烟草属花粉—叶肉原生质体的融合及杂种植株再生

用饥饿预处理分离烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）品系Ｎ３６４ Ｋｍ ＋ 二核早中期的花粉原生质体,用ＰＥＧ－高钙高ｐＨ 法诱导其与黄花烟草（Ｎ．ｒｕｓｔｉｃａ Ｌ．）叶肉原生质体融合。通过抗性筛选再生的４ 株小植株,经过氧化物酶同工酶、根尖染色体计数鉴定均为配子－体细胞三倍体杂种。未经筛选处理再生的２１株小植株,经鉴定有６ 株为配子－体细胞杂种。     

Regeneration of Hybrid Plantlets Via Pollen-Hypocotyl Protoplast Fusion in Brassica spp.

It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.     

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Summary Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10⁶ mature pollen protoplasts were fused with 7·10⁶ mesophyll protoplasts using a PEG/Ca²⁺ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l^-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l^-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.Abbreviations AAT aspartate aminotransferase - BCP bromocresol purple - EST esterase - MES 2-(N-morpholino) ethanesulfonic acid - AP acid phosphatase - PEG polyethyleneglycol - PER peroxydase     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Phenotypic Variation and Changes of Chromosome Number in Plantlets Regenerated from Potato Protoplasts

Morphological variation and change of chromosome number in the plantlets of potato (Solanum tuberosum L. cv. Xiao Yie Zi × Duo Zi Bai) regenerated from mesophyll protoplasts have been studied. The normal plantlets from protoplasts were similar to parent plants. Their chromosome numbers were 2n = 48±or 2n= 72 of euploid. The plantlets with distinctive phenotypic variation were likely to be aneuploid with increased chromosome numbers.     

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Summary Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.     

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt

Summary Green mesophyll protoplasts of the dihaploid potato line 1982 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 679 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%–4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Mesophyll Protoplast Culture and Plant Regeneration of Oriental Planetree (Platanus orientalis)

Large populations of mesophyll protoplasts were released from the leaves of 1.5–2 month old sterile seedlings, with a high protoplast yield (3.7× 10 6g-1FW) after protoplast purification. The purified protoplasts were cultured in a modified K8p liquid medium supplemented with 0.5 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L BA. Higher density (1× 106/ml) in the initial culture of protoplasts is favourable to the division of cultured mesophyll protoplasts of this woody species among the densities tested. The protoplasts started to divide after 6 days of culture, and achieved 26.8% division frequency by 14 days. Sustained divisions resulted in mass production of cell colonies and small calli in 8 weeks. The calli further grew to 2–3mm on the gelrite-solidified K8 medium supplemented with 0.2 mg/L NAA aud 0.5 mg/L BA. Then, they were transferred onto the MSB proliferation medium with 0.1 mg/L NAA and 0.25 mg/L BA, where compact and cream-coloured calli were formed. Shoot formation was initiated on MSB differentiation medium coraming 0.5 mg/L IAA, 1 mg/L each of BA and ZT. It was observed that the frequency of shoot formation was about 28.7%. Whole plantlets were regenerated upon transferring 3 cm shoots to 1/2MS medium with 0.5mg/L IBA and 0.1mg/L BA, from which they were already transplanted into pots and grew well in the phytotron of Shanghai Institute of Plant Physiology.     

Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Studies on Plant Regeneration from Mesophyll Protoplasts of Solanum tuberosum L.

Protoplasts from potato mesophyll of two strains (Solannum tuberosum L. cv. Xiao Yie Zi x Duo Zi Bia and Solanum tuberosum L. cv. Wu Meng 601) were induced to callus in culture medium of protoplasts. The callus derived from mesophyll protoplasts were transferred to MS medium with 2 mg/l ZT+0.1 mg/L IAA. Shoots regenerated from the callus were detected after 70 days of culture.The shoots which had grown to a height of 2–3 cm were transferred to MS medium with 0.05 mg/L NAA. Roots were coming out in a few days.Complete plantlets were achieved. Stern segments with 1–2 leaves were then transferred to a mixture of sterilized soil and grown, and produced tuber.     

Production of asymmetric somatic hybrid plants between Cichorium intybus L. and Helianthus annuus L.

In order to obtain male-sterile asymmetric somatic hybrids between chicory (Cichorium intybus L.) and a sunflower (Helianthus annuus L.) male-sterile cytoplasmic line, mesophyll chicory protoplasts inactivated with iodoacetic acid and hypocotyl sunflower protoplasts irradiated with γ-rays have been fused, using PEG and applying two different procedures. Thirty three plants were regenerated from putative hybrid calli. A cytological analysis of their root-tip cells indicated that most of them had 18 chromosomes, the same number as chicory. Through Southern hybridisation on total DNA using the maize mitochondrial specific gene probes Cox I, Cox II and Cob, three plants were identified as cytoplasmic asymmetric hybrids, as shown by hybridisation bands specific for both chicory and sunflower. One of the regenerated plants produced a novel pattern of hybridisation that was not detected in either parent. When hybridisation of total DNA was carried out with an atpA mitochondrial gene probe the same three cybrids presented both the fertile chicory fragment and the male-sterile sunflower fragment. Finally, Southern hybridisation with an ORF 522 probe, which in sunflower is co-transcribed with the atpA gene, confirmed the hybrid nature of the three plants. The morphology of the cybrids resembled the parental chicory phenotype, and at anthesis their anthers produced fewer pollen grains which could not germinate either ”in vitro” or ”in situ.” Cybrid plants grown in the field produced seeds when free-pollination occurred. Received: 26 April 2000 / Accepted: 28 August 2000     

Somatic hybridization between Lycopersicon esculentum and Lycopersicon pennellii

Summary Selection and screening methods were devised which resulted in the identification of a number of somatic hybrid callus clones following fusion of Lycopersicon esculentum protoplasts and L. pennellii suspension culture protoplasts. Visual selection for callus morphology combined with a high fusion frequency and irradiation of one parental protoplast type (¹³⁷Cs source, 1.5 Krads) resulted in selection of a callus clone population containing a high proportion of somatic hybrids. Analysis of a dimeric isozyme for the presence of a heterodimeric form was found to be satisfactory for distinguishing parental-type calli, somatic hybrid calli, and mixed calli derived from both types of unfused parental cells. No somatic hybrid calli produced shoots, although the sexual hybrid between L. esculentum and L. pennellii regenerated well under the culture conditions employed. This result suggests that the non-regenerable growth habit of the L. pennellii suspension culture was dominant in the somatic hybrid. The culture conditions described here are suitable for obtaining regenerated plants from L. esculentum mesophyll protoplasts. L. esculentum protoplast calli from fusion cultures gave rise to shoots with L. esculentum phenotype at higher frequency than calli from control unfused L. esculentum mesophyll protoplast cultures. The use of probes for species-specific organelle DNA fragments allowed identification of organelle DNA restriction fragments in digests of total DNA from small samples of individual callus clones. The callus clones analyzed either carried predominantly one parental plastid DNA type or mixtures of both types. Use of a mitochondrial DNA (mtDNA) probe which distinguishes two parental mtDNA fragments revealed that the L. pennellii-specific fragment was present in all clones examined, but the L. esculentum fragment was absent or in low proportion.     

Enrichment of heterokaryocytes between mesophyll and epidermis protoplasts by density gradient centrifugation after electric fusion

Summary Mesophyll protoplasts from Nicotiana glauca were fused with epidermal protoplasts from N. langsdorffii by an electric pulse. After the fusion products were centrifuged on stepwise density gradient centrifugation using Percoll and sea water, somatic hybrids were observed at 70%–80% in the fraction recovered from the intermediate specific gravity fraction between epidermis and mesophyll protoplasts. From offsprings of these somatic hybrids, teratomatous plants were regenerated. Since the difference of specific gravity between mesophyll and epidermis protoplasts is inherent, this procedure can be essentially applied to obtain somatic hybrids between any combination of plants. The significance of this study is discussed in relation to obtaining somatic hybrids between plant materials without any appropriate genetic markers.     

Somatic hybridization between birdsfoot trefoil (Lotus corniculatus L.) and L. conimbricensis Willd

Summary Somatic hybrid plants were produced by fusion of birdsfoot trefoil (Lotus corniculatus) cv Leo and L. conimbricensis Willd. protoplasts. Birdsfoot trefoil etiolated hypocotyl protoplasts were inactivated with iodoacetate to inhibit cell division prior to fusion with L. conimbricensis suspension culture protoplasts. L. conimbricensis protoplasts divided to form callus which did not regenerate plants. Thus, plant regeneration from protoplast-derived callus was used to tentatively identify somatic hybrid cell lines. Plants regenerated from three cell lines exhibited additive combinations of parental isozymes of phosphoglucomutase, and L. conimbricensis-specific esterases indicating that they were somatic hybrids. The somatic chromosome number of one somatic hybrid was 36. The other somatic hybrid exhibited variable chromosome numbers ranging from 33 to 40. These observations approximate the expected combination of the birdsfoot trefoil (2n=4x=24) and L. conimbricensis (2n=2x=12) genomes. Somatic hybrid flowers were less yellow than birdsfoot trefoil flowers and had purple keel tips, a trait inherited from the white flowered L. conimbricensis. Somatic hybrids also had inflorescence structure that was intermediate to the parents. Fifteen somatic hybrid plants regenerated from the three callus lines were male sterile. Successul fertilization in backcrosses with birdsfoot trefoil pollen has not yet been obtained suggesting that the hybrids are also female sterile. This is the first example of somatic hybridization between these two sexually incompatible Lotus species.Formerly USDA-ARS, St. Paul, Minn, USA     

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.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Regeneration Of Plantlets from Solanum nigrum L. Mesophyll Protoplasts

Using the “EA3-867” cellulase prepared in our laboratory, viable mesophyll protoplasts were isolated from Solanum nigrum L. The protoplasts grew and divided when cultivated in banging drops and thin liquid layer of NT medium, and calli formed. After transfering the calli on Dudits and MS solid medium (both supplemented with zeatin 1 mg/L, NAA 0.5 mg/L), regenerated plantlets had been obtained. The effects of different inositol amounts in NT medium on the growth of protoplasts were compared. The percentage of 1st and 2nd cell division after 5–8 days culture and the number of cell clusters increased in NT medium containing 250 mg/L inositol.