Genetic analysis of protoplast regeneration ability in Brassica oleracea

In Brassica oleracea L., plant regeneration from protoplasts is genotype-dependent and colony formation can be obtained routinely. In order to identify genes for regenerability, we performed a genetic analysis of the characteristic in the F₂ generation of a cross between two accessions selected for high and low regenerability. Regeneration frequencies were obtained from protoplast culture of 248 individual F₂ plants after 5, 10, and 15 weeks of culture on regeneration medium. Broad-sense heritability estimate was 0.492 at the early stage and lower (0.046–0.149) at advanced stages. The frequency distribution observed during short-term culture can be explained by two independent loci with duplicate dominant genes controlling regeneration. In long-term culture, one additional dominant gene can confer regeneration; i.e., three independent loci are responsible for regenerability. The results suggest that selection for high regeneration response may be efficient at early stages because of the lower environmental influence on the characteristic, and because fewer genes are involved at this stage than at advanced stages. The control of regenerability by a few dominant genes facilitates incorporation of the trait into breeding material. Molecular markers linked to the genes may assist in the selection of genotypes with high regeneration percentage in the future. This revised version was published online in June 2006 with corrections to the Cover Date.     

福建漳州青花菜栽培钾肥施用技术优选

以青花菜品种‘炎秀’Brassica oleracea var．italica ‘yanxiu’为材料,通过设置钾肥横向与纵向施肥的田间试验,探讨青花菜最佳钾肥用量和最佳钾肥运筹。结果表明,在设定氮、磷肥施用量分别为30.0 kg/667.7 m2、10.0 kg/667.7 m2的条件下,钾肥不同施用量对青花菜的株高、苗期功能叶片数、植株扩展度及全株重（去根）、单球重均有一定的影响,当钾肥施用量为19.1 kg/667.7 m2时,其产量最高,为1763.5 kg/hm2;在同样氮、磷肥的背景条件下,钾肥施用量以15.0 kg/667.7 m2,按基肥/追肥不同比例进行施用,青花菜所表现的株高、苗期功能叶片数、植株扩展度及全株重、单球重等形态及产量指标均有所差异,当基肥∶追肥按0.2∶0.8进行施用时,该产量指标均明显高于其它配比的处理。     

Suppression of the cysteine protease,aleurain, delays floret senescence in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

An aleurain-like protein, BoCP5, is up-regulated during harvest-induced senescence in broccoli floret and leaf tissue. BoCP5 is most closely related to an Arabidopsis protein (91%, AAF43041) and has 71% identity to barley aleurain (P05167). The mRNA for this gene accumulates within 6 h after harvest in broccoli florets, and its expression is reduced in tissue that has been held in senescence-delaying treatments (e.g. water, sucrose feeding, controlled atmosphere). The gene is also expressed in leaves during aging-related and harvest-induced senescence. Analysis of protein bands that cross-react with antibodies raised to the bacterial BoCP5 fusion protein, revealed prominent immunoreactive bands at ca. 26, 28, 31, and 38 kD in floret tissue. The 31 kD band was absent in protein extracts from leaf tissue. Agrobacterium-mediated transformation was used to produce transgenic broccoli plants with down-regulated BoCP5. A reduction in the postharvest expression of BoCP5 in floret tissue was achieved for four transgenic lines in the current study. In three of these lines postharvest floret senescence (yellowing) was delayed, and florets contained significantly greater chlorophyll levels during postharvest storage at 20 °C than wild-type plants. Line 4 showed the greatest down-regulation of BoCP5, and in this line postharvest protease activity remained at pre-harvest levels, and the yield of soluble proteins extracted from florets after harvest was significantly greater than that of wild-type tissue.     

Impact of single-gene and dual-gene Bt broccoli on the herbivore <Emphasis Type="Italic">Pieris rapae</Emphasis> (Lepidoptera: Pieridae) and its pupal endoparasitoid <Emphasis Type="Italic">Pteromalus puparum</Emphasis> (Hymenoptera: Pteromalidae)

Transgenic brassica crops producing insecticidal proteins from Bacillus thuringiensis (Bt) are being investigated as candidates for field release to control lepidopteran pests. Information on the potential impact of Bt brassica crops on pests and non-target natural enemies is needed as part of an environmental risk assessment prior to the commercial release. This first tier study provides insight into the tritrophic interactions among Bt broccoli plants, the herbivore Pieris rapae and its parasitoid Pteromalus puparum. We first evaluated the efficacy of three types of Bt broccoli plants, cry1Ac, cry1C and cry1Ac + cry1C, on different instars of P. rapae. Bt broccoli effectively controlled P. rapae larvae, although later instars were more tolerant. The efficacy of different Bt broccoli plants on P. rapae larvae was consistently cry1Ac > cry1Ac + cry1C > cry1C. When the parasitoid P. puparum developed in a P. rapae pupa (host) that had developed from Bt plant-fed older larvae, developmental time, total number and longevity of the P. puparum generated from the Bt plant-fed host were significantly affected compared with those generated from the non-Bt control plant-fed host. Simultaneously, negative effects on P. rapae pupae were found, i.e. pupal length, width and weight were significantly reduced after older P. rapae larvae fed on different Bt plants for 1 or 2 days. Cry1C toxin was detected using ELISA in P. rapae pupae after older larvae fed on cry1C broccoli. However, no Cry1C toxin was detected in newly emerged P. puparum adults developing in Bt-fed hosts. Only a trace amount of toxin was detected from entire P. puparum pupae dissected from the Bt plant-fed host. Moreover, no negative effect was found on the progeny of P. puparum developing from the Bt plant-fed host when subsequently supplied with a healthy host, P. rapae pupae. The reduced quality of the host appears to be the only reason for the observed deleterious effects on P. puparum. Our data suggest that the effects on P. puparum developing in Bt plant-fed P. rapae are mediated by host quality rather than by direct toxicity.     

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F₁ hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F₁ larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.     

青花菜两类雄性不育系主要农艺性状的研究

通过对青花菜显性细胞核雄性不育系(DGMS)、改良的萝卜细胞质雄性不育系(Ogura CMS)和由其与相同父本配制的F1杂交种植株的主要农艺性状的生长趋势和花球相关性状的研究,明确两类雄性不育系的应用价值,为青花菜两类雄性不育系的选育和利用及新品种的选育提供理论依据。本研究以结球甘蓝中的显性细胞核雄性不育材料DGMS 79-399-3和改良的细胞质雄性不育材料Ogura CMSR3629为原始不育源,利用优良的青花菜自交系8554和93219为转育父本,通过回交和杂交的方法获得的青花菜高代(n≥9代)两类雄性不育系和由不育系与相同父本配制的F1为试材,采取定期观测整个生长期内植株主要农艺性状和绘制生长曲线相结合的方法,系统地研究了植株的主要农艺性状的生长趋势和花球相关性状。结果表明,相同遗传背景下,保持系、DGMS、Ogura CMS及由不育系与相同父本配制的F1在株幅、株高、外叶长和外叶数方面的生长趋势基本一致,其中株幅、株高、外叶长,总体上表现出前期增长速度较快,之后变慢,最后趋于平缓或略呈下降的趋势;外叶数均表现出先增加后减少的趋势;侧枝数目因年份和遗传背景不同而异。由相同保持系获得的两类不育系的显球时间、采收时间基本相同,由其配制的F1显球和采收时间相同,花球产量因遗传背景的不同而异,花球外观品质性状表现一致。由DGMS和改良的Ogura CMS两类不育源转育的青花菜雄性不育系表现优良,该两类雄性不育源在青花菜杂交育种中具有重要的应用价值。     

Comparison of the effect of raw and blanched‐frozen broccoli on DNA damage in colonocytes

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched‐frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age‐matched and weight‐matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal‐based diet, unsupplemented (control) or supplemented with 600 g day^?1 of raw or blanched‐frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched‐frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes. Copyright © 2015 John Wiley & Sons, Ltd.