The Effects Of Humic Substances On Pinus Callus Are Reversed By 2,4-Dichlorophenoxyacetic Acid

The reversal of humic matter-induced inhibition of callus growth and metabolism by 2,4-dichlorophenoxyacetic acid (2,4-D) was studied in Pinus laricio. Two forest humic fractions (relative molecular mass (M_r) > 3500), derived from soil under Fagus sylvatica (Fs) and Abies alba (Aa) plantation, were used. Pinus laricio callus was grown for a subculture period (4 weeks) on Basal Murashige and Skoog (MS) medium plus forest humic matters (Fs or Aa), at a concentration of 1 mg C/l, and then was transferred, for an additional four weeks, to a MS medium culture without humic matter, but with different hormones: indole-3-acetic acid (IAA, 2 mg/1) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 mg/1) and/or 6-benzylaminopurine (BAP, 0.25 mg/1). Growth of calluse, glucose, fructose, and sucrose contents, and activities of soluble and bound invertases, glucokinase, phosphoglucose isomerase, aldolase, and pyruvate kinase were monitored. The results show a negative effect of humic fractions on callus growth, due to decreased utilization of glucose and fructose, and decreased activities of glycolytic enzymes. The effects are reversible. Substitution of humic fractions with 2,4-D+BAP or 2,4-D is followed by an increase of glycolytic enzyme activities and, consequently, by the utilization of glucose and fructose that induces a restart of growth. In contrast, the inhibitory effects of humic fractions persist when they are substituted with BAP alone, indicating that only the auxin 2,4-D is capable of reversing the negative effects. A possible competitive action on the auxin-binding site between 2,4-D and the chemical structures in the forest humic fractions is suggested.     

培养基成分和培养条件对黄芪愈伤组织中黄芪皂苷积累的影响

对提高黄芪愈伤组织中黄芪皂苷含量的人工调节技术进行了研究。生长在6,7-V基本培养基上的黄芪愈伤组织中黄芪皂苷含量比MS培养基上的高,而且光照比黑暗更有利于黄芪皂苷合成。调整6,7-V培养基中氨态氮和硝态碳的比例为1∶1(氮源总浓度为60 mmol.L-1)、KH2PO4浓度为1.5 mmol.L-1、CaCl2浓度为8.97 mmol.L-1时比较适合黄芪皂苷的积累。以蔗糖为碳源比葡萄糖和麦芽糖更有利于皂苷的积累。通过调节培养基成分和培养条件是改变黄芪细胞中黄芪皂苷积累趋势的一个重要手段。     

东北红豆杉组织培养的研究

以东北红豆杉（Ｔａｘｕｓｃｕｓｐｉｄａｔａ）的不同外植体进行愈伤组织诱导、继代和再分化培养，结果发现在ＭＳ培养基上，幼叶的出愈率最高，达７８３％，在Ｂ５培养基上，幼基的出愈率最高，达７６０％。诱导愈伤组织最适２，４－Ｄ浓度为２－３ｍｇ·Ｌ－１。经抗氧化剂处理，愈伤组织在改良ＭＳ培养基上经多次继代培养，褐化现象减轻或消失，生长速率最高可达００９０ｇ·ｋｇ－１·ｄ－１（鲜重）。在分化培养基上，由愈伤组织诱导出绿芽点。     

青蒿组织培养体系建立的研究

为建立青蒿品种改良及其快速繁殖技术体系,以青蒿试管苗子叶和胚轴、嫩叶、田间栽培青蒿的花序为外植体．研究其离体培养及试管苗再生途径。结果表明,以青蒿花序为外植体,以MS＋6-BA 0．5mg／l＋NAA 1．0mg／l为愈伤组织诱导培养基。愈伤组织在MS＋6-BA 4．0mg／l＋NAA 0．05mg／l培养基上分化,以MS＋6-BA 2．0mg/l＋6-KT 2．0mg/l为扩繁培养基．分化苗转到MS＋NAA 1．0mg／l培养基上生根。可以用较低成本,较短时间,比较简便地得到大量青蒿再毕试管苗．     

藏红花胚性愈伤组织的发生及其调控

为提高藏红花胚性愈伤组织的繁殖系数和出芽率,促进其生长和分化,以建立藏红花离体快繁体系,解决藏红花资源短缺问题,研究了藏红花胚性愈伤组织的发生及其调控. 结果表明,获得的藏红花球茎1细胞系具有良好的胚性愈伤组织发生能力. 胚性愈伤组织生长的优化条件为：在添加3.0 mg/L 6-BA, 0.25 mg/L NAA和400 mg/L CH的B5固体培养基上,22℃下全时暗培养25 d,繁殖系数达到9 g/g. 胚性愈伤组织出芽的优化条件为：在添加3.0 mg/L 6-BA, 0.25 mg/L NAA和400 mg/L CH的1/2 B5固体培养基上,在22℃及光照强度31.74 mmol/(m2×s)条件下,每天光照10 h,暗培养14 h,培养45 d出芽率达到44.7%,高于国外报道的20%.     

不同培养条件对银杏愈伤组织生长及黄酮含量的影响

以胚为外植体,通过固体培养,研究了培养基,激素和光照对银杏愈伤组织诱导的影响,以及继代培养中激1素和活性炭对愈伤组织生长和产黄酮的影响,结果表明:愈伤组织的诱导的最佳条件为DCR培养基+1.0mg/L2,4-D+0.5mg/LBA+完全暗条件培养。继代培养过程中既能促进愈伤组织生长又使黄酮含量最高的处理为0.3mg/LBA+2.0mg/L2,4-D,活性炭抑制愈伤组织生长但能促进黄酮的积累。     

响应面分析法优化西兰花离体细胞系萝卜硫素提取工艺

以西兰花子叶愈伤组织为实验材料,利用响应面法对西兰花愈伤组织萝卜硫素的提取工艺进行优化。在单因素实验基础上选取实验因素与水平,根据中心组合(Box-Benhnken)实验设计原理采用四因素三水平的响应面分析法,依据回归分析确定各工艺条件的影响因素,以愈伤组织中萝卜硫素提取率为响应值作响应面和等高线。在分析各个因素的显著性和交互作用后,得出浸提愈伤组织中萝卜硫素的最佳工艺条件为:温度25.7℃、固液比1:30(g/mL)、浸提时间2.8h、提取次数3次。在此条件下萝卜硫素产量实测值为0.0067%(理论值为0.0078%),说明采用响应面法优化得到的提取条件可靠。     

EMS诱变甘蔗愈伤组织的初步研究

为了探索EMS对甘蔗愈伤组织进行化学诱变的合适浓度和时间组合,以ROC22和粤糖93-159甘蔗愈伤组织为材料,研究了不同浓度(8、24、40μmol/L)、不同时间(2、4、6 h)诱变处理对愈伤组织的每克鲜重相对增重量和相对分化率的影响。并通过分化试验选择适合诱变后愈伤组织分化的培养基。结果表明,适合诱变处理后愈伤组织分化的培养基为:MS基本培养基+2 mg/L KT+2 mg/L NAA+3.0%蔗糖(W/V)+0.8%琼脂(W/V)。适合EMS诱变甘蔗的处理为:24μmol/L诱变处理2 h或8μmol/L诱变处理6 h。     

拳参愈伤组织的诱导及增殖效应的研究

目的研究适宜拳参愈伤细胞的诱导、组织分化及快速生长的培养基成分。方法以拳参的幼嫩叶柄为外植体,接种于附加2,4-D、N A A、6-B A、K T及其组合的M S培养基上,观察拳参愈伤组织的诱导、分化及愈伤细胞的生长状态。结果MS 0.4 mg/L 6-BA 2.0 mg/L NAA对拳参愈伤组织的诱导效果最好。MS 0.2 mg/L 6-BA 2.0 mg/L 2,4-D增殖培养的愈伤组织较适合于器官的分化,MS 0.2 mg/L 6-BA 4.0 mg/L NAA增殖培养的愈伤组织较适合于做细胞悬浮培养的材料。结论建立拳参愈伤组织诱导,愈伤组织分化和愈伤细胞快速增殖体系,为进一步研究药用植物拳参细胞的分子机制及次生代谢等提供资料。     

Study on Flax Genetic Transformation Mediated by Agrobacterium tumefaciens

Abstract

Flax is an important natural material for the linen spinning industry and as an oil crop in China. Flax products worth US $100 million are exported every year. Recently, with the development of a market economy and China's entry into the WTO, farmers and flax factories have shown a need for new varieties that have a short vegetation period, are resistant to herbicides and lodging, and have a high fiber content. In order to resolve these problems quickly, we have studied flax genetic transformation since 1998.

The paper is about flax genetic transformation by Agrobacterium tumefaciens strain EHA105. The explants were hypocotyl segments from 5-7 day old seedlings. The segments cannot form a callus when the concentration of kanamycin is 30 mg/L and 50 mg/L. Therefore, 50 mg/L of kanamycin was selected as an efficient concentration to select the transgenic plants. The segments of hypocotyl were treated with EHA105 and then were inoculated in different media (Ms, B5, N6). The experiment showed that Ms is a preference medium for flax genetic transformation. Agrobacteriumtieated segments of hypocotyl were cultured on the surface of modified Ms medium (Ms agar medium supplemented with 3.5 mg/L IAA, 2 mg/L KT, 150 mg/L LH). Callus was formed by 71.4%-81.6% of the hypocotyls. Callus inoculated in a regenerative medium of Ms with little BA and NAA could regenerate at the rate of 31.8%-40.2%, and 87.4% of regenerative plants could root in the medium of 1/2 Ms supplemented with 0.001 mg/L NAA. This transformation system has been tested by the GUS-INT gene. After co-cultivation of the segments of hypocotyl and GUS-INT, the segments of hypocotyl were moved to callus medium. After 3 days, the transformation rate of new callus was 72.0%; after 4 weeks the transformation rate of callus was 24.0%; the transformation rate of regenerative plants was 7.5%. The experiment allowed for the establishing of a flax genetic transformation system mediated by Agrobacterium tumefaciens.