Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

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.     

Clonal propagation ofGmelina arborea Roxb. byIn vitro culture

In vitro propagation technique ofGmelina arborea multipurpose and a fast growing tree species was studied. Nodal segment including axillary bud was used as a explant. They were cultured on MS media containing various concentrations (0–10 mg/l) of BAP alone or in combination with 0.002 mg/l of IBA. Nodal segments showed axillary bud proliferation in almost all media tested. MS media containing 0.22 mg/l of BAP alone and 2 mg/l of BAP in combination with 0.002 mg/l of IBA were effective for inducing multiple shoots and shoot elongation. MS medium supplemented with 0.02 mg/l of NAA and 1 mg/l of IBA gave the best result for rooting. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the green house. Adventitious shoots production from stem explants that were taken from regenerated plantletin vitro was also discussed. Stem segments were tested for their morphogenetic potential on MS media with various combinations and concentrations of BAP, zeatin and TDZ. Successfull result was obtained on MS media supplemented with 2 mg/l of BAP and 1 mg/l of zeatin or supplemented with 0.5 mg/l of BAP and 0.5 mg/l of TDZ. The shoots obtained on MS media containing 2 mg/l of BAP and 1 mg/l of zeatin rooted on MS media containing 0.02 mg/l of NAA and 1 mg/l of IBA, and plantlets were successfully obtained. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forest Society (1998).     

Embryo culture is an efficient way to conserve a medicinally important endangered forest tree species Strychnos potatorum

The present study reports a protocol for germination of Strychnos potatorum (ver. Tel. Chilla) using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog’s medium (MS) containing 20 g/L sucrose in comparison to McCown and Lloyd’s Woody Plant Medium (WPM). Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L-1 . WPM/MS medium containing glucose at levels 30, 20, 15 g·L-1 showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0 2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L-1 sucrose. Germination rates improved when the embryos were placed upright (vertically) in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65% 75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture.     

Preservation of Juniperus thurifera L.:a rare endangered species in Algeria through in vitro regeneration

Juniperus thurifera L.is an endemic Cupres saceae from the Aure`s Mountains of north eastern Algeria and endangered,in part,due to the scarcity of viable seeds It is threatened by other abiotic factors and the lack of an effective management strategy will increase its risk o extinction.The dearth of information on its in vitro regeneration impedes its application in forest managemen programs.We therefore developed a micropropagation protocol using microcuttings with auxiliary buds.Cuttings were grown on different combinations of media supplemented with plant growth regulators at different concentrations.The highest number of shoots and branches regenerated from original shoots was obtained on Woody Plant Medium(WPM)supplemented with 6-benzylaminopurine(BAP)(0.5 mg L^-1)and 2,4-dichlorophe noxyacetic acid(2,4-D)(0.25 mg L^-1).The best elongation of shoots was achieved with WPM supplemented with0.5 mg L^-1of BAP and 0.25 or 1 mg L^-1 of 2,4-D.On the second subculture,shoots had a higher number of branches than those of the first.The highest rooting rate,38.8%,was obtained with shoots cultured in 1/2 Murashige and Skoog(MS)medium supplemented with 5.0 mg L^-1each of indol-3-butyric(IBA)and naphthalene acetic acid(NAA).Similarly,the highest root numbers and lengths were produced on 1/2 MS medium supplemented with IBA and NAA(5.0 mg L^-1each).During transfer to acclimatization,rates of plant losses of 50% occurred.The second part of the experiment showed that the best shoot callusing was on WPM supplemented with BAP and 2,4-D,with either the combination 0.5+0.25 or 0.25+0.25 mg L^-1.The results of this research provide a starting point for further studies on in vitro regeneration of J.thurifera for the sustainable management of its unique ecosystem in the Mediterranean basin.     

Shoot multiplication and plant regeneration in <Emphasis Type="Italic">Caragana fruticosa</Emphasis> (Pall.) Besser

Different nutrient media can affect in vitro culturing protocols, and experimentation under varied growth conditions is valuable in plants where in vitro methods are in preliminary stages. We carried out the first in vitro propagation studies for the endangered species Caragana fruticosa (Fabaceae). We evaluated various nutrient media for their impact on shoot elongation and axillary bud proliferation using different concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). Shoot elongation was evaluated based on adventitious shoot primary culture and subculture regeneration from Caragana seedlings. Our goal was to improve both micropropagation and regeneration in C. fruticosa. MS nutrient media was superior to 1/2MS macronutrients, DKW, QL, and WPM for shoot elongation and axillary shoot proliferation. Shoots grown on 1/2MS and WPM exhibited some chlorosis, and shoots on QL produced larger leavers than plants growing on normal medium. The shoot proliferation coefficient on MS media supplemented with 2.22 μM BA and 0.44 μM BA + 2.69 μM NAA was significantly higher than that with other treatments in the primary culture. Shoots on 2.22 μM BA showed a higher proliferation coefficient (3.17) than others in the subculture. Shoots were rooted on 1/2MS medium with the addition of different concentrations of NAA. The optimal concentration for rooting was 0.27 μM NAA (74%). Roots exhibited many stout and long root hairs. Survivl of established plantlets was 82% at 30 days after transfer to soil. Plants established in the green house showed normal growth and displayed no apparent morphological differences compared to stock plants.     

The effect of isolation method on the chemical structure of residual lignin

Two methods are used for the isolation of residual lignin: acidolytic and enzymatic hydrolysis. Recently a two-step procedure that is a combination of enzymatic and acidic hydrolyses was proposed. In this paper, the structures of residual lignins isolated by these three methods are compared. Enzymatic hydrolysis gave lignin with the highest yield (83%); however, it contained high amounts of carbohydrates and protein. The molar mass of enzymatic lignin was the highest, indicating that no cleavage of lignin occurred. Acidolysis gave a significantly lower lignin yield (40%), but this lignin was practically free from impurities. The -aryl ether and lignin-carbohydrate linkages cleaved during the isolation, which was manifested in the decreased molar mass of the lignin as well as in increased phenolic hydroxyl group content. The new two-step isolation procedure gave properties between the preparations of enzymatic and acidolytic hydrolyses. The lignin yield was high (78%), but it contained some impurities, although less than the enzymatic lignin. The lignin-carbohydrate linkages cleaved to some extent, but the -aryl ether linkages remained intact.Y. Sun also affiliated with the Pulp and Paper Research Institute of Canada (PAPRICAN), Pulp and Paper Research Centre, McGill University.     

Clonal variation in root suckering ability of hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Current information on the root sucker ability of hybrid aspen (Populus tremula L. × P. tremuloides Michx.) is in most cases based on clone mixtures. In this study, we could separate the performance among clones by using two experimental sites with monoclonal plots of the crossing. The natural regeneration was followed for 2 years after harvest of the formerly planted stands, clear-cut at 22 and 25 years of age, respectively. We found that clonal differences were present in the number of root suckers produced per area unit and their biomass production. However, all included clones produced sufficiently many root suckers for a successful regeneration. To receive a more complete evaluation of the root sucker dynamics for future selection on the clonal level, further studies are needed where vitality, self-thinning and effects of thinning measures are coupled to the goal of the aspen forestry.     

Regeneration of <Emphasis Type="Italic">Melia volkensii</Emphasis> Gürke (Meliaceae) through direct somatic embryogenesis

Experiments were conducted to study plant regeneration through direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants from seeds of Melia volkensii stored for <3 and >12 months. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP, NAA and 2,4-D (0.5, 1.0 and 2.0 mg l⁻¹) alone, and BAP (0.5, 1.0, 2.0 and 4.0 mg l⁻¹) in combination with 2,4-D or NAA (0.2 and 0.5 mg l⁻¹). After 4 weeks in culture, up to 60% of cotyledonary explants from the seeds stored for <3 months produced direct somatic embryos on BAP (0.5–4.0 mg l⁻¹) in combination with 2,4-D (0.2 mg l⁻¹). The number of somatic embryos ranged from 5 to 14 per explant in BAP (0.5 mg l⁻¹) and 2,4-D (0.2 mg l⁻¹) combination. Only 20% of cotyledonary explants from seeds stored for >12 months produced somatic embryos. Mature zygotic embryos failed to produce any somatic embryos. Subcultures of somatic embryos from cotyledonary explants of seeds stored for <3 months formed clusters of shootlets on semi solid MS and 1/2 MS media. After 6 weeks of subculture on multiplication MS media augmented with BAP (0.5 mg l⁻¹) and IAA (0.2 mg l⁻¹), 70% of the shoot tips formed 4–7 shoots per explant. Up to 33% of the multiplied shoots were rooted in MS medium supplemented with 2.0 mg l⁻¹ IBA. Plantlets developed normally into seedlings in the greenhouse.     

Somatic embryogenesis and plant regeneration inAcanthopanax sciadophylloides

Somatic embryos ofAcanthopanax sciadophylloides Franch. et Sav. were differentiated from both zygotic and somatic embryos and calli, and plants were regenerated from these somatic embryos. A zygotic embryo, enclosed within a small portion of the endosperm, was incubated on Murashige and Skoog (MS) media supplemented with various combinations (range 0–10.0 mg/l) of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D). After 4 months, swelling of the zygotic embryos and callus formation was observed. When the swollen embryos were transferred to MS medium supplemented with 0.5 mg/l of 2,4-D, somatic embryos were formed in one to two months. After subculture on the same medium, new embryos were differentiated from various parts of the older somatic embryos. The calli were cultured on medium supplemented with 2.0 mg/l of 2,4-D and BAP for three weeks. Proliferated calli were transferred to medium supplemented with 1.0 mg/l of 2,4-D and BAP. Somatic embryos were differentiated from the calli within one to two months. Somatic embryos were germinated on half-strength MS medium without plant growth regulators and the plantlets were grown in soil. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995) & First Asia-Pacific Symposium on Forest Tree Genetic Improvement (Beijing).     

Plantlet regeneration from leaf protoplasts ofPopulus euphratica

Protoplasts were isolated from the leaves of sterile plants ofPopulus euphratica Oliv. by using 1% Cellulase “Onozuka” RS and 0.25% Pectolyase Y-23 in 0.6m of mannitol solution. Protoplasts were cultured in modified Murashige and Skoog's (MS) medium which contained no ammonium ions but was supplemented with BAP (6-benzylaminopurine), 2,4-D (2,4- dichlorophenoxy-acetic acid), and 1% sucrose at the cell density of 9×10⁴/ml. Cell divisions occurred in every culture medium, especially in the medium containing 0.5 mg/l of BAP and 0.1 mg/l of 2,4-D, in which callus was successfully induced by successive culture through cell cluster formation. Shoots were regenerated from the callus, and their growth was enhanced on 1/2 MS medium containing 0.8 mg/l of BAP. Finally, shoots were rooted and plantlets were regenerated on 1/2 MS medium without a hormone. A part of this paper was presented at the 106th Annual Meeting of the Jpn. For. Soc. (1995).     

Mechanical characterization of juvenile European aspen (Populus tremula) and hybrid aspen (Populus tremula × Populus tremuloides) using full-field strain measurements

Functional analysis of genes and proteins involved in wood formation and fiber properties often involves phenotyping saplings of transgenic trees. The objective of the present study was to develop a tensile test method for small green samples from saplings, and to compare mechanical properties of juvenile European aspen (Populus tremula) and hybrid aspen (Populus tremula × tremuloides). Small microtomed sections were manufactured and successfully tested in tension parallel to fiber orientation. Strain was determined by digital speckle photography. Results showed significantly lower values for juvenile hybrid aspen in both Young’s modulus and tensile strength parallel to the grain. Average Young’s moduli spanned the ranges of 5.9–6.6 and 4.8–6.0 GPa for European aspen and hybrid aspen, respectively. Tensile strength was in the range of 45–49 MPa for European aspen and 32–45 MPa for hybrid aspen. The average density (oven-dry) was 284 kg/m³ for European aspen and 221 kg/m³ for hybrid aspen. Differences in mechanical properties correlated with differences in density. Part of this article was presented at the 3rd International Symposium on Wood Machining, May 21–23, 2007, Lausanne, Switzerland     

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     

Clones No 1333 ofPopulus alba × P. davidiana and No 1132 ofP. davidiana × P. alba × P. daviana

The hybridization experiment was initiated in 1975, in which the parents ofP. davidiana were collected from Dailing, Heilongjiang Province,P. suaveolens were from Baicheng,P. simonii from Zhaodong of Heilongjiang Province, andP. tremula from Shanxi Province. Clones No 1333 ofP. Alba × P. davidiana and No 1132 ofP. davidiana × (P. alba × P. davidiana F1) had greater genetic variation and heritability in clones tested. (Responsible Editor: Dai Fangtian)     

Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.     

Lignin characterization of triploid clones of Populus tomentosa Carr.

In order to understand the structural characteristics of lignin in triploid clones of Populus tomentosa and its changes in the processes of pulping and bleaching, milled wood lignin (MWL), lignin carbohydrate complex (LCC) and the residual lignin from kraft pulp (KP) and sulfite pulp (SP) were isolated and analyzed by Fourier transform infrared (FTIR) spectrum and ¹³C nuclear magnetic resonance (NMR). The most diagnostic peaks were assigned and the differences were discussed. The spectral patterns reveal that triploid P. tomentosa shows the specific features of hardwood from temperate areas, but in the spectrum of FTIR, the strength ratio of A 1270 cm⁻¹ to A1226 cm⁻¹ is 0.88, higher than the average of hardwood from temperate areas, which will make the lignin delignification more difficult during pulping and bleaching. The LCC from triploid P. tomentosa is mainly composed of xyloglucan and glucuronic acid, and other glucides have much lower ratio. In LCC FTIR, there are three peaks at 1 427, 1 329 and 1 046 cm⁻¹, indicating that both semi-cellulose and cellulose could exist in LCC, and that there might be relationships between cellulose and lignin. Compared with the residual lignin from KP and SP, the condensed structure in KP is more than that in SP.     

Haploid breeding ofpopulus

After 14 year’s growth observation on the plant growth, it was found that obvious segregation appeared among the haploid pollen plants obtained from anther culture ofPopulus Xiaohei T.S. Hwany ex C. Wang et Tung in vitro. By vegetative propagation, 19 clones were selected and field contrast test was carried out. Statistical analysis result showed a significant difference in volume production. They are superior clones which could be selected from among the pollen plants for utilization. A haploid breeding procedure is suggested in the paper. This study was supported by the National Natural Science Foundation of China     

Micropropagation of valley oak shoots from seedling explants

Main and interactive effects of basal medium and cytokinin concentration on bud and shoot development of micropropagated valley oak explants were examined. Stem segments with axillary buds were placed on BTM (Broad Leaved Tree Medium), GD (Gresshoff-Doy) and WPM (Woody Plant Medium) media supplemented with 0.3, 0.7 and 1.5 mg 1^–1 of BAT (6-benzylaminopurine). Overall, BTM and GD media were superior for the micropropagation of this species, and both media promoted development of vigorous and relatively abundant shoots. Conversely, fewer shoots were initiated on WPM medium and those produced were frequently chlorotic. Irrespective of basal medium, the quantity of shoots produced after nine weeks generally increased as the concentration of BAP increased, but rosettes with short internodes and numerous leaves predominated on media supplemented with 0.7 or 1.5 mg 1^–1 of BAR Shoots on media with 0.3 mg 1^–1 of BAP, however, exhibited the long internodes and two or three leaves per shoot characteristic of typical valley oak sprouts, and were most suitable for subsequent micropropagation procedures.     

Somatic embryogenesis from cell suspension cultures of aspen clone

Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P. tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L–1 2,4-D and 0.05 mg·L–1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal me-dium with 10 mg·L–1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension cul-ture in a MS liquid medium supplemented with 10 mg·L–1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.