Preliminary studies on establishment of genetic transformation system in loblolly pine

A transformation system was established for loblolly pine (Pinus taeda L.) mature zygotic embryos usingAgrobacterium tumefaciens. The gene coding for the β-glucuronidase (GUS) gene was introduced into loblolly pine tissues and its transient expression was detected with histochemical staining. The influences of different genotypes.Agrobacterum concentrations. and cocultivation time on GUS expression and kanamycin resistant callus and shoot regeneration were investigated. The results showed that the highest GUS expression frequency (16.3%) and shoot regeneration frequency (78%) were obtained from genotype 9–1003 withAgrobacterium concentration decreased 9 times and cocultivation time of 56 hours respectively. GUS expression was obtained in all genotypes tested. The successful expression of the GUS gene in different genotypes suggested that it will be a useful transformation system for loblolly pine. (Responsible Editor: Chai Ruihai)     

The penetration rate of laser pulses transmitted from a small-footprint airborne LiDAR: a case study in closed canopy, middle-aged pure sugi (Cryptomeria japonica D. Don) and hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stands in Japan

Penetration rates of laser pulses transmitted from a small-footprint airborne light detection and ranging (LiDAR) system in closed-canopy, middle-aged (40–50 years old) hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) and sugi (Cryptomeria japonica D. Don) plantations that had similar levels of canopy openness were studied. The number of transmitted pulses was 107 427 points/ha for the hinoki cypress stand and 122 883 points/ha for the sugi stand. The penetration rates of the first (P_f) and second pulses (P_s) that reached the ground were 0.1% and 1.0% in the hinoki cypress stand, and 2.6% and 5.5% in the sugi stand, respectively. Penetration rates for the total number of pulses (P_f+s) that reached the ground within each stand were 1.1% and 8.1%, respectively, and values for P_f+s, P_f, and P_s for sugi and hinoki cypress stands were significantly different (P < 0.001). The results of this study indicated that the generation of accurate digital terrain models in dense hinoki cypress stands with complex topographies using only the data from laser pulses with poor laser penetration rates is likely to be difficult.     

Effects of Thidiazuron,basal medium and light quality on adventitious shoot regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured stem of <Emphasis Type="Italic">Populus alba</Emphasis>×<Emphasis Type="Italic">P. berolinensis</Emphasis>

The effect of Thidiazuron （TDZ）, basal media and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus albaxP berolinensis were determined to establish a high efficiency shoot regeneration system from stem explants of P. alba~P berolinensis. Stems ofPopulus alba~P berolinensis were collected from cultured shoots in vitro derived from dormancy buds of 3-year-old seedlings. The stem explants were cultured on MS medium containing 0.02-mg·L-1NAA （naphthaleneacetic acid）, and 0.1, 0.3, 0.5 and 1.0 mg·L-1 concentrations of TDZ to determine the effect of TDZ on shoot regeneration. Three basal media, i.e. MS, woody plant medium （WPM） and B5, were used to test their influences of different media on adventitious shoot regeneration. Green, red, blue and yellow plastic films in comparison with florescent light as control were used to observe their effects on shoot regeneration. The results showed that differ- ent concentrations of TDZ had an evident influence on shoot regeneration. Lower concentration of TDZ （0.1 mg·L-1） resulted in more ad- ventitious shoot regeneration and higher concentration of TDZ （〉0.1 mg·L-1） inhibited shoot regeneration. Among different media, MS medium exhibited a high efficiency for shoot regeneration, followed by WPM medium, while B5 medium inhibited shoot regeneration. Normal light and yellow light exhibited better effects on shoot regeneration, compared with other light.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

Responses of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa) seedlings to nutrient solutions containing various calcium/aluminum ratios

An attempt was made to examine the possible connection between the various ratios of calcium/aluminum (Ca/Al) in the nutrient solution of plant cultures and the active oxygen scavenging system of hinoki cypress (Chamaecyparis obtusa) seedlings. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in the needles were estimated at several stages during the 7-day treatment. The samples treated with the lower Ca/Al solutions show the highest SOD activities. The activities of APX and GR, both of them involved in the ascorbate-glutathione cycle, showed the same tendency with decrease to higher Ca/Al ratio. These results indicate that rhizospheric Ca might compete with Al and ameliorate Al toxicity on and in the roots, the Al stress is not transformed to the needles after a few days, and the ascorbate-glutathione cycle in the hinoki cypress needles might fluctuate and be suppressed by the rhizospheric Al stress during the 7 days. This work was supported in part by funding from the Japan Science & Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.     

Micropropagation of red sanders (Pterocarpus santalinus L.) using mature nodal explants

In vitro clonal multiplication of Pterocarpus santalinus L. was achieved using mature nodal explants of a 10-year-old elite quality tree. Combinations of serial transfer technique and incorporation of antioxidants (250 mg/l L-ascorbic acid and 50 mg/l citric acid) into the culture medium helped to minimize medium browning and improve explant survival during shoot sprouting. About 70% of explants were sprouted on Murashige and Skoog (MS) liquid medium containing 4.4 μM 6-benzyladenine (BA). The explant harvest period also influenced the bud break and shoot sprouting in nodal explants. The combination of 4.4 μM BA and 2.2 μM thidiazuron (TDZ) was found to be the most suitable growth regulator for obtaining the highest percentage of nodal segment sprouting (74%–75%), the number of secondary shoots per primary shoot (two or three), the shoot length (5–6 cm), the number of new nodal segments generated per active explant (four or five), and the multiplication coefficient (3.5) within 6 weeks. Repeated subculturing of nodal explants obtained from shoot cultures enabled continuous production of healthy axillary shoots. At the end of the sixth passage, about 90% of nodal explants produced five or six healthy green shoots, each being about 6.6 cm long with six or seven nodes. Multiplication coefficient was also increased from the first subculture (5.4) to the sixth subculture (8.3). The best rooting response was achieved on solidified half-strength MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 70% of the micropropagated plantlets were established successfully in 20-cm pots containing a mixture of soil and farmyard manure (4 : 1 ratio) and formed new leaflets.     

Effect of Ca on Al-induced activation of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa)

The effect of CA(NO₃)₂ on the active oxygen scavenging system in hinoki cypress (Chamaecyparis obtusa) seedlings cultured in a nutrient solution containing aluminum was examined. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The growth in height and dry matter allocation to each organ was little influenced over a period of 12 weeks by either Al or the concentration of Ca(NO₃)₂. The activity of superoxide dismutase (SOD) in the needles was stimulated by Al, and the effect of Al was lowered significantly by simultaneous application of 25 mM Ca(NO₃)₂. At week 1, the activity of catalase (CAT) in the needles was increased by Al, but the effect was no longer observed at week 12. The Al concentration in the roots was increased by treatment with Al, whereas the Al concentration in needles was not. These results indicate that rhizospheric Al stress stimulates antioxidative enzyme activities in hinoki cypress needles and the activation of the enzymes is suppressed by addition of Ca. The transmission of Al stress to the needles, which induced a change in the enzyme activity, is not caused by the transfer of the Al ion itself from roots to needles. This work was supported in part by funding from the Japan Science and Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.     

Butt rot ofChamaecyparis obtusa (Sieb. et Zucc.) Endlicher trees caused byPerenniporia subacida in Shikoku district, Japan: Pathogen, distribution of damaged trees in the stand, and soil investigation

The rot ofChamaecyparis obtusa (hinoki) trees was studied in the northern part of Kagawa Prefecture, Japan. Among 158 hinoki examined, butt rot was found in 28.6, 58.3, and 100% of trees in the 29, 30, and 34-year-old stands, respectively. All trees with butt rot, and 11 trees without it had rotted roots. Thirteen hinoki were peeled byCervus nippon (sika deer) and all of them were infected with butt rot. The maximum height of rot in deer-damaged trees was 2.6 m from the ground level, but was 1.9 m in undamaged trees. About eighty-two percent of rotted trees showed rotted areas of less than 40 cm² on the cross section of stems at the ground level. White mycelia and black flecks sometimes appeared in the rotted wood. Basidiocarps of polyporaceous fungus were often found on felled logs and rotted stumps of hinoki and identified asPerenniporia subacida. Basidiomycetous fungus was isolated frequently from rotted wood of roots and stems, and determined to beP. subacida by comparative study on cultural characteristics. An inoculation experiment and wood-decay test proved that the fungus was the cause of the rot of hinoki. Few absorbing roots of living trees were found in the clayey subsurface soil of the high dry bulk density and the less soil aeration. Some absorbing roots had root rot and the rot spread from the base of the absorbing root to the central part of the woody root. This is the first report on the rot of hinoki caused byP. subacida in Japan.     

Molecular evidence of the hybrid origins of leyland cypress (×Cupressocyparis leylandii)

Leyland cypress (×Cupressocyparis leylandii) has been regarded as an intergeneric hybrid between Monterey cypress (Cupressus macrocarpa) and Alaska cypress (Chamaecyparis nootkatensis) in the Cupressaceae. Each partial sequence of the 18S-rDNA from nuclear and therbcL from chloroplast DNA genomes was determined using above three species. At the 59th site from the 5′ end of the 152 bp of the determined 18S-rDNA sequence, one base difference was identified between Monterey cypress (T) and Alaska cypress (A). However, Leyland cypress showed either nucleotide A or T at this site. Since nuclear DNA is inherited by biparental mode, this result shows genetic evidence that the nucleic genome of Leyland cypress originates from Monterey cypress and Alaska cypress and that this species is of hybrid origin.     

In Vitro propagation of juvenileNothofagus leoni Espinosa (Fagaceae)

Nothofagus leoni has a restricted distribution in Chilean forests. This work determines suitable culture conditions forin vitro multiplication and rooting through shoots obtained from seedlings. Broadleaved Tree Medium was suitable for shoot multiplication. A medium with a pulse of 0.55 μM BA in the first subculture and two subcultures on BA-free medium resulted in a multiplication rate at day 63 of × 5.7, without callus growth or shoot neoformation. Rooted shoots of good quality (number and length of roots without callus growth) were obtained with 1.23 μM IBA (91.4% of rooting). The first roots appeared at day 11, reaching a higher speed of rooting at day 15.     

Water status of hinoki cypress, Chamaecyparis obtusa, attacked by secondary woodboring insects after typhoon strike

We estimated the water status of six 31-year-old hinoki cypress (Chamaecyparis obtusa) trees that were struck by a large typhoon. Measurements were made of the trunk water-conduction area of xylem, leaf water potentials, and diurnal changes in trunk diameters. The secondary woodboring insects, Callidiellum rufipenne and Ovalisia vivata, on the trees were captured by the sticky-trap banding method. In trees on which these wood borers were captured, water conduction areas were reduced, leaf water potentials were lower, and trunk diameter changes (R) fluctuated during the day, suggesting that the trees had been damaged by the typhoon and were under water stress. The secondary woodboring insects attacked the water-stressed trees.     

Agrobacterium-mediated transformation of lombardy poplar (Populus nigra L. var.italica Koehne) using stem segments

Genetically transformed lombardy poplar (Populus nigra L. var.italica Koehne) plants were regenerated by co-cultivation of stem segments withAgrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI121) which included genes for β-glucuronidase (GUS) and neomycin phosphotransferase. Successful transformation was confirmed by the ability of stem segments to produce calli in the presence of kanamycin, histochemical and fluorometric assays of GUS activity in plant tissues, and Southern blot analysis.     

Effects of patch cutting on leaf nitrogen nutrition in hinoki cypress (Chamaecyparis obtusa Endlicher) at different elevations along a slope in Japan

Leaf nitrogen nutrition of hinoki cypress (Chamaecyparis obtusa Endlicher) was investigated at three positions along a slope over a period of 3 years. At each slope position, nitrogen properties were compared in patch-cut plots (0.06–0.09 ha) and uncut control plots (0.04 ha). Nitrogen cycling at the lower slope was characterized by a higher rate of soil nitrogen mineralization, and higher nitrogen concentration in fresh leaves and leaf-litter. The soil nitrogen mineralization rate and fresh-leaf nitrogen concentration in the patch-cut plots were higher than those in the control plots. However, leaf-litter nitrogen concentration did not differ between the patch-cut and control plots. The results suggest that slope position strongly affects leaf nitrogen nutrition of hinoki cypress and soil nitrogen availability. By contrast, patch cutting does not affect leaf-litter nitrogen concentration. These findings indicated that hinoki cypress would not enhance forest nitrogen cycling through changes in leaf-litter nitrogen concentration after patch cutting.     

Comparison of gene delivery methods for transformation ofRobinia pseudoacacia L. by Ti-plasmid

The binary vector plasmid pIG121-Hm carrying the chimeric neomycin phosphotransferase (npt II), β-glucuronidase (gusA) and hygromycin phosphotransferase (hpt) genes was delivered intoRobinia pseudoacacia L. callus using theAgrobacterium tumefaciens-mediated transformation method and particle gun transformation method. It was determined that adding 10 mg/L of acetosyringone toAgrobacterium infection medium and callus-Agrobacterium co-culture medium could increaseAgrobacterium-mediated transformation efficiency. In contrast, particle gun transformation could successfully deliver plasmid DNA into the callus, but its transformation efficiency was lower and gene expression was transient in comparison with theAgrobacterium-mediated method, suggesting that substantially stronger promoters might be required.     

Progeny differences of hinoki (Chamaecyparis obtusa) and sawara (C. pisifera) against resinous stem canker disease and spatial distribution of damage (disease severity) in a progeny test

The incidence and severity of resinous stem canker disease were investigated in hinoki (Chamaecyparis obtusa) and sawara (C. pisifera) at a progeny test located in Yamatsuri Town, Fukushima Prefecture, Japan. Symptoms of the disease were observed in 307 trees out of 933 investigated trees (32.9%). The damage was more severe on lower slopes than on upper slopes, indicating that micro-environmental factors are causally associated with the occurrence of the disease. The severity of the disease varied both among nine open-pollinated progenies derived from hinoki plus-tree clones and among 13 progenies derived from pollination between hinoki plus-tree clones and the mixed-pollen of hinoki plus-tree clones. The severity of the disease also varied with height above the ground. This tendency was most obvious where the disease was most severe. An estimation of the narrow sense heritability,h ², of 0.214, was obtained from analysis of the 13 progenies derived from pollination with mixed-pollen. The heritability derived from material that had not been subjected to selection against the disease, indicated that breeding to enhance resistance would be possible to control the disease. The interaction between environments and progenies in relation to severity of the disease is also discussed. Ms. Setsuko Chiba and Mr. Mamoru Ueta gave us vigorous supports, and Mr. Haruki Orita, Dr. Tomiyasu Miyaura, and Dr. Shinichiro Ito gave us many helpful comments. We deeply appreciated their cooperation. The study was supported by a grant from the Ministry of Agriculture, Forestry. and Fisheries of Japan.     

Assessment of factors affectingin vitro shoot regeneration from axillary bud explant ofCamptotheca acuminata

Axillary buds from 3-yr.-old seedlings ofCamptotheca acuminata in the greenhouse were cultured on the different basal media with different concentrations of growth regulators for shoot regeneration for studying the effects of different basal media, different concentrations of growth regulators (BA or TDZ), sucrose, agar and pH value on shoot regeneration from axillary bud. The results showed that B5 and WPM media were the optimal basal media and the optimal phyotohormone was BA of 1.0 mg/L or TDZ of 0.1 mg/L; The concentrations of sucrose of 30g/L and agar of 6g/L were most suitable for the shoot regeneration; pH value from 5.8 to 6.6 were broadly effective, but the best at pH 5.8. Foundation item: The research was supported by the Key Project of Chinese Ministry of Education (03061) and Supported by Application Fund of Agricultural Research Production (03EFN216700297). Biography: Wang Hui-mei (1973-), female, Ph.D., Lecturer in Northeast Forestry University, Harbin 150040, P.R. China. Responsible editor: Zhu Hong     

杨树PtRRI基因的组织特异性表达模式分析

[目的]模式植物在木本植物中鉴定的许多重要调控因子家族在木本植物中出现了基因家族成员扩张,但ARRs家族作为细胞分裂素响应调节因子在杨树基因组中成员数量反而减少,其在木本植物中如何行使功能需要进一步研究。[方法]本研究通过生物信息学构建PtRRI启动子与GUS融合表达载体,检测植物激素处理后PtRRI表达量和检测PPtRRI::GUS转基因植株在生根过程中GUS信号等方法,对杨树PtRRI基因的组织特异性表达模式进行分析。[结果]表明:PtRRI在杨树根部、形成层、木质部表达量相对较高,PtRRI转录受6-BA激素诱导,与其在不定根发育过程中激素调控下的表达相一致。[结论]PtRRI基因可能参与杨树的次级生长。     

Thidiazuron-induced somatic embryos,their multiplication,maturation, and conversion in <Emphasis Type="Italic">Cinnamomum pauciflorum</Emphasis> Nees (Lauraceae)

Thidiazuron (TDZ) induced somatic embryogenesis from immature zygotic embryos in Cinnamomum pauciflorum Nees while 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) or picloram only induced callus and/or adventitious buds. The highest induction frequency for somatic embryogenesis was achieved with MS medium (Murashige and Skoog in Physiol Plant 15:473–497 1962) supplemented with 2.5 μM TDZ using torpedo-shaped embryos (3–5 mm in length) as explants. In addition, induction medium was supplemented with 0.8 g l⁻¹ casein, 0.4 g l⁻¹ glutamine, and 10 g l⁻¹ sucrose. Somatic embryos (SEs) initiated from root tips or hypocotyls without callus formation. SEs were maintained and multiplied via secondary somatic embryogenesis. Embryo maintenance medium was similar to induction medium except that TDZ was reduced to 0.5 μM. Secondary embryogenesis was enhanced by supplementation of 5 g l⁻¹ activated charcoal in the culture. The best medium for embryo maturation was MS medium containing 30 g l⁻¹ sucrose and 5 g l⁻¹ Phytagel without plant growth regulators. A typical mature SE consisted of two large cotyledons and a short embryo proper. Approximately 82% of selected mature SEs were able to germinate and 63% could convert into plantlets on germination medium that was composed of half strength MS medium salts, 10 g l⁻¹ sucrose, 3 g l⁻¹ Phytagel, and 5 g l⁻¹ activated charcoal.     

In vitro clonal propagation of mature Tectona grandis through axillary bud proliferation

An in vitro clonal propagation procedure for mature Tectona grandis (teak) trees is described. Multiple shoots were induced from nodal segments through axillary bud proliferation. A shoot multiplication rate of 6.33 was achieved on Murashige and Skoog's (MS) medium supplemented with 10 μM 6-benzyladenine BA and 1 μM 1-naphthalene acetic acid (NAA) during every subculture cycle of 4 weeks. In vitro raised shoots could be successfully rooted (66.66%) on liquid MS medium supplemented with 15 μM NAA, with 1.60 roots per shoot, every 6 weeks of culture. In vitro hardening was carried out in sand soaked with half-strength MS medium (organic free). The plantlets were acclimatized first in a mist chamber and then in polybags in a mixture of soil, sand, and farmyard manure (1 : 1 : 1 v/v) in a shade house.     

拟南芥AtFBDL 1 在植物顶端生长调节中的作用

拟南芥 AtFBDL1 基因是FBD-like基因家族的一员,其编码蛋白含有类似于F-box的结构域。表达模式网络预测结果显示该基因在茎顶端分生组织中高丰度表达,但对于FBD-like基因家族的研究还很少,其功能目前尚不明确。为此,本研究通过组织半定量表达分析和GUS染色显示 AtFBDL1 基因在拟南芥中具有时空表达特异性。结果表明:在真叶形成前和形成初期,该基因主要在茎顶端分生组织和下胚轴区域表达;真叶形成后,该基因在下胚轴的表达明显减少,而主要集中在茎顶端分生组织表达。遗传转化显示:与野生型植株相比,过表达 AtFBDL1 基因的植株生长发育缓慢,抽薹时间推迟3 4 d,莲座叶叶片面积减小,叶片数目平均增多10片,并且伴随有变态叶出现;过表达植株株高比野生型矮,株高最大差值达到12 cm。共表达网络预测 AtFBDL1 与多个与生长素和花发育相关的基因具有共表达关系。以上研究结果表明: AtFBDL1 基因在拟南芥的生长发育过程中,特别是在顶端分生组织分化过程中起重要作用。