The protoplasts isolation,culture and shoots regeneration of broccoli(Brassica oleracea var.italica)

Eight F1-hybrid cultivars of broccoli were studied.We obtained cell division,celled colonies and p-calli in 5 cultivars,roots and shoots regeneration in one cultivar.The leavesof propagated plantlets in vitro were cut into 1—2mm pieces,isolated with an enzyme solutioncontaining 2% cellulase and 1%macerase on a rotary shaker（50 rpm,21℃,3h,2500 lux light）,and purified with a 0.5M sucrose solution.The purified protoplasts were placed on a drop of 1%agarose.2—3 ml liquid medium was added around the agarose drops,and all of the cultures wereincubated at 25℃ under light（4000 lux）for 16 hours.3—5 days after isolation the cell divisionwas found.About 7 days after incubation 4 multicellular colonies were formed.After 3—5 wksome p-calli were developed.When the p-calli were 2—3 mm in diameter it was transferred to asolidified medium.Once they were developed to 1 cm in diameter they were transferred on a re-generation medium.About 5 months after incubation some roots and shoots grown from the calliwere

Review of Research Situation of Brassica oleracea var.italica

Broccoli(Brassica oleracea var.italica),also called green broccoli,green cauliflower,and calabrese,belongs to genus Brassica in family Cruciferae,and is annual or biennial herbaceous plant.Both broccoli and cauliflower are varieties of Brassica oleracea L.Broccoli is rich in nutrients.Its protein,amino acids and vitamins are higher than cauliflower,and broccoli is easy to grow,the supply period is long,and it has a good market and economic value.This paper introduced broccoli-related information from broccoli's nutritional value,cultivation techniques,existing problems and prospects.In addition,on the basis of existing studies,it discussed the future development prospects of broccoli,in order to promote the production research of broccoli in China.

花椰菜(Brassica oleracea var.botrytis L.)凝集素的细胞凝集和糖抑制作用的研究

花椰莱(Brassica oleracea var. botrytis L.)凝集素能凝集兔、大鼠和小鼠的红细胞,其中对兔的红细胞的凝集活性最高,最低凝集浓度为0.68μg/ml。但不凝集我们所测试的其它16种红细胞。浓度为0.1mg/ml时,花椰菜凝集素也能凝集小鼠艾氏腹水瘤细胞,S_(180)肉瘤细胞,大鼠W_(256)肿瘤细胞、人的MGC_(80-3)胃癌细胞、小鼠和大鼠的脾脏淋巴细胞、骨髓细胞以及牛精子细胞,而不凝集人的Hela细胞。该凝集素对免的红细胞的凝集活性可被L—鼠李糖和D—树胶醛糖所抑制,最低抑制浓度分别为33.3m mol/L和16.7m mol/L。

Transformation of insect-resistant gene into cauliflower (Brassica oleracea L.var. botrytis)

Cowpea Trypsin Inhibitor (CpTI) gene was transferred into the cotyle dons and hypocotyls of cauliflower by Agrobacterium-mediated transformation met hod. The best selective concentration of kanamycin (kan) was 15 mg L-1. The con centration of carbencillin (carb) was 500 mg L-1. 14 transgenic cauliflower pla nts were obtained. The putative transformants were assayed by PCR and Southern b lotting analysis. The results indicated that CpTI gene was transferred into caul iflower successfully.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.

BoMyrosinase plays an essential role in sulforaphane accumulation in response to selenite treatment in broccoli

Sulforaphane, a naturally specialized metabolite, plays significant roles in human disease prevention and plant defense. Myrosinase(MY) is a key gene responsible for the catalysis of sulforaphane formation, but the molecular mechanisms through which MY regulates sulforaphane biosynthesis in plants remains largely unknown. Here, we discovered that the change of sulforaphane content in broccoli sprouts caused by exogenous selenite treatments is positively related to BoMY expression. BoMY overexpression in the Arabidopsis thaliana tgg1 mutants could dramatically increase myrosinase activity and sulforaphane content in the rosette leaves of 35S::BoMY/tgg1 and rescue its phenotypes.Moreover, an obvious increase of myrosinase activity and sulforaphane content was displayed in transgenic BoMY-overexpressed broccoli lines.In addition, a 2 033 bp promoter fragment of BoMY was isolated. Yeast one-hybrid(Y1H) library screening experiment uncovered that one bHLH transcription factor, BoFAMA, could directly bind to BoMY promoter to activate its expression, which was further evidenced by Y1H assay and dual-luciferase reporter assay. BoFAMA is a selenite-responsive transcription factor that is highly expressed in broccoli leaves;its protein is solely localized to nucleus. Additionally, genetic evidence suggested that the knockdown of FAMA gene in Arabidopsis thaliana could significantly decrease sulforaphane yield by inhibiting the expression of myrosinase genes. Interestingly, exogenous selenite supply could partially restore the low level of sulforaphane content in transgenic Arabidopsis FAMA-silencing plants. Our findings uncover a novel function of FAMAMY module in the regulation of selenite-mediated sulforaphane synthesis and provide a new insights into the molecular mechanism by which selenite regulates the accumulation of sulforaphane in plants.

Comparison of carotenoid,chlorophyll concentrations and their biosynthetic transcript levels in different coloured cauliflower

Carotenoids and chlorophylls are among the most widely distributed pigments in nature that play essential roles in the photosynthetic apparatus and confer diverse colours in plants.Among all vegetables,cauliflower(Brassica oleracea L.ssp.var.botrytis)is rich in phytochemicals and is an important crop grown all over the world.This study investigates carotenoid and chlorophyll concentrations in differently pigmented cultivars and elucidates the role of transcriptional regulation of carotenoid accumulation including lutein andβ-carotene.Here,we characterised changes in pigments by UHPLC-DAD-ToF-MS and changes in transcript levels of carotenoid metabolic genes by qRT-PCR in florets and leaves of orange(‘Jaffa'and‘Sunset'),purple(‘Di Sicilia Violetto'and‘Graffiti'),green(‘Trevi')and white(‘Clapton')cultivars.Transcript levels of all carotenoid metabolic genes showed different transcript level patterns in the leaves and florets.Compared to the other cultivars,the orange cultivars had the highest levels ofβ-carotene in the florets and lutein in the leaves resulting in changes lutein/β-carotene ratios.In the green cultivar,higher transcript levels were also found,especially for phytoene synthase and phytoene desaturase genes of the core biosynthesis pathway.However,no increased carotenoid concentrations were observed,possibly due to a higher carotenoid turnover induced by the carotenoid cleavage dioxygenase 4 in the green cultivar.In the white(‘Clapton')and purple(‘Di Sicilia Violetto'and‘Graffiti')cultivars the phytoene desaturase transcript levels as well as carotenoid concentrations were low.Chlorophyll concentrations changed in trend comparable to the carotenoid concentrations and were only significantly lower in the leaves of the orange cultivar‘Jaffa'.Also,the chlorophyll a/b ratio changed in‘Jaffa'.In florets the highest chlorophylls concentrations were observed for the green cultivar(‘Trevi')and the purple cultivar(‘Di Sicilia Violetto').Taken together,the study demonstrates the complex source-sink relationship of carotenoid accumulation in different coloured cauliflower.

Growth and yield responses of broccoli cultivars to different rates of nitrogen in western Chitwan, Nepal

A field experiment was conducted with the objective to determine the optimum rate of nitrogen (N) fertilizer for effective growth and yield of two varieties of broccoli in southern plain of Nepal. The experiment was laid out with two-factorial completely random block design (RCBD) comprising two varieties of broccoli (Calabrese and Green Sprouting) and five N rates (0, 50, 100, 150 and 200 kg ha-1) with three replication in each treatment combinations. The effects of variety and N rate on total curd yield were significant but the interaction effect was non-significant. Green Sprouting produced 11% higher total curd than Calabrese. Similarly, curd production increased N rate up to 200 kg ha-1 reaching a maximum of 14.47 t ha-1. This indicated that optimum level of N could be beyond the rates tested in this study, which needs further experimentation.

Effect of H_2O_2 on Growth of Collard( Brassis oleracea L. ) Seedlings Under Salt Stress

Collard variety( Brassis oleracea L. var. acephala f. tricolor Hort.) as a research material was treated with exogenous H_2O_2 and H_2O_2 scavenger dimethyl thiourea under 100 mmol/L NaC l stress. Two days later,growth rate,dry weight,fresh weight and relative water content of the plants were determined. After 6h of treatment,the activity and gene expression of three antioxidant enzymes,superoxide dismutase( SOD),catalase( CAT) and ascorbate peroxidase( APX) in plants,were measured. The results showed that the growth rate,dry weight,fresh weight,relative water content,and the activity and gene expression of the three antioxidant enzymes in collard seedlings were higher in the treatment of salt stress with the addition of 0. 05 mmol/L exogenous H_2O_2 than in the simple salt stress treatment; and when endogenous H_2O_2 was removed,the growth rate,dry weight,fresh weight,relative water content,and the activity and gene expression of the three antioxidant enzymes in plant seedlings were lower than those under simple salt stress. It is speculated that under salt stress,H_2O_2 is involved in the regulation of antioxidant defense gene expression,and it might be an important regulator of salt-induced antioxidant system in collard leaves.

青花菜快速碱化因子RALF的克隆与序列分析

以一个与甘蓝显性核不育相关的差异表达片段序列为信息探针,在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR克隆与序列分析验证,获得了青花菜快速碱化因子RALF(Rapid Alkalinization Factors)基因的cDNA全长序列,命名为BoRALFL1(GenBank序列登录号DQ059310)。该cDNA全长240bp,编码79个氨基酸,与电子克隆获得的序列完全相同。序列分析表明,编码蛋白存在前导信号肽与多个磷酸化位点,与同源基因RALFL8核酸序列在88bp上有82%的一致性,推导的氨基酸序列在74个氨基酸上存在56%的一致性,不同植物间氨基酸序列N-端差异大,C-端具有较高的保守性。

L-精氨酸处理对青花菜贮藏过程中品质的影响

试验用0.2 mmol/L的L-精氨酸(L-Arg)溶液处理青花菜(Brassica oleracea var.italica)20 min,探讨在贮藏过程中对青花菜品质的影响。结果表明,L-Arg处理可有效抑制青花菜的黄化和细胞膜透性的增加,保持可溶性固形物含量,延缓叶绿素的降解和维生素C的流失。L-Arg处理能够有效延缓青花菜的衰老,抑制营养物质的流失。

甘蓝LFY基因的克隆及序列分析

以甘蓝ZQ启动抽薹的茎尖为材料,分别提取DNA和RNA,以两对引物扩增并序列拼接得到甘蓝LFYZQ基因DNA序列和cDNA完整编码区,长度分别为2560、1239bp,与花椰菜、拟南芥、芥菜和萝卜同源性分别达到91%、87%、86%、87%。该基因含有3个外显子(452、394、393bp)和2个内含子(514、807bp),共编码412个氨基酸,内含子剪接位点均符合经典GT-AG法则。将LFYZQ与网上公布的12种十字花科植物LFY氨基酸序列按分子进化分成两类:甘蓝LFYZQ与花椰菜以及Jonopsidium属植物Jonopsidium acaule这3个分为一类;其余10种植物分为第二大类。LFYZQ蛋白分子量为46kD,是一个不稳定的疏水蛋白,存在N-十四(烷)酰化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点、酰胺化位点共4种活性位点。

乙酰甲胺磷和NaCl交叉胁迫对花椰菜种子萌发及其幼苗生长的影响

为探究农药和盐交叉胁迫对植物生长影响的机制,试验研究了不同NaCl浓度(0.0%、0.2%、0.4%、0.6%、0.8%)下的不同浓度的乙酰甲胺磷处理液(0.00%、0.03%、0.06%、0.09%、0.12%)对花椰菜种子萌发及其幼苗生长的影响。结果表明:不同浓度乙酰甲胺磷、NaCl及二者的交叉胁迫对花椰菜种子萌发及其幼苗根长、苗高、侧根数目、鲜重和干重的增加均具有显著的抑制作用;乙酰甲胺磷加剧了NaCl对花椰菜的胁迫效应,NaCl也加剧了乙酰甲胺磷对花椰菜的胁迫效应,当乙酰甲胺磷的浓度≥0.09%、NaCl的浓度≥0.8%时,这种相互胁迫效应更加明显;花椰菜对0.2%NaCl处理具有抗性,但对0.03%乙酰甲胺磷处理非常敏感。

青花菜CC-NBS-LRR抗病基因BoCNL1的克隆与分析

根据已知序列设计PCR引物,从青花菜中克隆1个NBS-LRR(核苷酸结合位点-富含亮氨酸重复)抗病基因BoCNL1;在生物信息学分析的基础上,利用RT-PCR研究该基因在不同器官中的表达模式。测序结果表明,BoCNL1基因的编码区全长为2 550 bp,编码849个氨基酸;编码蛋白具CC(卷曲螺旋)、NBS和LRR结构域;进化分析结果表明,BoCNL1与不结球白菜的关系最近,在进化树上处于同一分支,与醉蝶花的关系最远;RT-PCR结果表明,BoCNL1在根、花茎、叶、花蕾、开放的花和嫩角果中均有表达,但表达量低。

Obtaining and Cytological Identification of a Set of Primary Trisomics in Cabbage

Selection of primary trisomics of the cabbage （Brassica oleracea var.capitata L） forms an important basis for gene chromosome mapping and for other genetic studies. The cabbage self-fertilization line - 9601 was used as material, using the root-tip cell chromosome number and pollen mother cell chromosome number identification and karyotype analysis to select the primary trisomics from the progenies of 3x x 2x in the cabbage. Many aneuploid plants with one or two extra chromosomes were obtained and a set of primary trisomics （Tri-1, Tri-2, Tri-3, Tri-4, Tri-5, Tri-6, Tri-7, Tri-8, and Tri-9, in which the Tri-1 and Tri-4 were from 2n＋2 plants and others from 2n＋ 1 plants） was acquired from these plants. Each trisomic exhibited some unique features, such as plant height, plant type, leaf type, size of flower bud, and inflorescence. The triploid crossing by the diploid is a convenient and effective way to select trisomics in the cabbage.

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。

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%。

甘蓝类蔬菜小孢子再生植株染色体倍性与气孔保卫细胞叶绿体数的相关性

【目的】研究甘蓝类蔬菜游离小孢子再生植株的染色体倍性与气孔保卫细胞叶绿体数的相关性,为甘蓝类蔬菜提供一种快速、简易、经济、实用而又可靠的倍性鉴定方法。【方法】采用分布型分析和t测验,对结球甘蓝、青花菜和芥蓝不同倍性的小孢子再生植株的气孔保卫细胞叶绿体数进行统计分析。以根尖染色体计数法和田间形态学观察法的倍性鉴定结果,对叶绿体数分界法的可靠性进行验证。【结果】同一倍性植株上的不同叶片间及同一叶片的不同部位间,气孔保卫细胞叶绿体数平均值及变异幅度非常接近。同一小孢子再生植株群体内的不同倍性植株的叶绿体数平均值差异极显著。不同倍性植株间的气孔保卫细胞叶绿体数均呈正态分布。本研究提出了一种根据甘蓝类蔬菜气孔保卫细胞叶绿体数进行染色体倍性鉴定的方法,即气孔保卫细胞叶绿体数≤10的为单倍体,10<叶绿体数≤15的为二倍体,>15的为多倍体。该方法经花期形态学观察和根尖染色体计数法验证,其吻合率达93.93%,且此倍性鉴定方法稳定,不受植株生长环境等外界因素影响。【结论】甘蓝类蔬菜游离小孢子再生植株的染色体倍性,可于幼苗期根据气孔叶绿体数目的多少进行鉴定,而且此倍性鉴定方法简单、快捷、经济而又可靠。

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。

高温胁迫对早熟花椰菜叶片抗氧化系统和叶绿素及其荧光参数的影响

在30～35℃高温胁迫条件下,对早熟花椰菜叶绿素含量、叶绿素荧光参数和叶片抗氧化系统进行研究。结果表明,35℃高温胁迫情况下,叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素含量比对照下降25％以上;叶绿素荧光参数,包括原初光能转换效率(Fv/Fm)、光合电子传递量子效率(φPs Ⅱ)、光化学猝灭系数(qP)均低于对照。另一方面,抗氧化酶,包括超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、愈创木酚过氧化物酶(GPX)、过氧化氢酶(CAT)的活性以及抗坏血酸(ASA、DASA)和谷胱甘肽(GSH、GSSG)等抗氧化剂的含量在高温胁迫下明显升高。