多年生黑麦草细胞分裂素信号通路B类ARR转录因子LpARR10的耐镉功能分析

镉（Cd）是矿区重金属污染的主要元素之一,严重限制了植物的生长、发育。细胞分裂素（CTK）能够缓解镉对植物的毒害,但具体机制尚不清楚。本研究克隆了多年生黑麦草CTK信号通路基因LpARR10,该基因具有6个外显子,编码598个氨基酸。进化分析表明,LpARR10与拟南芥B类ARR转录因子（B-ARRs）聚为一组,其中与AtARR10和AtARR12的亲缘关系较近。氨基酸序列分析结果显示,LpARR10具有B-ARRs典型的磷酸受体结构域、磷酸化和金属离子结合位点。表达模式分析表明,CTK（25 μmol·L^-1 6-BA）和Cd（200 μmol·L^-1 CdCl₂）处理0.5、2、6、12、24和48 h均显著提高了黑麦草根部LpARR10的表达;而在叶片中,LpARR10的表达仅在CTK和Cd处理2和6 h后显著升高。过量表达LpARR10转基因和野生型拟南芥在无Cd的MS培养基上生长10 d后,根长和鲜重没有显著差异;而在含有Cd（180 μmol·L^-1 CdCl₂）的MS培养基中,过量表达转基因株系的根长和鲜重显著高于野生型对照。综上,LpARR10在细胞分裂素调控的植物耐镉机理中发挥重要作用。     

断根与外源细胞分裂素诱导去叶黑麦草耐牧性的研究

为了研究牧草的持续再生性机制,通过盆栽试验,从黑麦草多次去叶后根系对叶片生长的影响方面,设4个处理：（1）不断根未喷施细胞分裂素,（2）断根未喷施细胞分裂素,（3）不断根喷施细胞分裂素,（4）断根喷施细胞分裂素。结果表明：断根和多次去叶均能降低新生叶片和根系的生物量。多次去叶能引起叶中的赤霉素和脱落酸含量的升高,生长素含量以及玉米素核苷(ZR)含量的降低。多次去叶条件下,喷施外源细胞分裂素能促进叶片中ZR含量及其生物量的增加。相关性分析表明,多次去叶条件下叶中ZR含量与其生物量呈显著性的正相关关系。总之,多次去叶条件下新生叶片中的细胞分裂素含量是影响去叶黑麦草持续再生性的关键因素。     

In Vitro propagation of juvenileNothofagus leoni Espinosa (Fagaceae)

Nothofagus leoni has a restricted distribution in Chilean forests. This work determines suitable culture conditions forin vitro multiplication and rooting through shoots obtained from seedlings. Broadleaved Tree Medium was suitable for shoot multiplication. A medium with a pulse of 0.55 μM BA in the first subculture and two subcultures on BA-free medium resulted in a multiplication rate at day 63 of × 5.7, without callus growth or shoot neoformation. Rooted shoots of good quality (number and length of roots without callus growth) were obtained with 1.23 μM IBA (91.4% of rooting). The first roots appeared at day 11, reaching a higher speed of rooting at day 15.     

Shortening the generation cycle in faba bean (Vicia faba) by application of cytokinin and cold stress to assist speed breeding

The aim of this study was to reduce the length of the breeding cycle for faba bean by accelerating seed setting. We examined the mode and time of exogenous 6-benzylaminopurine (BAP) (cytokinin) application, and cold treatments and their combinations in two faba bean genotypes. Acropetal node number of pod and seed set and pollen viability were recorded during the experiments. Application of BAP improved pollen germination. The application of 10^–5 M BAP 4 days after flowering increased seed set at the lower nodes. Cold treatment (8/4°C day/night for 2 days) after the onset of flowering induced the formation of more pods and faster pod set compared to the non-cold treatment. The time to first seed was significantly reduced, and pollen viability was increased in plants exposed to cold treatment. Increased pollen viability also showed a significant positive correlation with seed setting. The combinations of 10^–5 BAP and cold treatment together had similar and independent effects. These results will accelerate plant breeding in faba bean by providing additional tools for reducing generation time.     

Spontaneous induction of tetraploidy in hop using adventitious shoot regeneration method

In the process of breeding triploid hop cultivars, diploid varieties need to be raised to the tetraploid level. Existing methods are predominantly based on treatment of the apices with antimitotic substances, in vivo or in vitro , resulting in a reported maximum of 26% tetraploids and a large proportion (over 60%) of mixoploids. This study was set-up to test whether an adventitious shoot regeneration protocol can be used as an alternative. In the first part of the experiments, regeneration media and a choice of explants (internodes, petioles and leaf discs) were optimized using three hop cultivars with putatively low, medium or high adventitious shoot regeneration capacity. On an optimized induction medium supplemented with 6 mg/l N ⁶-(2-isopentenyl) adenine (2iP), a regeneration rate up to 17.7% was obtained from cultured internodes, even of the most recalcitrant 'Aurora', while the regeneration rate of the most responsive 'Tettnanger' was 56.9%. Flow cytometric analysis of ploidy level revealed a high frequency of tetraploid induction, the highest (58.6%) being in 'Savinjski golding'. In 616 regenerants of all three cultivars, 30.4% were found to be tetraploid, the others were diploid. Cytokinin content was found to be of minor importance in tetraploid induction. The high frequency of spontaneous tetraploid induction and, in particular, the complete absence of mixoploids makes this protocol an alternative to the currently established methods of chromosome doubling based on the use of antimitotic substances. The occurrence of a high proportion of tetraploid adventitious regenerants in hop is discussed in view of certain other biotechnological applications.     

Callus Induction of Aloe vera L var chinensis (Haw) Berg

Studies were given to the induction and growth of callus of Aloe vera L.var.chinensis(Haw.) Berg when the young stem was applied as explant.MS was taken as basic medium,added with various concentrations of auxins(IAA,IBA,NAA and 2,4-D),cytokinins(6-BA,KT and Zeatin) and the different combinations of these plant growth regulators.Results showed that the callus is barely induced by cytokinin alone,and the callus induction rates have significant difference when different concentrations of auxin and different combinations of auxin with cytokinin are used on the MS medium.In order to inhibit callus browning during culturing,various concentrations of vitamin C or active carbon are added into four combinations of auxin and cytokinin respectively,which can induce callus most effectively.The result showsd that vitamin C can alleviate callus browning more effectively than the active carbon.The most efficient medium for induction and growth of callus of Aloe vera L.var.chinensis(Haw.) Berg was MS+2.0 mg/L 6-BA+0.5 mg/L 2,4-D+10 mg/ L vitamin C,with which the callus induction rate can reach to 91.4% and callus grow vigorously.     

红花萱草的花茎花蕾组织快繁技术的研究

取红花萱草的花蕾、花茎外植体,在MS培养基上用不同浓度的细胞分裂素BA和生长素NAA进行培养。结果表明:花茎的分化率比花蕾高,而最适合的培养基配方是:MS+BA 0.5 mg/L+NAA 0.1 mg/L+蔗糖30 g/L+琼脂8 g/L。可诱导分化出愈伤组织,继代培养愈伤组织增埴的同时可获得健壮的再生植株。最适的生根培养基为:MS+NAA 0.3 mg/L+活性碳0.3 g/L+蔗糖30 g/L+琼脂8 g/L。     

葡萄细胞分裂素响应调节因子VvRR2互作蛋白筛选与鉴定

【目的】从葡萄中克隆细胞分裂素响应调节因子VvRR2,获得VvRR2的互作蛋白,为阐明VvRR2在欧洲葡萄抗病反应中的作用机制提供依据。【方法】对葡萄接种白粉病菌,提取总RNA后反转录,利用实时荧光定量PCR检测VvRR2转录本对白粉病菌的响应;构建瞬时表达载体pBI221-VvRR2-GFP,转化拟南芥原生质体进行亚细胞定位分析;构建酵母表达载体pGBKT7-VvRR2,转化酵母菌株AH109,检测VvRR2的转录激活活性;构建酵母表达cDNA文库,以VvRR2为诱饵,通过Mating法筛选互作蛋白,对获得候选序列进行Blast分析;将候选蛋白VvTGA的全长序列克隆至pGADT7载体形成重组载体pGADT7-VvTGA,与重组诱饵载体pGBKT7-VvRR2共转化酵母,进行双杂交验证VvRR2与VvTGA的相互作用;将VvTGA的全长序列克隆至pSPYNE（R）173载体,形成重组载体pSPYNE-VvTGA,将VvRR2的全长序列克隆至pSPYCE（M）载体,形成重组载体pSPYCE-VvRR2,然后将两个重组载体共转化拟南芥原生质体,利用双分子荧光互补技术验证VvRR2与VvTGA的相互作用。【结果】葡萄接种白粉病菌后,细胞分裂素响应调节因子VvRR2呈现受白粉病菌诱导表达模式。VvRR2定位在拟南芥原生质体的细胞核,转录激活试验结果表明VvRR2在酵母体内具有转录激活活性。在含有60 mmol·L^-1的3-AT培养基上可以抑制VvRR2诱饵载体的自激活活性,VvRR2诱饵载体对宿主酵母菌没有毒性。以VvRR2为诱饵,初步筛选到287个单克隆,在高严谨条件下进一步筛选获得23个有效序列,Blast分析显示这些基因参与蛋白质合成与降解、细胞分裂素信号传导、光反应和生物钟节律、生长发育和逆境响应。酵母回复双杂交试验结果显示含有空载体（pGADT7或pGBKT7）酵母在四缺培养基（含3-AT）上不能生长,含有两种重组质粒的酵母在四缺培养基（含3-AT）上能够生长,并在含有X-α-Gal的四缺培养基上能够显色。双分子荧光互补试验结果显示共转化pSPYCE-VvRR2与pSPYNE（R）173、pSPYNE-VvTGA与pSPYCE（M）的原生质体没有黄色荧光,而共转化pSPYCE-VvRR2与pSPYNE-VvTGA的原生质体显示黄色荧光。VvTGA的表达类似于VvRR2,呈现受白粉病菌诱导表达模式。【结论】葡萄细胞分裂素响应调节因子VvRR2是一个受白粉病菌诱导表达的转录因子,能够与VvTGA相互作用,并且VvTGA受白粉病菌诱导表达。     

杂交水稻再生芽萌发生长与植株内源细胞分裂素含量变化的关系

采用植物激素ELISA方法，研究了杂交水稻各器官内源细胞分裂素（iPA)的变化及其与再生芽萌发生长的关系。结果表明，头季稻齐穗后5－19d稻株叶和叶鞘中内源激素iPA的含量较低，再生芽生长缓慢或处于休眠状态；齐穗19d以后，内源激素iPA含量增加，再生芽萌发、伸长的速度也相应加快。前期（齐穗后19d)下部节位茎中iPA含量高于上部节位，低位芽长于高位芽；后期（齐穗后33d）上部节位茎、芽的iPA含量高于下部节位，上位芽生长速度则快于下位芽，可见，再生芽萌发生长与内源激素iPA变化关系密切。     

冬季修剪对苹果新根细胞分裂素含量的影响研究

本试验以盆载３年生新红星／海棠为试材，应用高效液相色谱分析方法，研究了冬季修剪对苹果新根内源细胞分裂素含量的影响。新根中玉米素核苷（ＺＲ）、玉米素（Ｚ）的含量，缓放植株高于短截植株；但ＺＲ／Ｚ的比值，缓放植株低于短截植株。