In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

Regeneration of plants from hypocotyl protoplasts of rapessed (Brassica napus L. var. oleifera) cultivars

Protoplast cultures were prepared from hypocotyls of ten spring rapeseed cultivars. Protoplasts from all genotypes tested formed calli, and shoots were regenerated from calli of nine of the genotypes at frequencies varying from 15 to 76%. The regenerating cultivars fell into a high regenerating group (>60% and a low regenerating group <25%).     

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.     

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.     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Efficient plant regeneration from hypocotyl protoplasts of broccoli (Brassica oleracea L. ssp. italica Plenck)

We have assessed the capacity of cultured protoplasts from two tissue sources of several commercially-grown broccoli cultivars to regenerate plants. A procedure that employs hypocotyl protoplasts and a culture medium with a high NAA:2,4-D auxin ratio was developed. The procedure permits highly efficient formation of colonies that regenerate shoots at frequencies of 8–17% with two of the four cultivars tested. The time required for the development of plants from protoplasts was 8–11 weeks. No mtDNA rearrangements were observed among any of 17 analysed regenerants. Double-stranded RNAs were detected in mitochondrial DNA (mtDNA) preparations of some, but not all, regenerants of one of the cultivars.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - IAA 3-indoleacetic acid - NAA 1-naphthaleneacetic acid - ZR zeatin riboside - CF colony formation - mtDNA mitochondrial DNA     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝(Brassica oleracea var. capitata)和青花菜(Brassica oleracea var. italica)小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一, 研究其小孢子胚植株再生频率的影响因素, 提高胚再生植株频率, 对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材, 对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明: 游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证, 结果显示, 游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

结球甘蓝和青花菜小孢子胚植株再生

结球甘蓝（Brassica oleracea var.capitata）和青花菜（Brassica oleracea var.italica）小孢子胚再生植株频率低是目前影响游离小孢子培养技术有效应用的关键问题之一,研究其小孢子胚植株再生频率的影响因素,提高胚再生植株频率,对促进游离小孢子培养技术在甘蓝类蔬菜育种中更好地应用具有重要意义。该文以结球甘蓝中甘11和青花菜TI-111等基因型为试材,对影响游离小孢子胚再生成植株的固体培养基类型、琼脂浓度、胚的类型及胚在液体培养基中的滞留时间等因素进行了研究。结果表明：游离小孢子培养25天的子叶胚在琼脂浓度为1%–1.25%的B5培养基上植株再生频率最高。进一步通过8个不同基因型对上述实验结果进行了验证,结果显示,游离小孢子培养25天的子叶胚在1%琼脂浓度的B5培养基上植株再生频率达77.8%–97.2%。     

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.     

Agrobacterium-mediated transformation and plant regeneration of Brassica oleracea var. capitata

An efficient protocol for Agrobacterium tumefaciens-mediated transformation of four commercial cultivars of Brassica oleracea var. capitata is described. A strain of A. tumefaciens LBA4404 with the neomycin phosphotransferase gene (nptII) and a CaMV 35S-peroxidase gene cassette were used for co-cultivation. Preliminary selection of regenerated transgenic plants was performed on kanamycin-containing medium. The frequency of transgenic plants was calculated on the basis of GUS (β-glucuronidase) activity detected by the histochemical X-gluc test. Tissue-specific GUS expression driven by the peroxidase gene promoter in transgenic plants was analysed by GUS staining. The transformation rates of the commercial cultivars of B. oleracea was higher than in previous reports. Southern blot analysis revealed that integration of marker genes occurred in single and multiple loci in the genome. All transgenic plants grew normally after a brief vernalization period and showed stable inheritance of the marker gene. The present study demonstrates that morphologically normal, fertile transgenic plants of B. oleracea can be obtained. Received: 24 August 1999 / Revision received: 23 November 1999 / Accepted: 3 December     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

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.     

In vitro culture of Cuminum cyminum regeneration of flowering shoots from calli of hypocotyl and leaf explants

Calli from hypocotyl explant of Cuminum cyminum L. (Cumin) grew rapidly on Gamborg's B₅ basal medium with the following supplements, (i) 0.5 mg/l — 2,4-D (ii) 4 mg/l — NAA plus 2 mg/l — Kinetin and (iii) 0.2 mg/l — NAA plus 0.2 mg/l — BAP, whereas calli from leaf explant in these media grew slowly. Hypocotyl and leaf calli produced roots when transferred to basal medium only and shoots in basal medium with 0.5 mg/l NAA and 0.1 mg/l BAP. Ninety percent of the shoots produced roots when they were transferred to half strength MS inorganic salts supplemented with 0.5 mg/l each of IBA and NAA.Fifty to sixty percent of rootless as well as rooted shoots produced terminal umbellate flowers on this medium.     

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.     

In vitro regeneration of shoot buds and plantlets from seedling root segments of Brassica napus L.

Root segments obtained from aseptically germinated seedlings of Brassica napus cv. Westar were used to optimize conditions for high-frequency shoot bud differentiation. The presence of low kinetin (0.5 M) and relatively high indole-butyric acid (1.0 M) levels facilitated optimum shoot bud differentiation. Modified MS medium (MMS) was superior to the other three basal media tested (MS, B5 and White's). Elevated sodium dihydrogen phosphate levels increased the differentiation of shoot buds. Increasing or decreasing the level of sucrose from 3% reduced the frequency of explants forming shoot buds. Addition of glutamine enhanced both the frequency of responding explants, as well as the number of shoots per responding explant. Root segments from 13-day-old seedlings produced the highest response (58%) in the presence of 100 mg l^-1 glutamine. The position of the segment on the main root, size, and the presence or absence of lateral roots altered the morphogenic response. Sealing of the donor seedling cultures with Parafilm^® instead of Stretch' n seal^® resulted in a higher production of shoot buds, although root segment cultures were not affected by the type of sealing. Spontaneous rooting occurred on all developed shoots.Dedicated to Dr. Friedrch Constabel on the occasion of his 60th birthday     

In vitro propagation of cauliflower,Brassica oleracea var. botrytis for hybrid seed production

Methods for obtaining heterotic F₁ and maintaining purebred lines for breeding of Brassica oleracea are limited by absence of male sterile lines and occurrence of inbreeding depression, respectively. The use of vegetative (stem, petiole, leaf, leaf rib) and floral (peduncle, pedicel, flower bud, curd) explants of cauliflower to regenerate purebred lines for crossing were examined. Of four growth regulator treatments and explant types used, best results were obtained with curd explants on MS medium with 6-benzyladenine (cytokinin) and gibberellic acid. Although 6-benzyladenine alone promoted formation of shoots in floral explants, both 6-benzyladenine and α-napthaleneacetic acid were required for vegetative explants. Use of α-napthaleneacetic acid, however, often increased callus formation. These culture techniques to maintain purebred regenerated plants will complement newly-derived nuclear-based male sterile lines obtained by the introduction of antisense copies of the gene BcpI, which is required for pollen fertility. This revised version was published online in June 2006 with corrections to the Cover Date.     

Taxonomy and evolution of broccoli (Brassica oleracea var. italica)

The origin and application of the name broccoli are discussed and a distinction between cauliflower and broccoli is proposed, based on their relative ontogeny at marketable maturity. The history and evolution of broccoli is considered in relation to cauliflower and its diversification into annual and biennial types is discussed. White-sprouting broccoli is considered to be closely related to English winter-hardy cauliflower. Calabrese, though representing only a small part of the italica gene pool, has been the most intensively developed, being currently represented by many cultivars, including F₁ hybrids. The potential for breeding new Cape and sprouting broccolis is discussed, and the need to conserve existing genetic variability of existing cultivars within Cape and sprouting broccoli is stressed.     

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.     

Comparative analysis of glucosinolate production in hairy roots of green and red kale (Brassica oleracea var. acephala)

Abstract

Glucosinolates (GSLs) are sulfur- and nitrogen-containing secondary metabolites that function in plant defense and provide benefits to human health. In this study, using Agrobacterium rhizogenes R1000, green and red kale hairy roots were established. The expression levels of GSLs biosynthesis genes and their accumulation in both kale hairy roots were analyzed by quantitative real-time PCR and HPLC. The results showed that the expression of most indolic GSLs biosynthesis genes was higher in the hairy roots of green kale than in that of red kale. In contrast, the expression of BoCYP83A1 and BoSUR1 encoding key enzymes aromatic GSL biosynthesis was significantly higher in red kale hairy root. The HPLC analysis identified six GSLs. The levels of 4-methoxyglucobrassicin, glucobrassicin, and 4-hydroxyglucobrassicin were 6.21, 5.98, and 2 times higher, respectively, in green kale than in red kale, whereas the levels of neoglucobrassicin and gluconasturtiin were 16.2 and 3.48 times higher, respectively, in red kale than in green kale. Our study provides insights into the underlying mechanisms of GSLs biosynthesis in kale hairy roots and can be potentially used as “biological factories” for producing bioactive substances such as GSLs.