经常问自己： Have I done something new and interesting? 低声说话；勤劳，踏实做事；心情平和，不让矛盾堆积，不计较得失；多吃水果蔬菜，植物油和不去麸的粮食，少吃肉类。 要善于在不断总结中变得聪明起来。 NB 基本培养基 KNO 3 2830 mg/L (NH 4 ) 2 SO 4 463 mg/L=374.8 mg/L NH 4 Cl KH 2 PO 4 400 mg/L MgSO 4 .7H 2 O 185 mg/L CaCl 2 .2H 2 O 166 mg/L FeSO 4 .7H 2 O 27.8mg/L Na 2 EDTA 37. 5 mg/L MnSO 4 .4H 2 O 10 mg/L H 3 BO 3 3 mg/L ZnSO 4 .7H 2 O 2 mg/L Na 2 MoO 4 .2H 2 O 0.25 mg/L CuSO 4 .5H 2 O 0.025 mg/L CoCl 2 .6H 2 O 0.025 mg/L KI 0.75 mg/L 盐酸硫胺素 thiamine CHL VB1 10 mg/L 盐酸吡哆醇 pyridoxine-CHL VB6 1 mg/L 烟酸 nicotinic acid 1 mg/L 肌醇 myo-inositol 100 mg/L 水解酪蛋白 300 mg/L 谷氨酰胺 500 mg/L 甘氨酸 2 mg/L 脯氨酸 2878 mg/L 蔗糖 30,000 mg/L PHytagel 2.6 mg/L pH 5.8 1. 硝酸盐是植物培养中重要的氮源，其代谢物亚硝酸盐是对植物有害的，所以亚硝酸盐还原酶 NiR 在组培中起重要作用。硝酸盐 nitrate 经 NR 催化为亚硝酸盐 nitrite ，再经 NiR 催化为氨，再经 GS 合成谷氨酸； 2. 钙离子利于胚细胞的产生和维持； 3. 银离子阻止乙烯诱导的坏死，促进器官发生及体细胞胚胎发生； 4. 铜离子提高过氧化物酶的活性，诱导胚性愈伤的产生和分化；高浓度的铜离子作为重金属离子使得 PPO 蛋白变性，抑制其活性，阻止褐化。 CuSO 4 .5H 2 O 的分子量为 250, 0.5mM 即 1.25mg/L 即可抑制 PPO 的活性到 20% 以下，培养基中 0.6mg/L 即可抑制 PPO 的活性到 50% 以下。另外， Vc 、硝酸银、 PVP 等抗氧化剂也能抑制 PPO 的活性。 5. 缺乏锌离子利于小麦胚细胞的产生； 6. 19-22 度低温利于共培养侵染；共培养的时间为 55 个小时侵染率最高； 7. 干燥和冷利于分化提高转化效率； 8. 硫代硫酸银和硝酸钙利于转化效率的提高； 9. promotion or prevention of shoot regeneration by cupric sulphate and high EDTA 10. 侵染后亚精氨 spermidine 利于愈伤的恢复； 11. 硝酸铵利于分化和胚细胞的产生； 12. The increase in ammonium nitrate concentration in regeneration medium resulted in substantial, concentration-dependent increase in homologous recombination frequency (HRF) (Boyko et al. 2009). ammonium-based salts induce conformational changes in human Rad51 leading to an increase in its activity and therefore promoting recombination. The transformation events we analyzed included better quality and more intact insertions of the transgene, achieved most probably due to the more frequent involvement of HR, an error-free repair mechanism rare earth elements during a tissue culture stage also holds much promise, as a positive effect of these elements on nitrogen metabolism has been documented 。 13. 氯化铈 CeCl3 or 氯化镧 LaCl3 诱导活性氧的产生，激发抗病反应， chloride ions had a positive influence on homologous recombination rates; removal of potassium ion from the MS medium results in a decrease in HRF. 14. 干燥利于侵染和愈伤分化；热击处理可降低或抑制农杆菌诱导的侵染细胞的死亡；饥饿处理利于侵染；钙离子利于适于转化的胚细胞的产生，银离子可以阻止乙烯诱导的组织坏死，硝酸铵、氯化钾和一些稀有元素（镧系）利于植物的生长和遗传转化。 15. 抗坏死物质（抗氧化剂）： antioxidants: 聚乙烯吡咯烷酮 PVP (0.5 and 1 g/L), 半胱氨酸 L-cysteine (150 mg/L), 没石子酸 gallic acid (1.5 mg /L), 谷胱甘肽 glutathione 400 mg /L; 二巯基苏糖醇 DTT (70 mg/L), 生物蝶呤 biopterin (15 mg/L), 抗坏血酸 ascorbic acid (150 mg/L 或 50μM ，分子量 176.12), and 柠檬酸 citric acid (150 mg/L), Sodium thiosulfate ( Na 2S2O3),158 mg/L, 其中 DTT 的效果很好。 16. 抗褐化：苯酚甲醛离子交换树脂 Amberlite XAD-4 is mainly used for the removal of 芳香族 aromatic hydrocarbons such as phenols and pesticides from wastes. However, they have also been utilized for both extraction of specific phytochemicals produced in vitro and removal of toxic compounds negatively affecting explant viability in plant cell cultures 。 17. 0.5 g/L 2-(4-morpholino)-ethanesulfonic acid (MES, 2- 吗啉乙磺酸 ), 调节氢离子浓度，促进植物生长。 18. 在侵染液和选择培养基中加入 100 μM L -cysteine 半胱氨酸（分子量 121.16 ）即 12.1 mg/L ，也可以 100-400 mg/L ，可以提高转化效率和利于抗性愈伤的恢复生长。 in the selection medium enhanced the frequency of transformation and transgenic plant recovery 。 大豆侵染：子叶节作为外植体， dip-wounding 手术刀沾菌后切掉主芽，去除顶端优势，促进 AM 的生长；再用菌液浸泡。 19. 恢复培养基 resting 和选择培养基 selection I II 也需要加 MES 0.5g/L +Vc+ 硝酸银 20. 侵染液： 1/2 NB 基本培养基 +2mg/L 2,4-D+proline 0.7g/L+CH 2000mg/L+myo-inositol 2g/L+ 谷氨酰胺 2 g/L ？ +68.5g/L sucrose （ 200 m M ） +36g/L glucose （ 200 m M ） pH 5.2 高温灭菌后 4 度保存，使用前加抽滤除菌的 L -cysteine 半胱氨酸 150 mg/L + silver nitrate 4.4 mg/L + Vc 150mg/L +100-200 μM AS+10 mL/L 10% Pluronic F68 osmotic treatment 不利于小麦的转化 硝酸银抑制农杆菌的生长，促进植物生长和恢复；需要在侵染后的共培养（ 0.85 mg/L ）、恢复培养和选择培养中添加。 Silwet L77, Pluronic acid F68, Tween 20 利于农杆菌吸附到愈伤表面，消除愈伤表面不利于吸附的物质；类似于真空渗入方法。 10 mm 2-morpholinoethanesulphonic acid (MES) and 50 mm potassium hydrogen phosphate was effective in retaining the pH within a range of ±0.1 pH units during 2 days of co-culture, 3 mm CaCl 2 可以大大提高侵染率 333 mg/L 侵染液，抑制农杆菌的生长。 低盐利于侵染， 1/10 盐浓度可以大大提高侵染率（高粱可达 17.5% ），但愈伤容易死亡， 1/2 盐浓度再加抗氧化剂 3.3mM(400 mg/L) cystein 和 1mM(154 mg/L)DDT 高粱可达 10% ，操作人 performer 和载体、基因、启动子等影响转化率，拷贝数和表达情况。 伤口和快速生长的时候分泌小分子酚类信号物质利于侵染；低温利于 4SS 型分泌通道的形成，所以集菌时要 15-20 ℃ ，悬浮菌液也要 10-20 ℃ 放置 20 分钟； 用 1M 蔗糖 342 g/L 处理愈伤 12 小时后，再侵染，会增加转基因的拷贝数。高渗透压预处理提高转化率，侵染前用 25% 的蔗糖处理 3-5 个小时。 培养愈伤时，培养基表面放上滤纸再放愈伤，可以防止褐化，可能是利于褐化物质扩散和干燥。 山梨醇氧化 --- 葡萄糖或果糖 --- 蔗糖或麦芽糖 转化效率主要是由受体基因型决定的，其中 T-DNA 的整合起主要的作用，愈伤的状态也很重要。愈伤的胚性，包括颜色、大小、 friability 易碎性、增殖速度、褐化倾向，愈伤的诱导周期、愈伤的分化能力，分化率以及每个愈伤产生芽的数量等等。 reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. Increased 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells. Effect of L-glutamine on Agrobacterium Growth The effect of L-glutamine on bacterial growth in presence of patchouli extract was examined. Among the concentrations of L-glutamine tested, 2.0 g/l was found effective in promoting bacterial growth in presence of inhibitory concentration (15 mg/ml) of patchouli callus extract (Fig. 5c, d). In the absence of callus extract, media supplemented with L-glutamine at all tested concentrations showed Agrobacterium growth similar to the growth observed in AB minimal media devoid of L-glutamine. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. Vir proteins suppress the host innate immune system; 农杆菌与植物互作的主要信号： phenolics (acetosyringone), aldose monosaccharides (glucose), acidic pH( ～ 5.5) and low phosphate , 其中酚类是必需的，其它信号能增加农杆菌对酚的敏感性，扩大信号作用。 农杆菌受到植物受伤信号分子（酚类）的诱导后，通过 VirA-VirG 信号系统激活毒性蛋白（ Vir ）的表达，诱导表达产生的 VirD1 、 VirD2 、 VirC1 和 VirC2 形成松弛体，具有松弛酶和核酸内切酶活性的 VirD2 能在特定的位点上 (LB 第 3 和 4 个碱基之间 ) 把 T-DNA 中的一条链从 Ti 质粒上切割下来，并且以共价键的形式结合在单链 T-DNA(RB) 的 5’ －端，形成 VirD2- 单链 T-DNA － nVirE2 复合物，该复合物在 VirD5, VirE2, VirE3 和 VirF 的作用下通过由 VirB 和 VirD4 组成的 type IV secretion system (T4SS) 从细菌进入到植物细胞内， T 链复合体在 VirE2 结合蛋白 VIP1 、 VirE3 和 VirE2 作用下形成超 T 链复合体并向细胞核靠近，到达植物细胞核后，在 VIP1 结合蛋白 VBF 和 VirF 的作用下， VIP1 和 VirE2 从复合体上解离下来，并通过蛋白酶体 SCF (Skp1-Cul1-F-box protein)ubiquitin E3 ligase complex 降解。 T-DNA 整合到植物基因组主要是通过宿主的 DNA 修复机制完成的。染色体修饰在 DNA 修复中起着重要的作用，构成核小体的八聚体的组蛋白 H2A, H2B, H3 和 H4 的 N- 端尾巴容易被进行翻译后的修饰，包括磷酸化、甲基化、乙酰化和泛素化等，这些修饰与 T-DNA 插入有关系。 染色质的状态感受高温（热击）： H2A-2 脱离染色质 DSB 诱导因子如 X 射线，能提高 T-DNA 的插入； 瞬时表达 rare-cutting restriction enzyme 能提高 T-DNA 的插入； Gram-negative bacterium is a broad-host range plant pathogen, tumour-inducing (Ti) plasmid transfer DNA (T-DNA), The expression of T-DNA-encoded bacterial genes in the host cell results in the production of enzymes that catalyse the synthesis of plant hormones, which are responsible for tumour growth and the formation of novel amino acid–sugar conjugates, termed as opines. As opines can serve as carbon and sometimes nitrogen sources for Agrobacterium to the exclusion of most other microorganisms, they provide a selective advantage for this species. Disarmed plasmids, the genes responsible for tumourous growth have been removed, ensuring that the transformed cells can be regenerated into fertile plants that transmit the engineered DNA to their progeny VirA is a membrane-bound sensor and VirG is the intracellular response regulator On signal sensing, the histidine kinase VirA activates VirG through transferring its phosphate to a particular aspartate of VirG, thereby activating VirG to function as a transcription factor. Phosphorylated VirG then binds at specific 12 bp DNA sequences of the vir gene promoters (vir boxes), thereby activating transcription. cells in the root elongation zone were found to be the most highly transformable (Yi et al, 2002). Cells of this non-meristematic zone are not undergoing a normal cell cycle, but endoreduplication 核内复制，有染色体的复制，而没有以后的细胞分裂。 拟南芥根伸长区的细胞易于转化，这是由于该非分裂区的细胞处于非正常的细胞周期中，而处于核内复制状态。 Cell division activity 对转化是很重要的。 Yi H, Mysore KS, Gelvin SB (2002) Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. Plant J32: 285–298. simple non-meristematic tissue is non-dividing tissue; they are cells in function 薄壁和厚壁组织 1)parenchyma cells- most abundant cells in plants; spherical cells which flatten at point of contact; alive at maturity; pliable, primary cell walls; large vacuoles for storage of starch, fats, and tannins (denature proteins); primary sites of the metabolic functions such as photosynthesis, respiration, and protein synthesis; they are ready reserves from which a plant makes specialized cells to meet its changing needs Specialized parenchyma: a)chlorenchyma- photosynthetic cells; have high density of chloroplasts b)aerenchyma- prominent intercellular spaces that improve gas exchange capacity of the tissue; provide maximum support with a minimum metabolic requirement c)transfer cells- specialized for short distance transfer of solutes between cells; have secondary cell walls; they are inner extensions of wall that increase surface area. Ex). mitochondria *transfer cells occur in areas of high solute transport, such as secretory tissues, which release substances that are produced within the protoplasm and are moved outside, i.e., nectar cells, mucilage in sundews, and resins 2)collenchyma cells- living protoplasm; unevenly thickened primary cell walls; elongate cells; longer than wide; just beneath epidermis; function to support growing organs, grass, floral parts, and border veins; their nonlignified cell walls can stretch 3)sclerenchyma cells- most are dead at maturity; rigid, thick, lignified, nonstretchable secondary cell walls. There are 2 types of sclerenchyma cells: a)sclereids or stone cells- short; variable shape; form hard layers such as the shells of nuts and seed coats; produce the gritty texture of pears b)fibers- long, slender; occur in strands or bundles; tiny cavity or lumen; the different hardnesses of fibers are used to make coarse rope, linen cloth, etc. chvA, chvB, and pscA (exoC), which are involved in the synthesis and/or localisation of periplasmic b-1,2 glucan ，利于农杆菌和植物附着； soluble pectic plant cell wall fractions decreases both the specific binding of Agrobacterium to plant cells and tumour-induction frequencies 。 The VirB complex belongs to the class of type IV secretion systems (T4SS), which are found across a broad range of Gram-negative bacteria and are involved in the conjugative transfer of plasmids between bacteria as well as the translocation of Vir factors from pathogens to host cells during infection 。 VirB1–11 and VirD4 and is required for virulence. Successful transformation is thought to depend on a combination of suitability for tissue culture and the ability to accept a transgene via Agrobacterium ( Boyko et al. 2009 ). In rice varieties recalcitrant to Agrobacterium transformation, reports indicating enhanced competence of transgene integration are limited, although improved transformation systems achieved by modifying tissue culture conditions and selection procedures for transformed cells have been reported ( Nishimura et al. 2006 , Hiei and Komari 2008 ). Transformation frequency was compared between indica and japonica varieties of rice that were recalcitrant and susceptible to Agrobacterium-mediated transformation, respectively ( Tie et al. 2012 ). In that report, it was estimated that the stable transformation frequency in indica varieties was 20–50 times lower than that in japonica varieties, although t ransient transformation frequency in indica varieties was half t hat in japonica varieties, suggesting that the low transformation efficiency in indica rice was due mainly to the low frequency of stable transformation . stable transformation ( Gelvin 2010 ), is indispensable for the subsequent clonal propagation of transformed calli under selection pressure such as antibiotics 。 农杆菌介导的遗传转化的成功通常是依赖于受体材料良好的组织培养和接受外源基因的能力的完美结合。通常情况下，粳稻稳定转化率是籼稻的 20-50 倍，而粳稻瞬时转化率仅仅是籼稻的两倍 ( Tie et al. 2012 ) ，由此可见 籼稻转化效率低主要是由外源基因稳定整合到基因组中的效率低决定的。抗性愈伤的扩繁生长是由稳定转化基因表达来实现的 ( Gelvin 2010 ) 。 It has been proposed that integration of the double-stranded T-DNA into double-strand breaks (DSBs) in the genome requires a non-homologous end-joining (NHEJ 非同源末端连接 ) process, which is one of the major pathways used to repair DNA DSBs. In yeast NHEJ mutants, such as ku70 and ligase4, the frequency of T-DNA integration is reduced dramatically ( van Attikum et al. 2001 ). In Arabidopsis, it was shown using a root transformation assay that a ku80 mutant—one of the proteins involved in NHEJ—was severely impaired in stable integration but not in transient expression ( Li et al. 2005 ). Moreover, T-DNA integration analysis has been reported in virE2 interacting protein2 (vip2) knockdown tobacco plants using the promoter trap system and the gusA gene ( Anand et al. 2007 ). On the other hand, there are reports that particular histones act to enhance transgene expression and result in improving transformation frequency ( Tenea et al. 2009 , Zheng et al. 2009 ). In our monitoring system, both the number of stable transformation events and the level of transient and/or stable transgene expression could be measured by counting the number of GFP-emitting cell sectors under a dissecting microscope, and the quantification of GFP fluorescence and ELuc luminescence levels, respectively ( Figs. 5 , 6 ), suggesting that our system could be applied to researching the correlation between transgene expression and the number of transformation events. We expect that further details of the molecular mechanism of T-DNA integration could be elucidated by the sequential observation of transient and/or stable transformation using our monitoring system in DNA repair mutants. In conclusion, we have established a sequential observation system to monitor stable transgene expression using ELuc. We believe that our system could be applied successfully to both the improvement of Agrobacterium-mediated transformation protocols and the analysis of the molecular mechanism of T-DNA integration in plants. the suppression of NHEJ-related gene expression causes an inhibition of Agrobacterium-mediated stable transformation, but not of import of T-DNA into the nuclei of infected cells. 抑制 NHEJ 相关基因的表达能有效的降低农杆菌的稳定转化效率，但不影响 T-DNA 进入细胞核和瞬时表达。 相反的报道 Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration 。 http://www.plantcell.org/content/24/10/4110.long Ku70/80 (X-ray cross complementation ptotein6/5), DNA ligase 4, Xrcc4 (X-ray cross complementation ptotein4) DNA 修复的非同源的末端连接， non-homologous end-joining (NHEJ) ， double-strand breaks (DSBs) Ku is a protein that binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair . Ku is evolutionarily conserved from bacteria to human. The ancestral bacterial Ku is a homodimer (two copies of the same protein bound to each other). Eukaryotic Ku is a heterodimer of two polypeptides , Ku70 (XRCC6) and Ku80 (XRCC5), so named because the molecular weight of the human Ku proteins is around 70 kDa and 80 kDa. The two Ku subunits form a basket-shaped structure that threads onto the DNA end . Once bound, Ku can slide down the DNA strand, allowing more Ku molecules to thread onto the end. In higher eukaryotes, Ku forms a complex with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the full DNA-dependent protein kinase , DNA-PK. Ku is thought to function as a molecular scaffold to which other proteins involved in NHEJ can bind. van Attikum H, Bundock P, Hooykaas PJJ. Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J. 2001; 20:6550-6558. 常用的培养基及特点如下： （1）MS培养基 它是1962年由Murashige和Skoog为培养烟草细胞而设计的。特点是无机盐和离子浓度较高，为较稳定的平衡溶液。其养分的数量和比例较合适，可满足植物的营养和生理需要。它的硝酸盐含量较其他培养基为高， 广泛地用于植物的器官、花药、细胞和原生质体培养，效果良好 。有些培养基是由它演变而来的。 （2）B5培养基 是1968年由Gamborg等为培养大豆根细胞而设计的。其主要特点是含有较低的铵，这可能对不少培养物的生长有抑制作用。从实践得知有些植物在B5培养基上生长更适宜，如 双子叶植物特别是木本植物 。 （3）White培养基 是1943年由White为培养番茄根尖而设计的。1963年又作了改良，称作White改良培养基，提高了MgSO4的浓度和增加了硼素。其特点是无机盐数量较低， 适于生根培养 。 （4）N6培养基 是1974年朱至清等为水稻等禾谷类作物花药培养而设计的。其特点是成分较简单，KNO3和(NH4)2SO4含量高。在国内已广泛应 用于小麦、水稻及其他植物的花药培养和其他组织培 养。 （5）KM-80培养基 它是1974年为原生质体培养而设计的。其特点是有机成分较复杂，它包括了所有的单糖和维生素， 广泛用于原生质融合的培养 。 分化培养基 MS 盐 + 烟酸 5 mg/L +VB6 10 mg/L+VB1 10 mg/L+ 肌醇 100 mg/L+CH 2 g/L+NAA 0.2 mg/L+kinetin 2 mg/L+sorbitol 30 g/L+sucrose 30g/L+gelrite 3 g/L Basic 培养基 B N6 （大量） 50ml （ 20 倍） A Ms-Fe 盐 10-20ml （ 100 倍） CH 0.3g/L S B5 macro 10ml （ 100 倍） phytogel 4g/L 或 agar 8g/L I B5 vita 10ml （ 100 倍） sucrose 30g/L C proline 0.5g/L glutamine 0.5g/L N6 ( 大量 梗稻种子 ) 终浓度 母液 (20 倍 ) 终浓度 母液 (20 倍 ) 培 KNO 3 2830 mg/L 56.6 g /L Mg SO 4 .7 H 2 O 185 mg/L 3.7 g/L 养 (NH 4 ) 2 SO 4 463 mg/L 9.26 g /L CaCl 2 .2H 2 O 166 mg/L 3.32 g /L 基 KH 2 PO 4 400 mg/L 8.00 g /L 制备 1 KNO 3 , (NH 4 ) 2 SO 4 , KH 2 PO 4 同时倒入烧杯 , 加水搅拌 , 使之完全溶解 . 2 Mg SO 4 .7 H 2 O 先溶于加了 100ML 水的烧杯中溶解后再缓慢倒入 1 中 , 边倒边搅 , 不能有沉淀 . 3 CaCl 2 .2H 2 O 的配制同 Mg SO 4 .7 H 2 O 4 配好后放于棕色瓶 4 ℃ 保存 MS( 大量 籼稻种子 ) 终浓度 母液 (20 倍 ) 终浓度 母液 (20 倍 ) 培 KNO 3 1900 mg/L 38 g /L Mg SO 4 .7 H 2 O 370 mg/L 7.4 g /L 养 NH 4 NO 3 1650 mg/L 33 g /L CaCl 2 .2H 2 O 440 mg/L 8.8 g /L 基 KH 2 PO 4 170 mg/L 3.4 g /L 制备 1 KNO 3 , NH 4 NO 3 , Mg SO 4 .7 H 2 O 同时倒入烧杯 , 加水搅拌 2 KH 2 PO 4 先溶于加了 100ML 水的烧杯中溶解后再缓慢倒入 1 中 , 边倒边搅 , 3 CaCl 2 .2H 2 O 同 KH 2 PO 4 4 配好后放于棕色瓶 4 ℃ 保存 Ms( 大量 花药用 ) 终浓度 母液 (20 倍 ) 终浓度 母液 元素 KNO 3 2830 mg/L 56.6 g /L Mg SO 4 .7 H 2 O 370 mg/L 7.4 g /L 培养 (NH 4 ) 2 SO 4 231 mg/L 4.62 g /L CaCl 2 .2H 2 O 160 mg/L 3.2 g /L 基 KH 2 PO 4 641 mg/L 12.82 g /L 制备 1 KNO 3 , (NH 4 ) 2 SO 4 , KH 2 PO 4 同时倒入烧杯 , 加水搅拌 2 Mg SO 4 .7 H 2 O 先溶于加了 100ML 水的烧杯中溶解后再缓慢倒入 1 中 , 边倒边搅 , 不能有沉淀 .CaCl 2 .2H 2 O 的配制同 Mg SO 4 .7 H 2 O 3 配好后放于棕色瓶 4 ℃ 保存 YM 脓杆菌培养基 KH 2 PO 4 0.5g/L Mannitol 10g/L L-Glutamine 2g/L NaCl 0.2g/L M g SO 4 0.2g/L Yeast extract 0.3g/L Agar 15g/L PH=7.0 诱导愈伤、继代培养基 basic+2,4-D(2mg/L) pH=5.8 培养基灭菌后有机物分解引起 pH 下降 0.2 ，温度升高 pH 会降低 H2O==H+ +OH- pH=lg C(H+) 共培养培养基 侵染液体 : NB + 肌醇 2 g/L+ 谷氨酰胺 2 g/L+ CH 500mg/L + 10 ml/L 10% Synperonic PE/F68 (Sigma, Pluronic F68) +0.1%AS( 终浓度 100 μ M AS ) 固体 :N6( 大 )+Fe+B5(micro)+B5(vita)+ sucrose( 前四项与 basic 相同 ) + 肌醇 2g /L + CH500mg/L+ 2,4-D (2mg/L) +phytogel +0.1%AS pH =5.5 加 2,4-D 前后都要调 PH 值 . 灭完菌 , 温度降下来后加 AS 共培养温度为 19 － 25 ℃ ， 22 ℃ 为适，温度高农杆菌生长太快，侵染能力下降。 冻的液体药品要完全溶解混匀后在使用。 筛选培养基 诱导愈伤 +cef(300mg/L)+hyg(50mg/ml) PH=5.5 一筛 , 二筛的 cef,hyg 的浓度依次逐渐下降 分化 1: basic+KT(4mg/L)+NAA(0.25mg/L)+cef(300mg/L)+hyg(50mg/L) 培养 2: basic+KT(0.5mg/L)+ NAA(0.25mg/L)+6-BA(2mg/L)+cef(300mg/L)+hyg(50mg/L) 基 PH=5.8 分化培养基 NB 培养基： 6-BA,2.5-3mg,NAA,0.25mg,KT,0.5mg ，每个培养皿放 8 个抗性愈伤，不可过多。干燥促进分化，脯氨酸减少褐化。 增加培养基的硬度和渗透压，加山犁醇 30 克 / 升。 medium supplemented with 3% (w/v) maltose 麦芽糖 ,2.0 mg kinetin and 0.5 mg NAA MS +BA 2 ． 0 mg ／ L+KT 0 ． 5 mg ／ L +NAA 0 ． 5 mg ／ L +CH 0 ． 5 g ／ L +sucrose 30 g ／ L+ agar 7 ． 0 mg ／ L 预分化： NB+6-BA 2 毫克 +NAA 1 毫克 +ABA 5 毫克 分化： NB+6-BA 3 毫克 +NAA 0.3 毫克 壮苗培养基 ( 生根 ) sucrose 30g/L N6 25ml/L MS-Fe 盐 10ml/L B5vita 10ml/L agar 7g/L 加 1.0 -5.0mg/L 的 Met, 起壮根作用。 水稻生根培养基中加入生长素吲哚丁酸 IBA 0.5 mg/L 或 NAA 2 毫克促进生根。 MS-Fe 盐 ( 必须单独配制 , 否则会沉淀 , 用鳌合铁 ) 终浓度 母液 (100 倍 ) FeSO 4 .7H 2 O 27.8mg/L 2.78g/L Na-EDTA 37.5mg/L 3.75g/L 制备 1 FeSO 4 .7H 2 O 溶解于水 , Na-EDTA 溶解于热水 , 将两者混合定容 , 于微波炉中加热 , 煮沸，颜色变深，冷却到室温，补水，棕色瓶 4 ℃ 保存 Sigma 的铁盐 100 倍母液为 4 克 / 升 2,4-D 母液 (1g/ml) 用无水乙醇溶解 2,4-D 后缓慢加入到 H 2 O 中 , 搅拌 , 如产生沉淀 , 则重配 . 配好后 4 ℃ 保存。溶解不彻底容易重新结晶。还可以用少量 1N 的氢氧化钠 ( 最好不要超过总体积的 10 ％ ) 在三角瓶中溶解，可以微波炉辅助加热和用枪吸打，充分溶解后再定容。 NAA 母液 (1g/ml) 用 1N KOH 溶解 NAA, 用水稀释定容 , 4 ℃ 保存 PAA 母液 (1g/ml) 用无水乙醇溶解加 H 2 O 搅拌 , 定容 , 4 ℃ 保存 6-BA 母液 (1g/ml) 用 HCl 溶解 , 加少量 HCl 后 , 用玻棒研磨成糊状 , 再加 HCl 使之完全溶解。还可以用少量 1N KOH 溶解，再加水定容即可。 AS （乙酰丁香酮） 用 DMSO 直接溶解定容 19.62mg/ml, 分装到无菌小管，用时稀释 1000 倍。 100 μ M AS. 分子量 196.20 KT 母液 (5mg/ml) 用 1NKOH 溶解，可以微波炉辅助加热，一定要溶解充分，用水稀释定容到 10ml ，过滤灭菌后分装到 EP 管中，冰冻保存。 B5 vitamin 终浓度 母液 (100 倍 ) 终浓度 母液 (100 倍 ) 肌醇 100mg/L 10g/l Nico-acid 1mg/l 0.1g/l pyrido(B6) 1 mg/L 0.1 g/l thiamin(B1) 10 mg/L 1 g/l 棕色瓶 4 ℃ 保存 ( 每次配 100ml 为好 ), 肌醇在配培养基时 , 加入到培养基中，时间久了会长菌，可以保存两个月。 B5(micro) KI 0.75mg/L H 3 BO 3 3.0mg/L MnSO 4 10mg/L ZnSO 4 7H 2 O 2.0mg/L Na 2 MnO 4 .2H 2 O 0.25mg/L CuSO 4 2H 2 O 0.025mg/L CoCl 2 0.025mg/L 影响 vir 基因活化的因素包 括酚类化合物、单糖分子和 pH 值等。 酚类化合物对苯二酚、没食子酸、对羟基苯甲酸、香草醛或乙酰丁香酮作为农杆菌 VIR 基因活化物，每种化台物的使用浓度均为 100μM 。对苯二酚、没食子酸对愈伤组织有很强的毒害作用，在分别加有这两种化合物的培养基中培养 3 d 后，愈伤组织很快变褐死亡；而在分别加有对羟基苯甲酸、香草醛、乙酰丁香酮的培养基中培养的愈伤组织生长正常。 同其它转化方法相比，根癌农杆菌介导的转化具有操作简便、不需特殊的仪器、比较短的培养周期、更重要的是外源基因 插入的低拷贝性和完整性以及转基因可育 植株比例高等优点。 目前粳稻的转化频率一般在 30 ％左右，最高可达到 64 ％。 抗性愈伤组织的分化是提高转化绿苗频率的关键，抗性愈伤组织如直接转入分化培养基，绿苗分化频率极低，而将抗性愈伤组织转入预分化培养基培养 15 d ，然后，再转入分化培养基培养，绿苗分化频率明显提高。尤其是预分化培养基中的渗透压的提高，能有效地促使抗性愈 伤组织的分化和绿苗的再生。 羧苄青霉素的除菌效果优于头孢霉素；但是，连续使用后，抗性愈伤组织分化 频率低于头孢霉素除菌后的抗性愈伤组织。因此，在实际使用中，宜选用头孢霉素除菌或将头孢霉素和羧苄青霉素交替使用，这样既能取得除菌效果又不影响抗性愈伤组织的分化能力。 5.1 水稻转化受体的准备 5.1.1 水稻幼胚愈伤组织的诱导培养 取开花后 12-15 天左右的水稻幼穗脱粒 , 用清水漂去秕粒 , 用 70% 乙醇浸泡 1-2 分钟，然后用加有几滴 Tween20 的 1.25% 的次氯酸钠溶液（活性氯含量为 1.25% ）浸泡 90 分钟，进行表面灭菌。 ( 灭菌时要经常搅拌 ) 用无菌水冲洗 3-4 次，沥去水备用。在无菌滤纸上用镊子和刮牙器挤出水稻幼胚置于固体诱导培养基（ NB 培养基）上， 26 ℃ 暗培养诱导愈伤组织。约 5-7 天后剥下愈伤组织，转入新鲜配制的继代培养基（ NB 培养基）上，在相同条件下继代培养 5 天左右，用于共培养。 5.1.2 水稻成熟胚愈伤组织的诱导培养 去壳的水稻成熟种子先用 70% 乙醇浸泡 1-2 分钟，然后用 0.1% 升汞浸泡 30 分钟，进行表面灭菌 ( 最好在摇床上进行 ) ，无菌水冲洗 3-4 次，再将种子放在无菌滤纸上吸干水分后，放在成熟胚愈伤诱导培养基上， 26 ℃ 暗培养。约 10-15 天后，剥下成熟胚盾片长出的愈伤组织 , 转入成熟胚继代培养基上，在相同条件下继代培养。以后每两周继代培养一次。挑选继代培养 4-5 天、色泽淡黄颗粒状的愈伤组织共培养。 成熟季节的天气；颖壳和种皮表面没有麻点 （病斑）；按成熟度分开（青米优于完熟米） 注 意：粳稻不适宜用 NaClO 灭菌。 5.2 农杆菌的培养 将含有目的基因载体的农杆菌 EHA105 在含有 50mg/L Kanamycin 的 YM 平板上划线， 28 ℃ 黑暗培养 2-3 天，用一金属匙收集农杆菌菌体 , 将其悬浮于共培养 CM 液体培养基中，调整菌体浓度至 OD 600 为 0.3-0.5 ，加入 AS ，使 AS 终浓度为 100mΜ ，即为共培养转化水稻用的农杆菌悬浮液。 5.3 水稻愈伤组织与农杆菌的共培养 挑选状态较好的继代到一定时间的水稻愈伤组织放入无菌三角瓶中 , 然后加入适量农杆菌悬浮液 ( 至少保证有足够的菌液与材料接触 ), 室温放置 20min, 并不时晃动。取出愈伤组织 , 在无菌滤纸上吸去多余菌液 , 随即转移到铺有一层无菌滤纸的固体共培养基上（将诱导愈伤和继代培养时始终紧贴培养基的那一面依然朝下放置，愈伤应摆放整齐，相互之间最好不要叠放）， 26 ℃ 黑暗培养 2-3 天。 仔细挑选无菌、状态较好（继代培养 4-5 天、色泽淡黄、颗粒状）的愈伤组织放入 100ml 无菌三角瓶中，加入适量农杆菌悬浮液，室温放置 20min ，并不时晃动。倒掉菌液，将愈伤组织放在无菌滤纸上吸去多余菌液，随即转移到铺有一层无菌滤纸的固体共培养基上， 25 ℃ 黑暗培养 2-3 天。 5.4 抗性愈伤组织的筛选 将共培养后的愈伤组织（也可以水洗除菌后）放在含有 50mg/lHygromycin 的筛选培养基上， 26 ℃ 暗培养 14 天，转到新鲜配制的筛选培养基上继续筛选 14 天。大部分愈伤组织在筛选后 10 天左右褐化，然后在褐化组织的边缘重新生长出乳白色的抗性愈伤组织。 现象：愈伤组织在选择培养基上出现脓状现象，导致培养失败。减少菌量和共培养后水洗并不能消除这一现象。 对策：需要从两方面把关：从继代培养开始，精心挑选愈伤组织，杜绝染菌的愈伤组织。另一方面，严把农杆菌关：农杆菌不应该多次传代，坚持用单菌落的农杆菌划线。 5.5 抗性愈伤组织的分化 从经两轮筛选后长出的抗性愈伤组织中，挑选乳黄色致密的抗性愈伤组织转至含有 50mg/L Hygromycin 的分化培养基上 , 先暗培养 3 天 , 然后转至 15h/d 光照条件下培养 , 一般经过 15-25 天左右 , 有绿点出现。 30-40 天后进一步分化出小苗。 从经两轮筛选后长出的抗性愈伤组织中，挑选乳黄色致密的抗性愈伤组织转至含有 50mg/l hygromycin B 的分化培养基上，先暗培养 3 天，然后转至 15h/d 光照条件下培养，一般经过 15-25 天左右，有绿点出现， 30-40 天后进一步分化出小苗。 只要愈伤组织的质量符合要求，预分化并非必需。我们省略了预分化，将抗性愈伤组织直接转到分化培养基上，不但节省了能源、昂贵的 hygromycin B 和 ABA ，大大降低了成本，而且使转基因的时间缩短了 10 天。 5.6 生根、壮苗和移栽 当抗性愈伤组织分化的芽长至约 50px 时 , 将小苗移到生根培养基上 , 培养两周左右。选择高约 250px 、根系发达的小苗，用温水洗去培养基，在温室内移栽入土。水面以不淹没小苗为度，如果天晴，需要遮荫到小苗成活（以吐水为准）。 A ， 再生的转基因苗要适时移到生根培养基上，保证转基因苗在试管中生长正常。 B ， 对过于细小的苗通过剪根、剪叶和再次转培，使转基因苗强壮。 C ， 选苗高约 10-375px ，根系旺盛的试管苗，选择时机直接移栽入土。 D ， 平整土地，保持水位，适当遮阴。 1 ，试管苗根系发达，株高约 250px 2 ，选择天气：春夏季选阴天、傍晚移栽； 冬季选晴天移栽，连续阴雨天不适合移栽 3 ，洗根时的水温要与土温基本一致 4 ，洗根和移栽时不要将成丛的试管苗分开 5 ，平整土地，控制水面，水不能淹没植株 程序 1. 种子去皮，挑成熟完整健康的种子 2. 用 70% 乙醇清洗浸泡 2-3 分钟，洗 3-4 次，用 0.1% 升汞浸泡 20-40 分钟 3. 无菌水洗 3 至 4 次 4. 置于虑纸上吹干 , 吹 3 小时以上 5. 摆放到诱导愈伤培养基上，黑暗中培养 15-20 天，直到长出黄色较大的愈伤 6. 剥下愈伤，转至继代培养基上，黑暗培养 2 周 7. 共培养。脓杆菌与愈伤在液体共培养基中轻轻摇培 30 分钟，转至共培养基（用虑纸盖住培养基）上， 吹风 3-4 小时。黑暗培养 3 天 8. 转至筛选培养基上，吹风吹干。黑暗培养 2 周。转至二筛培养基上，黑暗培养 2 周，直到长出新的愈伤。 9. 转至分化培养基上，黑暗培养一周。然后明暗交替（ 16 小时： 8 小时） 10. 剪苗后，转至生根壮苗培养基上 11. 移苗到温床，田间。地温要 15 ℃ 以上水稻才会生长，地温低于 12 ℃ 基本停止生长，移苗会死。气温要在 20 -40 ℃ 为好。 一种农杆菌介导的水稻转化方法，它包括下列步骤： 1. 愈伤的诱导：选取新鲜无病的栽培粳稻品系 H1493 成熟种子，去壳，用 70-75 ％乙醇浸洗 1-2min ，再用 0.1-0.15 ％ HgCl2 进行表面消毒 15-20min ，然后用无菌水充分浸洗，将消毒的种子放在灭过菌的滤纸上吸干后转到诱导培养基上，每皿放 10-12 粒种子，在 25 -28 ℃ 避光培养 13-15 天，诱导培养基为： N6 ，脯氨酸 0.8-1.2g/l ，水解酪蛋白 0.2-0.6g/l ， 2 ， 4-D1.5-2.5g/l ，蔗糖 40-50g/l ，组织培养胶 2.8-3.0g/l ， pH5.8-6.0 ，混匀即可； 2. 继代和预培养： 13-15 天后剥取新鲜、亮黄色的盾片愈伤并转到诱导培养基上继代培养 13-15 天，生长良好的胚性愈伤再转到预培养基上培养 3-6 天，预培养培养基为： N6 ，脯氨酸 0.4-0.8g/l ，水解酪蛋白 0.4-0.8g/l ， 2 ， 4-D1.5-2.5g/l ，麦芽糖 30-35g/l ，组织培养胶 2.8-3.0g/l ， pH5.4-5.6 ； 3. 农杆菌的准备与悬浮：从药用野生稻 BIBAC 基因组文库中选取一个 120Kb 的克隆 114G9 ，按常规方法提取该克隆的质粒 DNA ，通过电击转化的方法将该质粒转到含有毒性辅助质粒 pCH32 的 LBA4404 农杆菌菌株中，再将该菌株接种到含 50-60mg/l 卡那霉素的 LB 固体培养基上， 28 -30 ℃ 培养 2-3 天，刮取菌苔重新悬浮于农杆菌悬浮培养基，在 28 -30 ℃ 悬浮培养 2-3h 后使 OD600 为 1.0-1.2 ，将预培养 3-6 天的新鲜愈伤集中转到悬浮的菌液中，以 80-100r/min 浸染 15-20 分钟，农杆菌悬浮培养基的配方为： N6 ，脯氨酸 0.4-0.8g/l ，水解酪蛋白 0.4-0.8g/l ， 2 ， 4-D1.5-2.0g/l ，麦芽糖 30-35g/l ， pH5.5-5.7. 用前加 100-200μM 乙酰丁香酮； 4. 共培养：滤去菌液，将浸染的愈伤转到无菌的滤纸上吸干，再转到共培养基上， 21 -24 ℃ 黑暗共培养 2-4 天，共培养基为： N6 ，脯氨酸 0.4-0.8g/l ，水解酪蛋白 0.4-0.8g/l ， 2 ， 4-D1.5-2.0g/l ，麦芽糖 30-35g/l ， pH5.5-5.7 ，组织培养胶 2.8-3.0g/l. 用前加 100-200μM 乙酰丁香酮； 5. 农杆菌的去除与过渡培养：共培养后的愈伤用无菌水充分洗涤 4-6 次，直至洗过愈伤的水溶液变清亮，再用加入浓度为 300-400mg/L 的噻孢霉素的无菌水洗 2-3 次，每次 15-20min ，用无菌滤纸吸干愈伤并转到过渡培养基上培养 5-10 天，过渡培养基为： N6 ，脯氨酸 1.0-1.4g/l ，水解酪蛋白 0.4-0.6g/l ， 2 ， 4-D2.0-2.5g/l ，蔗糖 30-35g/l ，组织培养胶 2.8-3.0g/l ， pH5.8-6.0. 用前加 300-400mg/l 噻孢霉素； 6. 选择培养：过渡培养的愈伤再转到选择培养基上，随后每隔 13-15 天换一次培养基，选择一般持续 6-8 周，选择培养基为： N6 ，脯氨酸 1.0-1.4g/l ，水解酪蛋白 0.4-0.6g/l ， 2 ， 4-D2.0-2.5g/l ，蔗糖 30-35g/l ，组织培养胶 2.8-3.0g/l ， pH 5.8-6.0. 用前加 200-300mg/l 噻孢霉素和 40-60mg/l 潮霉素； 7. 预分化与分化再生：经选择后的抗性愈伤转到预分化培养基培养 7-10 天后再转到分化培养基进行光照培养，每天光照 16-20h ，光照强度 100-120μmolm-2 s-1 ，培养 2-4 周，预分化培养基为： MS ，水解酪蛋白 1.5-2.5g/l ， KT1.5-2.5mg/l ， NAA0.2-0.5mg/l ，麦芽糖 30-35g/l ，组织培养胶 2.8-3.0g/l ， pH5.8-6.0 ，分化培养基为： MS ， KT2.5-3.0mg/l ， NAA0.2-0.5mg/l ，麦芽糖 30-35g/l ，组织培养胶 2.8-3.0g/l ， pH5.8-6.0 ； 8. 生根与移栽：待小苗长至 2-100px 时转到装有生根培养基的试管中生长 2 -3 周，生长良好的小苗经 2-3 天的炼苗后便可移栽到温室中，生根培养基为： 1/2MS ， NAA0.2-0.5mg/l ，蔗糖 10-15g/l ，组织培养胶 2.5-2.8g/l ， pH5.8-6.0. 用前加 40-60mg/l 潮霉素。
Chemical modification of L-glutamine to alpha-amino glutarimide on autoclaving facilitates Agrobacterium infection of host and non-host plants: A new use of a known compound Indra Sandal , Amita Bhattacharya , Uksha Saini , Devinder Kaur , Shveta Sharma , Ashu Gulati , Jonnala K Kumar , Neeraj Kumar , Jyotsna Dayma , Pralay Das , Bikram Singh and Paramvir S Ahuja BMC Chemical Biology 2011, 11 : 1 doi:10.1186/1472-6769-11-1 Published: 31May2011 Abstract (provisional) Background Accidental autoclaving of L-glutamine was found to facilitate the Agrobacterium infection of a non host plant like tea in an earlier study. In the present communication, we elucidate the structural changes in L-glutamine due to autoclaving and also confirm the role of heat transformed L-glutamine in Agrobacterium mediated genetic transformation of host/non host plants. Results When autoclaved at 121 degrees C and 15 psi for 20 and 40 min, L-glutamine was structurally modified into 5-oxo proline and 3-amino glutarimide (alpha-amino glutarimide), respectively. Of the two autoclaved products, only alpha-amino glutarimide facilitated Agrobacterium infection of a number of resistant to susceptible plants. However, the compound did not have any vir gene inducing property. Conclusions We report a one pot autoclave process for the synthesis of 5-oxo proline and alpha-amino glutarimide from L-glutamine. Xenobiotic detoxifying property of alpha-amino glutarimide is also proposed.
经常问自己： Have I done something new and interesting? 低声说话；勤劳，踏实做事；心情平和，不让矛盾堆积，不计较得失；多吃水果蔬菜，植物油和不去麸的粮食，少吃肉类。 要善于在不断总结中变得聪明起来。 NB 基本培养基 KNO 3 2830 mg/L (NH 4 ) 2 SO 4 463 mg/L=374.8 mg/L NH 4 Cl KH 2 PO 4 400 mg/L MgSO 4 .7H 2 O 185 mg/L CaCl 2 .2H 2 O 166 mg/L FeSO 4 .7H 2 O 27.8mg/L Na 2 EDTA 37. 5 mg/L MnSO 4 .4H 2 O 10 mg/L H 3 BO 3 3 mg/L ZnSO 4 .7H 2 O 2 mg/L Na 2 MoO 4 .2H 2 O 0.25 mg/L CuSO 4 .5H 2 O 0.025 mg/L CoCl 2 .6H 2 O 0.025 mg/L KI 0.75 mg/L 盐酸硫胺素 thiamine CHL VB1 10 mg/L 盐酸吡哆醇 pyridoxine-CHL VB6 1 mg/L 烟酸 nicotinic acid 1 mg/L 肌醇 myo-inositol 100 mg/L 水解酪蛋白 300 mg/L 谷氨酰胺 500 mg/L 甘氨酸 2 mg/L 脯氨酸 2878 mg/L 蔗糖 30,000 mg/L PHytagel 2.6 mg/L pH 5.8 1. 硝酸盐是植物培养中重要的氮源，其代谢物亚硝酸盐是对植物有害的，所以亚硝酸盐还原酶 NiR 在组培中起重要作用。硝酸盐 nitrate 经 NR 催化为亚硝酸盐 nitrite ，再经 NiR 催化为氨，再经 GS 合成谷氨酸； 2. 钙离子利于胚细胞的产生和维持； 3. 银离子阻止乙烯诱导的坏死，促进器官发生及体细胞胚胎发生； 4. 铜离子提高过氧化物酶的活性，诱导胚性愈伤的产生和分化；高浓度的铜离子作为重金属离子使得 PPO 蛋白变性，抑制其活性，阻止褐化。 CuSO 4 .5H 2 O 的分子量为 250, 0.5mM 即 1.25mg/L 即可抑制 PPO 的活性到 20% 以下，培养基中 0.6mg/L 即可抑制 PPO 的活性到 50% 以下。另外， Vc 、硝酸银、 PVP 等抗氧化剂也能抑制 PPO 的活性。 5. 缺乏锌离子利于小麦胚细胞的产生； 6. 19-22 度低温利于共培养侵染；共培养的时间为 55 个小时侵染率最高； 7. 干燥和冷利于分化提高转化效率； 8. 硫代硫酸银和硝酸钙利于转化效率的提高； 9. promotion or prevention of shoot regeneration by cupric sulphate and high EDTA 10. 侵染后亚精氨 spermidine 利于愈伤的恢复； 11. 硝酸铵利于分化和胚细胞的产生； 12. The increase in ammonium nitrate concentration in regeneration medium resulted in substantial, concentration-dependent increase in homologous recombination frequency (HRF) (Boyko et al. 2009). ammonium-based salts induce conformational changes in human Rad51 leading to an increase in its activity and therefore promoting recombination. The transformation events we analyzed included better quality and more intact insertions of the transgene, achieved most probably due to the more frequent involvement of HR, an error-free repair mechanism rare earth elements during a tissue culture stage also holds much promise, as a positive effect of these elements on nitrogen metabolism has been documented 。 13. 氯化铈 CeCl3 or 氯化镧 LaCl3 诱导活性氧的产生，激发抗病反应， chloride ions had a positive influence on homologous recombination rates; removal of potassium ion from the MS medium results in a decrease in HRF. 14. 干燥利于侵染和愈伤分化；热击处理可降低或抑制农杆菌诱导的侵染细胞的死亡；饥饿处理利于侵染；钙离子利于适于转化的胚细胞的产生，银离子可以阻止乙烯诱导的组织坏死，硝酸铵、氯化钾和一些稀有元素（镧系）利于植物的生长和遗传转化。 15. 抗坏死物质（抗氧化剂）： antioxidants: 聚乙烯吡咯烷酮 PVP (0.5 and 1 g/L), 半胱氨酸 L-cysteine (150 mg/L), 没石子酸 gallic acid (1.5 mg /L), 谷胱甘肽 glutathione 400 mg /L; 二巯基苏糖醇 DTT (70 mg/L), 生物蝶呤 biopterin (15 mg/L), 抗坏血酸 ascorbic acid (150 mg/L 或 50μM ，分子量 176.12), and 柠檬酸 citric acid (150 mg/L), Sodium thiosulfate ( Na 2S2O3),158 mg/L, 其中 DTT 的效果很好。 16. 抗褐化：苯酚甲醛离子交换树脂 Amberlite XAD-4 is mainly used for the removal of 芳香族 aromatic hydrocarbons such as phenols and pesticides from wastes. However, they have also been utilized for both extraction of specific phytochemicals produced in vitro and removal of toxic compounds negatively affecting explant viability in plant cell cultures 。 17. 0.5 g/L 2-(4-morpholino)-ethanesulfonic acid (MES, 2- 吗啉乙磺酸 ), 调节氢离子浓度，促进植物生长。 18. 在侵染液和选择培养基中加入 100μM L -cysteine 半胱氨酸（分子量 121.16 ）即 12.1 mg/L ，也可以 100-400 mg/L ，可以提高转化效率和利于抗性愈伤的恢复生长。 in the selection medium enhanced the frequency of transformation and transgenic plant recovery 。 大豆侵染：子叶节作为外植体， dip-wounding 手术刀沾菌后切掉主芽，去除顶端优势，促进 AM 的生长；再用菌液浸泡。 19. 恢复培养基 resting 和选择培养基 selection I II 也需要加 MES 0.5g/L +Vc+ 硝酸银 20. 侵染液： 1/2 NB 基本培养基 +2mg/L 2,4-D+proline 0.7g/L+CH 2000mg/L+myo-inositol 2g/L+ 谷氨酰胺 2 g/L +68.5g/L sucrose+36g/L glucose pH 5.2 高温灭菌后 4 度保存，使用前加抽滤除菌的 L -cysteine 半胱氨酸 150 mg/L + silver nitrate 4.4 mg/L + Vc 150mg/L +100-200 μM AS+10 mL/L 10% Pluronic F68 低盐利于侵染， 1/10 盐浓度可以大大提高侵染率（高粱可达 17.5% ），但愈伤容易死亡， 1/2 盐浓度再加抗氧化剂 3.3mM(400 mg/L)cystein 和 1mM(154 mg/L)DDT 高粱可达 10% ，操作人 performer 和载体、基因、启动子等影响转化率，拷贝数和表达情况。 伤口和快速生长的时候分泌小分子酚类信号物质利于侵染；低温利于 4SS 型分泌通道的形成，所以集菌时要 15-20 ℃ ，悬浮菌液也要 10-20 ℃ 放置 20 分钟； 用 1M 蔗糖 342 g/L 处理愈伤 12 小时后，再侵染，会增加转基因的拷贝数。高渗透压预处理提高转化率，侵染前用 25% 的蔗糖处理 3-5 个小时。 培养愈伤时，培养基表面放上滤纸再放愈伤，可以防止褐化，可能是利于褐化物质扩散和干燥。 Effect of L-glutamine on Agrobacterium Growth The effect of L-glutamine on bacterial growth in presence of patchouli extract was examined. Among the concentrations of L-glutamine tested, 2.0 g/l was found effective in promoting bacterial growth in presence of inhibitory concentration (15 mg/ml) of patchouli callus extract (Fig. 5c, d). In the absence of callus extract, media supplemented with L-glutamine at all tested concentrations showed Agrobacterium growth similar to the growth observed in AB minimal media devoid of L-glutamine. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. Vir proteins suppress the host innate immune system; 农杆菌与植物互作的主要信号： phenolics (acetosyringone), aldose monosaccharides (glucose), acidic pH( ～ 5.5) and low phosphate , 其中酚类是必需的，其它信号能增加农杆菌对酚的敏感性，扩大信号作用。 农杆菌受到植物受伤信号分子（酚类）的诱导后，通过 VirA-VirG 信号系统激活毒性蛋白（ Vir ）的表达，诱导表达产生的 VirD1 、 VirD2 、 VirC1 和 VirC2 形成松弛体，具有松弛酶和核酸内切酶活性的 VirD2 能在特定的位点上 (LB 第 3 和 4 个碱基之间 ) 把 T-DNA 中的一条链从 Ti 质粒上切割下来，并且以共价键的形式结合在单链 T-DNA(RB) 的 5’ －端，形成 VirD2- 单链 T-DNA － nVirE2 复合物，该复合物在 VirD5, VirE2, VirE3 和 VirF 的作用下通过由 VirB 和 VirD4 组成的 type IV secretion system (T4SS) 从细菌进入到植物细胞内， T 链复合体在 VirE2 结合蛋白 VIP1 、 VirE3 和 VirE2 作用下形成超 T 链复合体并向细胞核靠近，到达植物细胞核后，在 VIP1 结合蛋白 VBF 和 VirF 的作用下， VIP1 和 VirE2 从复合体上解离下来，并通过蛋白酶体 SCF (Skp1-Cul1-F-box protein)ubiquitin E3 ligase complex 降解。 T-DNA 整合到植物基因组主要是通过宿主的 DNA 修复机制完成的。染色体修饰在 DNA 修复中起着重要的作用，构成核小体的八聚体的组蛋白 H2A, H2B, H3 和 H4 的 N- 端尾巴容易被进行翻译后的修饰，包括磷酸化、甲基化、乙酰化和泛素化等，这些修饰与 T-DNA 插入有关系。 染色质的状态感受高温（热击）： H2A-2 脱离染色质 DSB 诱导因子如 X 射线，能提高 T-DNA 的插入； 瞬时表达 rare-cutting restriction enzyme 能提高 T-DNA 的插入； Gram-negative bacterium is a broad-host range plant pathogen, tumour-inducing (Ti) plasmid transfer DNA (T-DNA), The expression of T-DNA-encoded bacterial genes in the host cell results in the production of enzymes that catalyse the synthesis of plant hormones, which are responsible for tumour growth and the formation of novel aminoacid–sugar conjugates, termed as opines. As opines can serve as carbon and sometimes nitrogen sources for Agrobacterium to the exclusion of most other microorganisms, they provide a selective advantage for this species. Disarmed plasmids, the genes responsible for tumourous growth have been removed, ensuring that the transformed cells can be regenerated into fertile plants that transmit the engineered DNA to their progeny VirA is a membrane-bound sensor and VirG is the intracellular response regulator On signal sensing, the histidine kinase VirA activates VirG through transferring its phosphate to a particular aspartate of VirG, thereby activating VirG to function as a transcription factor. Phosphorylated VirG then binds at specific 12 bp DNA sequences of the vir gene promoters (vir boxes), thereby activating transcription. cells in the root elongation zone were found to be the most highly transformable (Yi et al, 2002). Cells of this non-meristematic zone are not undergoing a normal cell cycle, but endoreduplication. Cell division activity 对转化是很重要的。 chvA, chvB, and pscA (exoC), which are involved in the synthesis and/or localisation of periplasmic b-1,2 glucan ，利于农杆菌和植物附着； soluble pectic plant cell wall fractions decreases both the specific binding of Agrobacterium to plant cells and tumour-induction frequencies 。 The VirB complex belongs to the class of type IV secretion systems (T4SS), which are found across a broad range of Gram-negative bacteria and are involved in the conjugative transfer of plasmids between bacteria as well as the translocation of Vir factors from pathogens to host cells during infection 。 VirB1–11 and VirD4 and is required for virulence. 分化培养基 MS 盐 + 烟酸 5 mg/L +VB6 10 mg/L+VB1 10 mg/L+ 肌醇 100 mg/L+CH 2 g/L+NAA 0.2 mg/L+kinetin 2 mg/L+sorbitol 30 g/L+sucrose 30g/L+gelrite 3 g/L
引用出处： http://www.51xuewen.com/Blog/B_aShow.aspx?blog=wangleleID=9497 Marine Agar (DSMZ Medium 123) Composition per liter: Agar ..................................................................15.0g Tryptone............................................................10.0g Peptone ...............................................................5.0g Yeast extract ........................................................1.0g Synthetic seawater ............................................. 1.0L pH 7.8 0.2 at 25C Synthetic Seawater Composition per liter: NaCl ..................................................................24.0g MgCl26H2O .....................................................11.0g Na2SO4................................................................4.0g CaCl26H2O.........................................................2.0g KCl......................................................................0.7g KBr......................................................................0.1g SrCl26H2O .......................................................0.04g H3BO3 ...............................................................0.03g NaSiO39H2O...................................................5.0mg NaF...................................................................3.0mg NH4NO3 ...........................................................2.0mg Fe3PO44H2O ...................................................1.0mg Preparation of Synthetic Seawater: Add components to distilled water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add agar, tryptone, peptone, and yeast extract to synthetic seawater and bring volume to 1.0L. Mix thoroughly. Adjust pH to 7.8. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Halobacillus halophilus, Halomonas spp., Vibrio harveyi,Cobetia marina, and Ruegeria atlantica. Marine Agar 2216(DSMZ Medium 604) Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g Peptone................................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract ........................................................1.0g KCl....................................................................0.55g NaHCO3 ............................................................0.16g Ferric citrate........................................................0.1g KBr....................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4 ..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg pH 7.6 0.2 at 25C Source: This medium is available as a premixed powder from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Hyphomonas spp., Oceanospirillum spp., Hyphomicrobium indicum, Psychroflexus gondwanensis=Flavobacterium gondwanense, Salegentibacter salegens=Flavobacterium salegens, Psychromonas antarctica, Sulfitobacter mediterraneus, Thalassomonas viridans,Vibrio spp., Marinospirillum minutulum=Oceanospirillum minutulum, Terasakiella pusilla=Oceanospirillum pusillum, Pseudoalteromonas atlantica=Alteromonas atlantica, Pseudomonas atlantica,Roseobacter spp., Erythrobacter longus, Pseudospirillum japonicum=Oceanospirillum japonicum,Marinobacter hydrocarbonoclasticus (Pseudomonas nautica), Psychrobacter spp., and Moritella japonica. For the isolation, cultivation, and maintenance of a wide variety of heterotrophic marine bacteria. Marine Agar with Biphenyl Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g Peptone ...............................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract........................................................1.0g KCl....................................................................0.55g NaHCO3............................................................0.16g Ferric citrate........................................................0.1g KBr ...................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4..........................................................8.0mg Na2SiO3............................................................4.0mg NaF ..................................................................2.4mg NH4NO3 ...........................................................1.6mg Biphenyl...........................................................1.0mg pH 7.6 0.2 at 25C Preparation of Medium: Add components, except biphenyl, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. After agar solidifies, aseptically add a few crystals of biphenyl to each plate. Use: For the cultivation and maintenance of biphenyl-utilizing marine bacteria, such as Cycloclasticus pugetii. Marine Agar with -Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B ..................................................... 60.0mL Solution C ..................................................... 10.0mL pH 7.2 0.2 at 25C Solution A: Composition per liter: NaCl ..................................................................25.0g Agar ..................................................................15.0g MgSO47H2O......................................................5.0g Casamino acids ...................................................2.5g Carrageenan.....................................................2.5g NaNO3.................................................................2.0g CaCl22H2O.........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Solution B: Composition per 100.0mL: Na2HPO42H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Solution C: Composition per 100.0mL: FeSO47H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of ATCC strain 43554. Marine Agar with - and -Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B..................................................... 60.0mL Solution C..................................................... 10.0mL pH 7.2 0.2 at 25C Solution A: Composition per liter: NaCl ..................................................................25.0g Agar ..................................................................15.0g MgSO47H2O......................................................5.0g Casamino acids ...................................................2.5g NaNO3 ................................................................2.0g -Carrageenan...................................................1.25g -Carrageenan...................................................1.25g CaCl22H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Solution B: Composition per 100.0mL: Na2HPO42H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Solution C: Composition per 100.0mL: FeSO47H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation and maintenance of Pseudomonas carrageenovora. Marine Agar with Naphthalene Composition per liter: NaCl ................................................................19.45g Agar ..................................................................15.0g MgCl2..................................................................8.8g Peptone ...............................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract ........................................................1.0g KCl....................................................................0.55g NaHCO3 ............................................................0.16g Ferric citrate........................................................0.1g KBr....................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4 ..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg Naphthalene ........................................................1mg pH 7.6 0.2 at 25C Preparation of Medium: Add components, except naphthalene, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. After agar solidifies, aseptically add a few crystals of naphthalene to each plate. Use: For the cultivation and maintenance of naphthalene-utilizing marine bacteria Marine Agar with Sulfur (ATCC Medium 1922) Composition per liter: NaCl ................................................................19.45g Sulfur ................................................................10.0g MgCl2..................................................................8.8g Peptone................................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract ........................................................1.0g KCl....................................................................0.55g NaHCO3 ............................................................0.16g Ferric citrate........................................................0.1g KBr....................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4 ..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg pH 7.6 0.2 at 25C Preparation of Sulfur: Autoclave sulfur for 15 min at 0 psi pressure100C on three successive days. Preparation of Medium: Prepare anaerobically under a gas phase of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Cool to 5055C. Aseptically add 10.0g of sterile sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes. Use: For the cultivation and maintenance of Thermococcus litoralis. Marine Ameba Medium Composition per liter: Agar ..................................................................10.0g Malt extract .........................................................0.1g Yeast extract........................................................0.1g Artificial seawater.............................................. 1.0L Artificial Seawater: Composition per liter: NaCl ..................................................................27.5g MgSO4 7H2O...................................................6.78g MgCl26H2O .....................................................5.38g KCl....................................................................0.72g NaHCO3............................................................. 0.2g CaCL22H2O.......................................................1.4g Preparation of Artificial Seawater: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add components to artificial seawater and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Cochliopodium clarum, Heteramoeba clara, Ling***oeba leei, Paramoeba pemaquidensis, and Vannella species. Marine Broth 2216 (LMG Medium 164) Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g Peptone ...............................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract........................................................1.0g KCl....................................................................0.55g NaHCO3............................................................0.16g Ferric citrate........................................................0.1g KBr ...................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg pH 7.6 0.2 at 25C Source: This medium is available as a premixed powder from BD Diagnostic Systems. Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Use: For the cultivation of Vibrio liquefaciens and for the isolation, cultivation, and maintenance of a wide variety of heterotrophic marine bacteria. Marine Broth with Biphenyl Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g Peptone................................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract ........................................................1.0g KCl....................................................................0.55g NaHCO3 ............................................................0.16g Ferric citrate........................................................0.1g KBr....................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4 ..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg Biphenyl ...........................................................1.0mg pH 7.6 0.2 at 25C Preparation of Medium: Add components, except biphenyl, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Aseptically add a few crytals of biphenyl to each tube or flask. Use: For the cultivation of biphenyl-utilizing marine bacteria. Marine Broth with -Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B ..................................................... 60.0mL Solution C ..................................................... 10.0mL pH 7.2 0.2 at 25C Solution A: Composition per liter: NaCl ..................................................................25.0g MgSO47H2O......................................................5.0g Casamino acids ...................................................2.5g -Carrageenan .....................................................2.5g NaNO3 ................................................................2.0g CaCl22H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Solution B: Composition per 100.0mL: Na2HPO42H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Solution C: Composition per 100.0mL: FeSO47H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of ATCC strain 43554. Marine Broth with - and -Carrageenan Composition per 1070.0mL: Solution A.......................................................... 1.0L Solution B..................................................... 60.0mL Solution C..................................................... 10.0mL pH 7.2 0.2 at 25C Solution A: Composition per liter: NaCl ..................................................................25.0g MgSO47H2O......................................................5.0g Casamino acids ...................................................2.5g NaNO3 ................................................................2.0g -Carrageenan...................................................1.25g -Carrageenan...................................................1.25g CaCl22H2O ........................................................0.2g KCl......................................................................0.1g Preparation of Solution A: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Solution B: Composition per 100.0mL: Na2HPO42H2O.................................................3.56g Preparation of Solution B: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Solution C: Composition per 100.0mL: FeSO47H2O........................................................0.3g Preparation of Solution C: Add component to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Preparation of Medium: Aseptically add 60.0mL of sterile solution B and 10.0mL of sterile solution C to 1.0L of sterile solution A. Mix thoroughly. Distribute into sterile tubes or flasks. Use: For the cultivation and maintenance of Pseudomonas carrageenovora. Marine Broth with Naphthalene Composition per liter: NaCl ................................................................19.45g MgCl2..................................................................8.8g Peptone................................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract ........................................................1.0g KCl....................................................................0.55g NaHCO3 ............................................................0.16g Ferric citrate........................................................0.1g KBr....................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4 ..........................................................8.0mg Na2SiO3............................................................4.0mg NaF...................................................................2.4mg NH4NO3 ...........................................................1.6mg Naphthalene ........................................................1mg pH 7.6 0.2 at 25C Preparation of Medium: Add components, except biphenyl, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Aseptically add a few crytals of naphthalene to each tube or flask. Use: For the cultivation of naphthalene-utilizing marine bacteria. Marine Broth with Sulfur Composition per liter: NaCl ................................................................19.45g Sulfur ................................................................10.0g MgCl2..................................................................8.8g Peptone ...............................................................5.0g Na2SO3..............................................................3.24g CaCl2...................................................................1.8g Yeast extract........................................................1.0g KCl....................................................................0.55g NaHCO3............................................................0.16g Ferric citrate........................................................0.1g KBr ...................................................................0.08g SrCl2..................................................................0.03g H3BO3 ...............................................................0.02g Na2HPO4..........................................................8.0mg Na2SiO3............................................................4.0mg NaF ..................................................................2.4mg NH4NO3 ...........................................................1.6mg pH 7.6 0.2 at 25C Preparation of Sulfur: Autoclave for 15 min at 0 psi pressure100C on three successive days. Preparation of Medium: Prepare anaerobically under a gas phase of 80% N2 + 10% CO2 + 10% H2. Add components, except sulfur, to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Autoclave for 15 min at 15 psi pressure121C. Cool to 50C. Aseptically add 10.0g of sulfur. Mix thoroughly. Aseptically and anaerobically, under a gas phase of 80% N2 + 10% CO2 + 10% H2, distribute into sterile tubes. Use: For the cultivation of Thermococcus litoralis. Marine Caulobacter Medium Composition per liter: Proteose peptone...............................................10.0g Yeast extract........................................................3.0g Artificial seawater.............................................. 1.0L pH 7.27.4 at 25C Artificial Seawater: Composition per liter: Commercially available marine aquarium salts mixture ...........................variable Preparation of Artificial Seawater: Add commercially available marine aquarium salts mixture to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Combine components. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Use: For the cultivation of Caulobacter halobacteroides and Caulobacter maris. Marine Chlorobiaceae Medium 2 Composition per 1051.0mL: Solution 1.................................................... 950.0mL Na2S9H2O solution...................................... 60.0mL NaHCO3 solution .......................................... 40.0mL Vitamin B12 solution ....................................... 1.0mL pH 6.8 0.2 at 25C Solution 1: Composition per 950.0mL: NaCl ..................................................................20.0g MgSO47H2O......................................................3.0g KH2PO4...............................................................1.0g NH4Cl .................................................................0.5g CaCl22H2O.......................................................0.05g Trace elements solution SL-8 ......................... 1.0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Cool to 4550C. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA .................................................5.2g FeCl24H2O.........................................................1.5g CoCl26H2O ......................................................0.19g MnCl24H2O .......................................................0.1g ZnCl2.................................................................0.07g H3BO3 ...............................................................0.06g NaMoO42H2O..................................................0.04g CuCl22H2O ......................................................0.02g NiCl26H20........................................................0.02g Preparation of Trace Elements Solution SL8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Na2S9H2O Solution: Composition per 100.0mL: Na2S9H2O..........................................................5.0g Preparation of Na2S9H2O Solution: Add Na2S9H2O to distilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure121C. Cool to 4550C. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ..............................................................5.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ......................................................2.0mg Preparation of Vitamin B12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 6.8 with sterile H2SO4 or Na2CO3 . Aseptically distribute into sterile 50.0mL or 100.0mL bottles with metal screw-caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble. Use: For the isolation and cultivation of marine members of the Chlorobiaceae. Marine Chromatiaceae Medium 2 Composition per 1051.0mL: Solution 1.................................................... 950.0mL Na2S9H2O solution...................................... 60.0mL NaHCO3 solution.......................................... 40.0mL Vitamin B12 solution ....................................... 1.0mL pH 7.3 0.2 at 25C Solution 1: Composition per 950.0mL: NaCl ..................................................................20.0g MgSO47H2O......................................................3.0g KH2PO4...............................................................1.0g NH4Cl .................................................................0.5g CaCl22H2O ......................................................0.05g Trace elements solution SL-8 ......................... 1.0mL Preparation of Solution 1: Add components to distilled/deionized water and bring volume to 950.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Cool to 4550C. Trace Elements Solution SL-8: Composition per liter: Disodium EDTA.................................................5.2g FeCl24H2O.........................................................1.5g CoCl26H2O ......................................................0.19g MnCl24H2O .......................................................0.1g ZnCl2.................................................................0.07g H3BO3 ...............................................................0.06g NaMoO42H2O..................................................0.04g CuCl22H2O ......................................................0.02g NiCl26H20........................................................0.02g Preparation of Trace Elements Solution SL8: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Na2S9H2O Solution: Composition per 100.0mL: Na2S9H2O..........................................................5.0g Preparation of Na2S9H2O Solution: Add Na2S9H2O to distilled/deionized water and bring volume to 100.0mL. Autoclave for 15 min at 15 psi pressure121C. Cool to 4550C. NaHCO3 Solution: Composition per 100.0mL: NaHCO3 ..............................................................5.0g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Vitamin B12 Solution: Composition per 100.0mL: Vitamin B12 ......................................................2.0mg Preparation of Vitamin B12 Solution: Add vitamin B12 to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: To 950.0mL of cooled, sterile solution 1, aseptically add 60.0mL of sterile Na2S9H2O solution, 40.0mL of sterile NaHCO3 solution, and 1.0mL of sterile vitamin B12 solution. Mix thoroughly. Adjust pH to 7.3 with sterile H2SO4 or Na2CO3 . Aseptically distribute into sterile 50.0mL or 100.0mL bottles with metal screw-caps and rubber seals. Completely fill bottles with medium except for a pea-sized air bubble. Use: For the isolation and cultivation of marine members of the Chromatiaceae. Marine Cytophaga Agar Composition per liter: Agar ..................................................................15.0g Nutrient broth......................................................8.0g Yeast extract ........................................................5.0g Salt solution ....................................................... 1.0L Salt Solution: Composition per liter: NaCl ................................................................12.86g MgCl2................................................................2.48g KCl....................................................................0.75g CaCl2.................................................................0.56g Fe(SO4)2(NH4)2...............................................0.048g Preparation of Salt Solution: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Preparation of Medium: Add solid components to 1.0L of salt solution. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure 121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation and maintenance of Cytophaga species. Marine Cytophaga Medium Composition per liter: NaCl ..................................................................24.7g Agar ..................................................................15.0g MgSO47H2O......................................................6.3g MgCl26H2O .......................................................4.6g Tryptic digest of casein.......................................1.0g Yeast extract........................................................1.0g KCl......................................................................0.7g NaHCO3 solution .......................................... 10.0mL CaCl22H2O solution..................................... 10.0mL pH 7.2 0.2 at 25C NaHCO3 Solution: Composition per 10.0mL: NaHCO3..............................................................0.2g Preparation of NaHCO3 Solution: Add NaHCO3 to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. CaCl22H2O Solution: Composition per 10.0mL: CaCl22H2O ........................................................1.2g Preparation of CaCl22H2O Solution: Add CaCl22H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Autoclave for 15 min at 15 psi pressure121C. Preparation of Medium: Add components, except NaHCO3 solution and CaCl22H2O solution, to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 min at 15 psi pressure 121C. Cool to 5055C. Aseptically add 10.0mL of sterile NaHCO3 solution and 10.0mL of sterile CaCl22H2O solution. Mix thoroughly. Pour into sterile Petri dishes or distribute into sterile tubes. Use: For the cultivation of Cytophaga species, Flexibacter species, Microscilla species, and Saprospira grandis. Marine Cytophaga Medium A Composition per liter: Agar ..................................................................15.0g Pancreatic digest of casein..................................2.0g Beef extract .........................................................0.5g Yeast extract........................................................0.5g Sodium acetate....................................................0.2g Seawater...................................................... 700.0mL pH 7.2 0.2 at 25C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Flexibacter maritimus. Marine Cytophaga Medium B Composition per liter: Agar ..................................................................15.0g Pancreatic digest of casein..................................2.0g Beef extract .........................................................0.5g Yeast extract ........................................................0.5g Sodium acetate....................................................0.2g Seawater...................................................... 500.0mL pH 7.2 0.2 at 25C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Vibrio ordalii. Marine Cytophaga Medium C Composition per liter: Agar ..................................................................15.0g Pancreatic digest of casein..................................2.0g Beef extract .........................................................0.5g Yeast extract ........................................................0.5g Sodium acetate....................................................0.2g pH 7.2 0.2 at 25C Preparation of Medium: Add components to seawater and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure 121C. Pour into sterile Petri dishes or leave in tubes. Use: For the cultivation of Cytophaga agarovorans, Cytophaga fermentans, and Cytophaga salmonicolor. Marine Desulfovibrio Medium Composition per liter: Solution A................................................... 980.0mL Solution B ..................................................... 10.0mL Solution C ..................................................... 10.0mL pH 7.8 0.2 at 25C Solution A: Composition per 980.0mL: NaCl ..................................................................25.0g DL-Sodium lactate...............................................2.0g MgSO47H2O......................................................2.0g Na2SO4................................................................1.0g NH4Cl .................................................................1.0g Yeast extract........................................................1.0g K2HPO4...............................................................0.5g CaCl22H2O ........................................................0.1g Resazurin .........................................................1.0mg Preparation of Solution A: Add components to distilled/deionized water and bring volume to 980.0mL. Mix thoroughly. Gently heat and bring to boiling. Continue boiling for 3-4 min. Allow to cool to room temperature while gassing under 100% N2. Solution B: Composition per 10.0mL: FeSO47H2O........................................................0.5g Preparation of Solution B: Add FeSO47H2O to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Solution C: Composition per 10.0mL: Ascorbic acid ......................................................0.1g Sodium thioglycolate ..........................................0.1g Preparation of Solution C: Add components to distilled/deionized water and bring volume to 10.0mL. Mix thoroughly. Preparation of Medium: To 980.0mL of cooled solution A, anaerobically add 10.0mL of solution B and 10.0mL of solution C. Mix thoroughly. Adjust pH to 7.8 with NaOH. Distribute into tubes or flasks. During distribution, swirl the medium to keep the precipitate in suspension. Autoclave for 15 min at 15 psi pressure121C. Use: For the cultivation and maintenance of Desulfovibrio desulfuricans, Desulfovibrio salexigens, and Desulfovibrio vulgaris. Marine Flagellate Medium Composition per 15.0mL: Rice grains ..........................................................2.0g Seawater........................................................ 15.0mL Preparation of Medium: Autoclave rice grains for 15 min at 15 psi pressure121C. Add 2.0g of sterile rice grains to 15.0mL of filter-sterilized seawater. Aseptically distribute into T-25 tissue culture flasks. Use: For the cultivation of Acanthoecopsis unguiculata, Amastigomonas species, Bicosoeca vacillans, Bodo designis, Bodo variabilis, Caecitellus parvulus, Choanoeca perplexa, Codosiga gracilis, Diaphanoeca grandis, Entosiphon species, Goniomonas species, Procryptobia species, Pseudobodo tremulans, Rhynchomonas nasuta, Salpingoeca urceolata, Stephanoeca diplocostata, and Stephanopogon apogon. Marine Flagellate Medium with B-Vitamins Composition per liter: Seawater...................................................... 990.0mL Vitamin solution............................................ 10.0mL Vitamin Solution: Composition per 100.0mL: ThiamineHCl ...................................................0.15g Calcium D-(+)-pantothenate..............................0.05g Nicotinamide.....................................................0.05g PyridoxalHCl ...................................................0.05g Riboflavin .........................................................0.05g Folic acid.........................................................0.025g PyridoxamineHCl ..........................................0.025g Biotin .............................................................12.5mg Preparation of Vitamin Solution: Add components to distilled/deionized water and bring volume to 100.0mL. Mix thoroughly. Filter sterilize. Preparation of Medium: Allow natural seawater to age for 2 months. Filter sterilize. Aseptically add 100.0mL of sterile vitamin solution. Mix thoroughly. Aseptically distribute into sterile tubes or flasks. Use: For the cultivation of Oikomonas species. Marine Glucose Trypticase Yeast Extract Agar (MGTY Agar) Composition per liter: Agar ....................................................................8.0g Glucose ...............................................................2.0g Pancreatic digest of casein..................................1.0g Yeast extract ........................................................1.0g L-CysteineHClH2O............................................0.5g Seawater...................................................... 750.0mL Tris-HCl buffer (5.0 mM, pH 7.5) ................ 50.0mL Resazurin (0.1% solution)............................... 1.0mL pH 7.5 0.2 at 25C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks under 97% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave for 15 min at 15 psi pressure121C with fast exhaust. Use: For the cultivation and maintenance of Spirochaeta isovalerica. Marine Glucose Trypticase Yeast Extract Broth (MGTY Broth) Composition per liter: Glucose ...............................................................2.0g Pancreatic digest of casein..................................1.0g Yeast extract........................................................1.0g L-CysteineHClH2O............................................0.5g Seawater...................................................... 750.0mL Tris-HCl buffer (5.0 mM, pH 7.5) ................ 50.0mL Resazurin (0.1% solution) .............................. 1.0mL pH 7.5 0.2 at 25C Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat while stirring and bring to boiling. Distribute into tubes or flasks under 97% N2 + 3% H2. Cap with rubber stoppers and place tubes in a press. Autoclave for 15 min at 15 psi pressure121C with fast exhaust. Use: For the cultivation and maintenance of Spirochaeta isovalerica. Marine Methanogenium Alcohol Medium Composition per 1003.0mL: NaCl ..................................................................21.0g MgCl26H2O .......................................................3.0g NaCl ....................................................................1.0g KCl......................................................................0.5g MgCl26H2O .......................................................0.5g NH4Cl .................................................................0.4g Sodium acetate3H2O..........................................0.4g KH2PO4...............................................................0.2g CaCl22H2O ........................................................0.1g NaHCO3 solution.......................................... 60.0mL 2-Propanol....................................................... 5.0mL Na2S9H2O solution........................................ 3.0mL Cyanocobalamin solution ............................... 1.0mL Selenite-molybdate-tungstate solution............ 1.0mL Thiamine solution ........................................... 1.0mL Trace elements solution .................................. 1.0mL
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