马铃薯种间体细胞杂种植株的细胞学观察和过氧化物同工酶谱分析

对马铃薯双单倍体品系81－15（2n＝2x－24）和南美二倍体栽培种Solanumphureja（2n＝2x＝24）体细胞融合获得的15个林系、81－15和二倍体野生种S．chacoense（2n＝2x＝24）体细胞融合获得的10个株系进行了细胞学鉴定和过氧化物同工酶谱分析。结果表明,杂种植株除一个株系为非整倍体外（染色体数为37）,其余株系均为四倍体（2n＝2x＝48）,是两个亲本的染色体数之和。与二倍体的双亲相比,杂种植株的叶下表皮气孔保卫细胞内叶绿体数目较多,但单位面积气孔数目较少,明显地表现出了四倍体的特征。杂种植株的过氧化物同工酶谱是其双亲酶谱谱带的总和。     

马铃薯体细胞杂种的青枯病抗性鉴定

本试验对马铃薯四倍体栽培种(Solanum tuberosum)的双单倍体系81-15与二倍体野生种S.cha-coense的原生质体融合株系进行了青枯病抗性鉴定,其目的是对利用体细胞融合技术获得抗青枯病种质的有效性及抗性鉴定技术进行评价,获得抗病育种材料。结果表明:在鉴定的18个体细胞融合株系中,抗性分离表现为从感病(S)到抗病(R),多数表现为中感(MS)到中抗(MR)水平。田间病圃鉴定、人工接种鉴定和分子标记鉴定的结果显示:田间病圃鉴定的病情指数(DI)和人工接种鉴定病级(DS)的相关性达到极显著水平(DI=-0.39+4 DS,r2=0.921),双侧翼SCAR标记SCA07446和SCA12980鉴定同时具有两个标记特异带的株系均为前两种鉴定表现为中感(MS)以上的材料。综合分析同时利用3种方法鉴定的结果:CHT-3、CHT-5、CHT-6、CHT-10、CHT-15等5个株系为抗病材料,证明体细胞融合是利用野生种质资源抗性的有效途径。     

马铃薯种间体细胞杂种植株的细胞学观察和过氧化物同工酶谱分析

对马铃薯双单倍体品系８１－１５（２ｎ＝２ｘ＝２４）和南美二倍体栽培种Ｓｏｌａｎｕｍ ｐｈｕｒｅｊａ（２ｎ＝２ｘ＝２４）体细胞融合获得的１５个株系、８１－１５和二倍体野生种Ｓ．ｃｈａｃｏｅｎｓｅ（２ｎ＝２ｘ＝２４）体细胞融合获得的１０个株系进行了细胞学鉴定和过氧化物同工酶谱分析。结果表明,杂种植株除一个株系为非整倍体外９染色体数为３７）,其余株系均为四倍体（２ｎ＝２ｘ＝４８）,是两个亲本的染色体数之     

普通小麦与Elymus rectisetus衍生后代的细胞遗传学和形态学研究

披碱草（Elymus rectisctus,以下简写为E．rectiselus)具有二倍性无融合生殖特性,为了获得小麦-E．rectisetus稳定的异附加系或异代换系,对(小麦-E．rectiselus)BC2F2衍生后代的细胞学和形态学进行了研究。结果表明,在BC2F5～BC2F7镜栓的单株中,体细胞染色体数目在22～50条之间,其中42条和44条染色体所占的比例最大,分别占鉴定植株总数的38．0％和35．9％。三个2n=42=21″的稳定株系与普通小麦Fukuhokomugi杂交F1的花粉母细胞染色体构型为2n=18″ 6′,表明有三对E．rectiselus染色体代换到普通小麦中;三个2n=44=22″的稳定株系与Fukuhokomugi杂交F1的花粉母细胞染色体构型为21″ 1′,表明它们为二体异附加系。此外,衍生后代的形态学特征趋向于普通小麦。     

马铃薯可育联会消失突变体产生的第一次分裂重组配子

已经发现二倍体马铃薯(2n=2x=24)联会消失突变体可以相当高的频率产生能育的2n 花粉粒。可以认为这样的2n 花粉粒是通过整个染色体组均衡分裂产生的,所以是第一次减数分裂重组(FDR)的产物。在联会销失突变体中,第二次减数分裂重组(SDR)配子的染色体数不平衡,导致其败育,所以可育的2n 配子就构成了一个同型的 FDR 配子群体。在6个杂交组织的306个后代个体中,发现了76个联会消失突变体,其中有23个花粉育性超过了20%。一些这样可育的突变体已成功地用作父本材料。几乎所有可育突变体均来自其双亲都可产生2n 配子的组合。另一种情况是在两个以正常亲本(只产生 n 花粉)与2n 花粉亲本的组合中,几乎所有突变体或是雄性不育,或是花粉育性低于20%。已经发现联会消失是由单隐性基因控制的,而减数分裂核重组的遗传机制还不清楚。尽管在育种上应用联会消失基因有一定困难,但还是有积极意义的。     

同源四倍体水稻与非洲栽培稻杂交的后效性研究

利用亚洲栽培稻中的4份同源四倍体水稻(O.sativa,2n=4x=48)和相应的4份二倍体水稻(O.sativa,2n=2x=24)为母本,以4份非洲栽培稻(O.glaberrima,2n=2x=24)为花粉供体进行远缘杂交后,对其杂种后代的分离动态进行了研究.结果表明,不同倍性的普通栽培稻与非洲栽培稻之间杂交后代的结实率以二倍体普通栽培稻的较高.在配制的32个杂交组合中,其杂种第1代群体均表现出明显的营养生长优势.从群体的生长势来看,杂种第2代群体比杂种第1代群体要弱一些;在杂种第2代群体中,以同源四倍体水稻为母本的杂交组合的分离现象比以二倍体水稻为母本的杂交组合的分离现象更明显.在各杂交组合的第3代群体中,从植株的株叶形态和生育期来看,株系间的差异和株系内的变异依然很明显,变异频率更宽,变异种类更多.在普通栽培稻与非洲栽培稻的杂交组合中,育性变异、生长势变异、株叶形态变异、染色体变异和结实性变异等是较易发现的变异现象.     

甘蓝型油菜与诸葛菜属间杂交F12群体的品质及遗传分析

以甘蓝型油菜（AACC,2n=38）品种Oro与诸葛菜（OO,2n=24）属间杂交F4世代中出现的五倍体单株（AACCO,2n=50）为起始材料连续自交选育、鉴定,直至获得F12群体。各F12株系间表现一定的形态差异, 但株系内植株形态特征一致。细胞学观察表明85.3%的F12单株具有与甘蓝型油菜相同的染色体数目,花粉母细胞(PMCs) 减数分裂中正常配对和分离;其余植株的染色体数目分别为37、39和40;红紫色植株在减数分裂中有落后染色体,四分体时期会出现微核。基因组原位杂交(Genomic in situ hybridization,GISH)分析未检测出整条的诸葛菜染色体;但AFLP分析表明存在渗入的诸葛菜DNA片段。F12植株的脂肪酸组成及硫苷含量表现较大的变异, 有的植株油酸含量高于Oro而硫苷降低。     

3个甘蔗与斑茅远缘杂交后代BC1的染色体遗传分析

对3个甘蔗与斑茅远缘杂交后代BC1进行真实性鉴定及染色体核型分析,以探讨甘蔗与斑茅BC1的染色体传递方式。利用2对鉴定斑茅真实杂交后代的特异引物对3个甘蔗与斑茅BC1进行鉴定,采用根尖分生区细胞去壁低渗涂片法制片,显微拍照计数染色体数目,并进行染色体核型分析。3个BC1材料均为斑茅的真实杂交后代,崖城01-69体细胞染色体核型公式为2n=121=120 m+1 sm,其染色体按2n+n方式传递;崖城01-116的体细胞染色体核型公式为2n=122=118 m+4 sm,其染色体传递方式为2n+n;崖城01-134的体细胞染色体核型公式为2n=121=120 m+1 sm,其染色体传递为2n+n。推断甘蔗与斑茅BC1的染色体以2n+n的方式传递。     

小麦单细胞再生植株后代染色体与DNA变异的初步研究

为了使体细胞无性系变异更好地应用于作物品种改良,以小麦单细胞培养再生植株后代为材料,研究了其染色体和DNA的变异.结果表明,再生植株后代与未经培养的亲本相比,花粉母细胞减数分裂染色体行为发生异常,终变期染色体数目和体细胞染色体数目发生变化,而且早期世代变异幅度大.但随着繁殖世代的增加,花粉母细胞减数分裂染色体行为和体细胞染色体数目的变化渐趋稳定,至第五代基本稳定.对农艺性状已稳定的再生植株第九代不同株系总DNA进行RAPD-PCR扩增,结果表明,不同株系与未经培养的亲本相比在DNA水平上存在变异,出现亲本特异性谱带缺失,该类株系表现为矮秆、早熟.     

大麦（Hordeum vulgareL.）花药培养再生植株染色体的遗传分析和酯酶同工酶

通过观察８２株大麦花药培养再生植株根实细胞有丝分裂和５６株花粉母细胞（ＰＭＣ）减数分裂过程中染色体的结构和分离情况，结果表明，单倍体和二倍体植株各为１７．１％和３５．４％，混倍体植株４７．５％，在混倍体植株中，有二倍体，三倍体，四倍体甚至六倍体细胞和一些非整倍体细胞，但仍以单倍体，二倍体和四倍体细胞为主，根尖细胞有线分裂过程中有染色体结构的变异，如染色体断片，环状染色体等，二倍体ＰＭＣ减数分裂过程     

Production and characterization of “second generation” somatic hybrids derived from protoplast fusion between interspecific somatohaploid and dihaploidSolanum tuberosum L.

Protoplasts were fused to produce somatic hybrids between a triploid (2n=3x=32-34) interspecific somatohaploid betweenSolanum brevidens Phil. andS. tuberosum L., and a dihaploid (2n=2x=24) anther-derived line ofS. tuberosum cv. Van Gogh. A total of 265 plants were regenerated from protoplast fusion derived calli and their hybridity was verified using fusion partner specific RAPD markers. These “second generation” somatic hybrids were aneuploid pentaploids (2n=5x=51-65) with a 2C DNA content ranging from 3.36 to 4.43 pg, which corresponded to the sum of the 2C values of each of the fusion partners (somatohaploid: 2.22 pg; and the dihaploid line of cv. Van Gogh: 1.87 pg). Most of the “second generation” somatic hybrids were vigorous, but variable in morphology. They were extremely resistant to PLRV and they had tolerance to PVY infection derived from the somatohaploid fusion partner. Even though most of the “second generation” hybrids tuberized, the tuber morphology was variable and most were poorly shaped. InErwinia soft rot resistance tests, the tubers showed higher level of resistance than the tetraploidS. tuberosum cultivars, the dihaploidS. tuberosum fusion partners and the hexaploid somatic hybrids betweenS. brevidens andS. tuberosum. The “second generation” somatic hybrids were all male sterile and failed to produce berries or seeds.     

Sexual progeny of somatic hybrids between potato andSolanum brevidens: Potential for use in breeding programs

Hexaploid somatic hybrids have been obtained by fusion of protoplasts fromSolanum brevidens (PI 218228, 2x = 2n = 24) andS. tuberosum (PI 203900 or cv. Russet Burbank; 2x = 4n = 48). In the work reported here, pentaploid progeny derived from sexual crosses between the somatic hybrids and the potato cultivar, Katahdin were assessed for transfer of disease resistances and improvement of agronomic traits. Segregation was noted in disease resistances to race 0 ofPhytophthora infestans and to tuber soft rot caused byErwinia spp., indicating that sexual transfer of traits captured by protoplast fusion had occurred. The pentaploid progeny showed substantial variation in and often improvement of some agronomic traits as compared to the somatic hybrids. The results indicate that interspecific somatic hybridization can produce material with potential for use in a potato breeding program     

Clubroot resistance introgression in interspecific hybrids between Raphanus sativus and Brassica napus

Clubroot is a prevailing soil-borne disease affecting rapeseed production worldwide.However,few clubroot resistant rapeseed accessions were available for breeding.Identification and introgression of new clubroot resistant genes from closely related species by distant hybridization is an effective strategy.In the present study,9 radish(Raphanus sativus L.,2n=18,RR)lines resistant to Plasmodiophora brassicae pathotype 4 were used as donors to transfer clubroot resistance into a susceptible rapeseed(Brassica napus L.,2n=38,AACC)line by distant hybridization combined with embryo rescue.Nine intergeneric crosses were made but only 1(411×93039)produced F1 plants both from embryo rescue and natural seed-setting.Authenticity of triploid F1 hybrids(2n=28,ACR)were verified by flower color,cytological observation and molecular marker analysis,and 2 genuine F1 hybrids were identified.After chromosome doubling,these synthetic allohexaploid plants(2n=56,AACCRR)became partially fertile(pollen viability rate=35%)and were backcrossed with rapeseed parent to generate a BC1 population(2n=47,AACCR).Totally 178 BC1 plants were obtained,of which the majority(96.1%)were resistant to clubroot.These backcrossing progenies could be used for the breeding of new rapeseed varieties resistant to clubroot.     

Interspecific crossability and cytogenetic analysis of sexual progenies of Mexican wild diploid 1EBN speciesSolanum pinnatisectum andS. cardiophyllum

Mexican wild diploid species,Solanum pinnatisectum (S. pnt) (2n=2x=24, 1EBN: endosperm balance number) is a useful germplasm source of late blight and Colorado potato beetle (CPB) resistance in potato improvement. However, it is very difficult to cross this species with other 1EBNSolanum species. Sexual hybrids among three accessions ofS. pnt and two accessions ofS. cardiophyllum (S. cph) (2n=2x=24, 1EBN) were studied. There were large differences in the cross-compatibility among the genotypes and accessions from these two 2x-1EBN species. Interspecific incompatibility existed in the crosses betweenS. pnt andS. cph, in whichS. cph functioned only as the male parent. The crosses with accessions PI 275236 ofS. pnt produced 35 hybrids following the conventional propagation procedures. Seven hybrids were obtained with theS. pnt accession PI 275233 with the aid of embryo rescue, while hybridization involving accession PI 253214 resulted in no fruit. The cytogenetic analysis indicated that all of the hybrids derived from crosses of accession PI 275233 ofS. pnt withS. cph had 2n = 24 chromosomes and were pollen fertile. Crosses involving accession PI 275236 ofS. pnt produced both 2x (2n=24) and 3x (2n=36) hybrids. Meiotic analysis at metaphase I of pollen mother cells (PMCs) showed a fairly high rate of chromosome pairing that averaged between 10.10 and 11.10 bivalents per cell in most of the 2x hybrids, indicatingS. pnt andS. cph have the similar genome. A high frequency of trivalents and quadrivalents were observed in 3x hybrids derived fromS. pnt 2 ×S. cph 1, indicating homology exists among these chromosomes. These results suggest that specific gene(s) and not either differences between genomes or EBN controlled interspecific crossability and embryo development. The disease and insect tests on the 2x and 3x hybrids revealed that all have high levels of resistance to both late blight and CPB. Thus it could appear that selection of genotypes is a key for successful interspecific hybridization when using Mexican wild diploid species as a source of economic important traits.     

Differences between hybrids ofSolanum tuberosum L. andSolanum circaeifolium Bitt. obtained from symmetric and asymmetric fusion experiments

Summary Potato plants obtained by symmetric and asymmetric protoplast fusion experiments withSolanum tuberosum andSolanum circaeifolium were compared and their potential use for plant breeding was investigated. For asymmetric hybridisation wild species tissue was irradiated with X-rays. Irradiation enhanced regeneration frequency. RFLP analysis showed that both techniques gave approx. 95% hybrids among the regenerates. Asymmetric protoplast fusion gave 22% regenerates with a ploidy level between 2x and 4x or 4x and 6x. None of the regenerates obtained from symmetric fusion had these ploidy levels. The tetraploid hybrids showed good male and female fertility. Male fertility was highest for the eutetraploid regenerates. With increasing asymmetry and with increasing ploidy level fertility decreased. Berry induction and seed production were observed for the eutetraploid and, to a lesser degree, for the hypotetraploid and hypohexaploid hybrids.     

Selection of monoploids for protoplast fusion and generation of intermonoploid somatic hybrids of potato

The union of potato monoploid genotypes (2n=1x =12) through protoplast fusion may result in vigorous somatic hybrids due to a reduction of the “genetic load” normally present in this highly heterozygous tetraploid (2n=4x=48) crop. More than 100 androgenic monoploids derived from diploid (2n=2x=24)Solanum phureja Juz. & Buk. andS. chacoense Bitt. xS. phureja clones were evaluated in field trials during 1996 and 1997 to identify the most promising genotypes for protoplast fusion experiments. Compared to the total population, the 1996 selected genotypes had higher means for tuber number (30.1 vs 11.2 tubers/plant), average tuber weight (3.0 vs 1.8 g/tuber) and total yield (66.1 vs 20.4 g/plant). Similarly, the 1997 selected genotypes had higher means for tuber number (42.8 vs 25.4 tubers/plant), average tuber weight (3.6 vs 2.5 g/tuber) and total yield (114.0 vs 63.4 g/plant) compared to the total population. The 31 selected monoploid genotypes from 1996-97 varied in their response to protoplast isolation and culture from no growth (9), cell enlargement (5), limited cellular divisions (8), callus formation (5) to plant regeneration from callus (4). Chemical fusion and electrofusion produced three groups of intermonoploid somatic hybrids. Polymorphic simple sequence repeat (SSR) loci enabled distinction of somatic hybrids from parental somaclones. Rapid DNA extraction with SSR analysis enabled screening of calluses to identify somatic hybrid tissue prior to plant regeneration. The somatic hybrids were highly polyploid, mostly hexaploid (2n=6x=72), possibly due to fusion of endopolyploid protoplasts and/or chromosomal doubling during plant regeneration.     

Interspecific somatic hybridization betweenSolanum tuberosum L. andS. bulbocastanum dun. as a means of transferring nematode resistance

Interspecific somatic hybrids were produced between tetraploidSolanum tuberosum and a nematode-resistant accession of the diploid speciesSolanum bulbocastanum by protoplast fusion. Hybrid cells were selected using dual fluorescent labeling of protoplast preparations prior to fusion. Hybridity of regenerated plants was confirmed with a combination of morphological assessment, chromosome counting and isozyme analysis. Somatic hybrids had the same level of resistance to infection by race 1 of the nematodeMeloidogyne chitwoodi as theS. bulbocastanum parent used in the fusion. Some of the somatic hybrids were fertile as females when crossed with tetraploidS. tuberosum breeding lines. Thus, these hybrids can be used in a potato improvement program to incorporate a valuable pest resistance     

Utilizing the frost resistance of diploid solanum species

Introductions of 13 frost-resistant diploid (2n=2x=24)Solanum species (2 non-tuberous) were inter-crossed to produce F₁ hybrids involving two frost resistance sources. Successful F₁ combinations were: (i) inter-crossed to produce hybrids involving 3 to 4 frost resistance sources, (F₁×F₁); and (ii) crossed with haploids (2n=24) of frost-susceptible tetraploid (2n=4x=48)S. tuberosum subsp.tuberosum, (F₁×tbr). Thirty-five of the 75 crosses between the 13 species produced viable seed. The F₁ and F₁×F₁ progenies segregated to yield plants more resistant to freezing temperatures than the parental species. All attempts to cross these hybrids with 24-chromosometbr were successful. Although tuber production improved following the first cross totbr, the frequency of resistant plants decreased approximately 50%. Thus far, all first back-cross attempts, (F₁×tbr)×tbr, have succeeded. Utilizing hybrids synthesized from several sources of frost resistance as the donor parents, and clonal selections of 24-chromosometbr as the recurring parents, may provide a high order of resistance throughout the backcrossing program and implement selection of frost-resistant, long-day tuberizing parental stocks acceptable to the potato breeder.     

Segregation of leptines and other glycoalkaloids inSolanum tuberosum (4x) ×S. chacoense (4x) crosses

A clone, 8380-1, selected fromSolanum chacoense (PI 458310) for its high foliage content of the leptine glycoalkaloids, a factor in resistance to Colorado potato beetle, was doubled in chromosome number from 2n=2x=24 to 2n=4x=48. Three 4x clones were crossed with sixS. tuberosum (4x) clones. Foliage glycoalkaloid contents were measured for 452 F1 hybrids from 15 crosses. The 4xchacoense parental clones were not different in respect to glycoalkaloid contents and were similar to the original 2x clone. All F1 hybrids synthesized foliage leptines ranging from 9 to 369 mg/100 g fresh weight (fw) with a mean content of 113 mg/100 g fw. The proportion of leptines in the total glycoalkaloid content ranged from 1% to 62% with a mean of 25%. The 4xchacoense parent mean leptine content was 1482 mg/ 100 g fw which was 90% of the total glycoalkaloid content. Tubers from 136 hybrids and the threechacoense parental clones were tested for glycoalkaloid contents. The tuber solanine + chaconine contents of the 136 hybrids ranged from 30 to 180 mg/100 g fw with a mean of 79. The mean tuber content of the threechacoense parental clones was 157 mg/100 g fw. Leptines were not found in any of the tubers.