Comparative study on antioxidative system in normal and vitrified shoots of Populus suaveolens in tissue culture

To explore the physiological and biochemical mechanism of the occurrence of vitrified shoots of Populus suaveolens in tissue culture, the changes in water, chlorphyll, lignin, H₂O₂, phenylalanine ammonialyase (PAL), malonaldehyde (MDA), protective enzymatic systems, and some key enzymes involved in the ascorbate- glutathione cycle were comparatively studied in both normal and vitrified shoots of P. suaveolens. The results show that the lower activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and PAL, and the less contents of chlorphyll, lignin, ascorbate (ASA) and reduced glutathione (GSH) as well as the lower ratios of ASA / DHA and GSH / GSSG are observed in vitrified shoots than in normal ones during the whole culture period. While in comparison with normal shoots, the higher activity of superoxide dismutase (SOD) and the more concentrations of water, H₂O₂, MDA, dehydroascorbate (DHA) and oxidized glutathione (GSSG) are found in vitrified shoots. Statistical analysis indicates that the enhanced activity of SOD and the decreased activities of CAT and POD as well as some enzymes involved in the ascorbate-glutathione cycle might be closely correlated to the accumulation of H₂O₂. The less regeneration of ASA and GSH and the lower capacity of the ascorbate-glutathione cycle observed in vitrified shoots might be due to a significant decrease in APX, MDAR, DHAR and GR activities and a decline in redox status of ASA and GSH. The decreases in chlorphyll content might result in a decline in photosynthesis. The lower activities of POD and PAL could result in the decrease of lignin synthesis and cell wall ligination, which might be the key factor leading to the increase in water content. It is concluded that the deficiency of detoxification capacity caused by the lower capacity of the ascorbate-glutathione pathway and the decreased activity of protective enzymatic system might lead to the large accumulation of H₂O₂ and the enhancement of membrane lipid peroxidation, which might be the main cause leading to the occurrence of vitrifying shoots of P. suaveolens in tissue culture. [Supported by National Natural Science Foundation of China (Grant No. 30271093) and the Foundation of State-designated Base for Biology Researching and Teaching in Beijing Forestry University]     

甜杨优良雄性无性系的苗期选择

论述了大兴安岭地区甜杨优良无性系选育的早期成果．从135个供选群体中选育出了8个优良雄性无性系，分别是03，10，59．145，156，160，193，226。     

Characterization and Role of Glucose-6-phosphate Dehydrogenase of Populus suaveolens in Induction of Freezing Resistance

Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) was purified from the leaves of 8-week-old Populus suaveolens cuttings. The enzyme activity in the absence and presence of reduced dithiothreitol (DTTred) was determined. The results show that the G6PDH activity is not inactivated by pre-incubation with DTTred, indicating that the purified enzyme probably presented in cytosol of P suaveolens. The catalytic characteristics and kinetic parameters of cytosolic G6PDH purified from P.suaveolens cuttings were also studied. The results show that G6PDH is characterized by Km value of 360/amol.L^-1 for G6P and 16μmol.L^-1 for NADP, a pH range of 7.3-8.9, and the maximum activity around pH 8.2. The enzyme activity is inhibited by various metabolites such as NADPH, NADH, GTP, UTP, ATP, AMP, ADP, CoA, acetyl CoA, fructose-6-phosphate (F6P), erythrose-4-phosphate (E4P), ribose-5-phosphate (R5P) and 3-phosphoglycerate (3-PG) (all at 1 mmol‘L i except for NADPH and NADH)to different extents. NADPH is the most effective inhibitor of enzyme activity, with an inhibition of 72.0%. The addition of metal ions such as MgC12, CaC12 and KC1 (all 1.0 mmol.L^-1) to the standard reaction mixture has no remarkable influence on the cytosolic G6PDH activity. However, CdCl2 (1.0 mmol.L-1) causes high inhibitory effect on the enzyme activity. To explore the role of G6PDH on the enhancement of freezing resistance induced by freezing acclimation, the changes in the cytosolic G6PDH activity and freezing resistance (expressed as LTs0) of P. suaveolens cuttings during freezing acclimation at -20℃ were investigated. The results reveal that freezing acclimation decreases LTs0 of cuttings, and increases the activity of cytosolic G6PDH compared with control ones,while 2 d of de-acclimation at 25 ~C result in a decrease in cytosolic G6PDH activity, and caused an increase in LT50. Furthermore,the change in cytosolic G6PDH activity is found to be closely correlated to the degree of freezing resistance of cuttings during freezing acclimation. It is suggested that cytosolic G6PDH may be involved in the induction of freezing resistance of cuttings.     

三倍体毛白杨组培快繁技术研究

笔者从试验材料、外植体的灭菌与接种、培养条件,分化与生根培养,小植株的移栽与定植3个方面介绍了三倍体毛白杨的组培快繁技术,指出采用增殖与生根同步进行的方法进行快繁,年增殖次数可达8次,每次增殖倍数为4.4倍,培养出的组培苗质量好,移栽成活率高,基本达到了工厂化生产的指标要求。在此基础上,进行了三倍毛白杨组培快繁的成本核算,从成本核算过程中可以看出,人工费用和能源消耗费是制约苗木成本的两大因素,其中,人工费用约占全部支出的40%.     

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

High Level Expression of Glucose-6-phosphate Dehydrogenase Gene PsG6PDH from Populus suaveolens in E. coli

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-a-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol·L–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

三倍体欧洲山杨组培技术的研究

通过对三倍体欧洲山杨组培技术的研究试验，采用增殖与生根同步进行的方法进行快繁，其年增殖次数可达8次，平均每次增殖培数为4．4倍，可以达到工厂化生产的指标要求。培养出的组培苗质量好，移栽成活率高，而且可以降低苗木的成本。     

胡杨文化探讨

在我国传统文化里,胡杨文化已经根植在人类发展的历史进程中,随着社会的发展,不断衍生出新的形式和内容,并与人们的生产和生活息息相关。胡杨保护着荒漠生态,是耐人寻味的历史景观。文章通过胡杨的物态文化、行为文化、精神文化来系统阐述人们对胡杨崇拜及胡杨精神对人类的影响。     

毛白杨离体快速繁殖简报

以河北省易县毛白杨为试材，对其离体快速繁殖过程中的各阶段进行了较为系统的观察分析。结果表明： 幼树半木质化新梢中部单芽茎段为最佳外植体材料； 启动培养基以改良培养基 Ｂ５＋ ０２ ｍ ｇ／ Ｌ Ｎ Ａ Ａ为最好； 试管苗的叶片可与腋芽及茎尖同时作为继代培养的材料， 最佳分化培养基为 Ｍ Ｓ＋ ０２５ ｍ ｇ／ Ｌ Ｂ Ａ＋ ０２５ ｍ ｇ／ Ｌ Ｚｔ＋ ０２５ ｍ ｇ／ Ｌ Ｉ Ａ Ａ；最佳生根培养基为１／２ Ｍ Ｓ＋ ００２ ｍ ｇ／ Ｌ Ｎ Ａ Ａ＋ １０ ｍ ｇ／ Ｌ Ｖ Ｂ１＋１５％ 蔗糖， 生根率可达１００％ ， 生根数量为４ 条。     

蓝桉组织培养的研究

用蓝桉（Ｅｕｃａｌｙｐｔｕｓｇｌｏｂｕｌｕｓ）离体芽器官诱导培养，分化形成丛生芽，年繁殖系数３￣（１２）。０．１～０．５ｍｇ／Ｌ的６－ＢＡ或０．５～０．８ｍｇ／Ｌ的ＫＴ诱导外植体（带节茎段）腋芽萌动的效果最佳，诱导率分别达８０．３％和８１．５％。１．５～２０ｍｇ／Ｌ的６～ＢＡ或２０～２．５ｍｇ／Ｌ的ＫＴ分别与０．５～１．０ｍｇ／Ｌ的ＮＡＡ组合，对于促进腋芽分化形成丛生芽及继代培养中芽的增殖具有最佳效果。培养基中的无机盐浓度、蔗糖含量对蓝桉试管苗的生根具有显著影响；ＩＢＡ促进蓝桉试管苗的生根。至目前为止，在１／２ＭＳ无机盐培养基＋ＩＢＡ１．２～１．４ｍｇ／Ｌ＋Ｓ５ｇ／Ｌ中诱导生根，生根率最高可达２６．４％。     

The role of antioxidant system in freezing acclimation-induced freezing resistance of Populus suaveolens cuttings

We investigated the changes in the contents of H2O2, malonaldehyde (MDA) and endogenous antioxidants, the activities of protective enzymes and some critical enzymes involved in the ascorbate-glutathione (ASA-GSH) cycle as well as freezing resistance (expressed as LT50) and correlations mentioned above, in detail using Populus suaveolens cuttings. The purpose was to explore the physiological mechanism of the enhancement of freezing resistance induced by freezing acclimation at –20°C, and to elucidate the physiological mechanisms by which trees adapt to freezing. The results showed that freezing acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), monodehydroascorbate reductase (MDAR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). And it increased the contents of reduced ascorbate (ASA), reduced glutathione (GSH), dehydroascorbate (DHA) and oxidized glutathione (GSSG). However, H2O2 and MDA contents and LT50 of cuttings were decreased. LT50 in cuttings was found to be closely correlated to the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, H2O2, MDA, ASA, GSH, DHA and GSSG during freezing acclimation. This suggested that the enhancement of freezing resistance of cuttings induced by freezing acclimation may relate to the distinct increase for the levels of SOD, POD, CAT, APX, DHAR, MDAR, GR, ASA, GSH, DHA, and GSSG. In addition, the observed levels of APX, DHAR, MDAR, GR, ASA, DHA, GSH and GSSG were higher than those of SOD, POD and CAT during freezing acclimation. It indicated that a higher capacity of the ASA-GSH cycle is required for H2O2 detoxification, and growth and development of cuttings. Based on the obtained results, it can be concluded that the ASA-GSH cycle plays an important role in enhancement of freezing resistance of P. suaveolens cuttings during freezing acclimation.     

四倍体刺槐的组织培养研究

本文对四倍体刺槐组织培养中的各种影响因素进行了试验分析 ,确定了最佳分化培养基和生根培养基的成分 ,并分析讨论了激素、光照、温度等因子在其培养中的作用及健化时的注意事项等 ,为四倍体刺槐的进一步快繁提供了科学依据     

驱蚊香草的组织培养技术

利用驱蚊香草茎段进行组织培养,研究了添加在培养基中不同激素组成、浓度、培养条件等因素对不定芽诱导、继代增殖、防止玻璃化以及生根的影响.结果表明:适于驱蚊香草不定芽诱导的培养基为MS 6-BA1.5 mg/L NAA0.05 mg/L,适于继代增值的培养基为MS 6-BA1.5 mg/L NAA0.05mg/L,适于生根的培养基为配方为1/2MS IBA0.5mg/L NAA0.1 mg/L;用透气设施的封口材料明显地降低驱蚊香草的玻璃化;基质培养基生根优于琼脂培养基.     

秃杉组织培养研究

本试验取秃杉种子刚萌发的苗端作外植体进行了不定芽的诱导,确定了最佳分化和根培养基的成分,分析讨论了激素种类及浓度和光照、温度等因子在培养中的作用以及移栽注意事项,为秃杉的快速繁殖提供了科学依据。     

照山白杜鹃组织培养研究

以照山白杜鹃(Rhododendron micranthum)试管苗的茎段为外植体,研究不同激素组合对其外植体诱导、丛生芽增殖及试管苗生根的影响.结果表明:最适丛生芽诱导培养基为Read+ ZT 4.0 mg/L+ NAA 0.05 mg/L+蔗糖(3％),诱导率达92.41％;最适丛生芽增殖培养基为Read+ ZT 3.0 mg/L+ NAA 0.1 mg/L+蔗糖(3％),增殖系数达7.56;生根培养基为Read+ IAA 0.5mg/L+蔗糖(2％),生根率达92.5％;NAA不适宜照山白杜鹃生根诱导;将试管苗移栽到松毛土∶泥炭土∶河沙=1∶2∶1的基质中,成活率达89％.     

无花果组织培养再生系统的研究

以无花果展叶腋芽、休眠腋芽、室外嫩茎和水培芽为外植体，通过初始培养获得无菌苗后，选取无菌苗的不同部位作外植体，如茎段、叶片、愈伤组织等，经过诱导培养、分化培养及生根培养，形成再生植株。研究了激素种类及浓度、培养基种类对诱导的影响，提出了无花果的最佳培养条件，并讨论了影响移栽成活的一些因素。     

黄色马蹄莲的组织培养研究

以黄色马蹄莲块茎为外植体组培试验结果:最适合的灭菌时间为0.1%的HgCl2消毒15min;在MS添加2.0mg·L-16-BA+0.1mg·L-1 NAA的培养基上可以很好的诱导产生愈伤组织,并且从愈伤产生丛生芽的数量也最多;在MS添加1.0mg·L-1 6-BA+0.2mg·L-1 NAA的培养基上经过初代培养的芽丛可以健壮生长同时继续增殖出新的愈伤组织;在不添加任何激素的MS培养基上马蹄莲生根率均可以达到90%以上,且试管苗根系良好,生长健壮。     

铁皮石斛组织培养研究

以铁皮石斛（Dendrobium catenatum）嫩茎为试验材料,建立组织培养体系。结果表明,铁皮 石斛嫩茎用 HgCl 2 试剂消毒 5~7 min 效果最好,污染率和死亡率极低。最佳诱导培养基为 MS 6-BA1.5 mg/L NAA0.5 mg/L KT0.2 mg/L 3% 蔗糖,诱导率达到 96%。最佳继代培养基是 MS 6-BA1.5 mg/ L NAA0.5 mg/L KT0.2 mg/L 5% 香蕉提取液 5% 水解酪蛋白 3% 蔗糖,增值系数达到 8.0。最适宜铁 皮石斛组培苗生根的培养基为 1/2MS 6-BA0.1 mg/L IBA1.0 mg/L 5% 香蕉提取液 2% 蔗糖,生根率为 97%。移栽 30 d 后,成活率达到 90% 以上。     

邓恩桉组织培养技术研究

邓恩桉是一种优良的耐寒桉树,它在我国的推广利用因受无开花结实所限,邓恩桉组培生根困难,为使其能尽快得以推广本文进行了邓恩桉的组培生根试验,试验结果表明:6-BA0.5mg.L-1是最适于启动邓恩桉丛生芽的发生。激素组合6-BA0.5mg.L-1 NAA0.1mg.L-1和6-BA0.3mg.L-1 NAA0.2mg.L-1的交替使用可用于邓恩桉的继代培养,IBA0.5mg.L-1可用于邓恩桉的生根培养,在生根试验中观察到温度低于15℃时邓恩桉根尖变黑,组培苗植株上下部停止生长。大量元素对邓恩桉生根的影响最大,其次是微量元素和有机物,对其影响最小的是蔗糖。