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.     

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。     

紫薯愈伤组织诱导和培养条件优化及绿原酸类化合物的积累

以紫薯叶片为外植体,通过筛选培养基和培养条件建立了紫薯愈伤组织培养体系。研究6-苄基氨基嘌呤（6-BA）及萘乙酸（NAA）不同浓度组合对紫薯愈伤组织诱导的影响,之后分别考察基础培养基MS、1/2MS、B5、WPM、White及NAA、6-BA、2,4-二氯苯氧乙酸（2,4-D）三种激素对紫薯愈伤组织继代生长的影响。确定最适基础培养基后,对激素进行单因素实验后再做正交实验。结果表明1/2MS基本培养基中添加1.5 mg/L 6-BA和0.4 mg/L NAA适合紫薯愈伤组织诱导,诱导率为81.82%。MS为优选基础培养基,通过正交实验优化的激素配比组合为1.0 mg/L NAA,0.4 mg/L 6-BA,1.0 mg/L 2,4-D。最佳继代培养基为MS+1.0 mg/L NAA+0.4 mg/L 6-BA+1.0 mg/L 2,4-D+6 g/L琼脂+30 g/L蔗糖,此优化激素条件下愈伤组织的生物量增长倍数为23.46倍,3-咖啡酰奎宁酸、3,5-二咖啡酰宁酸、4,5-二咖啡酰宁酸三种绿原酸类化合物含量分别为0.30%、1.91%、1.10%,三者总含量3.31%,因此,通过紫薯愈伤组织培养生产绿原酸类化合物是可行的。      

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.     

培养条件对杜仲愈伤组织形成及次生代谢过程的影响

对杜仲愈伤组织诱导研究表明,诱导出愈率在不同外植体类型、外植体年龄与部位、外植体接种方式间存在显著差异,5 mm×5 mm大小、叶龄为15 d左右的糙皮杜仲叶片的中下部外植体在采用叶片背面贴置于培养基的培养方式出愈率最高;培养基类型、激素水平和光照条件对出愈效果均有显著的影响,在附加2,4-D(0.5 mg* L -1 )和BA(0.6 mg*L -1)的Harada培养基和附加2,4-D(0.5 mg*L -1 ) 、BA(0.6 mg*L -1 )和BA(0.6 mg*L -1)或附加NAA(0.5mg*L -1 )的Bourgin & Nistc h 培养基上,以24 h的黑暗启动培养加以光暗交替培养(光照14h+黑暗10 h),并在接种后12 d 内更换培养基,均能诱导出杜仲愈伤组织,以叶片作外植体时,BA的浓度建议不高于1.0 mg*L -1,KT的存在不利于愈伤组织的诱导;在附加了KT(0.7 mg*L -1 )、NAA(0.8 mg *L -1 )和10 mg*L -1葡聚糖的MS培养基上,通过向培养基中添加递进浓度的丙酮酸研究表明,杜仲愈伤组织中的次生代谢产物桃叶珊瑚甙的产量;通过继代培养的4因素3水平正交试验发现,培养温度、碳源、培养基类型及激素水平对杜仲叶片愈伤组织生长速率及愈伤组织中的氯原酸含量均有显著的影响,适宜愈伤组织生长的培养条件有别于适宜于氯原酸积累的培养条件;添加了5 mg*L -1的苯丙氨酸的White培养基比不含苯丙氨酸的 MS培养基和Bourgin & Nistch培养基,更有利于杜仲愈伤组织中氯原酸的积累,表明硝态氮的存在有利于氯原酸的积累,而铵态氮的存在则较有利于愈伤组织的生长.     

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%.     

Prediction of fracture healing under axial loading,shear loading and bending is possible using distortional and dilatational strains as determining mechanical stimuli

Numerical models of secondary fracture healing are based on mechanoregulatory algorithms that use distortional strain alone or in combination with either dilatational strain or fluid velocity as determining stimuli for tissue differentiation and development. Comparison of these algorithms has previously suggested that healing processes under torsional rotational loading can only be properly simulated by considering fluid velocity and deviatoric strain as the regulatory stimuli. We hypothesize that sufficient calibration on uncertain input parameters will enhance our existing model, which uses distortional and dilatational strains as determining stimuli, to properly simulate fracture healing under various loading conditions including also torsional rotation. Therefore, we minimized the difference between numerically simulated and experimentally measured courses of interfragmentary movements of two axial compressive cases and two shear load cases (torsional and translational) by varying several input parameter values within their predefined bounds. The calibrated model was then qualitatively evaluated on the ability to predict physiological changes of spatial and temporal tissue distributions, based on respective in vivo data. Finally, we corroborated the model on five additional axial compressive and one asymmetrical bending load case. We conclude that our model, using distortional and dilatational strains as determining stimuli, is able to simulate fracture-healing processes not only under axial compression and torsional rotation but also under translational shear and asymmetrical bending loading conditions.     

苦参愈伤组织诱导及继代培养条件研究

目的：研究苦参愈伤组织诱导与继代培养,为药用成分苦参碱及氧化苦参碱的来源提供新途径.方法：考察了不同外植体和激素组合对苦参愈伤组织诱导的影响和苦参愈伤组织增殖继代的最佳培养条件.结果：分别以幼芽、根、茎段、叶片为外植体,在MS＋1.0mg/L 2,4-D＋1.0mg/L 6-BA＋1.0mg/L NAA激素组合下苦参愈伤组织的诱导率分别为94.12%、74.19%、35.71%、23.08%;在MS＋0.5mg/L 2,4-D＋0.1mg/L 6-BA＋0.5mg/L NAA激素组合下,苦参愈伤组织增殖率最高,为25.3%.结论：幼芽为苦参愈伤组织诱导最佳外植体.不同激素组合对愈伤组织诱导率的影响不大,但对其增殖的影响较大,其中NAA具有显著影响.