Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Somatic embryogenesis in leaf callus from cauliflower (Brassica oleracea var. Botrytis)

Embryogenesis was induced in leaf callus of cauliflower (Brassica oleracea var. botrytis) maintained on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA, 1.0 mg l⁻¹) and kinetin (0.5 mg l⁻¹). The callus first developed meristematic nodules on the surface of which embryoids were initiated superficially. The callus masses whene transferred to the same medium with a lower concentration of IAA (0.1-0.01 mg l⁻¹) developed a much larger number of embryoids, which presumably developed from single superficial cells. All the stages of embryoid formation viz. globular, heart-shaped and torpedo-shaped were observed. A number of abnormalities were also noted. Precocious proliferation of superficial cells of the embryoids resulted in accessory embryoid development. Some of the embryoids showed a reversed polarity with respect to the tissue of origin. The origin, development and organisation of induced embryoids is discussed.     

Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the β -glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.     

Transformation of rapid cycling cabbage (Brassica oleracea var. capitata) with Agrobacterium rhizogenes

Summary Genetically transformed cabbage (Brassica oleracea var. capitata) roots were obtained after inoculation with two engineered Agrobacterium rhizogenes strains, each harbouring a plant selectable marker gene in their T-DNA. Axenic root clones resistant to kanamycin or hygromycin B were established, most of which did not exhibit the phenotypic characteristics of Ri-transformed roots. Shoot regeneration was induced from roots after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting plants exhibited various phenotypes: some looked normal, while others showed the transformed phenotype observed in other species. Direct evidence for genetic transformation was obtained by molecular hybridization. The trait was transmitted to the progeny. Transformed cabbage plants can be obtained within 6 months using this approach.     

Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica)

 An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II (NOS-NPTII-NOS) gene for kanamycin resistance and a β-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes the use of 200 μm acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mm mannopine and 50 μm acetosyringone in the co-cultivation medium and 1 mm arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13. Received: 2 July 1998 / Revision received: 9 February 2000 / Accepted: 17 February 2000     

Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) and cabbage (B. oleracea var. capitata)

Transgenic broccoli (Brassica oleracea var. italica) was produced by two Agrobacterium tumefaciens-mediated transformation methods. One used flowering stalk explants from mature plants; the other used hypocotyl and petiole explants from in vitro-grown seedlings. Several hundred transformants containing a Bacillus thuringiensis -endotoxin gene (CryIA(c)-type) and the neomycin phosphotransferase gene were recovered. Rooted transformants were obtained in as little as 3 months using seedling explants. Transgenic cabbage was also obtained by the seedling explant method. Parameters important for high efficiency regeneration and transformation rates included use of a tobacco nurse cell layer, sealing of petri dishes with a porous surgical tape instead of Parafilm, preculture of seedling explants and appropriate length of co-cultivation with Agrobacterium. Advantages and disadvantages of each transformation procedure are discussed.     

Agrobacterium rhizogenes-mediated transformation of Brassica oleracea var. sabauda and B. oleracea var. capitata

Agrobacterium rhizogenes A4M70GUS-mediated transformation of Savoy cabbage (Brassica oleracea L. var. sabauda) and two local lines of cabbage (B. oleracea L. var. capitata) was obtained using hypocotyl and cotyledon explants. The percentage of explants which formed roots was very high in all genotypes: 92.3 % in Savoy Gg-1, 64.4 % in cabbage P₂₂I₅, and 87.2 % in P₃₄I₅. Spontaneous shoot regeneration of excised root cultures grown on the hormone-free medium occurred in all three genotypes. In cabbage lines P₂₂I₅ and P₃₄I₅ shoot regeneration was higher (9.3 and 2.6 % respectively) than in Savoy cabbage Gg-1 (1.3 %). Transgenic nature of hairy root-derived plants was evaluated by GUS histological test and PCR analysis. All the tested cabbage shoots were GUS positive whilst in a Savoy cabbage GUS expression was registered only in 55 % of tested clones. PCR analysis demonstrated the presence of the GUS gene in regenerated shoot clones and in T₁ progeny.     

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis)

Summary. The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of -carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as membranous chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.     

Purification and characterization of peroxidase from cauliflower (Brassica oleracea L. var. botrytis) buds

Peroxidases (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) are part of a large group of enzymes. In this study, peroxidase, a primer antioxidant enzyme, was purified with 19.3 fold and 0.2% efficiency from cauliflower (Brassica oleracea L.) by ammonium sulphate precipitation, dialysis, CM-Sephadex ion-exchange chromatography and Sephadex G-25 purification steps. The substrate specificity of peroxidase was investigated using 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulphonic acid) (ABTS), 2-methoxyphenol (guaiacol), 1,2-dihydroxybenzene (catechol), 1,2,3-trihyidroxybenzene (pyrogallol) and 4-methylcatechol. Also, optimum pH, optimum temperature, optimum ionic strength, stable pH, stable temperature, thermal inactivation conditions were determined for guaiacol/H(2)O(2), pyrogallol/H(2)O(2), ABTS/H(2)O(2), catechol/H(2)O(2) and 4-methyl catechol/H(2)O(2) substrate patterns. The molecular weight (M(w)) of this enzyme was found to be 44 kDa by gel filtration chromatography method. Native polyacrylamide gel electrophoresis (PAGE) was performed for isoenzyme determination and a single band was observed. K(m) and V(max) values were calculated from Lineweaver-Burk graph for each substrate patterns.     

Plant regeneration from cotyledonary protoplasts of cauliflower (Brassica oleracea L. var.botrytis L.)

Summary Protoplasts isolated enzymatically from precultured cotyledonary leaves ofB. oleracea var.botrytis and cultured in KM8p medium (Kao andMichayluk 1975) underwent sustained divisions in about 0.1% population to eventually produce callus, whereas mesophyll protoplasts from either field grown orin vitro raised plants failed to divide. The callus readily differentiated on Murashige-Skoog medium as modified for shoot culture (Binding 1974) to give rise to shoot and roots.     

Genetic Transformation of Brassica juncea var. tsatsi Induced by Ri T-DNA of Agrobacterium rhizogenes

Hairy roots of mustard (Brassica juncea var. tsatsi) cv. "Paoye' were obtained from in vitro inoculation of reversely inserted petioles with Agrobacteriurn rhizogenes strain LBA9402 harbouring the agropine-type Ri plasmid (pRi1855). The root inducing rate was 100%. Transformed roots grew rapidly on hormone-free MS medium and showed typical hairy root phenotype. Transformed plantlets regenerated from hairy roots on MS medium supplemented with BA 8.0 mg/L and NAA 0.6 mg/L. Opine analysis evidenced the integration and expression of TR-DNA, PCR analysis and Southern hybridization confirmed the integration of TL-DNA including 862 bp rol B sequence in the transformed plants.     

Temporal and spatial root development of cauliflower (Brassica oleracea L. var. botrytis L.)

Row crops are often inefficient in utilizing soil resources. One reason for this appears to be inefficient rooting of the available soil volume. Five experiments were performed to study the temporal and spatial root development of cauliflower (cv. Plana). The crop was grown with 60 cm between rows, and root development was followed in minirhizotrons placed under the crop rows, 15 cm, and 30 cm from the crop rows. Soil was sampled and analyzed for nitrate content at the final harvest and once during growth. In two of the experiments N fertilizer rate was varied and in two of the other experiments two cultivars were compared (cv. Plana and Siria).The rooting depth of cauliflower was found to be linearly related to temperature sum, with a growth rate of 1.02 mm day^-1 °C^-1. Depending on duration of growth this leads to rooting depths at harvest of 85–115 cm. Soil analysis showed that the cauliflower was able to utilize soil nitrogen down to at least 100 cm.With Plana differences in root growth between row and interrow soil were only observed during early growth, but with Siria this difference was maintained until harvest. However, at harvest both cultivars had depleted row and interrow soil nitrate equally efficient. Nitrogen fertilizer did not affect overall root development significantly.The branching frequency of actively branching roots was increased in all soil layers from about 6 to 10 branches cm^-1 by increasing N fertilizer additions from 130 to 290 kg N ha^-1. Increasing N supply increased the number of actively branching roots in the topsoil and reduced it in the subsoil.The average growth rate of the roots was always highest in the newly rooted soil layers, but fell during time. At 74 days after planting very few roots were growing in the upper 60 cm of the soil whereas 70% of the root tips observed in the 80–100 cm soil layer were actively growing. Within each soil layer there was a large variation in growth rate of individual root tips.     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.     

A structure-specific endonuclease from cauliflower (Brassica oleracea var. botrytis) inflorescence.

A protein with structure-specific endonuclease activity has been purified to near homogeneity from cauliflower ( Brassica oleracea var. botrytis) inflorescence through five successive column chromatographies. The protein is a single polypeptide with a molecular mass of 40 kDa. Using three different branched DNA structures (flap, pseudo-Y and stem-loop) we found that the enzyme, a cauliflower structure-specific endonuclease, cleaved the single-stranded tail in the 5'-flap and 5'-pseudo-Y structures, whereas it could not incise the 3'-flap and 3'-pseudo-Y structures. The incision points occur around the single strand-duplex junction in these DNA substrates and the enzyme leaves 5'-PO4 and 3'-OH termini on DNA. The protein also endonucleolytically cleaves on the 3'-side of the single-stranded region at the junction of unpaired and duplex DNA in the stem-loop structure. The structure-specific endonuclease activity is stimulated by Mg2+ and by Mn2+, but not by Ca2+. Like mammalian FEN-1, the protein has weak 5'-->3' double-stranded DNA-specific exonuclease activity. These results indicate that the cauliflower protein is a plant structure-specific endonuclease like mammalian FEN-1 or may be the plant alternative.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l^?1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.     

Effects of temperature and irradiance on vegetative growth of cauliflower (Brassica oleracea L. botrytis) and broccoli (Brassica oleracea L. italica)

Cauliflower (Brassica oleracea L. botrytis) and broccoli (Brassicaoleracea L. italica) plants were grown in large pots in growthchambers for a range of temperatures (mean air temperaturesfrom 7.0-25.3 C) and irradi-ances (from 9.3-50.8 mol m^–2d^–1 or 4.7-25.4 MJ m^–2 d^–1). The extinctioncoefficient for PAR decreased with plant size reaching a valueof 0.55 in cauliflower and 0.45 in broccoli at plant leaf areasof 0.235 m² and 0.227 m², respectively. The leaf area expansionrate was unaffected by irradiance when compared at identicalleaf surface temperatures. The response of expansion rate tosurface temperature was fitted to a broken stick model witha base temperature of –0.7C and an optimum temperatureof 21.0C. The radiation conversion coefficient increased withair temperature below 13.8C and remained constant above this.The estimated radiation conversion coefficient above 13.8Cand for a PPFD of 20 mol m^–2 d^–1 was 0.77 g mol^–1in cauliflower and 0.87 g mol^–1 in broccoli. The radiationconversion coefficient declined with increasing irradiance levelfrom a maximum of 1.89 g mol^–1 at near nil irradiancein cauliflower. Key words: Leaf area, dry matter, radiation use efficiency, extinction coefficient