somatic hybrids with potato cultivars. Because no progeny was obtained from the BC₂ genotypes, alternative approaches were sought to overcome the sexual crossing barrier. Five potato genotypes, one of which contains the hygromycin resistance gene, were used in the fusion experiments. All vigorous regenerants were used for the estimation of nuclear DNA content using flow cytometry. Plants with a DNA content higher than that of the BC₁‐6738 or BC₂ genotypes were considered potential somatic hybrids. Forty‐nine potential somatic hybrids resulted from fusion experiments with BC₁‐6738, from which 20 grew vigorously in the greenhouse and flowered. After pollination with several 4x potato cultivars, eight genotypes produced seeded berries and five genotypes gave seedless berries. In addition, 11 of these 13 somatic hybrids were selected for genomic in situ hybridization (GISH) analysis to determine their genomic composition. Nine had exactly or approximately the expected number of 36 S. nigrum and 60 potato chromosomes. In one genotype, only 22 instead of 36 S. nigrum chromosomes were found and one potato chromosome was possibly missing. Only five potential somatic hybrids were detected among the 79 regenerants from BC₂‐9017 (+) 2x potato fusion experiments that were analysed by flow cytometry. Two of these hybrids were rather vigorous and did flower, but pollinations with potato have not yet set any berries.     

Nuclear genome constitution and other characteristics of somatic hybrids between dihaploid Solanum acaule and tetraploid S. tuberosum

Eleven somatic hybrids (2n = 68 to 74) obtained between S. tuberosum ssp. tuberosum cv. Dejima (2n = 48) and ATDH-1 (2n = 24), an anther-culture-derived dihaploid of S. acaule (Yamada et al., 1997), were characterized by nuclear RFLP markers using 49 single-copy DNA probes distributed throughout the potato genome (2 to 6 probes per chromosome). One of the somatic hybrids, DA8-2, had 72 chromosomes and all the Dejima- and ATDH-1-specific markers (124 and 103 bands, respectively), suggesting the presence of a whole set of both parental chromosomes. The other somatic hybrids lost varying numbers of markers up to seventeen. The pattern of the loss of markers indicated the elimination of five chromosomes among four somatic hybrids. A nucleolar organizer region of chromosome 2 was often eliminated in the somatic hybrids. The somatic hybrids studied here had higher frequencies of multivalent formation than the S. tuberosum parent. They had reasonably good seed set when pollinated with S. tuberosum pollen. Hence, homoeologous recombination between S. acaule and S. tuberosum chromosomes is possible and useful traits from S. acaule may be transferred to the S. tuberosum gene pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

Chromosome numbers in potato cultivars hypersensitive to late blight

The chromosomes of twenty-four blight resistant potato cultivars produced from S. demissum x S. tuberosum hybrids were counted. Twenty-two cultivars had 2n=4x=48 chromosomes and two cultivars had 2n=4x+1=49 chromosomes. The implications of these results are discussed briefly in relation to previous studies and to potato breeding methods.     

异源三倍体水稻原生质体培养植株再生

本试验利用二倍体栽培稻（Oryza stativa L.）品种8204,染色体组型为AA,2n=24作为母本,四倍体小粒野生稻（Oryza minuta）染色体组型为BBCC,2n=4x=48作父本杂交,获得异源三倍体杂交种,染色体组型为ABC。F1根尖检查,染色体数目为2n=3x=36。用该异源三倍体杂交种的幼穗诱导愈伤组织,经长期细代培养进行原生质体游离,获     

克服甘薯组(Section batatas)种间杂交不亲和性研究再报

甘薯组Ａ系列野生种Ｉ．ｔｒｉｌｏｂａ和Ｂ系列栽培种徐薯１８杂交授粉后１１～２０天的胚珠培养获得了试管苗和杂种植株，观察比较了杂种和两亲的形态特征，进行了细胞学鉴定。同时一植株的不同细胞中，染色体数均多于３０，但有变异，变动在３８～５６范围内，杂种植株的花器中，雌雄蕊结构发生变异，全部花朵没有出现雄蕊，雌蕊却有１～５套。用Ｂ系列２个野生种和栽培种的６个品种的花粉与杂种植株回交授粉，均不结实，这些异常     

甘薯和Ipomoea lacunosa的种间体细胞杂种植株再生及鉴定

用ＰＥＧ融合法融合甘薯品种高系１４号和近缘野生种Ｉｐｏｍｏｅａ ｌａｃｕｎｏｓａ的原生质体，将融合原生质体培养在含有０．０５ｍｇ／Ｌ２，４－Ｄ和０．５ｍｇ／Ｌ ＫＴ的ＭＳ培养基上，愈伤组织迅速增殖。将其中的７０个愈伤组织培养在添加３．０ｍｇ／Ｌ ＢＡＰ的ＭＳ培养基上，并进一步培养在ＭＳ基本培养基上，获得９株再生植株。过氧化物酶同工酶、酯酶同工酶和ＲＡＰＤ分析表明，其中２株再生植株（ＫＬ１和ＫＬ３）