Intra- and interspecific gametosomatic hybridisation within the genus Petunia

Following PEG and high pH induced fusion, intraspecific gametosomatic hybrid plants (pollen tetrad protoplasts of a normal purple flowered variety of P. hybrida fused with cell suspension protoplasts of a nuclear albino mutant of the variety Blue Lace) and interspecific gametosomatic hybrid plants (tetrad protoplasts (as above) fused with cell suspension protoplasts of a nuclear albino mutant of P. parviflora) were recovered. Hybrid plants of both combinations possessed an intermediate vegetative and floral morphology with chromosome numbers of 2n=3x=21 and 2n=3x=25 respectively. Hybrid cells were in both systems identified as green colonies against an albino background as a result of complementation to chlorophyll proficiency. Pollen tetrad protoplasts did not divide. The production of such plants at the intra- and interspecific level in Petunia has shown that the concept of gametosomatic hybridisation can be extended to genera other than Nicotiana. An alternative selection strategy is available to that as used earlier for Nicotiana.     

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     

The production of fertile,triploid somatic hybrid plants (Nicotiana glutinosa (n) + N. tabacum (2n)) via gametic: somatic protoplast fusion

Summary The fusion of gametic protoplasts with somatic protoplasts giving rise to gametosomatic hybrid plants was investigated. Gametosomatic hybrid plants were regenerated following the fusion of nitrate reductase deficient (Nr^–) Nicotiana tabacum Nia-130 leaf mesophyll protoplasts with N. glutinosa tetrad protoplasts. The resulting plants were confirmed as hybrids, based on leaf and floral morphology, chromosome number, leaf esterase and leaf callus peroxidase zymograms and Fraction-1-protein analysis. The five gametosomatic hybrid plants had the expected pentaploid, but functionally triploid chromosome number of 3n=5x=60. The relevance of triploid gametosomatic hybrids in facilitating limited gene transfer, is discussed. The utilisation of tetrads as a generally available source of haploid protoplasts for fusion studies is proposed.     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon

Intergeneric somatic hybrid plants between Hamlin sweet orange [Citrus sinensis (L.) Osbeck] and Flying Dragon trifoliate orange (Poncirus trifoliata Raf.) were regenerated following protoplast fusion. Hamlin protoplasts, isolated from an habituated embryogenic suspension culture, were fused chemically with Flying Dragon protoplasts isolated from juvenile leaf tissue. The hybrid selection scheme was based on complementation of the regenerative ability of the Hamlin protoplasts with the subsequent expression of the trifoliate leaf character of Flying Dragon. Hybrid plants were regenerated via somatic embryogenesis and multiplied organogenically. Hybrid morphology was intermediate to that of the parents. Chromosome counts indicated that the hybrids were allotetraploids (2n=4x=36). Malate dehydrogenase (MDH) isozyme patterns confirmed the hybrid nature of the regenerated plants. These genetically unique somatic hybrid plants will be evaluated for citrus rootstock potential. The cell fusion, selection, and regeneration scheme developed herein should provide a general means to expand the germplasm base of cultivated Citrus by intergeneric hybridization with related sexually incompatible genera.Abbreviations MDH malate dehydrogenase - CTV citrus tristeza virus - MT Murashige and Tucker basal medium - BH3 protoplast culture medium, Grosser and Chandler, 1987 - PEG polyethylene glycol - GA3 giberellic acid - BA N-(phenylmethyl)-1 H-purin-6-amine - HCl hydrochloric acid Florida Agricultural Experiment Station Journal Series No. 7972     

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens harbouring pGV 38501103 neo (dimer) containing chimaeric kanamycin resistance reporter genes. Transformed protoplasts were regenerated to fertile and phenotypically normal plants. Transformation was confirmed by kanamycin resistance, nopaline production, neomycinphosphotransferase II activity, and Southern blot hybridization. Seed progeny from self-pollinated transformants expressed the introduced kanamycin resistance as a Mendelian trait.Abbreviations BAP 6-benzylaminopurine - Cf Claforan^R - 2.4D 2,4-dichlorophenoxy acetic acid - Km kanamycin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - NPT II neomycinphosphotransferase - npt II neomycinphosphotransferase II gene - NOS nopaline synthase - nos nopaline synthase gene - ocs octopine synthase gene - IAA indole-3-acetic acid     

Protoplast fusion and the cell cycle

Protoplasts isolated from rapidly dividing cell suspension cultures of either Nicotiana sylvestris or tumor derived cultures of Crepis capillaris were fused by PEG or liposome treatments to form homokaryons. Analysis of binucleates by Feulgen microspectrophotometry, and autoradiography, has revealed that whereas fusion products of all cell cycle combinations occur, protoplasts of certain cycle phases participate in fusions more frequently than expected, and there is a slight predominance of like-with-like cycle combinations. It is argued that this tendency towards specificity of fusion may be explained by cycle related variation in surface charge on protoplasts, and the mechanisms of action of the fusogens used.Abbreviations PEG polyethylene glycol - CAPT tumorous cell culture of Crepis capillaris - NS-1 cell culture of Nicotiana sylvestris     

Plant regeneration from leaf protoplasts of Brassica oleracea var. italica CV Green Comet broccoli

A procedure is described for regeneration of plants from leaf protoplasts of the hybrid broccoli cultivar, Green Comet (Brassica oleracea var italica). The totipotency of protoplasts isolated from plants regenerated from hypocotyl explants (GCR) was greater than that of protoplasts from plants grown directly from seed (GC). Using medium B developed by Pelletier et al (1983), division efficiencies greater than 70% were obtained in leaf protoplasts isolated from GCR. Approximately 1% of these protoplasts formed calli on solidified medium; 77% of the calli regenerated shoots. In contrast, protoplasts from seed-grown material showed a lower division efficiency (15–22%) and fewer protoplast-derived calli produced shoots. Some of the 178 protoplast-derived plants grown to maturity had variant phenotypes.Abbreviations NAA napthalene acetic acid - BA 6-benzylaminopurine - MES 1-morpholino-ethane sulfonate This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree     

Plant regeneration from callus-derived protoplasts of Pelargonium x domesticum

A protocol for regenerating plants from callus-derived protoplasts of Pelargonium x domesticum (rega l geranium cv. Melissa) has been developed. Protoplasts were isolated from leaf-derived callus tissue on MS medium supplemented with 3.0 mg/l naphthalene acetic acid, 2.0 mg/l 6- benzylaminopurine, and 3.0% sucrose. This callus yielded 2.7×10⁵ protoplasts/gram of tissue after a 6 hr incubation in an enzyme solution consisting of 2.0% cellulysin, 0.5% macerase, and 0.5 M sucrose. Protoplasts were plated at 1×10⁵ protoplasts/ml in a mixture (11 v/v) of KMP8/KP liquid medium layered on the same medium solidified with 0.6% agarose. Protoplast division was initiated within 2 days, and colonies of 15 to 50 cells developed 8 wk after plating. P-calli 1–2 mm³ developed 15 wk after plating, and plants regenerated from the p-calli have been transferred to the greenhouse.Abbreviations NAA naphthaleneacetic acid - 6-BAP 6-benzylaminopurine - CW Calcofluor White - FDA fluorescein diacetate     

Does pretreatment by rhodamine 6-G affect the mitochondrial composition of fusion-derived Nicotiana cybrids?

Rhodamine-6G(R6G), a lipophilic dye which degrades mammalian mitochondria, was shown to arrest the division of Nicotiana protoplasts. When albino recipient-protoplasts were treated with R6G and fused with X-irradiated (green) donor- protoplasts, only green cybrid plants were obtained. The mtDNA of the cybrids was analyzed by Southern-blot hybridization. We found that cybrids which resulted from N. rustica (donor) protoplasts, fused with R6G-treated albino protoplasts, had only parental-type mtDNA. When another donor, with N. undulata mtDNA, was used, most of the resulting cybrids contained non-parental mtDNA. Only one cybrid (out of 12) had N. undulata -type (donor) mtDNA.Abbreviations big N. bigelovii - IAA indoleacetic acid - mtDNA mitochondrial DNA - R6G rhodamine 6G - tbc N. tabacum und, N. undulata     

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Summary Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82–120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33–78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.     

Somatic hybridization in the genus Solanum: S. tuberosum and S. brevidens

Somatic hybrid plants were regenerated from fused mesophyll protoplasts of an albino potato (Solanum tuberosum spp. tuberosum) variant and Solanum brevidens, a non-tuber bearing species which is sexually incompatible with S. tuberosum. These somatic hybrid plants represent the first example of direct hybridization between potato and members of the taxonomic group Etuberosa, and offer the potential for introgressing valuable germplasm from Solanum species outside the sexually compatible range into a worldwide crop species.     

Callus formation from leaf mesophyll protoplasts of Malus Xdomestica Borkh. cv. Greensleeves

Large yields (1.85 × 10⁷/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. Greensleeves. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.Abbreviations BAP 6-benzylaminopurine - K kinetin - Z zeatin - GA₃ gibberellic acid - IBA 3-indole butyric acid - NAA 1-naphthalene acetic acid - IAA 3-indole acetic acid - ABA abscisic acid - f.wt. fresh weight - MS Murashige and Skoog (1962)     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

Callus formation from Malus x domestica cv. ‘Jonathan’ protoplasts

Protoplasts could be successfully isolated and cultured from callus and suspension cultures of Malus xdomestica cv. Jonathan. Protoplast-derived colonies were recovered when the osmoticum (glucose) was gradually reduced in semi-solid 8p medium or by the use of feeder plates. Formation of embryo-like structures was induced from the protoplast-derived callus on media supplemented with IAA and BA. These structures formed roots but plants failed to develop. Protoplasts could be isolated from leaves, but not from stems or petioles. The leaf protoplasts failed to divide.List of abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ABA abscisic acid - IAA indole acetic acid     

Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): factors important for efficient protoplast culture and plant regeneration

Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 l droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).Abbreviations BAP 6-Benzylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - IAA indole acetic acid - MES 2-(N-morpholino)- ethane sulfonic acid - NAA naphthalene acetic acid - PE plating efficiency     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Conditions for isolation and regeneration of viable protoplasts of oil palm (Elaeis guineensis)

Protoplasts were isolated from cell cultures of oil palm (Elaeis Guineensis). The protoplasts were cultured on a nurse medium containing oil palm cells in the presence of which protoplasts formed cell walls and divided to form cell cultures.Abbreviations NAA -naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - TEM thin-section electron microscopy     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid