Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10⁶ / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl₂ and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.Abbreviations BAP benzylaminopurine - NAA naphthaleneacetic acid - PCR Polymerase Chain Reaction - fw fresh weight     

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.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×10⁴ calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by Southern hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation

Protoplasts of the nicotinamide-deficient Hyoscyamus muticus cell line nic^– IVH2 and of the nitrate reductase-deficient Nicotiana tabacum cell line NR^– cnx 68 were induced to fuse. Selection for putative interspecific hybrid clones was via auxotroph complementation. Controls included tests for cross-feeding, cross inhibition, PEG-induced variation, culture-induced variation, reversion, viability, delayed selection. Protrotrophic cell lines were recovered exclusively from PEG-treated mixtures of both protoplast types. The putative hybrid clones grew independent of exogenously supplied auxins and cytokinins, and at a faster rate than either parent. The morphogenic potential of different clones varied from non-morphogenic to regeneration of fertile plants. Indirect evidence for the hybrid nature of the clones is provided from a) tight selection, b) hormone-independent growth, c) hybrid vigour, d) extreme morphological variation, e) isoenzyme bands from both parents, f) morphogenic potential. Definite proof for the hybrid nature was, however, provided from species-specific DNA hybridization. Chimerism could be excluded since only the large subunit of Hyoscyamus muticus ribulose bisphosphate carboxylase was found and since species-specific DNA hybridization identified clones which gave no Nicotiana tabacum signal.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BAP 6-Benzylaminopurine - PEG Polyethyleneglykol - MES (2[N-morpholino]ethane sulfonic acid - Tris-HCl Tris(hydroxymethyl)-aminoethane-HCl % is given throughout in w/v - FMI Friedrich Miescher-Institut     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR⁺) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR⁺ calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5–9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1–25.8% of the tomato genome. Thus, DNA contained in 3.8–13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.     

Root hair protoplasts ofLotus corniculatus L. (birdsfoot trefoil) express their totipotency

Treatment of the roots of 24–48 h old seedlings of the forage legumeLotus corniculatus with 1.0% Cellulase YC, and 0.1% Pectolyase Y-23 in 4.2% mannitol solution released protoplasts from the tips of root hairs within 30–40 sec of enzyme incubation. Roots from approximately 1000 seedlings yielded 1.7×10⁵ protoplasts. Ten percent of protoplasts divided to form cell colonies when cultured at 1.0×10⁵ ml^–1 in droplets of KM8P medium with 0.6% Sea Plaque agarose. Colonies formed callus on UM agar medium; protoplast-derived tissues produced shoots on B5 medium containing 0.05 mg 1^–1 of BAP. Regenerated plants were phenotypically and cytologically normal (2n=2x=24±2), and produced nitrogen fixing root nodules following inoculation withRhizobium. These results confirm the totipotency of protoplasts isolated from specialised epidermal cells of seedling roots ofLotus corniculatus.     

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.     

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.     

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     

Clonal propagation of Enset (Ensete superbum (Roxb.) Cheesman) through shoot tip cultures

Summary The morphogenetic potential of the shoot tip explants ofEnsete superbum (Roxb.) Cheesman, a wild relative of the cultivated bananas, was investigated and an effective clonal propagation method devised. Shoot tip explants grown in modified MS medium containing 1.5 mg l^–1 BAP and 1 mg l^–1 KIN developed corms which on transfer to medium containing 3 mg l^–1 IBA and 1.5 mg l^–1 BAP, regenerated a large number of shoots from the surface of the corm, the origin of which was traced to single hypodermal cells. Shoots were rooted on a half-strength MS medium salts containing 3 mg l^–1 IBA and 0.1 mg l^–1 BAP. The rooted plantlets were hardened and planted in the field where the plants looked normal.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

Molecular analysis of single copy and repetitive genes on chromosome 2 in intergeneric tomato somatic hybrid plants

Restriction fragment polymorphisms were used to identify and quantify the nuclear contributions from each parent to somatic hybrid plants between tomato (Lycopersicon esculentum Mill.) cv. Sub-Arctic Maxi and Solanum lycopersicoides Dun. Three single-copy clones, 2–13, 2–17, and 3–288, and a clone for the 45s ribosomal RNA, pHA2, all mapped to chromosome 2 of tomato, were used in analysis of 47 somatic hybrids. The amount of hybridizing probe for each parental band was quantified by densitometry of the autoradiograph film. Analyses with the three single-copy clones indicated that there were more than two S. lycopersicoides copies in most somatic hybrid plants. For at least one somatic hybrid there was a loss of one tomato copy. No evidence was found for more than two copies donated from tomato or loss of a copy from S. lycopersicoides. Most of the observed variation in copy number of the single-copy clones was consistent with chromosomal changes occurring in the suspension cells from which S. lycopersicoides parental protoplasts were derived.The number of copies of rDNA derived from each parent varied independently of the number of copies of single-copy clones from each parent. Changes in the copy number of rDNA occurred in both tomato and S. lycopersicoides genomes.     

Asymmetric somatic hybrid plants between Medicago sativa L. (alfalfa,lucerne) and Onobrychis viciifolia Scop. (sainfoin)

This paper reports on the production of intergeneric somatic hybrid plants between two sexually incompatible legume species. Medicago sativa (alfalfa, lucerne) leaf protoplasts were inactivated by lethal doses of iodoacetamide. Onobrychis viciifolia (sainfoin) suspension-cell protoplasts were gamma-irradiated at lethal doses. Following electrofusion under optimized conditions about 50,000 viable heterokaryons were produced in each test. The fusion products were cultured with the help of alfalfa nurse protoplasts. Functional complementation permitted only the heterokaryons to survive. A total of 706 putative heterokaryon-derived plantlets were regenerated and 570 survived transplantation to soil. Experimentation was aimed at the introduction of proanthocyanidins (condensed tannins) from sainfoin, a bloat-safe plant, to alfalfa, a bloat-causing forage crop; however, no tannin-positive regenerant plants were detected. Most regenerant plants have shown morphological differences from the fusion parents, although, as expected, all resembled the recipient parent, alfalfa. Southern analysis using an improved total-genomic probing technique has shown low levels of sainfoin-specific DNA in 43 out of 158 tested regenerants. Cytogenetic analysis of these asymmetric hybrids has confirmed the existence of euploid (2n=32; 17%) as well as aneuploid (2n=30, 33–78; 83%) plants. Pollen germination tests have indicated that the majority of the hybrids were fertile, while 35% had either reduced fertility or were completely sterile.     

In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson)

Endosulfan tolerant lines of mustard (Brassica campestris cv. Brown Sarson) have been developed through tissue culture methods. Cotyledonary expiants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus was then transferred to MS medium containing (0.1–2.0 ugl^–1) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endosulfan tolerant regenerated plants were also tolerant to 3.0 ugl endosulfan, thereby, suggesting that tolerance has been acquired at the gene level.Biochemical investigation revealed higher levels of total free sugar, free amino acids, protein and activity of peroxidase in the tolerant cell line.Abbreviations MS Murashige and Skoog medium (1962) - NSM non-selective medium - SM selective medium - BAP 6-Benzylaminopurine - NAA -naphthaleneacetic acid - Z zeatin