Regeneration and gene transformation systems of Robinia pseudoacacia ‘Idaho’ mediated by Agrobacterium tumefaciens

Robinia pseudoacacia ‘Idaho’ is one of several multi-purpose trees used in ornamental, soil and water conservation, fodder and nectar sources. Plant abiotic stress tolerance transformed by genes could meet the requirements for reclamation of arid or alkalid lands and vegetation restoration. For this paper, we studied the effects of auxin and cytokine on Idaho locust in vitro regeneration and the establishment of gene transformation systems for plants mediated by Agrobacterium tumefaciens. Results showed that the ratios of cytokinin and auxin were the major factors affecting adventitious bud differentiation on a MS medium; the concentration of 0.5 mg•L^–1 6-BA benefitted callus proliferation and 0.25 mg•L^–1 IBA promoted shoot rooting; however, a higher IBA concentration will inhibit rooting. The most effective antitoxin for screening transgenic Idaho locust shoots was G418 and the most sensitive concentra-tion of it was 8 mg•L^–1.     

Production of protoplasts from cultures of <Emphasis Type="Italic">Ophiostoma picea</Emphasis>

A method is described for producing viable protoplasts from germinating conidia (germlings) of Ophiostoma picea. The use of MgSO₄-based osmotic stabilizers significantly improved protoplast release, as did the use of 20-h-old germlings. Protoplast release rates also increased with higher enzyme concentration and incubation times, but there were associated negative effects, including reduced regeneration. The results indicate that the use of young germlings, low lytic enzyme levels, and short exposure periods produced the most viable protoplasts.     

Regulation of phytohormones on root primordium initiation and adventitious root formation in the etiolated shoots of Paeonia suffruticosa ‘Yinfen Jinlin’

Tree peony is well known and sought after for its large, colorful flowers. Its propagation is via vegetative methods. Mechnisms of the adventitious rooting and the regulation of rooting processes are the principles and techniques of plant propagation and improvement. Microstructures and fluctuations of phytohormones in the adventitious rooting were studied with the etiolated softwood shoots of Paeonia suffruticosa ‘Yinfen Jinlin’. There are no pre-primordia in the shoots of the cultivar. Adventitious roots are produced in five stages: shoot selection, primordium initiation, primordium growth, conducting tissue differentiation and root protrusion. Primordia initiated in the cortex. The contents of the endogenous hormones, IAA, ABA and GA, were 5.842, 0.873 and 1.043 nmol&#8226;g^–1 FW on the bases of shoots, respectively. CTKs which included isopentenyl adenine (iPA), zeatin riboside (ZR) and dihydrozeatin riboside (DHZR) were 0.949, 0.695 and 2.034 nmol&#8226;g^–1 FW, respectively. DHZR is active among CTKs. The ratio of IAA to GA, CTK and ABA clearly increased at the stage of primordium initiation, while they showed low levels at the stages of primordium growth. The ratios were restored at the shoot levels at the stage of root protrusion. IBA provoked primordia initiation in the cortex, the vascular cambium, the pith and even in the callus induced on the base of shoots. IAA levels in the treated shoots increased gradually to its highest level (three times of control) at the stage of conducting tissue differentiation. The ratios of IAA to GA, CTK and ABA clearly decreased at the stage of primordium initiation. The ratio of IAA to ABA is regulated at 10:1.     

Large electro-fused protoplasts ofPopulus alba selected by a micromanipulator: Techniques and some characteristics of cells and their regenerants

An improved method for selection of large electro-fused protoplasts ofPopulus alba by a micromanipulator was developed. The conditions for electric cell fusion treatment were optimized. For the best result, protoplasts with a cell density of 5 × 10⁵/mL were treated with an alternate current (1 MHz, 200 V/cm) and pulsed with a direct current (2 kV/cm) for 100μs in 2.5 mM CaCl₂ and 0.55 M mannitol. The electo-fused protoplasts were cultured in NH₄NO₃-free Murashige and Skoog’s medium containing 0.6 M of mannitol, 0.09 M sucrose, 1μM of 2,4-dichlorophenoxyacetic acid and 0.1μM of benzyladenine, the same medium used for protoplast culture, but at a very low cell density of 5–10 × 10²/mL in a well of a 96-well culture plate. When cell aggregates derived from individual fused protoplasts were transferred to fresh medium with 0, 0.3 or 0.6 M mannitol, large colonies developed. In the shoot differentiation medium, the reaction of the calluses derived from large fused protoplasts towards the growth regulators differed from the non-fused ones. In medium containing 1μM each of naphthalene acetic acid andN-(2-chloro-4-pyridyl)-N′-phenylurea, growth of callus from electro-fused ones was not reduced by much compared to the control, but shoot differentiation was inhibited. Gibberellic acid (0.1–10μM) was beneficial to shoot regeneration; however, irregularly shaped leaves appeared at high gibberellic acid concentrations. Shoots regenerated were rooted in Murashige and Skoog’s medium containing 4μM of indole-3-butyric acid. Some plantlets obtained had a varied morphology. Based on the characteristics of growth, some cells derived from electro-fused protoplasts appear to be physiologically different from the non-fused one.     

Nitraria sibirica cell suspension culture:establishment,characterization and application

Nitraria sibirica Pall.is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value.The objectives of this study include induction and multiplication of callus,establishment of a suspension cell line,and isolation of protoplasts from cell suspensions.Murashige and Skoog(MS) medium was used for callus induction from mature seeds of N.sibirica.Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mgL~(-1) 6-benzylaminopurine(6-BA) and 1.0 mgL~(-1) 2,4-dichlorophenoxy(2,4-D) acetic acid.Suspension cultures of N.sibirica were initiated by transferring friable calli to the same liquid multiplication medium.Characterization of the suspension culture was assessed based on fresh mass,dry mass,cell viability and p H value of the culture.A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium,including a lag phase,an exponential growth phase,a stationary phase,and a negative acceleration phase.The effect of factors such as pre-plasmolysis,enzyme combination,enzymolysis time and mannitol concentration,on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures.The maximum yield(9.79 9 106 cells/g) and highest viability(79.97%) of protoplast were reached when approximately 1 g of cell suspension(cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 molL~(-1) mannitol mixture solution,cellulose onozuka R-10 2%(w/v),hemicellulose 0.2%,macerozyme R-10 1%,and pectolyase Y-23 0.5%.Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10(P0.05).     

Vegetative propagation of Dalbergia sissoo: effect of growth regulators, length, position of shoot and type of cuttings on rooting potential in stem cuttings

The effect of auxins (IAA or IBA at 100,200,500 mg L 1) on rooting and sprouting parameters differed significantly (p < 0.05).Rooting and sprouting percentages were higher in some treatments while other parameters were significantly higher in other treatments.The results of the present study revealed that rooting was significantly (p < 0.05) affected by the length of cuttings,as well as the position of shoots and type of cuttings.On average,86.0 per cent rooting was observed in the 30-cm long cuttings compared to the other two lengths of cuttings,i.e.,20 and 25 cm,irrespective of any auxin treatment.In a control set,without any auxin treatment,a maximum of 60.0% rooting was recorded in the 30-cm long cuttings and a minimum of 40.0% of the cuttings rooted in the 20-cm long cuttings.Similarly,cuttings collected from the lower and upper portions of shoots resulted in 72.5 and 52.5 per cent rooting respectively,irrespective of auxin treatments.Maximum rooting was observed in the lower portion (40.0%) of cutting compared to the upper portion (30.0%) in the control set without auxin treatment.Leafless branches produced maximum (72.0%) rooting compared to the leafy cuttings (35.0%),irrespective of the auxin treatment.The leafy cuttings produces a minimum of 15.0% sprouting and rooting in the control set.     

Regeneration of protoplasts from hyphal strands ofVolvariella volvacea

A series of experiments on the preparation and regeneration of protoplasts from hyphal strands ofVolvariella volvacea (Bull. ex. Fr.) Singer were conducted with the aim of optimizing the conditions for its efficient regeneration. One commercial (Vvcl) and two wild (EAAC-0001 and EAAC-0002) strains ofV. volvacea from the Philippines were used and subjected to varying conditions to determine the most efficient means for regeneration of their protoplasts. The effects of age and type of strain, pH, type and concentration of osmotic stabilizer, enzymatic composition, treatment time, temperature, reciprocal frequency during enzymatic lysis of the cell wall, and centrifugation conditions were investigated. Results showed that the three strains ofV. volvacea had varying responses in terms of yield, size, and ability of their protoplasts to regenerate into the protoplast regeneration medium. Among the three strains, EAAC-0002 had the highest rate of regeneration. The 5-day-old culture ofV. volvacea, when subjected to a combination of 2% Novozyme 234 and 0.2% chitinase in 0.6M mannitol (pH 6.0) for 3 h at 30°C, 90 strokes/min and centrifuged at 1100 g for 10 min; produced an efficient yield of protoplasts with a relatively high regeneration rate.Part of this paper was presented at the 47th annual meeting of the Japan Wood Research Society, Kochi, Japan, April 2–5, 1997     

Osmotic regulation in leaves and roots of olive trees during a water deficit and rewatering

We evaluated the osmotic adjustment capacity of leaves and roots of young olive (Olea europaea L.) trees during a period of water deficit and subsequent rewatering. The trials were carried out in Basilicata (40 degrees 24' N, 16 degrees 48' E) on 2-year-old self-rooted olive plants (cv. 'Coratina'). Plants were subjected to one of four drought treatments. After 13 days of drought, plants reached mean predawn leaf water potentials of -0.45 +/- 0.015 MPa (control), -1.65 +/- 0.021 (low stress), -3.25 +/- 0.035 (medium stress) and -5.35 +/- 0.027 MPa (high stress). Total osmotic adjustment increased with increasing severity of drought stress. Trees in the high stress treatment showed total osmotic adjustments ranging between 2.4 MPa at 0500 h and 3.8 MPa at 1800 h on the last day of the drought period. Osmotic adjustment allowed the leaves to reach leaf water potentials of about -7.0 MPa. Active osmotic adjustment at predawn decreased during the rewatering period in both leaves and roots. Stomatal conductance and net photosynthetic rate declined with increasing drought stress. Osmotic adjustment in olive trees was associated with active and passive osmotic regulation of drought tolerance, providing an important mechanism for avoiding water loss.     

<Emphasis Type="Italic">Nitraria sibirica</Emphasis> cell suspension culture: establishment,characterization and application

Nitraria sibirica Pall. is a shrub that grows in saline-alkali soil and has traditional medicinal value and potential commercial value. The objectives of this study include induction and multiplication of callus, establishment of a suspension cell line, and isolation of protoplasts from cell suspensions. Murashige and Skoog (MS) medium was used for callus induction from mature seeds of N. sibirica. Seed-derived calluses were further multiplied on MS medium augmented with 0.5 mg L^?1 6-benzylaminopurine (6-BA) and 1.0 mg L^?1 2,4-dichlorophenoxy (2,4-D) acetic acid. Suspension cultures of N. sibirica were initiated by transferring friable calli to the same liquid multiplication medium. Characterization of the suspension culture was assessed based on fresh mass, dry mass, cell viability and pH value of the culture. A typical growth curve was observed after inoculating 1.5 g of callus in 40 mL liquid medium, including a lag phase, an exponential growth phase, a stationary phase, and a negative acceleration phase. The effect of factors such as pre-plasmolysis, enzyme combination, enzymolysis time and mannitol concentration, on the isolation of cell-derived protoplasts were evaluated to determine the usefulness of suspension cultures. The maximum yield (9.79 × 10⁶ cells/g) and highest viability (79.97%) of protoplast were reached when approximately 1 g of cell suspension (cultured for 6 days) was inoculated for 12 h in cell and protoplast washing solution made of 0.8 mol L^?1 mannitol mixture solution, cellulose onozuka R-10 2% (w/v), hemicellulose 0.2%, macerozyme R-10 1%, and pectolyase Y-23 0.5%. Protoplast yield was significantly influenced by pre-plasmolysis and cellulose onozuka R-10 (P < 0.05).     

Stand structure and dynamics of four native Scots pine (Pinus sylvestris L.) woodlands in northern Scotland

Stands in four native pinewoods (Glenmore, Black Wood of Rannoch,Glen Garry and Glen Affric) dominated by Scots pine (Pinus sylvestrisL.) with contrasting management histories and climates wereassessed for differences in age, structure and dynamics. Treeage, height, diameter at breast height (d.b.h.), basal area,stem density and x, y coordinates were used to compare the recruitmentof trees (>1.3 m height, >7 cm d.b.h.), saplings (>1.3m height, <7 cm d.b.h.) and seedlings (<1.3 m) followingdisturbance events and protection from browsing. There was a20-year lag between localized intensive cultivation and treerecruitment on sites that were protected from deer browsing(Glenmore and Glen Garry). Recruitment was low in sites withdisturbance but no protection (Black Wood of Rannoch). The oldestpopulation, Glen Affric, showed signs of initial intense recruitmentfollowed by a long period of nil recruitment. Abundant standingdead trees were recorded only in Glen Affric, and prolific birchand rowan only in Glen Garry. Managers should consider localizedintense cultivation in conjunction with a complete reductionin browsing pressure for rapid seedling recruitment and increasedstructural diversity.     

白灵菇菌丝生长因子的研究

研究了影响白灵菇菌丝生长的主要因素,比较了不同菌株在不同培养基含水量、酸碱度和温度条件下菌丝生长的情况。试验结果表明:白灵菇菌丝最适合生长的培养基含水量为65%~75%,Pl.n0028在最适合含水量的培养基中菌丝生长与其它菌株有差异,生长速度较快;最适合生长的培养基pH值为6~8,Pl.n0029在最适合pH的培养基中菌丝生长与其它菌株有差异,生长速度较快;最适合生长的温度为25~30℃,Pl.n0029在适合培养温度下菌丝生长与其它菌株有差异,生长速度较快。     

蓝花楹组织培养研究

文章以蓝花楹（Jacaranda mimosifolia）种子无菌育苗获得的外植体为材料,进行增殖和生根培养条件优化筛选研究,以建立其高效稳定的离体再生技术。结果表明,泥炭土中蓝花楹种子萌发最快,芽最健壮;最适宜的增殖培养基为 MS ＋6-BA1．0 mg／L ＋IBA 0．1 mg／L ＋蔗糖30 g／L ＋琼脂6 g／L,继代周期20 d,增殖系数达4．77;最佳生根培养基为1／2 MS ＋IBA0．5 mg／L,20 d 生根率达100％;移栽到泥炭土∶珍珠岩＝2∶1混合基质上,30 d 移栽成活率达92％以上。     

都百凤桃树离体植株再生培养的研究

以从日本引进的桃树品种都百凤为试材,采用正交设计,研究了6-BA,NAA,接种芽数,活性炭对不定芽增殖与壮苗的影响,探讨了激素、培养基盐浓度、活性炭对都百凤组培苗生根的影响。结果表明,都百风桃树离体植株再生继代增殖培养基为MS＋6BA3mg／L～5mg／L＋NAA0．1mg／L～0．2mg／L＋蔗糖30g／L,培养基pH值为5.9,以3芽为1个转接单位,培养温度为25℃,光照强度2000Lx,光照时间16h／d;生根培养基为1／2MS＋IBA0.2mg／L＋蔗糖20g／L＋琼脂7g／L＋活性炭0．4g／L,培养基pH值为6．0,培养温度为22℃,光照强度1600Lx,光照时间16h／d;组培苗转入生根培养基舌暗处理2d,再转到光照下培养有利于生根。     

防治人参土传病害生防菌株X1和B59培养基筛选及发酵条件的优化研究

通过正交试验和单因素试验对防治人参土传病害的生防菌B59和X1菌株的培养基组成和培养条件进行优化研究。B59菌株培养基成分和最适培养条件为:葡萄糖15g/L,牛肉膏5.0g/L,NaH2PO4+Na2HPO45g/L,pH8,250mL三角瓶中装入25mL培养基,接种量为1.5%,30℃,160r/min培养。X1菌株的最佳培养基成分和培养条件为:葡萄糖15g/L,牛肉膏+酵母膏(1∶1)10.0g/L,NaH2PO4+Na2HPO42.5g/L,pH8,250mL三角瓶中装入25mL培养基,接种量为1%,25℃,160r/min培养。优化培养的枯草芽孢杆菌X1和B59菌株对人参腐皮镰刀菌抑菌能力显著提高。     

人心果原生质体分离初探

以人心果幼嫩叶片组织为材料,研究了酶液组合、酶解时间、酶液中甘露醇含量、pH值、温度等因素对其原生质体分离的影响,结果表明:以3%纤维素酶R-10+0.4%果胶酶Y-23为混合酶液,0.6 mol.L-1甘露醇为渗透压稳定剂,pH 5.6的酶液组合,在28℃的黑暗条件下振荡,酶解时间2 h,分离出的原生质体产量最佳,可达到12.4×106个.g-1。     

Growth and mortality in underplanted tree seedlings in response to variations in canopy closure of Norway spruce stands

Six broadleaved tree species and Picea abies (L.) Karst. wereplanted under spruce plantations of varying densities, in Sweden.Treatments included control (994 stems ha^–1), dense (538stems ha^–1), sparse (294 stems ha^–1) and gap (0stems ha^–1) overstory treatments. There was an increasein height and diameter growth from control to sparse overstorytreatment of all underplanted tree species except for ash (Fraxinusexcelsior L.) and Norway maple (Acer platanoides L.). Site conditionsmay have hampered the growth of these species, as well as wildcherry (Prunus avium L.). Both oak (Quercus robur L.) and sprucehad greater growth in the gap treatment, relative to the othertreatments. Insecticide application did not influence seedlinggrowth or survival. The performance of beech (Fagus sylvaticaL.), lime (Tilia cordata Mill.), spruce and oak was consistentwith shade tolerance ranking. Beech and lime had a very highsurvival rate, even under the densest canopy. The growth andmortality of ash, maple and wild cherry differed significantlyfrom what was expected. This experiment demonstrated significantvariation in interspecific growth and mortality between sevenplanted tree species in relation to canopy density. Correctsite and species selection is crucial when underplanting inshelterwood systems.     

晚松悬浮细胞系的建立和原生质体的分离

植物悬浮细胞系的建立和原生质体的分离已被广泛地应用于生理学、生物化学、细胞学、遗传学及分子生物学的研究 ,它是生物技术中进行原生质体培养、杂交、基因转移、突变系筛选等项研究的较理想手段[1] 。针叶树在这方面的研究起步虽晚 ,但至今已取得令人瞩目的进展[2 ] 。晚松     

红掌的组织培养与快速繁育

选生长健壮的红掌盆苗,研究其组培快繁技术,结果表明：MS＋BA 1 mg/L＋NAA 0.2 mg/L＋葡萄糖30 g/L诱导率最高;1/2MS＋BA 2 mg/L＋2,4-D 0.5 mg/L＋椰汁100 mL/L＋蔗糖30 g/L培养基愈伤组织及芽苗增殖率最高;壮苗最佳培养基MS＋BA 0.5 mg/L＋IBA 0.05 mg/L＋椰汁100 mL/L＋蔗糖30 g/L;最佳生根培养基1/2MS＋NAA 0.3 mg/L＋蔗糖20 g/L,生根率达95%以上,当生根苗有3条-4条时即可移栽。     

Use of stump diameter to estimate diameter at breast height and tree volume for major pine species in El Salto, Durango (Mexico)

Foresters may require to estimate the diameter at breast height(d.b.h.) and the volume of trees that have been cut, and mayonly have available the stumps as an indicator of the size ofthe trees. In the present study, equations for predicting bothd.b.h. and volume from stump diameter inside bark were developedfor major pine species in the forest region of El Salto, Durango(Mexico). The d.b.h. was estimated with relatively high accuracywith a simple linear model. The tree volume was also estimatedwith high precision by use of an allometric equation. Weightedlinear and non-linear least squares methods were used to takeinto account the problem of heteroscedasticity observed in thevolume–stump diameter relationships. The results of thenon-linear extra sum of squares method and of the F tests indicatedthat species-based equations for estimating both d.b.h. andvolume from stump diameter are required.     

Root anchorage and stem strength of black spruce (Picea mariana) trees in regular and irregular stands

A tree winching experiment was conducted, simulating wind actionand resulting damage, in order to assess mechanical resistanceof black spruce (Picea mariana (Mill.) B.S.P.) trees in standsof regular and irregular structures. The critical bending moment(M_c) of winched trees was determined and the relationship betweenM_c and tree and stand characteristics was investigated throughstatistical analyses. Stem mass (SW), average spacing betweentrees (S) and height:diameter ratio (H/d.b.h.) were initiallyselected by a screening procedure to predict M_c. Potential differencesbetween stand structures were tested in mixed models using differentsubsets of the data. Included fixed effects varied between thedifferent models. SW was the most useful and significant variablein all models and H/d.b.h. was significant only when snappedtrees were included in the analysis. When decayed samples wereexcluded, resistance to uprooting was higher in irregular stands.Decay seemed to play an important role in irregular stands andshould be investigated further. Since no difference was observedin the relationship between stem mass and critical turning momentbetween distant sites, relationships should be applicable acrosswide regions. This study provides some of the basic relationshipsrequired to model windthrow risk in irregular stands. However,the effects of stand structure on wind load for individual treeswill also need to be considered.