Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

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.     

Cytological analysis of tetraploid hybrids between sweet potato and diploid Ipomoea trifida (H. B. K.) Don.

Summary Tetraploid F₁ hybrids between Ipomoea batatas, sweet potato (2n = 6x = ca. 90), and diploid (2n = 2x = 30) I. trifida (H. B. K.) Don. showed various degrees of fertility reduction. The present study aimed to clarify its causes by cytological analysis of meiotic chromosome behavior in the diploid and sweet potato parents and their tetraploid hybrids. The diploid parents showed exclusively 15 bivalents, and the sweet potato parents exhibited almost perfect chromosome pairing along with predominant multivalent formation. Their hybrids (2n = 4x= 57–63) formed 2.6–5.0 quadrivalents per cell, supporting the autotetraploid nature. The meiotic aberratios of the hybrids were characterized by the formation of univalents, micronuclei, and abnormal sporads (monad, dyad, triad, and polyad). The causes underlying these aberrations were attributed in part to the multivalent formation, and in part to a disturbance in the spindle function. Three hybrids showing serious meiotic aberrations were very low in fertility. The utilization of the sweet potato-diploid I. trifida hybrids for sweet potato improvement is described and, further, the role of interploidy hybridization in the study of the sweet potato evolution is discussed.     

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)

Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readilyisolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N₆ medium and and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots.     

Transgenic sweet potato expressing mammalian cytochrome P450

Sweet potato [Ipomoea batatas (L.) Lam] is considered to be recalcitrant to transformation and regeneration because of its genotype-dependent in vitro responses. The lack of a genotype-independent transformation and regeneration system limits biotechnological applications in this plant species. To establish a transformation system for a diverse group of sweet potato genotypes, we examined sweet potato regeneration after transformation in five cultivars. An Agrobacterium tumefaciens transformation system was used for the introduction of mammalian cytochrome P450 genes, which are capable of conferring herbicide tolerance. Among the different factors studied, including explant type, plasmid vectors, and auxin type in the initiation media, auxin type had the greatest effect on the regeneration response. Of the auxins tested, only 4-fluorophenoxyacetic acid (4FA) induced regeneration from all cultivars. In terms of the quality of calli, 4FA promoted the induction of type I calli, which were capable of somatic embryo formation, whereas type II calli fail to produce somatic embryos. The frequency of somatic embryo formation was also affected by the composition of the embryo-induction media. Transgenic plants were regenerated from all cultivars. The stable integration and expression of transgenes was confirmed by several approaches. This Agrobacterium-mediated transformation system should be applicable to a wide range of sweet potato cultivars.     

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1^–1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1^–1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.Abbreviations MS Murashige and Skoog basal medium - IAA Indol-3-acetic acid - NAA naphthaleneacetic acid - 2,4-D dichlorophenoxyacetic acid - Mes 2-(N-morpholino)-ethanesulfonic acid - Cpw cell and protoplast washing solution     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum and S. bulbocastanum

The mesophyll protoplasts were isolated from the Solanum tuberosum (S. tbr) clones of different ploidy level (4x Bzura cv., 2x H-8105, and 2x ZEL-1136) as well as from the wild species: S. bulbocastanum (S. blb, 2x) and two accessions of S. nigrum (S. ngr, 6x). Additionally, the protoplasts were isolated from the cell suspensions of Bzura cv. and H-8105 clone. The conditions of protoplast isolation as well as the media for their culturing and regeneration, were selected and optimized for the studied genotypes. For mesophyll protoplasts, the shooting calli were produced by all the cultured protoclones except that of S. bulbocastanum. The shoots excised from the protoplast-derived calli developed into whole plants in all the studied potato clones but only in one accession of S. nigrum, i.e. S. ngr var. gigantea. As for suspension-cell-derived protoplasts, only H-8105 clone produced the regenerative type of calli, though normal shoots could not be obtained. The regenerative capacity of the protoplasts isolated from leaves and cell suspensions is compared and discussed. We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.     

Expression of green-fluorescent protein gene in sweet potato tissues

Green-fluorescent protein (GFP) gene expression, transient and stable after electroporation and particle bombardment, was analyzed in tissues of sweet potato cv.Beauregard. Leaf and petiole tissues were used for protoplast isolation and electroporation. After 48 h, approximately 25–30% of electroporated mesophyll cell protoplasts regenerated cell walls, and of these, 3% expressed GFP. Stable expression of GFP after four weeks of culture was observed in 1.0% of the initial GFP positive cells. In a separate experiment, we observed 600–700 loci expressing GFP 48 h after bombarding leaf tissue or embryogenic calli, and stable GFP-expressing sectors were seen in leaf-derived embryogenic calli after four weeks of protoplast culture without selection. These results demonstrate GFP gene expression in sweet potato tissues. Screening for GFP gene expression may prove useful to improve transformation efficiency and to facilitate detection of transformed sweet potato plants.     

Survey of genome sequences in a wild sweet potato,Ipomoea trifida (H. B. K.) G. Don

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log₂ ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Sweet potato (Ipomoea batatas (L.) Lam.) breeding has been hampered by self-and cross-incompatibilities that are frequently encountered among the plants in the section Batatas. Ovule culture techniques were developed to assist in overcoming some of these incompatibilities. Ovules that contain embryos at the late globular to heart shaped stage of development were cultured on MS medium containing full strength or one-half strength salts with 3%, 8% or 12% sucrose. Ovules were cultured either intact or after slicing. Ovules of I. triloba and I. trifida were successfully cultured as early as 3 and 4 days after pollination while sweet potato ovules were successfully cultured 5 and 6 days after pollination. The percentage of ovules with developing embryos on the media tested ranged from 27.8% to 50.2%. The highest percentage of embryos developed when the ovules were sliced and cultured on medium containing one-half MS salts and 8% sucrose. Three plants were recovered from cultured ovules of incompatible interspecific crosses.Abbreviations DAP days after pollination - MS medium Murashige and Skoog (1962) medium     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.     

Ultrastructure of fusion products from soybean cell culture and sweet clover leaf protoplasts

Summary Protoplasts from cultured cells of soybean (Glycine max L.) and from sweet clover (Melilotus officinalis L.) mesophyll cells were fused with polyethylene glycol and subsequently cultured for six days. The resulting fusion products as well as unfused protoplasts of each parental species regenerated cell walls and divided. The fusion products were characterized by the presence of soybean leucoplasts and sweet clover chloroplasts. The chloroplasts appeared to be degenerating but other cytoplasmic organelles were typical of actively growing plant cells. The fate of individual nuclei could not be determined.Supported by National Research Council of Canada, Grant A6304     

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.     

Somatic hybridization between Dianthus chinensis and D. barbatus through protoplast fusion

Summary Protoplasts isolated from leaf mesophyll cells of Dianthus chinensis and D. barbatus were fused by polyethylene glycol (PEG). Calli exhibiting vigorous growth were selected from the PEG-treated protoplasts and shoots were regenerated from one of these calli after 5 months of culture. These shoots readily rooted and continuously produced flowers in the in-vitro condition. The data on flower color, chromosome number, and esterase isozyme patterns indicated that this plantlet was an interspecific somatic hybrid. The hybridity of the plantlet was also confirmed by nuclear rDNA analysis. This report provides the possibility of applying the somatic hybridization technique for the genetic improvement of the genus Dianthus.     

黑果枸杞原生质体培养及植株再生

该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明：(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL^-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10⁶个·g^-1,活力为92%。(2)改良MS培养基 固体液体双层培养(MS₂ 固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^-1 6 BA+0.1 mg·mL^-1 IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。     

Somatic hybridization between selected Lycopersicon and Solanum species

Summary Mesophyll protoplasts of an interspecific Lycopersicon esculentum Mill, (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 10⁵/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium.Abbreviations ABA abscisic acid - BAP 6 benzylaminopurine - 2,4-D 2,4 dichlorophenoxy acetic acid - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - GOT glutamate oxaloacetate - IAA indoleacetic acid - IBA indolebutyric acid - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - MES morpholinoethane-sulfonic acid - PEG polyethylene glycol - 6-PGDH 6 phosphogluconate dehydrogenase - PGI phosphoglucoisomerase