农杆菌介导的楸树遗传转化体系

【目的】以楸树胚性愈伤组织为受体,建立有效的楸树遗传转化体系,为今后楸树性状的遗传改良奠定基础。【方法】通过农杆菌EHA105介导以胚性愈伤组织作为外植体进行遗传转化,通过正交试验获得最优的遗传转化条件,进而将外源基因转入到楸树基因组中。【结果】在1/2 MS培养基中添加不同梯度浓度的卡那霉素(Kana)进行选择压力筛选,在添加了60 mg·L~(-1)Kana的1/2 MS培养基中,楸树胚性愈伤组织的分化率为0.00%,存活率仅为5.71%,因此确定60 mg·L~(-1)为遗传转化的选择压。采用正交设计L18(37)进行农杆菌介导的楸树遗传转化试验,通过GUS化学组织染色统计瞬时表达率,正交试验直观分析和单因素方差分析结果表明:在预培养时间为2天,采用农杆菌菌株EHA105、菌液浓度OD600值为0.7、添加乙酰丁香酮(AS)浓度为300μmol·L~(-1)、侵染时间为10 min,共培养时间为5天的条件下,农杆菌介导的转化效率最高,且对转化效率影响最大的2个因素是乙酰丁香酮浓度和预培养时间。对浸染后的胚性愈伤组织进行8个月的筛选培养,共获得32个抗性组织团,对其中15个增殖较多的抗性愈伤组织进行PCR检测,表明86.67%的抗性组织团中有外源基因整合到楸树基因组中。内源激素水平会对植物体细胞胚分化产生影响,细胞分裂素(CTK)和脱落酸(ABA)促进体胚发生,生长素(IAA)和赤霉素(GA)对体胚发生有抑制作用。通过测定内源激素可知,转基因的抗性组织中内源CTK和ABA水平显著低于野生型的楸树胚性愈伤组织,而内源IAA和GA则显著高于野生型胚性愈伤组织,推测内源激素水平可能是转基因抗性组织体胚分化能力比较差的原因。【结论】建立了农杆菌介导的楸树胚性愈伤组织的遗传转化体系,对筛选获得的15个抗性愈伤组织进行PCR检测,其中13个抗性愈伤组织中有外源基因的整合。内源激素水平的变化可能是导致楸树转基因抗性愈伤组织难以分化的原因。     

甜椒抗菌蛋白基因(harp)转化桉树的研究

用不同浓度的2,4-D与IAA对桉树叶盘进行了愈伤组织的诱导及植株再生的试验,建立了新的桉树再生体系.进一步用含甜椒抗菌基因(hrap)的农杆菌侵染桉树叶盘,发现侵染后对愈伤组织诱导率无明显影响,却抑制了桉树的植株再生.对再生苗的PCR和Sou thern杂交检测证实已将hrap基因已转入桉树植株的基因组中.     

农杆菌介导慈竹4CL基因遗传转化梁山慈竹

以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天(共培养基表面加一层无菌滤纸),在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。     

高羊茅组织培养基的选择

用不同种类的基本培养基及不同浓度2,4-D、6-BA,对本地高羊茅的种子进行愈伤诱导.结果表明:基本培养基种类对愈伤诱导无明显的效果;MS培养基下不同浓度2,4-D、6-BA对高羊茅愈伤诱导时间及出愈率差别很大,以9 mg/L效果最佳,愈伤诱导率达70.2%左右;过高浓度2,4-D、6-BA对萌芽和愈伤的形成有负作用;愈伤组织分化以MS-BA2.0 mg/L-NAA0.5 mg/ L为最佳 MS基本培养基,2,4-D浓度以9.0 mg/L为最佳,最适合高羊茅愈伤组织诱导的培养基为MS 9.0 mg/L2,4-D.     

桉属植物子叶培养的形态发生及植株再生

愈伤组织的培养物是从柠檬按幼苗的下胚轴及子叶培养中取得。在麦雷许戈(Murashige)和斯高(Skoog)基本培养基内加入玉米素(1毫克/升)和吲哚乙酸(0.2毫克/升)从子叶愈伤组织上在六周内成功地产生了小植株0从一年生苗木上取得的叶愈伤组织和茎愈伤组织上并没有分化。本文所报导的研究成果提供了对愈伤组织生长和小植株形成的最适宜条件。     

农杆菌介导的杜仲叶片愈伤组织遗传转化体系

【目的】优化杜仲叶片愈伤组织的再生体系,研究愈伤组织对抗生素和抑菌剂的敏感性,探索影响农杆菌介导杜仲愈伤组织遗传转化的最适转化因子水平,构建农杆菌介导的杜仲愈伤组织瞬时转化体系,使杜仲成年植株外植体为受体的遗传转化成为可能,为杜仲基因功能的研究与定向改良奠定基础。【方法】以杜仲成年植株叶片为材料诱导愈伤组织,通过添加不同浓度植物生长调节剂与大量元素的MS培养基进行不定芽的诱导与增殖,确定最适培养基。在此基础上,在培养基中添加不同浓度抗生素及抑菌剂,研究愈伤组织对其敏感性。以获得的叶片愈伤组织受体系统为基础,通过L_(16)(4~5)的正交试验,探索不同转化因子对农杆菌介导叶片愈伤组织遗传转化的瞬时转化效率的影响,建立最适瞬时转化体系。使用获得的瞬时转化体系对愈伤组织进行遗传转化操作,筛选抗性芽,对抗性芽进行GUS组织化学染色与PCR检验。【结果】再生体系优化的结果表明,3/4大量元素浓度的MS培养基能够促进杜仲愈伤组织不定芽的诱导及生长;愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,诱导率为83%±10. 0%;不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,平均伸长长度为(2. 47±1. 33) cm。抗生素与抑菌剂敏感性试验表明,遗传转化的选择培养基中,抑菌剂头孢霉素的最适浓度为200 mg·L~(-1),筛选用的抗生素卡那霉素最适浓度为70 mg·L~(-1)。转化因子的正交试验表明,最适的农杆菌介导杜仲叶片愈伤组织遗传转化的转化因子组合为:预培养5天、侵染10 min和共培养3天。使用最适瞬时转化体系对约200个愈伤组织进行遗传转化操作,共筛选获得3个抗卡那霉素的抗性芽; GUS组织化学染色显示GUS基因在抗性芽中得到了表达,PCR检测证明这些抗性芽中存在NPTⅡ基因。【结论】杜仲叶片愈伤组织诱导不定芽的最适培养基为3/4MS+0. 27μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA,不定芽复壮的最适培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA。农杆菌介导的杜仲叶片愈伤组织瞬时转化体系为:预培养5天、侵染10 min和共培养3天,筛选培养基为3/4MS+0. 054μmol·L~(-1)NAA+4. 4μmol·L~(-1)6-BA+200 mg·L~(-1)Cef+70 mg·L~(-1)Km。利用此体系共获得3个抗性芽,PCR分析和GUS组织化学染色都表明T-DNA已整合到抗性芽基因组中。     

GUS基因导入杨树杂交品系NC5331的研究

对杨树杂交品系NC5331进行基因转导研究。采用土壤肿瘤杆菌品系ASE9749(pMON9749)为基因载体与无菌试管苗叶片和温室里试管苗的叶片进行协同培养,然后进行选择培养诱导转导愈伤组织,诱导率为6.7％～8.0％。转导愈伤组织通过增殖并诱导芽的形成,诱导率为100％,并对芽诱导生根,培育完整植株,最后移栽到土壤并在温室里正常生长。对所形成的愈伤组织和植株叶片进行GUS组织化学分析均显示杆菌中β-葡萄糖醛酸酶基因已被转导到植物体细胞中,并得到表达。该杨树杂交品系的转导研究,在中国国内尚未见报导。     

几种作用因子对多年生黑麦草组织培养影响的研究

以多年生黑麦草成熟种子为外植体进行了愈伤组织诱导和分化的研究。结果表明:dicamba替代2,4 D,蔗糖替代麦芽糖可以显著提高愈伤组织诱导率和植株再生率;在一定的浓度范围内(3～9mg·L-12,4 D)2,4 D浓度的升高可明显提高愈伤组织的诱导率,但同时却降低了分化率;在愈伤诱导培养基中同时使用两种生长激素(2,4 D和NAA)的效果要好于单独使用一种生长激素(2,4 D)的效果;水解酪蛋白、脯氨酸和谷氨酰胺浓度的增加并没有促进植株再生率的升高。     

热激启动子控制的FT基因诱导杨树早期开花体系的优化

为实现杨树早期开花,缩短育种周期,利用农杆菌介导的转化方法,探讨影响热激启动子控制的FT1基因转化白杨派杂种无性系的因素,优化其转化条件,并实现FT1基因的转化,采用热激诱导方法诱导2～3个月大的转基因植株早期开花。结果表明:叶盘转化前的预培养、菌液浓度、侵染时间及共培养时间对卡那霉素抗性植株再生率的影响均达到极显著水平;叶盘在愈伤组织诱导培养基上暗培养6天,然后用活化至OD600=0.5的菌液侵染60min,再在愈伤组织诱导培养基上共培养2天,该条件下FT1基因转化白杨派杂种的卡那霉素抗性植株再生率可达29.84%;37℃每天1h持续热激3周可使FT1基因顺利表达,促进转基因植株开花;热激植株大小是影响转基因植株热激诱导开花的限制因素,低于20cm的植株不能诱导开花;热激诱导可产生相对较多的正常花器,但花器变异仍然十分广泛。     

白桦愈伤组织染色体制片方法及数目变异研究

该文探讨白桦愈伤组织染色体的制片方法,并对白桦愈伤组织及其再生植株的染色体数目变异进行了研究。结果表明：取继代培养1周的白桦愈伤组织,用0.2%的秋水仙素溶液预处理1.5～2 h,1 mol/L HCl室温（18～20℃）解离10～15 min或2%纤维素酶和果胶酶混合液室温解离30 min,压片后易获得分散、染色效果好的细胞分裂中期染色体图像。随着继代次数增加,白桦愈伤组织染色体数目变异的频率及范围增大。在愈伤组织分化过程中,二倍体细胞占有优势,能够分化为正常的植株,愈伤组织再生植株的变异频率低于愈伤组织。     

Browsing Damage on Pine (Pinus sylvestris and P. contorta) by a migrating moose (Alces alces) Population in Winter: Relation to Habitat Composition and Road Barriers

In Scandinavia, moose (Alces alces L.) sometimes cause severe browsing damage to economically-important pine. Moose-vehicle accidents have spurred construction of fences along roads, and these may interfere with moose migration between summer and winter ranges, or the road alone may be a barrier. If this happens and moose build up along roads, landowners may suffer economically. Therefore, this study investigated whether roads, fences or other factors influence the use of young pine stands by moose. Eighty stands along roads in northern Sweden were evaluated in which individually-browsed branches were counted on 9972 pines. Moose browsing was not significantly related to birch (Betula pendula Roth, B. pubescens Ehrh.) density, nor did it differ between pines (Pinus contorta Douglas or P. sylvestris L.). However, increased pine density, site productivity and proximity to a highway were associated with increased browsing. Further large-scale studies are needed to understand moose habitat selection and the effects of roads.     

Effects of Root Freezing on the Physiology and Growth of Picea glauca,Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes

The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings were submitted to various frost temperatures during an artificial frost to induce different levels of root damage. Frost-damaged and control seedlings were placed in a greenhouse under high and low soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedling survival was reduced when root damage reached levels of 60-80%. Root systems of all three species showed partial to total recovery by the end of the experiment. In general, root freezing damage caused reductions in seedling growth, with these reductions becoming less significant over time. Root damage had little to no effect on black spruce and jack pine seedling physiology, while white spruce CO 2 uptake decreased with increasing root damage. Shoot nitrogen content of all three species decreased slightly with increasing root damage.     

Stand Structure in Pine,Spruce and Deciduous Forests: A Comparison Between Finnish and Russian Karelia

This study investigated the stand structure in pine, spruce and deciduous forests in the border district of Finland and Russia. A total of 46 mature forest stands was selected as pairs, the members of each pair being as similar as possible with respect to their forest site type, age, moisture and topography. The stands were then compared between the two countries by means of basal areas and number of stems. The proportions of dominating tree species were 2-12% lower, and correspondingly the proportions of secondary tree species higher, in Russian forests. The density of the forest stock was also higher in each forest type in Russia. The forests in the two countries differed most radically in terms of the abundance of dead trees. The amount was two to four times higher in Russian deciduous and spruce forests, and in pine forests the difference was 10-fold. The stand structures indicated that Russian coniferous stands, in particular, were more heterogeneous than intensively managed pine and spruce stands in Finland.     

Growth and Terminal Bud Formation in Picea abies Seedlings Grown with Alternating Diurnal Temperature and Different Light Qualities

The root collar diameter and the height:diameter ratio are of particular importance in container-grown seedlings where a high density in the containers may produce spindly seedlings. Temperature regimes and light quality are known to affect plant growth. The aim of this study was to identify responses in Picea abies (L.) Karst. seedlings grown with light providing different red:far-red ratios and under temperature regimes with alternating day (DT) and night temperature (NT) from negative (DT < NT) to positive (DT > NT) difference (DIF) between DT and NT. Experiments were conducted in controlled environment chambers and in a daylight phytotron. Only limited thermoperiodic responses appeared in P . abies seedlings with respect to seedling height and dry weight accumulation. The formation of terminal buds, however, was clearly delayed in seedlings grown at negative DIF. The results indicate a requirement for day extension light that is high in far-red, to prevent terminal bud formation under natural short-day conditions. An extended study should be conducted to clarify the minimum level of light intensity and the optimal light quality needed to prevent terminal bud formation under natural short-day conditions.     

CAD与建筑设计的关系

本文分析了CAD在设计中引起的正面和负面影响,并进行了系统的阐述,从而使设计者在应用中能够保持客观的态度。     

Natural Regeneration in Clear-cuts: Effects of Scarification,Slash Removal and Clear-cut Age

The effects of soil scarification (mounding), slash removal and clear-cut age on the natural regeneration in clear-cuts was evaluated using data from four sites in southern Sweden. The treatments were carried out during a good seed and establishment year for birch ( Betula pubescens Ehrh. and B. pendula Roth). Scarification had the strongest positive effect on the density of naturally regenerated seedlings, especially in birch, but also in pine ( Pinus sylvestris L.) and spruce [ Picea abies (L.) Karst]. Slash removal had a positive effect on birch density. No statistically significant effect of clear-cut age was found. In addition, no statistically significant interactions between clear-cut age and scarification or slash removal were found. The ingrowth of field vegetation was the fastest in areas that were not scarified, less rapid in areas scarified on old clear-cuts, and the slowest after scarification in fresh clear-cuts. In conclusion, it may be possible to control the density of birch during a good establishment year for birch. If birch is desired, the best combination of treatments is to remove the slash and scarify; otherwise, these treatments should be avoided.     

Risk Perception Among Non-industrial Private Forest Owners

Based on an enquiry, risk perception among non-industrial private forest owners is described in relation to climate change and forestry hazards. Of the respondents, 11% took action to remedy the effects of climate change. Out of a given set, hazards were ranked according to each respondent's experience of recent substantial financial loss to the estate and in relation to his or her willingness to make investments aimed at risk reduction. For each hazard, the respondent assessed the risk in four classes ranging from very high to negligible risk. Six hazards were considered most problematic in all three aspects: browsing damage, falling timber prices, damage by wind, spruce bark beetle, root rot and pine weevil. A majority of the respondents claimed to take action to reduce the risk associated with at least one hazard, while 35% did not know whether they did. Excluding climate change, the need for decision support was the largest in relation to damage by wind owing to a combination of perceived high risk and a high level of ignorance in relation to whether risk-reducing measures were taken.     

Interactions in decomposition and N mineralization between tropical legume residue components

In situ produced plant residues contain a mixture of different plant components of varying quality. To assess synergistic or antagonistic interactions occurring during the decomposition and mineralization of such mixtures, individual plant parts (stems, leaves, leaf litter and roots) or the mixture of stems, leaves and leaf litter of the agroforestry species pigeonpea (Cajanus cajan) or of crop residues of peanut (Arachis hypogaea) or of the weed hairy indigo (Indigofera hirsuta) were incubated in pots for 19 weeks. Periodically, remaining plant residues were sieved out (>2 mm), weighed and N content as well as soil mineral N determined. Above- and below-ground residues of peanut decomposed fastest and showed the largest N release in agreement with their high N concentration and low-acid detergent fibre (ADF) : N ratio. Hairy indigo was hypothesized to be of lower quality than pigeonpea because of its high-polyphenol content. However, it decomposed faster than pigeonpea, largely because of the higher N and lower lignin concentration of its components. Ranking of individual plant components for N mineralization resulted in the following pattern, leaves > leaf litter > roots > stems. In mixtures of the different plant components a similar species order in decomposition was obtained, e.g. peanut > hairy indigo > pigeonpea. The amount of N released from the mixture was dominated by stem material that comprised 46–61% of the mixture. The interactions in mixtures were relatively small for peanut (generally high-quality components) as well as for pigeonpea (low proportion of high-quality components, i.e. N rich leaf material). However, a positive interaction occurred during later stages of N mineralization in the mixture of hairy indigo as it had a significant proportion of N rich components and absence of highly reactive polyphenols. Thus, for plants with low to intermediate chemical quality attributes, manipulating plant composition (e.g. by varying harvest age, affecting stem and leaf proportions) will be important to obtain significant interactions during decomposition when its components are mixed.     

Pasture production and tree growth in a young pine plantation fertilized with inorganic fertilizers and milk sewage in northwestern Spain

The use of organic waste materials such as milk sewage as an organic fertilizer could have the dual advantages of organic-waste disposal and reduced dependence on inorganic fertilizers. The effects of fertilization with (1) conventional mineral fertilization, (2) milk sewage sludge at 40 kg N ha⁻¹ target rate and (3) no fertilization on pasture production and tree growth were examined in an experiment consisting of two pasture mixtures under a one-year-old Pinus radiata plantation with a density of 2500 trees ha⁻¹. The two pasture mixtures were: (1) Dactylis glomerata L. var. saborto (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹); (2) Lolium perenne L. var. Tove (25 kg ha⁻¹) + Trifolium repens L. group Ladino (4 kg ha⁻¹) + Trifolium pratense L. var. Marino (1 kg ha⁻¹). The experiment began in the spring of 1995 using a randomized block design with three replicates in Castro Riberas de Lea (Lugo, Galicia, north-western Spain). Plot size was 12 × 8 m², with a 1 m buffer strip between plots. Two-year data showed that fertilization with either material had a positive effect on pasture production, with no significant difference between the two fertilization treatments. Tree growth in the milk sewage sludge plot was significantly higher than in the control plots. Inorganic fertilization increased pasture production, but affected tree growth negatively. The results show that milk sewage sludge could be used as a fertilizer in silvo-pastoral systems. This revised version was published online in June 2006 with corrections to the Cover Date.