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%。     

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.     

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.     

Boron Mobility and Nutrition in Broccoli (Brassica oleracea var. italica

Broccoli (Brassica oleracea var. italica cv Premium Crop) plantswere germinated in soil, transferred to vermiculite three weekslater and grown in the glasshouse, then either supplied continuouslywith boron levels ranging from 0.0 (deficient) to 12.5 (toxic)mg l^–1 of nutrient solution or transferred from 2.5 to0.0 mg B l^–1 at the initiation of inflorescence development.At commercial maturity the concentrations of various inorganicand organic solutes in phloem exudates and xylem saps, as wellas plant characteristics and elemental composition of the variousplant parts, were determined. Under deficient B levels leaf midrib and stem corkiness wereevident, together with signs of stem pith breakdown, symptomswhich resemble the initiation of the hollow stem disorder. Thexylem sap B concentration declined by about 50 % when B wasnot supplied or was removed after a period of adequate supply;the phloem concentration was unaffected. Also, the decreasingB concentration gradients from mature transpiring tissues toyoung developing sinks disappeared. Therefore, it is concludedthat when B is deficient, it is retranslocated from source leavesin the phloem stream supplying the developing leaves and inflorescence.The data also suggested that at toxic levels B undergoes extensivelateral transfer, probably from xylem to xylem, thereby enhancingthe B concentration of developing sinks. The B regime influenced dry-matter partitioning, retranslocationof some elements, and the synthesis and distribution of aminoacids and sugars, reflecting the general nature of B involvementin plant processes. Brassica oleracea var., italica, broccoli, phloem, mobility, retranslocation, boron nutrition, transport fluids, concentration gradients     

High temperature arrest of inflorescence development in broccoli (Brassica oleracea var. italica L.)

High temperature causes unevenly-sized flower buds on broccoli inflorescences. This deformity limits production of broccoli to areas where summer temperatures rarely exceed 30 C. The stage of development sensitive to heat was determined by exposing plants of 'Galaxy' broccoli at varying developmental states to 35 C day temperature for 1 week, and subsequently analysing the head structure. During the high temperature exposure, the development of certain flower buds was arrested. There was no corresponding cessation of bud initiation at the apex. No injury resulted if heat was applied before the reproductive induction, or was their injury to differentiated flower buds. Meristems were affected only if heat was applied during inflorescence production or the floral initiation process. Shorter heat exposures produced little injury, and longer exposures were lethal. The plant's development at this sensitive period still appeared vegetative externally, but the youngest leaves had just begun to reorientate as a consequence of the reduced stem elongation rate. The meristem was less than 1 mm wide, and floral primordia were just forming, still subtended by leaf primordia. The injury was fully expressed by the time the head was first exposed (approximately 5-10 mm wide), though it became more apparent as the head matured. The buds that were delayed in development by the high temperature developed into normal flowers.Key words: Brassica oleracea, broccoli, flowering, heat injury, developmental arrest      

Genotype-specific response of cultured broccoli (Brassica oleracea var. italica) anthers to cytokinins

Anther culture medium was prepared with different types and concentrations of cytokinins to gain greater insight into the control of embryo formation during Brassica oleracea L. var. italica (broccoli) anther culture. The independent addition of the four cytokinins tested had widely divergent effects dependent upon cytokinin concentration and the genetic background of the test plants. All cytokinins were generally inhibitory at high concentrations, however, individual plants showed significant stimulation of embyro formation at typical physiological levels. The influence of cytokinins was highly cultivar-specific, some lines were stimulated, others inhibited and still other test lines were largely unaffected. Although the addition of cytokinins was needed for embryo formation for some plants, in no instance were cytokinins able to replace the inductive effect of high-temperature treatments.     

Two novel bioactive glucosinolates from Broccoli (Brassica oleracea L. var. italica) florets

Two novel glucosinolates along with one known glucosinolate were isolated from Broccoli (Brassica oleracea L. var. italica) florets. Their structures were established mainly by 1D ((1)H and (13)C NMR), 2D NMR ((1)H-(1)H COSY, DEPT 135°, HSQC and HMBC), and Tandem MS-MS spectrometric data as 2-mercaptomethyl sulfinyl glucosinolate [(Z)-4-(methylsulfinyl)-N-(sulfooxy)-2-((2'S,3'R,4'S,5'S,6'R)-3',4',5'-trihydroxy-6'(hydroxylmethyl)-2'-mercapto tetrahydro-2H-pyran-2-yl) butane amide] 1, (Z)-1-((2S,5S)-5-hydroxytetra-hydro-2H-pyran-2-ylthio)-2-(1H-indol-3-yl) ethylidene amino sulfate 2 and a known cinnamoyl [6'-O-trans-(4″-hydroxy cinnamoyl)4-(methylsulphinyl)butyl glucosinolate] 3. Compound 1 exhibited scavenging activity against DPPH with an inhibitory concentration IC(50) of 20mM, whereas compound 3 was a weak antioxidant when compared to the standard quercetin (5mM) as a positive control. Both the compounds showed a significant and similar antimicrobial activity against Staphylococcus aureus with an IC(50) of <625μg/mL when compared to antibiotic duricef. Against Salmonella typhimurium the IC(50) of 1 and 3 was determined as <625μg/mL and <1250μg/mL, respectively, when compared to ampicillin (IC(50) ?39μg/mL) as a positive control.     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

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     

青花菜SRAP-PCR体系优化与品种分子鉴定

本试验对青花菜基因组DNA的SRAP-PCR体系中主要影响因子Mg2+、dNTPs和引物浓度进行了优化。结果表明,反应体系中适宜浓度为Mg2+1.5-3.0mmol/L,dNTPs0.4mmol/L,引物0.25-0.50μmol/L(模板DNA约20ng,16μL反应体系)。运用优化的反应体系,对20个青花菜品种进行分子鉴定,从10个引物组合中筛选到7个多态性引物组合,获得60个多态性位点,平均每个引物组合在供试品种中产生8.6个多态性位点,鉴别品种数4.3个。双引物组合me1/em6与me2/em9可以区分所有供试材料。     

Transgene expression in broccoli (Brassica oleracea var. italica) clones propagated in vitro via leaf explants

We have developed an efficient protocol for the in vitro propagation of transgenic broccoli plants using leaf explants as starting material. A high frequency of shoot formation from leaf explants was obtained on Murashige and Skoog medium containing benzyladenine (BA, 5 mg/l) and naphthaleneacetic acid (0.5 mg/l). Frequent subcultures of existing shoots and shoot clusters to medium containing only BA (2 mg/l) promoted rapid shoot multiplication. The use of a 1:1 mixture of Agargel and Gelrite in the rooting medium increased the number of healthy roots per rooted plant. Applying this protocol, we obtained thousands of clonal rooted plantlets within 6 months from a transgenic broccoli plant carrying the cry1Ac and cry1C genes from Bacillus thuringiensis associated with kanamycin and hygromycin selectable markers, respectively. Thirty randomly selected clones that had been propagated for 1 year on medium containing kanamycin (50 mg/l) all showed resistance to both kanamycin and hygromycin. Genomic DNA and total soluble proteins were isolated from 16 of these clones. Polymerase chain reaction analysis indicated that the cry1Ac and cry1C genes were both maintained. ELISA assays showed that all of the clones produced a high level of Cry1Ac protein similar to the original transgenic plant; however, most clones had significantly lower levels of Cry1C protein than the original plant. This variation indicates that it is important to evaluate transgene expression in transgenic clones propagated long-term in vitro. In vitro propagation starting from leaf explants was also successful with other transgenic and non-transgenic Brassica oleracea materials, including broccoli, cauliflower, and collard.     

甘蓝和青花菜杂种小孢子培养

对甘蓝(Brassicaoleraceavar.capitata)×青花菜(Brassicaoleraceavar.italica)的20个杂种及相应的父母本进行游离小孢子培养,并对影响甘青杂种小孢子胚胎发生的主要因子进行探讨,适于小孢子培养的培养基为1/2NLN,附加0.5mgL-1NAA、0.05mgL-1BA、5mgL-1AgNO3、0.2mgL-12,4-D和0.1mgL-1活性炭。结果有14个杂种能产生胚状体,诱导率70%;不同杂种间小孢子胚胎发生频率存在很大差异,最高的是绿洲808×夏宝,平均每蕾16.2个胚。诱导杂种胚状体发生的最佳时期是小孢子单核靠边期至双核期,34℃热激2d有利于小孢子细胞对称分裂。在含糖170gL-1的液体培养基中培养3d,添加低糖(含糖110gL-1)的培养液,可显著提高出胚率。     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Transformation of broccoli (Brassica oleracea var. italica) with isopentenyltransferase gene via Agrobacterium tumefaciens for post-harvest yellowing retardation

Transgenic plantlets with a retarding effect on post-harvest yellowing in broccoli have been generated via Agrobacterium tumefaciens-mediated transformation of cytokinin synthesizing ipt (isopentenyltransferase) gene. The ipt gene is constructed under the control of senescence-associated gene promoters from Arabidopsis in the forms of pSG529(+) and pSG766A, which were the gifts from Dr R.M. Amasino at University of Wisconsin, Madison. Evidence of transgene integration was confirmed by assays on neomycin phosphotransferase II (NPTII) activity of selection markers, PCR and Southern hybridization. Based on the chlorophyll retention rate (>50%) after 4 days of post-harvest storage at 25 °C, it was found that 31% of transformants exhibited the effect of retarding yellowing in detached leaves, with 16% having the effect on florets and 7.2% on both leaves and florets. RT-PCR revealed that ipt gene expression occurred early on the day of detachment. Factors such as vacuum aid infiltration, plasmid differences, explant types, seedling ages and kanamycin concentrations were also studied. Putative transformation frequencies tended to vary with plasmids and explant types. The advantage of vacuum aid infiltration depended on explant types. The optimal kanamycin concentration should be determined experimentally for each study to avoid the high escape rate of kanamycin selection. Flow cytometric analysis of explant nuclear DNA phases was found to be helpful for selecting suitable explants for transformation and minimizing the polyploid transformants. A reproducible transformation protocol without any pre-culture was established for explants of hypocotyl, cotyledon, and peduncle. Most of the ipt transformants with a retarding effect on yellowing had a chimeric nature but showed little or no serious morphological abnormality in comparison with their parental line. Through proper selection, transformation lines with the capability of retarding post-harvest yellowing in broccoli should be feasible.     

Indole-3-acetonitrile Is a Critical Molecule with Weed Allopathic Suppression Function in Broccoli (Brassica oleracea var. italica)

Cruciferous plants are frequently used for ecologically benign weed control in agricultural production. Most effective Broccoli varieties were screened using the entropy method-based topsis model at first. Result showed that varieties of Lvwawa and Lvbaoshiwere most effective in allelopathic suppression on radishes. Column and thin-layer chromatography were used to extract the allelopathic compounds from broccoli residues, which contained various herbicidal active substances; among them, purified single-molecule indole-3-acetonitrile has a stronger inhibitory effect than pendimethalin (commercial herbicide). The weed inhibition rate increased with increasing broccoli residue dosage, with a 40 g/m² broccoli residue dose yielding the highest suppression rate. Its effect was similar to that of indole-3-acetic acid. Too much of this substance leads to the plant's death. Moreover, broccoli residues had effective control effect on weeds in natural soils in greenhouse and field trials. The results demonstrated that broccoli residue could be used for weed management in field for abundant allopathic suppression molecules to weeds, and that Indole-3-acetonitrile is one of the most important allopathic molecule.     

Regulation of Harvest-induced Senescence in Broccoli (Brassica oleracea var. italica) by Cytokinin, Ethylene, and Sucrose

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. The two plant hormones ethylene and cytokinin are known to act antagonistically on harvest-induced senescence in broccoli: ethylene by accelerating the process, and cytokinin by delaying it. To determine the level at which these hormones influenced senescence, we isolated and monitored the expression of genes normally associated with senescence in broccoli florets treated with exogenous 6-benzyl aminopurine (6-BAP), 1-aminocyclopropane-1-carboxylic acid (ACC), a combination of 6-BAP and ACC, and sucrose, in the five days following harvest. Exogenous 6-BAP caused both a reduction (BoACO) and an increase (BoACS) in ethylene biosynthetic gene expression. The expression of genes used as senescence markers, BoCP5 and BoMT1, was reduced, whereas BoCAB1 levels were maintained after harvest in response to exogenous 6-BAP. In addition, the expression of genes encoding sucrose transporters (BoSUC1 and BoSUC2) and carbohydrate metabolizing enzymes (BoINV1 and BoHK1) was also reduced upon 6-BAP feeding. Interestingly, the addition of ACC prevented the 6-BAP-induced increase in expression of BoACS, but 6-BAP negated the ACC-induced increase in expression of BoACO. The culmination of these results indicates a significant role for cytokinin in the delay of senescence. The implication that cytokinin regulates postharvest senescence in broccoli by inhibiting ethylene perception and/or biosynthesis, thus regulating carbohydrate transport and metabolism, as well as senescence-associated gene expression, is discussed and a model presented.     

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