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     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

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     

Studies of protoplast culture and plant regeneration from commercial and rapid-cyclingBrassica species

Protoplasts were isolated from aseptic shoot cultures of commercial cultivars ofBrassica napus, B. oleracea andB. campestris, and from the six rapid-cycling brassica species. Of the rapid-cycling species, onlyB. napus responded well to the culture conditions used; 2% of protoplasts formed calli and up to 5% of calli regenerated shoots. Regeneration was also achieved from commercial cultivars ofB. napus andB. oleracea. For these two species the plating density, time of dilution with fresh medium and the composition of the shoot-inducing medium were all found to have an important influence on the efficiency of plant regeneration. Both responded better to maltose than to sucrose-based media. Under the optimum conditionsB. napus showed a plating efficiency of 7.8% and shooting efficiency of 17%; forB. oleracea the figures were 2% and 56%, respectively.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Expression of the C3-C4 intermediate character in somatic hybrids between Brassica napus and the C3-C4 species Moricandia arvensis

The wild crucifer Moricandia arvensis is a potential source of alien genes for the genetic improvement of related Brassica crops. In particular M. arvensis has a C₃-C₄ intermediate photosynthetic mechanism which results in enhanced recapture of photorespired CO₂ and may increase plant water-use efficiency. In order to transfer this trait into Brassica napus, somatic hybridisations were made between leaf mesophyll protoplasts from cultured M. arvensis shoot tips and hypocotyl protoplasts from three Brassica napus cultivars, Ariana, Cobra and Westar. A total of 23 plants were recovered from fusion experiments and established in the greenhouse. A wide range of chromosome numbers were observed among the regenerated plants, including some apparent mixoploids. Thirteen of the regenerated plants were identified as nuclear hybrids between B. napus and M. arvensis on the basis of isozyme analysis. The phenotypes of these hybrids were typically rather B. napus-like, but much variability was observed, including variation in flower colour, leaf shape and colour, leaf waxiness, fertility and plant vigour. CO₂ compensation point measurements on the regenerated plants demonstrated that 3 of the hybrids express the M. arvensis C₃-C₄ intermediate character at the physiological level. Semi-thin sections through leaf tissues of these 3 plants revealed the presence of a Kranz-like leaf anatomy characteristic of M. arvensis but not found in B. napus. This is the first report of the expression of this potentially important agronomic trait, transferred from Moricandia, in M. arvensis x B. napus hybrids.     

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.Abbreviations PEG Polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - IBA Indole-3-butyric acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962)     

Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare X Triticum aestivum hybrids

Summary Embryogenic callus cultures were obtained by culturing young inflorescence tissues of Hordeum vulgare cv. PF51811 (2x)XTriticum aestivum cv. Chinese Spring (6x) hybrids on 2,4-D-containing N₆ medium. After subculture for about 10 months the calli retained a high potentiality for somatic embryogenesis and plant regeneration. Of about 300 regenerated plants, approximately 100 were transplanted to potting soil. Eight embryoids and three regenerated plants examined had 28 chromosomes identical to the original hybrid plants, while one regenerated plant was found to be a mixploid composed of cells with 28 and 56 chromosomes. The possibility for obtaining amphiploid hybrids through tissue culture is discussed.     

Improved protoplast culture and stability of cytoplasmic traits in plants regenerated from leaf protoplasts of cauliflower (Brassica oteracea ssp.botrytis)

Nearly 1000 plants have been regenerated from leaf protoplasts of two cauliflower (Brassica oleracea ssp.botrytis) alloplasmic inbred lines. One line (7642A) carried the Ogura (R1) cms cytoplasm derived from radish; the other line (7642B) carried a normalBrassica cytoplasm and was the fertile maintainer for the cms line. The majority of regenerated plants displayed normal vegetative morphology; they formed normal cauliflower heads and retained the floral characteristics of seed-grown plants from which they were derived. We found no change in either male sterility or in the low temperature-induced chlorosis associated with the 7642A line. Mitochondrial DNA analysis by hybridization with five cloned mtDNA probes revealed no apparent alteration in 75 regenerated plants of both lines. These results indicate that cytoplasmic traits inBrassica oleracea are stable after one cycle of in vitro culture and regeneration.     

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Summary An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 M atrazine. Its chromosome number was 36–38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.     

Turgor-dependent efflux of assimilates from coats of developing seed of Phaseolus vulgaris L.: water relations of the cells involved in efflux

The turgor-homeostat model of assimilate efflux from coats of developing seed of Phaseolus vulgaris L. was further characterised. The turgor pressure (P), the volumetric elastic modulus () and hydraulic conductivity (L_p) of the seed coat cells responsible for assimilate efflux and cotyledon storage parenchyma cells were determined with a pressure probe. In addition, turgor of the seed coat and cotyledons was estimated by measuring the osmolalities of symplastic and apoplastic fluids extracted by centrifugation. Osmolality of symplastic and apoplastic saps collected from the seed coat declined significantly over the period of seed development from a cotyledon water content of 80% to 50%. However, the difference in osmolalities of the apoplastic and symplastic saps remained relatively constant. For cotyledons, osmolality of the apoplastic sap exhibited a significant decline during seed development, while the osmolality of symplastic sap did not change significantly. Hence cotyledon P increased as the water content dropped from 80% to 50%. For both detached and attached empty seed coats, a small decrease (ca. 40mOsmol·kg^–1) in the osmolality of the bathing solution, led to a rapid increase in P of cells involved in assimilate efflux (efflux cells) by about 0.07 MPa. Thereafter, cell P exhibited a rapid decline to the original value within some 20–30 min. When P of the efflux cells was reduced by increasing the osmolality of the bathing solution, P exhibited a comparable rate of recovery for attached empty seed coats but there was no P recovery to its original value in the case of detached seed coats. In contrast, the cotyledon storage parenchyma cells did not exhibit P regulation when the osmolality of the bathing solution was changed. The observations that the efflux cells of P. vulgaris seed coats can rapidly adjust their P homeostatically in response to small changes in apoplastic osmolality are consistent with the operation of a turgor-homeostat mechanism. The volumetric elastic modulus () of the seed coat efflux cells exhibited a mean value of 7.3±0.8 MPa at P=0.15 MPa and was found to be linearly dependent on cell P. The e of the cotyledon storage parenchyma cells was estimated to be 6.1±1.0 MPa at P=0.41 MPa. Hydraulic conductivity (L_p) of the seed coat cells and the cotyledon cells was (8.2±1.5) × 10^–8m·s^–1·MPa^–1and (12.8±1.0) × 10^–8 m·s^–1·MPa^–1, respectively. The relatively high , i.e., low elasticity, for the seed coat cell walls would ensure that small changes in water potential of the seed apoplast will be reflected in large changes in cell P. The high L_p values for both the seed coat and the cotyledon cells is consistent with the rapid changes in P in response to changes in water potential of the seed apoplast.Abbreviations LYCH Lucifer Yellow CH - volumetric elastic modulus - L_p hydraulic conductivity - P turgor pressure - osmotic pressure - t_1/2 half-time for water exchange The investigation was supported by funds from the Australian Research Council. We are grateful to Louise Hetherington for competent technical assistance and to Kevin Stokes for raising the plant material.     

Protoplast culture and regeneration from Brassica oleracea ‘rapid cycling’ and other varieties

The results are reported for the first time on successful plant regeneration from mesophyll-derived protoplasts of rapid cycling B. oleracea. Comparative data were also presented on plant regeneration from mesophyll-derived protoplasts of two other varieties namely var. botrytis and var. gemmifera. It was found that a modified Pelletier (Pelletier et al. 1983) protocol is highly beneficial for protoplast culture and plant regeneration from mesophyll-derived protoplasts. The plating efficiency of B. oleracea rapid cycling protoplasts was, in the best combination of isolation method, culture technique and culture media 4.5%±0.4% and the plant regeneration frequency approximately 15%. Plant regeneration was further improved by transferring the calli from the D medium of Pelletier to a callus growth medium (MS11) and subsequently to the K₃ regeneration medium of Glimelius (Glimelius 1984). Various factors influencing plating efficiency and plant regeneration from mesophyll protoplasts of B. oleracea are discussed.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - IBA 3-indole butyric acid - NAA 1-naphtylacetic acid - 2iP N⁶-(2-isopentyl)adenine     

Prolific direct plant regeneration from cotyledons of white clover

Summary A facile procedure has been developed to regenerate white clover (Trifolium repens L.) plants, rapidly and directly from cotyledon explants of 3 day old seedlings. Scanning electron microscopy and histological sectioning demonstrated that shoot meristems developed from individual epidermal cells on the adaxial surface of the cotyledonary stalk, proximal to the site of excision. Initial cell divisions occurred after 2 days of culture and regenerated plants were transferred to soil within 6–8 weeks. Regenerated plants were normal, flowered and set seed. The highest shoot regeneration frequency (an average of 20 shoots per cotyledon) was obtained using an MS based medium containing 1.0 mg 1^-1 6-benzylaminopurine and 0.05 mg 1^-1 -napthaleneacetic acid. A similar regeneration frequency was obtained from cotyledon explants taken from eight different white clover cultivars.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - MS Murashige and Skoog medium     

In vitro multiplication of a sterile interspecific hybrid,Brassica fruticulosa x B. campestris

In vitro methods for plant multiplication of a sterile interspecific hybrid between Brassica fruticulosa and B. campestris through either micropropagation or callus regeneration is described. Shoot-tip, single-node and leaf explants, obtained from in vitro-grown hybrids, regenerated on media containing NAA and BA. In vitro application of colchicine induced chromosome doubling in in vitro-regenerated shoots resulting in the production of fertile amphidiploids. Comparative studies on regeneration potential of the hybrid and its parents were also carried out using callus from leaf explants. The explants of B. fruticulosa and the hybrid were capable of shoot and root formation while those of B. campestris failed to form shoots but produced profuse roots. The results demonstrate the efficacy of an in vitro method in producing a large number of hybrid plants and fertile amphidiploids from incompatible crosses that yield very few hybrid seeds/seedlings.Abbreviations BA benzyladenine - CMS cytoplasmic male sterile - AA diploid genome of B. campestris - FF diploid genome of B. fruticulosa - NAA -naphthaleneacetic acid     

Plant regeneration from petal protoplast culture ofPetunia hybrida

Morphologically normal plants have been regenerated from petal protoplasts of petunia (Petunia hybrida) flower. Maximum protoplast yields from petal tissues were obtained within 2 days after anthesis. Protoplasts were cultured on modified Murashige and Skoog's medium in which NH₄NO₃ and Fe·EDTA concentrations were reduced to 1/3 (7mM) and 1/10 (10 M), respectively. After plating, protoplasts gradually reduced pigment density, and plastids developed near the nucleus. In premitotic petunia petal cells, the nucleus moved from the periphery to the central region of the cell. The first cell divisions were detected after 6–10 days of culture initiation, and the average division frequency was 15% in the best culture condition. The results indicated that the time of the first cell division and cell division frequency were closely related to flower age after anthesis. More than a hundred plants with morphologically normal shoots and roots have been obtained. Those plants grew vigorously in soil.Abbreviations BA benzylaminopurine - DAPI 4, 6-diamidino-2-phenylindole - 2, 4-d dichlorophenoxyacetic acid - Fe·EDTA Fe·ethylenediaminetetraacetate - IAA indole-3-acetic acid - MtSB microtubule stabilizing buffer - NAA -naphthaleneacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid     

Transformation of Nicotiana tabacum,N. debneyi,and N. rustica: Inheritance and protoplast expression of antibiotic resistance

Agrobacterium tumefaciens strains harbouring plasmid vectors pBCAT1, pVU1011 or pMON806 were used to transform leaf explants of Nicotiana tabacum cultivars Delgold and Candel, N. debneyi, and N. rustica var. NRT. Transgenic plants resistant to the selective agents kanamycin, hygromycin or methotrexate were regenerated and used as sources of leaf mesophyll protoplasts. Protoplasts divided and regenerated plants in the presence of selective agents at levels inhibitory to protoplasts of non-transformed plants. Cross-resistance of protoplasts to more than one selective agent was not observed in this study which suggests that this approach may lead to an efficient interspecific somatic hybrid selection system.     

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were Corbit (first subculture); GAF/Park and 88Ab3073 (second subculture); and GAF/Park and 87Ab5932 (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. GAF/Park, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes GAF/Park, 88Ab3073, and Tibor. The smaller clusters only regenerated plants for GAF/Park and 88Ab3073. From one gram of callus used to initiate suspensions of GAF/Park and 88Ab3073, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of GAF/Park, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Somatic embryogenesis,plant regeneration and somaclonal variation in barley

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25–60%). For Dissa and Ingrid, young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of Golden Promise responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of Golden Promise and Ingrid, most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alteractions were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1- to 6- month-old cultures of barley.     

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     

“Erussica”, the intergeneric fertile somatic hybrid developed through protoplast fusion between Eruca sativa Lam. and Brassica juncea (L.) Czern.

Summary Hypocotyl calli-derived protoplasts of two cultivars of Brassica juncea (2n=36), a major oil-seed crop, were fused with normal as well as -irradiated mesophyll protoplasts of Eruca sativa (2n=22). The irradiation of the Eruca fusion partner increased the plating efficiency as well as the morphogenic potentiality of the fusion products over the normal fusion. Fertile plants could be regenerated from such fusion products. Analysis of 63 out of 181 plants regenerated showed that, indeed, 11 somatic hybrids (2n=58) and 10 partial somatic hybrids (chromosome number ranged between 50 and 56) had been obtained. Pollen viability (0%–82.9%) and seed set (0%–50%) of the hybrids indicated them to be useful for future studies.