农杆菌介导霞多丽葡萄胚性细胞系遗传转化条件的优化

为建立葡萄(Vitis vinifera)遗传转化技术体系,以霞多丽葡萄(Chardonnay)胚性细胞系为靶组织,采用GUS检测法,对影响农杆菌介导葡萄遗传转化效率的主要因素进行了研究。结果表明,超声波处理时间长短对转化效率有较大影响,在所试的0.5、1、5、10 min 4种不同时间的超声波处理中,以5 min时转化效率较好,平均达到9.11个蓝色斑点;AS浓度对转化效率有明显差异,当浓度为50μmol/L时,平均蓝色斑点数为8.89个,100μmol/L时,达到12.44个;DTT质量浓度对转化效率也有较大影响,在所试的3种质量浓度(1,2,3 mg/L)中,以3 mg/L为最佳,有16.67个蓝色斑点。通过GUS瞬时表达检测,确立了农杆菌介导葡萄胚性细胞系遗传转化的几个最适影响因素,从而为葡萄遗传转化技术体系的建立奠定了基础。     

Evaluation of ultrasonic aggregate stability and rainfall erosion resistance of disturbed and amended soils in the Lake Tahoe Basin, USA

Application of organic soil amendments to disturbed soil has been shown to improve aggregate stability and reduce soil susceptibility to erosion. Employing ultrasonic aggregate stability assessment techniques described earlier [Fristensky, A. and Grismer, M.E., 2008. A simultaneous model for ultrasonic aggregate stability assessment. Catena, 74: 153–164.], we assess the effect of two experimental organic soil amendments – a compost and a woodchip mulch incorporated at a rate of 2000–6000 kg ha^− 1 N-equivalence – on soil aggregation and aggregate stability at four drastically disturbed sites within the Lake Tahoe Basin, USA. Experimental plots were established 1–3 years prior to testing. The soils were of granitic or volcanic origin, and disturbed by either ski run or road development. Soil treatments were observed to significantly (p < 0.05) increase both aggregation (300% average increase) and ultrasonic aggregate stability (600% average increase) relative to the untreated soil. However, at the two sites disturbed by ski run development, the control treatment (tilling and surface application of pine–needle mulch) performed comparably to the two incorporated compost treatments, suggesting that the effects of the experimental amendments on aggregation were negligible at these sites, or their effective duration was shorter than the evaluation period.Rainfall simulations (72–120 mm h^− 1) were performed on the treatment plots, and results were compared with the ultrasonic aggregate stability indices. Significant (p < 0.05) positive correlations were obtained between the measurements of aggregate instability and indices of soil susceptibility to runoff, including steady-state infiltration rate (measured values between 1 and 120 mm h^− 1), and the level of kinetic energy of applied rainfall at which runoff commences (EBR, measured values between 12 and 224 J m^− 2). However, no correlation was found between the ultrasonic aggregate stability indices and observed soil erosion variables. Interestingly, positive relationships (p < 0.05) were observed between both infiltration rate and EBR and the proportion of 2–20 μm and < 2 μm particles liberated from the largest aggregates detected in each soil. Our results suggest that ultrasonic aggregate stability indices may be useful indicators of soil susceptibility to runoff and erosion under rainfall.     

大豆子叶节再生影响因素的研究

为了建立一个大豆子叶节高效再生体系用于大豆的遗传转化，选用10个东北主栽大豆品种（Glycine max(L.)Merr.）的子叶节作为外植体，研究了基因型、植物激素的浓度、超声波辅助处理时间…等6种影响大豆子叶节再生的因素。结果表明合丰35、合丰25、黑农40再生效率较高，平均每个外植体分化出的丛生芽数分别为6.69、6.32和5.98；确定了丛生芽分化阶段所需的BA浓度，合丰35、合丰25为1.5mg/l，黑农40为1.2mg/l。适宜的外植体大小为保留全部子叶。作为遗传转化时的除菌剂，头孢唑啉钠（Cef）在500mg/l时对子叶节的再生无显著影响。当遗传转化时使用的超声波辅助处理时间小于12秒时，对子叶节再生无显著影响。     

大豆悬浮细胞培养及作为外源基因转化受体的研究

以吉林小粒7号大豆品种为材料,切取无菌幼苗下胚轴诱导愈伤组织,初步建立了大豆悬浮细胞系,测定不同硼酸浓度下悬浮细胞生长变化;采用农杆菌介导法对大豆悬浮细胞进行外源GUS基因的转化,先通过不同程度的超声处理确定最佳超声时长,然后在最佳超声处理时长的基础上,探讨了硼酸浓度对转化效率的影响;最后采用PCR和Southern blot杂交对抗性愈伤做分子鉴定。结果表明:大豆悬浮细胞正常生长硼酸浓度在2.0~10 mg·L-1,过高或过低均会抑制大豆细胞正常的生长,高于100 mg·L-1时抑制作用最显著。但在转化过程中,适当增加硼酸浓度(10~30 mg·L-1)则能够提高悬浮细胞的转化效率,并且有助于转化组织的筛选,借以3 min超声的辅助作用,30 mg·L-1硼酸浓度下转化效率达到1 mg转化细胞团中含28个转化体。     

负压和超声波处理对农杆菌介导的花生遗传转化效率的影响

为了提高根癌农杆菌介导的花生遗传转化效率,研究了不同强度的负压和超声波处理时间对外植体芽诱导率的影响以及对花生遗传转化的作用.结果表明,较低强度(抽拉30次)的负压处理和短时间(2min)的超声波处理对花生遗传转化有促进作用,虽然超声波处理延长了芽再生时间,但是有助于提高花生外植体芽点诱导率.     

Sonic dispersion of soil DTPA solution suspension as an extraction procedure for DTPA micronutrient test

Abstract

To examine sonic dispersion as an extraction procedure for DTPA micronutrient test, soil DTPA solution suspensions were sonicated to extract Fe, Zn, Mn, and Cu. The elements extracted after 15 or 60 seconds sonication were compared with those extracted after 1/2 or 2 hours shaking.

The mean differences between Fe, Zn, Mn, or Cu extracted after 15 seconds sonication and those extracted after 1/2 hour shaking were not significant. However, the extractions obtained after 2 hours shaking were significantly higher than those obtained after 15 or 60 seconds sonication.

The elements extracted after 15 or 60 seconds sonication correlated significantly with those extracted after 1/2 or 2 hours shaking. Therefore, 15 or 60 seconds sonication can replace 2 hours shaking in the extraction procedure for DTPA micronutrient test. However, 15 seconds sonication is more convenient than 60 seconds and does not require cooling of soil solution suspension during sonication. Therefore 15 seconds sonication ‐ a replacement for 2 hours shaking to extract Fe, Zn, Mn, and Cu ‐ is recommended.     

超声波辅助农杆菌介导八棱海棠转rolC基因

应用超声波辅助农杆菌介导法,对八棱海棠进行rolC基因转化,以期提高转化效率并获得转基因植株。利用gus基因瞬间表达的方法研究了超声波处理时间、处理时期和农杆菌悬浮液中乙酰丁香酮(As)浓度对rolC基因转化率的影响。结果表明,叶盘在D600nm为0．6且含有75 mg／L As的农杆菌悬浮液中侵染2 min后,用功率为100 W的超声波处理30 s,再浸泡2．5 min,然后放到再生培养基上共培养3 d,能获得最佳的gus基因瞬间表达率。最佳处理条件下转化683枚八棱海棠叶片,共得到138个抗性愈伤组织和15株抗性苗,转化率为2．2％。GUS染色、PCR及Southern blotting检测结果显示,有12个八棱海棠株系的基因组中整合了完整的外源rolC基因。     

超声波对镉胁迫鸭茅和冰草种子萌发的影响

为探讨超声波和重金属镉二因素对鸭茅（Dactylis glomerata L.）和冰草（Agropyron cristatum L.）种子发芽率、胚芽生长和生理活性的影响,设置五种不同时长的超声波处理两种种子,处理后的种子分别用不同浓度的氯化镉溶液进行处理,通过培养皿纸上发芽法培养20d。结果表明：单一超声波处理,均提高鸭茅种子的发芽率且在5 min时发芽率最高,冰草种子仅在超声波5 min的处理下发芽率提高;超声波时长为5 min时显著提高鸭茅幼苗中POD的活性;各超声波处理均使鸭茅幼苗中MDA含量增加。单一镉溶液处理均抑制鸭茅和冰草种子的发芽和胚芽的生长（除浓度为0.04%的镉溶液提高冰草种子的发芽率）;0.02%镉显著提高鸭茅幼苗POD的活性,镉溶液对鸭茅幼苗中MDA的含量无显著作用。超声波与镉溶液组合处理下,在0.05%的镉胁迫下,超声波处理10 min显著提高鸭茅和冰草种子的发芽率;当镉溶液浓度为0.01%时,各超声波显著促进鸭茅胚芽生长。综合来看,超声波处理可以有效减缓鸭茅和冰草种子的镉胁迫效应,提高种子和成苗活力。     

利用农杆菌介导法转化大豆子叶节的影响因素研究

以3个大豆品种为材料,采用GUS瞬时表达的方法,对适合于大豆子叶节和胚尖转化的基因型进行筛选,结果表明:适合大豆子叶节转化的品种为东农50,适合胚尖转化的品种为黑农41。在子叶节转化系统中,摸索了农杆菌侵染浓度与侵染时间的最佳配比组合、乙酰丁香酮浓度和超声波辅助处理对大豆转化效率的影响。优化后的条件为:农杆菌侵染的浓度OD600=0.6,侵染时间30～40 min,乙酰丁香酮浓度200μmol.L-1,超声波辅助转化时间5 s。在上述条件下,成功获得了转基因植株,转化效率为2.3%。