Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

A comparative study of DNA synthesis in cotyledon protoplast cultures of Brassica napus,B. campestris and B. oleracea using an immunocytochemical technique

The present study was designed (1) to observe the characterization of 5-bromo-2′-dexoyuridine (BrdU) incorporation into cultured Brassica cotyledon protoplasts and (2) to investigate the genetic differences in the levels of nuclear DNA synthesis (expressed by the percentage of nuclei labelled with BrdU) in cotyledon protoplast cultures from 12 cultivars of three Brassica species (Brassica napus, B. campestris and B. oleracea) at an early stage using immunocytochemistry. Nuclei labelled with BrdU were different from those showing only staining with 4′-6′-diamidino-2-phenylindole (DAPI) under fluorescence and light microscopy. Two to 5% of nuclei were labelled with BrdU after 1 h of culture, indicating that nuclear DNA synthesis occurred at a very early stage of culture. The percentage of nuclei labelled with BrdU increased with time over the length of the culture period. The mean percentage of nuclei labelled with BrdU in the 12 cultivars was about 25% at 24 h after culture initiation. The curve of the increase in percentage of nuclei labelled with BrdU exhibited an S-shape from 1 to 24 h. However, cultivar differences in percentages of nuclei labelled with BrdU were very significant over the time course of 1-24 h from initial culture, with cultivars Eureka (B. napus), Global (B. napus), Narc 82 (B. napus), Bunyip (B. campestris) and Sugar Loaf (B. oleracea) having a consistently higher percentage of nuclei labelled with BrdU than the other cultivars. Species differences were also significant, with cultivars of B. napus showing much higher percentages than the tested cultivars of B. campestris and B. oleracea. The results indicate that the differences in nuclear DNA synthesis in Brassica cotyledon protoplast cultures were most likely at both intra- and interspecies levels.     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple,versatile feeder layer system for Brassica oleracea protoplast culture

Protoplasts from cauliflower (Brassica oleracea ssp. botrytis) and broccoli (ssp. italica) leaves and hypocotyls were successfully cultured on membrane filters over a feeder layer of cells from a B. campestris suspension culture. Cells from rice, tomato and tobacco suspensions were not as effective as the B. campestris cells. Plants were recovered from protoplasts of previously recalcitrant Brassica genotypes. Protoplasts cultured in low numbers (10–100) on the feeder layer divided and formed colonies capable of plant regeneration, as did fused protoplasts.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - PCV packed cell volume     

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

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

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Asymmetric somatic hybrids of Brassica: partial transfer of B. campestris genome into B. oleracea by cell fusion

Summary To examine the possibility of producing asymmetric somatic hybrids of Brassica having a complete genome of one species and a part of the other, we fused inactivated B. oleracea protoplasts with X-irradiated B. campestris protoplasts. The plants obtained were studied with regard to their morphology, isozymes and chromosomes. The morphology of the hybrids was similar to B. oleracea in 9 out of 22 hybrids studied and the rest showed the intermediate phenotype of the parents. Analysis of three isozymes, leucine aminopeptidase, acid phosphatase and esterase indicated that ten hybrids lost B. campestris-specific bands in one or more of the three isozymes examined. The chromosome analysis showed that 90% of the hybrids were aneuploids. In addition, abnormal chromosomes were often found in root tip cells. These results suggested that the hybrids obtained were asymmetric in nature and resulted from elimination of B. campestris chromosomes by X-ray irradiation.     

Intergeneric (intersubtribe) hybridization between Moricandia arvensis and Brassica A and B genome species by ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (C₃–C₄ intermediate species) and Brassica A and B genome species (B. campestris and B. nigra) were produced via ovary culture. When M. arvensis was used as a female parent, the hybrid embryo yield (0.25–0.45 embryo per pollination) was similar between two genomes, regardless of the male parent. The reciprocal hybrid using B. campestris as a female was also obtained, although yield of embryo was lower (0.02 embryo per pollination). On the other hand, no hybrids were obtained without the in vitro technique. As most hybrid embryos could not develop normal shoots, plants were regenerated by inducing shoots on the cultured hypocotyl. The hybrid nature of the regenerated plant was confirmed morphologically and cytogenetically. A certain amount of bivalents (2.52-2.71) in the hybrids indicated the existence of partial chromosome homology between two genera. The present results indicate that ovary culture is an effective technique for overcoming the crossing barrier between M. arvensis and Brassica cultivated species.     

Study of factors affecting the culture of Brassica napus L. and B. juncea Coss. mesophyll protoplasts

Various factors affecting the culture of Brassica napus and B. juncea mesophyll protoplasts were examined in order to develop suitable culture media for these species. The basic components (salts and vitamins) of culture media K3 and Kao best supported cell division and colony development in protoplast culture of both species. The addition of casamino acids to Kao's medium resulted in colony browning in B. napus genotypes. B. napus protoplasts grew well with glucose as the osmotic stabilizer, whereas B. juncea protoplasts responded better to sucrose. High NAA and low 2,4-D combinations were effective in stimulating colony growth. Colony development was rapid for a range of genotypes cultured with these recommendations in these media and plant regeneration was obtained from protoplast-derived calli in both species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-Benzylaminopurine - MES 2(N-Morpholino)ethane sulfonic acid Contribution No. 931.     

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.     

Initiation of morphogenic cell-suspension and protoplast cultures of barley (Hordeum vulgare L.)

Cell-suspension cultures were initiated from embryogenic calli of various barley cultivars. Seven fast-growing suspension lines were obtained from four different cultivars (cvs. Dissa, Emir, Golden Promise and Igri). Two of these cell suspensions showed morphogenic capacity. From a cell suspension of cv. Dissa, albino plantlets were regenerated when aggregates were cultured on solid medium. Aggregates of cv. Igri usually stopped differentiation at the globular stage, but occasionally formed scutellum-like structures. Five suspension lines were used for protoplast isolation and culture. Dividing protoplasts were obtained from all lines, but with cv. Igri a few divisions only and no further development were observed. Protoplasts from the various lines differed in the time of first division (2–14 d), division frequency (0.09–70.9%) and efficiency of colony formation (0.09–7.3%). Protoplasts isolated from the morphogenic cell suspension of cv. Dissa developed compact calli which sporadically regenerated albino plantlets.Abbreviations CC, MS, N6, SH, Kao8p culture media; see Material and methods - cv chltivar - dicamba 3,6-dichloro-o-anisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes

Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Sweden and 1 from India, and 5 cultivars of B. oleracea from Sweden and 1 from China (B. alboglabra). A total of 17 isozyme loci was studied, 10 of these were polymorphic in B. campestris and 6 were polymorphic in B. oleracea. The level of heterozygosity seemed to be reduced in the Swedish cultivars compared to the Chinese landraces and cultivars of B. campestris. The level of heterozygosity in B. oleracea was even lower than that in the Swedish cultivars of B. campestris. A phylogeny of the cultivars and landraces of B. campestris showed that the B. campestris var yellow sarson cultivar, originating from India, deviated significantly from the other cultivars of B. campestris. A phylogeny of the cultivars of B. oleracea confirmed the expectations that the cultivar B. alboglabra was not closely related to the cultivated forms of B. oleracea.     

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     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

Regeneration of plants from protoplasts of rapid cycling Brassica oleracea L.

Rapid cycling Brassica species have great potential in plant genetic research because of their short life cycles and their minimal space requirements. Rapid cycling B. oleracea can be grown with up to six generations per year. Protoplast culture of this genotype can be applied for gene transfer by direct DNA uptake and by protoplast fusion. We here report on fast regeneration of flowering plants from protoplasts of rapid cycling B. oleracea. Regeneration frequencies of 27–65% were achieved with multiple shoots developing from individual calli. The regenerated plants were grown to maturity, and flowering and other morphological characteristics were monitored. The regenerants flowered within a similar time frame as plants grown from seeds. The ploidy level of regenerated and seed-grown plants was measured by flow cytometry. Many (20–45%) of the regenerants were tetraploid. Although only few seeds could be obtained from the tetraploids, large numbers of seeds with good germination were recovered from the diploid regenerants.Abbreviations MS-3,0 Murashige and Skoog medium containing 3% sucrose and no growth regulators - MES 1-morpholino-ethane sulfonate     

In situ hybridization with species-specific DNA probes gives evidence for asymmetric nature of Brassica hybrids obtained by X-ray fusion

Summary We have previously reported production of somatic hybrids between B. oleracea and B. campestris by fusion of B. oleracea protoplasts with X-irradiated B. campestris protoplasts, in order to transfer a part of the B. campestris genome into B. Oleracea. Our previous analysis of morphology, chromosome number, and isozyme patterns of the hybrids suggested that they are asymmetric in nature. To obtain further evidence for the asymmetric nature of the hybrids, we isolated B. campestris-specific repetitive sequences and used them for in situ hybridization of the chromosomes of the hybrids. The repetitive DNA probes could specifically identify 8 out of 20 chromosomes of the B. campestris genome, and analysis of the hybrids indicates that 1–3 chromosomes of B. campestris are lacking in all five hybrids examined, giving clear evidence for the asymmetric nature of the hybrids. Furthermore, in situ hybridization revealed that some of the abnormal chromosomes observed in the hybrids are generated by rearrangements of B. Campestris chromosomes caused by X-irradiation. Altogether, our study indicates that in situ hybridization using species-specific repetitive sequences is a useful tool to analyze chromosomal compositions of various types of hybrids obtained by cell fusion or conventional methods.     

Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol