Novel flowering and fatty acid characters in rapid cycling Brassica napus L. resynthesized by protoplast fusion

Novel rapid cycling Brassica napus lines have been produced by protoplast fusion between rapid cycling B. oleracea and rapid cycling B. rapa. Fusion products were selected based on iodoacetate inactivation and regeneration ability. A total of 36 plants was recovered from 3 regenerating calli. All were confirmed as somatic hybrids by morphological features, flow cytometric estimation of nuclear DNA content, RAPD analysis and/or DNA hybridization. Plants from two of the calli contained chloroplasts from B. rapa, and plants from the third contained B. oleracea chloroplasts. Some plants flowered in vitro, but on average flowering was initiated 22 days after transfer to soil. Although seed set was fairly low after self pollination, more seeds were obtained from pollination of open flowers than from pollination of buds. Seeds of the somatic hybrid B. napus showed novel fatty acid compositions, different from the mean of the two parental lines. Flowering was monitored in plants grown from seeds of the somatic hybrids, rapid cycling B. napus (CrGC 5-1) and the two diploid parental genotypes. Progeny of the somatic hybrids flowered faster and were more vigorous than rapid cycling B. napus (CrGC 5-1). The improved lines contain chloroplasts from B. rapa, unlike rapid cycling B. napus (CrGC 5-1), which has B. oleracea chloroplasts. The somatic hybrid lines produced may be useful for genetic studies or further in vitro manipulations.Abbreviations CrGC Crucifer Genetics Cooperative, University of Wisconsin-Madison - MES 1-morpholino-ethane sulfonate - MS-3,0 Murashige and Skoog medium containing 3% sucrose and no growth regulators - RAPD random amplified polymorphic DNA - RC rapid cycling - RFLP restriction fragment length polymorphism - std standard deviation - TE 10mM Tris, 1 mM EDTA, pH 8     

Plasticity in Sex Expression of Spinach (Spinacia oleracea) Regenerated from Root Tissues

The sex expression of spinach (Spinacia oleracea L.) was modified by a procedure where plants were regenerated from root callus of spinach over three successive generations. Somatic embryos obtained from the root callus of female or male plants were grown in vitro under long day conditions until flowering. Although the regenerants derived from somatic embryos derived from female plants were all female at the first generation, few progenies produced male flower organs and expressed gynomonoecy in subsequent generations. Once the female plants regenerated into gynomonoecy, they never expressed complete femaleness at post generation. However, the male plants easily altered into andromonoecy at subsequent generations, and several andromonoecious plants showed the sex reversion at post generation, although this phenomenon did not occur in the female progenies. No absolute sex conversion was found in both female and male progenies. The plasticity of the sex expressions of spinach indicates that female and male individuals might essentially be capable of generating both flower organs.     

Mapping loci controlling flowering time in Brassica oleracea

The timing of the transition from vegetative to reproductive phase is a major determinant of the morphology and value of Brassica oleracea crops. Quantitative trait loci (QTLs) controlling flowering time in B. oleracea were mapped using restriction fragment length polymorphism (RFLP) loci and flowering data of F₃ families derived from a cabbage by broccoli cross. Plants were grown in the field, and a total of 15 surveys were made throughout the experiment at 5–15 day intervals, in which plants were inspected for the presence of flower buds or open flowers. The flowering traits used for data analysis were the proportion of annual plants (PF) within each F₃ family at the end of the experiment, and a flowering-time index (FT) that combined both qualitative (annual/biennial) and quantitative (days to flowering) information. Two QTLs on different linkage groups were found associated with both PF and FT and one additional QTL was found associated only with FT. When combined in a multi-locus model, all three QTLs explained 54.1% of the phenotypic variation in FT. Epistasis was found between two genomic regions associated with FT. Comparisons of map positions of QTLs in B. oleracea with those in B. napus and B. rapa provided no evidence for conservation of genomic regions associated with flowering time between these species.     

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     

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.     

An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants

A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L. Mill). More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1–0.5% glucose. Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation. The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048. The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry. The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population. The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes. Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82–100%), followed by cotyledon petiole-derived plants (63–78%). Transgenic plants produced from hypocotyls contained 22–58% diploids depending on the genotype used in transformation. Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.Abbreviations BAP Benzylaminopurine - MS Murashige-Skoog - MsCHI Medicago sativa chalcone isomerase - PMI Phosphomannose isomerase     

The influence of plant growth regulator concentrations and callus age on somaclonal variation in callus culture regenerants of strawberry

The effect of plant growth regulator concentrations and ageing of callus on the extent and nature of variation among callus culture regenerants of strawberry (Fragaria × ananassa) cv. Redcoat was examined. Plants regenerated from callus culture had reduced plant vigour, shorter petiole length and smaller leaf size, but more leaves and runners under greenhouse conditions. These responses appeared to be due to a physiological influence of plant growth regulators. No distinct phenotypic variants were observed at plant growth regulator concentrations in the range of 1–10 M each of BA and 2,4-d combination, but the highest concentration (20 M each) of this combination produced a high frequency (10%) of dwarf type variants. The dwarf nature of these variants was maintained in the runner plants produced by the primary regenerants. The plants regenerated from 8-week-old calli did not show any distinct morphological variants. However, a significant proportion of deformed leaf shape (6–13%) and yellow leaf (21–29%) variants was obtained among plants regenerated from 16 and 24-week-old calli. The primary regenerants of the leaf shape variants were established as chimeras. The chimeric plants produced runner progeny with normal plants and plants with completely distorted leaf morphology. Both leaf shape and yellow leaf variants remained stable through runner propagation. Isozyme analysis failed to distinguish any of the variants from the standard runner plants. Flow cytometric analysis indicated the aneuploid nature of leaf shape variants but it could not distinguish dwarf and yellow leaf variants from standard runner plants.     

Quantitative trait loci affecting plant regeneration from protoplasts of<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

Quantitative trait loci (QTLs) controlling the plant-regeneration ability of Brassica oleracea protoplasts were mapped in a population of 128 F₂ plants derived from a cross between the high-responding, rapid-cycling line and a low-responding, broccoli breeding line of B. oleracea. A modified bulked segregant analysis with AFLP markers identified two QTLs for plant regeneration. In a multiple regression analysis, the two QTLs explained 83% of the total genetic variation for regeneration recorded 15 weeks after initial transfer of microcalli to regeneration medium. Both QTLs showed additive effects, and the alleles contributing to the high regeneration frequencies were derived from the high-responding, rapid-cycling line. Using microsatellites with known location, the two QTLs were mapped to linkage groups O2 and O9 on the map published by Sebastian et al. [(2000) Theor Appl Genet 100:75–81] or to chromosomes C8 and C7 on the map published by Saal et al. [(2001) Theor Appl Genet 102:695–699]. QTLs for the early flowering trait of the rapid-cycling parent have previously been mapped to the same two linkage groups. Association between flowering time and regeneration ability was, however, not found in the present material, indicating that plant-regeneration ability can be transferred between cultivars independently of the early flowering trait. The detection of two major QTLs for plant regeneration in B. oleracea may provide the initial step towards the identification of markers suitable for marker-assisted selection of regeneration ability.     

Isozyme Variability in Plants Regenerated from Calli of Cereus peruvianus (Cactaceae)

Morphological and isozyme variation was observed among plants regenerated from callus cultures of Cereus peruvianus. Different morphological types of shoots (68%) were observed in 4-year-old regenerated plants, while no distinct morphological variants were observed in plants grown from germinated seeds. Isozyme patterns of 633 plants regenerated from calli and of 261 plants grown from germinated seeds showed no variation in isocitrate dehydrogenase isozyme, and the differential sorbitol dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, acid phosphatase, and peroxidase isozyme patterns observed in regenerated plants were attributed to nonallelic variation. Allelic variation was detected at three isoesterase loci. The proportion of polymorphic loci for both populations was 13.6% and the deviation from Hardy–Weinberg equilibrium for the Est-1 and Est-7 loci observed in somaclones was attributed to the manner in which the regenerant population was established. The high values for genetic identity among regenerant and seed-grown plant populations are in accordance with the low levels of interpopulation genetic divergence. In somaclones of C. peruvianus, morphological divergence was achieved within a short time but was not associated with any isozyme changes and also was not accompanied by biochemical genetic divergence.     

RAPD and ISSR analyses of regenerated pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L. plants

Long-term pea callus cultures of different genotypes (mutants R-9 and W-1 and cultivar Viola) were used to regenerate plants (generation R₀). The regenerants displayed changes both in qualitative and in quantitative traits. The most dramatic morphological alterations and complete sterility were observed in regenerants of the cultivar Viola. To estimate the genetic differences, regenerants were compared with the original lines with the use of RAPD (random amplified polymorphic DNA) and ISSR (inter simple sequence repeat) analyses. The extent of divergence varied among regenerants and depended mostly on the original genotype. The genetic difference from the original line was no more than 1% in W-1 regenerants, 0.7–5.3% in R-9 regenerants, and 10–15% in sterile regenerants of the cultivar Viola. The genetic variation of plants regenerated from a callus culture maintained for ten years did not exceed that of plants obtained from a culture maintained for two years.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 71–77.Original Russian Text Copyright © 2005 by Kuznetsova, Ash, Hartina, Gostimskij.     

Chromosome variation in wheat plants regenerated from cultured immature embryos

Summary A cytological study has been made of plants regenerated from cultured immature embryos of four wheat cultivars (Triticum aestivum, 2n = 6x = 42). In total, 29% of the 192 plants examined were aneuploid with a range in chromosome numbers of 38–45. Evidence of chromosome structural changes was also found. This variation occurred in regenerants of all four cultivars, but there were large differences in the proportions of aneuploids arising from individual cultures which meant that no significant differences could be demonstrated between cultivars. Chromosome abnormalities were present in plants regenerated both from embryogenic cultures and from cultures in which the origin of shoots could not be distinctly defined.     

In vitro culture of isolated microspores and regeneration of plants in Brassica campestris

A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.     

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.     

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.)

Summary Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.     

Production of new CMS Brassica oleracea by transfer of `Anand' cytoplasm from B. rapa through protoplast fusion

New types of cytoplasmic male sterility (CMS) in Brassica oleracea would be useful for F₁ hybrid seed production. The `Anand' cytoplasm derives from the wild species B. tournefortii. Rapid cycling stocks of B. rapa and B. oleracea were used in cybridization experiments as donor and recipient of `Anand' (=`tour') CMS, respectively. Prior to fusion with PEG, donor protoplasts were inactivated with 30 krad γ-rays and recipient ones with 3 mM iodoacetate, respectively. No calli were obtained from the pre-treated protoplasts. The frequency of shoot regeneration was 21–43% in untreated B. oleracea controls, but only 0–0.5% in `Anand' B. rapa. Putative cybrids were regenerated from about 3% of the calli from fused protoplasts. Regenerated plants were analyzed for nuclear DNA content, plant and flower morphology, pollen production, female fertility, cold tolerance, and organelle composition. Eighty-one percent of the regenerated controls and 63% of fusion-derived plants were diploid. The rest showed DNA contents corresponding to 2x–4x, 4x, or higher ploidy levels, presumably due to somatic doubling in vitro and/or fusions in which the donor nucleus was not completely eliminated. Sixty-four percent of the cybrids had stamens and petals varying in size and shape and were male-sterile, with indehiscent anthers. Their phenotype was otherwise similar to that of B. oleracea. The remaining plants had normal flowers and were male-fertile. Data from crosses with fertile pollinators indicated good female fertility in some of the sterile lines, both after hand and insect pollinations in cages. Mitochondrial (mt) segregation in the cybrids was slightly biased towards `Anand' mitochondria, and the presence of `Anand' mtDNA fragments was strongly associated with male sterility. Evidence of mtDNA rearrangements was obtained in some cybrids. Segregation of chloroplasts was slightly biased towards B. oleracea. The presence of `Anand' chloroplasts with a B. oleracea nucleus did not result in cold temperature chlorosis, as seen in `Ogura' CMS plants. Received: 22 February 1996 / Accepted: 10 May 1996     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

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.     

Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency.

Summary An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed. This procedure yielded an average transformation rate of 76% for ecotype C24, and 15–20% for ecotypes Landsberg-erecta and Columbia. A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium. Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection. Transformed cells developed calli and regenerated shoots within 4–5 weeks of culture. A total of 1500 fertile transgenic plants were regenerated. In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and -glucuronidase) as well as by genetic segregation tests. R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria. Short duration (7–8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis