A cytogenetic and phenotypic characterization of somatic hybrid plants obtained after fusion of two different dihaploid clones of potato (Solatium tuberosum L.)

Summary Somatic hybrid plants of various ploidy levels obtained after chemical fusion between two dihaploid clones of potato Solanum tuberosum L. have been analysed by cytological, morphological and molecular methods. The hybrid nature of tetraploid and hexaploid plants and the genome dosage in hexaploid hybrids were confirmed by Giemsa C-banding. Tetraploid and hexaploid hybrids showed numerical as well as structural chromosome mutations. The latter occurred mainly in the nuclear organizing chromosome. The tetraploid hybrids were more vigorous than the dihaploid parents as demonstrated by an increase in height, enlargement of leaves, increase in the number of internodes, restored potential for flowering and increased tuber yield. The grouping of tetraploid somatic hybrids into various classes on the basis of leaf morphology revealed that plants with a full chromosome complement were more uniform than aneuploids. Many hexaploid somatic hybrids were also more vigorous than the dihaploid parents and could be grouped into two different classes on the basis of floral colour and tuber characteristics, the differences being due to their different dosage of parental genomes. Most of the tetraploid somatic hybrids showed pollen development halted at the tetrad stage as one of the parental clones contained a S. Stoloniferum cytoplasm. However, one tetraploid plant produced pollen grains with high viability. The chloroplast genome in the hybrid plants was determined by RFLP analysis. All of the hybrids had a cpDNA pattern identical to one parent, which contained either S. Tuberosum or S. Stoloniferum cpDNA. A slight preference for S. Tuberosum plastids were observed in hybrid plants. No correlation between pollen development and plastid type could be detected.     

Analysis of somatic hybrids between two sterile dihaploid Solanum tuberosum L. breeding lines. Restoration of fertility and complementation of G. pallida Pa2 and Pa3 resistance

Fourteen somatic hybrids generated by electrofusion of mesophyll protoplasts from a non-flowering dihaploid S. tuberosum clone, DHAK-11, and a male-sterile dihaploid clone S. tuberosum, DHAK-33, were grown in the greenhouse and subjected to morphological assessments and tests for fertility and resistance to the white potato cyst nematode Globodera pallida pathotypes Pa2 and Pa3. The ploidy level of the hybrids ranged from 38 to 63 chromosomes. All hybrids developed flowers with violet petals except for one, hy-56, that possessed red petals. The colour of the tuber skin was purple in all hybrids except in hy-56 where the tuber skin was red. All of the hybrids were female fertile and generated viable seeds. Near-tetraploid hybrids produced the highest number of seeds per fruit and these seeds had a normal size. Hybrids with 58 or more chromosomes produced smaller seeds and less seeds per fruit. The germination frequency of the seeds was not influenced by the chromosome number of the hybrids. Pollen viability was determined and the male fertility of three hybrids was tested. Pollination with these three hybrids gave rise to fruit development, but only one produced viable seeds. The hybrids were tested for resistance to G. pallida pathotypes Pa2 and Pa3. A high level of resistance to Pa3, inherited from one parental clone, DHAK-11, and a high level of resistance to Pa2, inherited from the other parental clone, DHAK-33, was combined in four hybrids. These results demonstrate, that protoplast fusion is an efficient method for restoring the fertility of somatic hybrids generated from sterile parent clones, and is a powerful procedure for the complementation of multigenetic disease resistance traits in potato breeding lines.     

Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides

Summary Leaf mesophyll protoplasts of Lycopersicon esculentum (2n=2x=24) were fused with suspension culture-derived protoplasts of Solanum lycopersicoides (2n=2x=24) and intergeneric somatic hybrid plants were regenerated following selective conditions. A two phase selection system was based on the inability of S. lycopersicoides protoplasts to divide in culture in modified medium 8E and the partial inhibition of L. esculentum protoplasts by the PEG/DMSO fusion solution. At the p-calli stage, putative hybrids were visually selected based on their hybrid vigor and lime-green coloration in contrast to slower growing parental calli characterized by a watery, whitish-brown coloration. Early identification of the eight hybrid plants studied was facilitated by isozyme analysis of leaf tissue samples taken from plants in vitro at the rooting stage. Regenerated plants growing in planting medium were further verified for hybridity by 5 isozymes marking 7 loci on 5 chromosomes in tomato. These included Skdh-1 mapped to chromosome 1 of tomato, Pgm-2 on chromosome 4, Got-2 and Got-3 on chromosome 7, Got-4 on chromosome 8, and Pgi-1 and Pgdh-2 both on chromosome 12. Fraction I protein small subunits further confirmed the hybrid nature of the plants with bands of both parents expressed in all hybrids. The parental chloroplasts could not be differentiated by the isoelectric points of the large subunit. Seven of the eight somatic hybrids had a chromosome number ranging from the expected 2n=4x=48 to 2n=68. Mixoploid root-tip cells containing 48, 53, 54 or 55 chromosomes for two of the hybrids were also observed.Michigan Agricultural Experiment Station Journal Article No. 11736. Supported by Grant No. I-751-84R from BARD — The United States — Israel Binational Agricultural Research and Development Fund     

Somatic hybrids between potato <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and wild species <Emphasis Type="Italic">Solanum verneï</Emphasis> exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum verneï (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.     

Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments—cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.Abbreviations GISH Genome in situ hybridization - RAPD Random amplified polymorphic DNA - SCF Small chromosome fragment - SSR Simple sequence repeat     

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。     

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.     

Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L.

Summary In order to produce fertile somatic hybrids, mesophyll protoplasts from eggplant were electrofused with those from one of its close related species, Solanum aethiopicum L. Aculeatum group. On the basis of differences in the cultural behavior of the parental and hybrid protoplasts, 35 somatic hybrid plants were recovered from 85 selected calli. When taken to maturity either in the greenhouse or in the field, the hybrid plants were vigorous, all rapidly overtopping parental individuals. The putative hybrids were intermediate with respect to morphological traits, and all of their organs were larger, particularly the leaves and stems. DNA analysis of the hybrids using flow cytometry in combination with cytological analysis showed that 32 were tetraploids, 1 hexaploid and 2 mixoploids. The hybrid nature of the 35 selected plants was confirmed by a comparison of the isoenzyme patterns of isocitrate dehydrogenase (Idh), 6-phosphogluconate dehydrogenase (6-Pgd) and phosphoglucomutase (Pgm). Chloroplast DNA (ctDNA) restriction analysis using Bam HI revealed that among the 27 hybrid plants analyzed, 10 had S. aethiopicum patterns and the 17 remaining hybrids exhibited bands identical with those of eggplant without any changes. All of the somatic hybrid plants flowered. Both parental plants had 94% stainable pollen, while the hybrids varied widely in pollen viability ranging from 30% to 85%. The somatic hybrids showed high significant variation in fruit production. Nevertheless, there was a tendency for low fertility to be associated often with S. aethiopicum chloroplast type and/or with an abnormal ploidy level, while good fertility was mostly associated with the tetraploid level and eggplant chloroplasts. Interestingly, 2 tetraploid somatic hybrid clones were among the most productive, yielding up to 9 kg/plant. As far as the fertility of the F₁ sexual counterpart was concerned, only 2 fruits of 50 g were obtained. Hybrid fertility in relation to phylogenetic affinities of the fusion partners is discussed.     

RAPD analysis of the interspecific somatic hybrids: Solanum bulbocastanum (+) S. tuberosum

The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H-8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids.     

柑桔细胞电融合再生两个种间体细胞杂种

朋娜脐橙(Citrussinensis Osbeck)胚性细胞悬浮系原生质体分别与粗柠檬(C.jambhiri Lush)、枸头橙(C.aurantium)叶肉原生质体经电场诱导而融合。经培养,两组合均获得再生植株。对朋娜脐橙+粗柠檬的再生胚状体进行染色体计数,随机取样的52个胚状体中,26个为四倍体,另外26个为二倍体;对74棵再生植株进行染色体计数,由此说明都为四倍体;表明体细胞杂种在植株再生过程中具有明显的竞争优势。朋娜脐橙+枸头橙再生的14棵植株都为四倍体。对朋娜脐橙+粗柠檬部分植株进行POX同工酶和RAPD分析,表明所有检测植株都为杂种。朋娜脐橙+枸头橙再生植株经RAPD分析,表明也为杂种。     

Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.Abbreviations CrGC Crucifer Genetics Cooperative, University of Wisconsin-Madison - MES 1-morpholino-ethane sulfonate - MS-3,0 Murashige and Skoog medium containing 3% sucrose and no growth regulators - RAPD random amplified polymorphic DNA - RC rapid cycling - RFLP restriction fragment length polymorphism - std standard deviation - TE 10mM Tris, 1 mM EDTA, pH 8     

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybridization can be an interesting alternative for the selection of heterozygous and vigorous potato plants through combination of dihaploid genomes. The resulting hybrids can harbour interesting characters and thus can be used in agriculture if they are in agreement with agronomic criteria.

In this report, we used an intraspecific somatic hybridization technique for the production of tetraploid potato lines. Two parental combinations were used in protoplast electrofusion procedure: Aminca-Cardinal and Cardinal-Nicola. The selection of somatic hybrids was based on in vitro plant vigour. Therefore, among the 75 regenerated plants obtained from Aminca-Cardinal fusion, 3 putative hybrids were retained and 2 plant lines were selected among the 54 regenerated from the Cardinal-Nicola fusion. Heterosis was observed in the larger hybrid tuber size compared to the parents’. Our results also showed a precocity in the in vitro tuberization for the hybrids. Moreover, all of the regenerated putative hybrids were tetraploid (2n=4x=48 chromosomes). Isocitrate dehydrogenase and malate dehydrogenase isoenzyme analyses confirmed the hybrid nature of these lines. A molecular characterization performed by PCR amplification of simple sequence repeats and inter-simple sequence repeats confirmed that all these lines were somatic hybrids.

The effect of potato virus Y infection on these hybrid lines was tested by mechanical inoculation of plants cultivated in a greenhouse. The majority displayed a reduction of infection rate associated with a delayed appearance of symptoms compared to the parents. Moreover, complete resistance was noted for one hybrid line (CN2). All hybrids also showed improved tolerance to Pythium aphanidermatum infection during tuber storage or after plant inoculation.     

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.     

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

Summary DNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

An examination of hybridization between the cattail species typha latifolia and typha angustifolia using random amplified polymorphic DNA and chloroplast DNA markers

Typha glauca represents a significant portion of the biomass of the wetlands surrounding the Great Lakes, USA. It is generally accepted to be a form of hybrid between T. latifolia and T. angustifolia, which itself appears to be an exotic introduction from Europe. Based on morphological and isozyme data, conflicting theories have been proposed for the hybrid nature of T. glauca: it has been described as a hybrid swarm, a distinct hybrid species and an F1 hybrid. Therefore, we developed random amplified polymorphic DNA (RAPD) and chloroplast DNA markers, specific to the parental species, to assess hybrids. Ten RAPD primers gave 17 fragments specific to T. angustifolia and 13 fragments specific to T. latifolia. All of the interspecific hybrids contained each of the species-specific markers, indicating an F1 hybrid status. Furthermore, all hybrids tested contained the T. angustifolia chloroplast haplotype, which is consistent with differential interspecific crossing success found previously. Additional confirmation of an F1 hybrid status was gained by examining seedlings from T. glauca. These progeny were expected to be advanced-generation hybrids, as opposed to the F1 hybrid parent. Analysis of the seedlings revealed segregating marker patterns consistent with patterns observed in experimental advanced-generation hybrids, although these advanced hybrids do not appear to be a significant part of mature stands. Our data do not provide support for extensive gene flow between T. latifolia and T. angustifolia. However, our results suggest that hybridization between the native and introduced Typha species has impacted the native population through the spread of the F1 hybrid, T. glauca.     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

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。     

Regeneration and analysis of interspecific asymmetric potato –Solanum ssp hybrid plants selected by micromanipulation or fluorescence-activated cell sorting (FACS)

 Recipient protoplasts from three Solanum tuberosum genotypes, cv ‘Folva’ (2n=4x=48), cv ‘Matilda’ (4n) and ‘161 : 14’ (2n), were electrofused with X-ray-irradiated donor protoplasts from two wild species S. spegazzinii (2n) or S. microdontum×S. vernei (2n). Prior to fusion, protoplasts were fluorescence-labelled with either fluorescein diacetate or scopoletin. Fusion products were identified by dual fluorescence and selected by micromanipulation or fluorescence-activated cell sorting (FACS). All putative hybrid plants were analysed by the random amplified polymorphic DNA (RAPD) technique. Our analysis demonstrates that each asymmetric hybrid plant has an individual and stable profile of donor-specific RAPD bands. The irradiation of donor protoplasts hampered the growth of selected heterofusion products in a dose-dependent way. Irradiation resulted in donor chromosome elimination, but not in a dose dependent way, in the tested interval. In asymmetric hybrids with the S. spegazzinii donor 33–68% of the donor-specific RAPD bands were missing, indicating a similar level of chromosome elimination. In asymmetric hybrid plants with the S. microdontum×S. vernei donor 74–95% of the donor RAPD bands were missing. Chromosome countings revealed that these hybrids had chromosome numbers equal to or below the chromosome numbers found in the tetraploid recipients. This is the first time that highly asymmetric hybrid plants between two tetraploid potato recipients and the donor S. microdontum×S. vernei have been obtained. Received : 16 December 1996 / Accepted: 21 February 1997     

Morphological, molecular and cytological analyses of Carica papaya×C. cauliflora interspecific hybrids

 Morphological, molecular and cytological analyses were performed to assess the hybridity of 120 putative interspecific hybrids of Carica papaya L.×C. cauliflora Jacq. In the putative interspecific hybrids the number of main leaf veins was intermediate between the two parents while the hermaphrodite flower sex form and the low vigour were distinctive features of these hybrids. Petiole length, stem diameter, leaf length, leaf width and flower colour were similar to C. papaya, whereas leaf shape, type, serration, venation, petiole hairiness and flower shape were similar to C. cauliflora. Markers generated by the polymerase chain reaction using 72 10-mer primers (random amplified polymorphic DNA) revealed a high level of polymorphism (64%) between C. papaya and C. cauliflora. Seventeen of these primers yielded reliable and easily scorable polymorphic banding patterns that were further screened to reveal hybrids. A range of 1–5 RAPD primers consistently confirmed that all 120 plants were genetic hybrids, with all of them containing at least one band from the male parent. Cytological analysis revealed that 7–48% of the cells in many of the interspecific hybrids were aneuploid suggesting that chromosome elimination was occurring. The frequency of aneuploid cells was negatively associated (r=0.88) with the number of bands from the male parent integrated into the hybrid. Pollen fertility of the hybrids was from 0.5 to 14.0% while C. papaya and C. cauliflora had 88.0–99.0% and 90.0–97.0% fertile pollen, respectively. Received: 28 February 1996 / Accepted: 27 September 1996