Cloning and analysis of telomere-associated sequences of <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Total genomic DNA was extracted from leaves of Ginkgo biloba L. by the method of hot CTAB. After determining quantification of DNA sample by microclorimetric spectrophotography, Arabidopsis-type telomere primer and Sau3A I cassette primer were used to isolate telomere-associated sequences from G. biloba L. by the method of cassette-ligation-mediated polymerase chain reaction (PCR). Using this method, two telomere-associated sequences, TAS1 and TAS2, were isolated. The authors preformed Southern hybridization ofEcoR I-treated G. biloba genomic DNA with each clone. The hybridization pattern showed that the clones obtained were derived from G. biloba genomic DNA. There are the Arabidopsis-type TTTAGG tandem repeats in telomeres of G.biloba.     

Monitoring genetic stability inQuercus serrata Thunb. somatic embryogenesis using RAPD markers

Genetic stability of propagules regeneratedvia somatic embryogenesis is of paramount importance for its application to clonal forestry. Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability in somatic embryogenesis ofQuercus serrata Thunb. (Japanese white oak). Forty samples from an embryogenic line, consisting of regenerated plantlets, somatic embryos, and embryogenic calli, were examined using 54 decanucleotide primers. A total of 6520 clear reproducible bands obtained from these studies exhibited no aberration in RAPD banding pattern among the tested samples. Our results show that somaclonal variation is absent in our plant propagation system. The genetic stability is discussed in terms of the origin of somatic embryos.     

银杏枝条部位和年龄对不定根形成的影响及其与非结构碳水化合物含量的关系

[目的]研究银杏枝条部位、年龄对不定根形成的影响,并探讨其相应的叶、茎段中非结构碳水化合物(NSC)含量变化与扦插生根的关系。[方法]以50年生银杏树冠上、下部枝条的1、2和3年生位置的茎段为材料截取插穗,500 mg·L-1IBA与纯水对照处理基部60 s,在全光喷雾状态下研究扦插生根情况,并利用HLPC法分析相应部位叶和茎段中NSC含量。[结果]表明:银杏扦插不定根形成过程分为4个阶段,即0～4周愈伤形成期、4～6周愈伤膨大期、6～8周不定根开始形成期、8周后不定根伸长;生根部位主要在切口处。树冠上、下部位插穗的生根率未见明显差异,但随着枝龄的增加而明显降低。NSC分析表明:下部叶片NSC组分中多糖和总糖含量明显高于上部的,但茎段NSC的所有组分在上下部均无明显差异;随着枝龄的增加,相应位置叶片NSC各组分含量没有明显差异,但相应茎段NSC组分中葡萄糖、果糖、多糖、总糖、淀粉含量却明显降低,与生根率的变化趋势一致。此外,500 mg·L-1IBA处理并未明显提高生根率,还需进一步优化处理浓度。[结论]银杏树冠上部和下部枝条对不定根形成无影响;未处理枝条的枝龄与生根率呈负相关,而茎段NSC含量与生根率呈正相关;叶片NSC含量对不定根形成影响较小;生根方式以愈伤生根类型为主。该结论将为今后银杏扦插生根及其机制研究提供参考。     

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).     

Development of transgenic hybrid sweetgum (<Emphasis Type="Italic">Liquidambar styraciflua</Emphasis> × <Emphasis Type="Italic">L. formosana</Emphasis>) expressing γ-glutamylcysteine synthetase or mercuric reductase for phytoremediation of mercury pollution

Using Agrobacterium-mediated gene transfer, we generated transgenic hybrid sweetgum (Liquidambar styraciflua × L. formosana) overexpressing two types of genes to enhance plant remediation of mercury-contaminated soil and water: bacterial γ-glutamylcysteine synthetase gene (ECS), the first and most important enzyme in phytochelatin synthesis, or various genes encoding a mercuric ion reductase (merA9, merA18, merA77). Hybrid sweetgum proembryogenic masses (PEMs) constitutively overexpressing ECS were able to grow in the presence of 50 μM HgCl₂, which inhibited wild-type PEMs, but plantlets regenerated from the PEMs had abnormal form and did not survive for more than a few weeks following germination. In contrast, mature somatic embryos generated from PEMs constitutively overexpressing merA9 and merA18 converted to normal plantlets on germination medium containing 25 μM HgCl₂, while control embryos were killed on 25 μM Hg(II)-medium. Transgenic merA plantlets displayed enhanced resistance to Hg(II) and released Hg(0) two to three times more efficiently than the wild-type plantlets.     

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that: 1) germination rate of grade 3 embryos in immature seeds with 0.6–0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4–0.6 cm diameter was 77.8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2) The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3) The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata var. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.     

Characteristics of reproductive biology forLarix originating in Japan

The paper summarized the life cycle, environmental features and distributions ofLarix origination in Japan. The time of blooming and fruiting and the ways of cone collection and seeds storage were introduced. The treating methods of seeds germination and the sowing time for both Japanese larch (Larix kaempferi Carr) and Dahurian larch (Larix gmelinii var.japonica Pilger) were discussed. Responsible Editor: Zhu Hong     

Somatic embryo production and plant regeneration of Japanese black pine (Pinus thunbergii)

Somatic embryogenesis in Pinus thunbergii was initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of somatic embryos were obtained on maturation media containing maltose, activated charcoal, abscisic acid, and polyethylene glycol as osmotic agent. The best result among the cell lines tested was achieved with the cell line T-205-3. More than 900 somatic embryos per petri dish, on average, were obtained after about 8 weeks of culture on maturation medium. Sixty percent of somatic embryos tested germinated after transfer to plant growth regulator-free medium and then 85% of them converted into plantlets.     

北京城区银杏行道树生长现状与健康状况研究

The planting site, size, and health condition of street tree Ginkgo biloba in Beijing urban area were investigated and the influence factors were analyzed based on filed survey. The results showed that G. biloba were mainly planted in tree pit within strip planting median. The average height, diameter at breast height and canopy diameter were 8.11 m, 13.22 cm and 4.07 m, respectively, 82.9% trees were less than 10 m in height, and most G. biloba trees were still in the initial stage of height and diameter increment. The tree health status was described with three classes: class I (healthy), class II (fair) and class III (unhealthy). The percentage of class I, II and III for G. biloba trees in Beijing urban area accounted for 16.0%, 37.2% and 42.9% respectively. The results of analysis showed that the health status of G. biloba was associated with the site condition, tree size and management practices. The trees planted in urban forest patch grew better than the trees planted in green belts and tree pits; meanwhile, the bigger trees were healthier than the smaller ones. In addition, the effects of urban heterogeneous environment and human activities on the health of Ginkgo biloba were analyzed and the suggestions on management were launched.     

Transgenic poplar with enhanced growth by introduction of the ugt and acb genes

The transgenic poplar (Populus tremula L.) was obtained by transfer of the ugt and acb genes via triparental mating, which was employed to deliver large fragments of TDNA as a cluster. Freshly harvested seeds of local poplar were placed on MS agar medium and plantlets were obtained. After 1 year of subcultivation, plantlets were infected with a transconjugant of triparental mating with target ugt and acb genes into axillary buds. The transformed sprouts so obtained were cut and subcultivated on agar medium with an addition of 0.6 mg/l indole-3-butyric acid as an auxin source. The transformed sprouts showed GUS activity and resistance to gentamycin and kanamycin. The integrity of the target ugt and acb genes into poplar genome was demonstrated via PCR and Southern blot hybridisation. The transgenic poplar plants revealed a higher growth energy, corresponding to a higher content of IAA as opposed to control plants. Both transgenic and non-transformed plants were potted into soil for outdoor acclimatisation and subsequently transferred to earth in beds. Growing outside during 3 years, the transgenic poplar demonstrated a higher growth rate with fast bud and branch development.     

Plant regeneration by somatic embryogenesis from mature seeds of <Emphasis Type="Italic">Magnolia obovata</Emphasis>

We attempted to develop a method for the regeneration of plantlets from mature seeds of medically important Magnolia obovata via the induction of somatic embryogenesis in vitro. We initially cultured halves of mature seeds on either Murashige and Skoog (MS) medium or B₅ medium that contained 0, 1, 5 or 10 μM gibberellic acid (GA₃) for 1 month and then transferred the half-seeds to half-strength MS basal medium or B₅ basal medium for further culture in the absence of GA₃. Proembryogenic masses (PEMs) were observed 1 month after the transfer of the halved mature seeds to the medium without GA₃. The frequency of formation of PEMs was higher (28%) after initial culture in MS basal medium plus 1 μM GA₃ than in other tested media (0 or 4%). Somatic embryos that had been developed from PEMs were cultured on half-strength MS basal medium or B₅ basal medium for completion of maturation and then transferred to fresh aliquots of the same medium for initiation of germination. The frequency of germination, with the formation of normal primary leaves and roots, was above 80%. We transferred the somatic embryo-derived plantlets to soil for acclimatization and the plantlets continued to thrive.     

Selection, regeneration and protein profile characteristics of NaCl-tolerant callus ofRobinia pseudoacacia L.

Changes in callus growth, differentiation potency and protein profile induced by salt stress were investigated in the calli induced from a hypocotyl ofRobinia pseudoacacia L. The NaCl treatment evidently obstructed the callus growth and the influences seemed to continue to be present in the selected calli even after they were restored in NaCl-free medium that could partially recover the callus growth, and some polypeptides were found to restore. The calli selected and restored intermittently every two weeks for one year completely lost their differentiation potency. The NaCl-tolerance test of the regenerated plantlets revealed that part of the plantlets that directly regenerated from the selected NaCl-tolerant calli were able to survive in 0.2 M NaCl, while most of the plantlets regenerated from the NaCl-nonselected calli or from restored calli were obviously damaged by this treatment. The SDS-PAGE assay indicated that a 54 kDa polypeptide increased distinctly to a very high level while a 41 kDa polypeptide decreased to a lower level in almost all the NaCl-tolerant plantlets. This observation led to the suggestion that the changes may play an adaptive role in allowing plantlets to survive under saline conditions.     

Caulogenic callus induction and adventitious bud formation from embryos of long-term stored seeds ofPicea jezoensis

Caulogenic calli with a high differentiation potency were induced from mature embryos ofPicea jezoensis seeds stored over a long time, for 29 years, resulting in the active formation of adventitious buds. Embryos began to induce calli within 3 weeks of cultivating on LP medium containing 3 μM BAP and 1 μM 2,4-D. Then, the calli proliferated and transformed into caulogenic calli with bud primordia in 8 weeks. The caulogenic calli increased actively with the addition of 500 mg/l ofl-glutamine in the medium. Furthermore, caulogenic calli, induced on LP medium containingl-glutamine, resulted in the formation of adventitious buds, which elongated after transferring the calli into LP medium with 0.1 μM BAP, but withoutl-glutamine. It appears that the number of adventitious buds and the process of shoot elongation are influenced by the kind of nitrogen contained in the medium for callus induction. A part of this study was presented at the 107th Annual Meeting of the Japanese Forestry Society (1996).     

In vitro shoot development ofEucalyptus citriodora on Rockwool in the film culture vessel under CO2 enrichment

An efficient system for growingin vitro plantlets ofEucalyptus citriodora Hook was developed. In the conventional closed system of culture with 2% sugar-containing gellan gum Murashige and Skoog (MS) medium supplemented with 0.02 mg/l indole-3-isobutyric acid, a serious defoliation of shoots was observed after three weeks. In contrast, plantlets grown on the sugar-free MS medium in the aerated bottle under 3,000 ppm CO₂ enriched condition did not show any defoliation. A marked enhanced growth of plantlets and no defoliation were observed on rockwool with the sugar-free liquid MS medium in the “Culture Pack”, made of fluorocarbon polymer film, under CO₂ enriched condition. CO₂ enrichment for this sugar-free “Culture Pack”-Rockwool system was also found to contribute to an improved growth of the plants in acclimatization. A part of this paper was presented at the 106th Annual Meeting of the Japanese Forestry Society (1995).     

Relationship between resistance to vole browsing and content of ether extract in the bark of larch species and hybrids

We determined the survival rate of tree families produced by the intra- and interspecific crossing of larch species damaged by browsing of vole (Clethrionomys rufocanus bedfordiae Thomas) and the contents of ether extracts (EE) in their bark. The average survival ofLarix gmelini var.japonica Pilg. (L. gmelini)×L. gmelini (G×G) was highest, followed by the hybridL. gmelini×L. leptolepis Gordon (G×L). The families of the hybrid G×L, which is considered suitable for silviculture in Hokkaido, Japan, showed significant differences in survival rates, suggesting that it is possible to produce improved varieties of the hybrid which are more resistant to vole browsing. The EE contents of the bark were under strong hereditary control, and the rank of the EE content among the various families hardly fluctuated between the location of the test fields. The correlation coefficient between the survival rates and the EE contents was highly significant (r=0.89,n=24,p<0.01), indicating that the EE content in the bark may be used as a possible index of resistance or susceptibility to vole browsing in larch plantations. A part of this report was presented at the 106th Annual Meeting of the Japanese Forestry Society, at Sapporo, April, 1995.     

New parasitoids of<Emphasis Type="Italic"> Saperda populnea</Emphasis> (L.) (Col. Cerambycidae) in Bulgaria

The parasitoid complex of Saperda populnea (L.) (Col., Cerambycidae) was studied during the period 2002–2003 at different sites in Bulgaria. Galls containing cerambycid larvae were collected on branches of aspen (Populus tremula L.), goat willow (Salix caprea L.) and hybrid poplars and were studied under laboratory conditions. As a result, four new species for the parasitoid complex of S. populnea in Bulgaria were recorded: Dolichomitus tuberculatus (Geoffroy), Xylophrurus lancifer (Gravenhorst), Gelis ornatulus (Thomson) (Hym., Ichneumonidae) and Dendrocerus serricornis (Boheman) (Hym., Megaspilidae). D. serricornis and G. ornatulus are new species for Bulgarian fauna. D. tuberculatus and X. lancifer were found as primary parasitoids, and G. ornatulus and D. serricornis as hyperparasitoids. The primary parasitoids developed solitarily in the host larvae. D. serricornis was reared gregariously from a puparium of Billaea irrorata (Meig.) (Dipt., Tachinidae). The host of G. ornatulus was not identified, but the hyperparasitid could be connected with Iphiaulax impostor (Scop.) (Hym., Braconidae). The mortality of S. populnea caused by the parasitoids was 2.0–6.3%.     

北京市3种道路防护林春季滞尘规律研究

[目的]研究不同道路防护林的滞尘能力及滞尘的动态变化和空间分布规律。[方法]在春季对3种道路防护林(油松林、圆柏林、银杏林)距道路不同宽度的滞尘量进行连续观测,对比3种道路防护林滞尘能力,分析降雨、极大风速、相对湿度、PM10等因子对滞尘动态的影响以及3种道路防护林滞尘的空间分布特征。使用单位叶干质量滞尘量(mg·g-1)表征叶面滞尘能力。[结果]表明:(1)3种植物叶面滞尘能力差异显著,圆柏银杏油松,分别为4.79±0.20、2.48±0.07、1.42±0.04 mg·g-1,单株和单位林分面积滞尘量均为圆柏林油松林银杏林;(2)3种道路防护林在外界影响下滞尘量处于动态变化之中,油松林具有比银杏和圆柏林更高的滞尘稳定性;(3)降雨量较低时3种道路防护林滞尘量均增加,降雨量较高时3种道路防护林滞尘量均降低,油松和银杏林的滞尘量更容易受降雨影响而降低;随着风速增大,3种道路防护林滞尘作用不断加强,风速继续增大时,油松和圆柏林滞尘量均有减少,银杏林滞尘量仍有显著增加。(4)3种道路防护林滞尘量在五环路侧(北)均高于香山路侧(南),油松和圆柏林均呈现为道路防护林中间位置为最低点,银杏林中间位置滞尘量最高。(5)油松和圆柏林滞尘量外部比内部变化大,银杏林滞尘量内部比外部变化大。[结论]道路防护林的滞尘效益受树种、林分结构、所处环境、天气条件等多方面因素共同影响,在营建和管理过程中应充分考虑各种因素,充分发挥滞尘作用。     

Comparative pharmacokinetics and bioavailability studies of quercetin, kaempferol and isorhamnetin after oral administration of Ginkgo biloba extracts, Ginkgo biloba extract phospholipid complexes and Ginkgo biloba extract solid dispersions in rats

The aim of this study was to improve the oral bioavailability of Ginkgo biloba extract (GBE) through preparing G. biloba extract phospholipid complexes (GBP) and G. biloba extract solid dispersions (GBS). Firstly we prepared the GBP and GBS and studied their physicochemical properties by differential scanning calorimetry (DSC), powder X-ray diffraction (XRD) and dissolution. Then we studied the pharmacokinetic characteristics and bioavailability in rats. The results showed that the bioavailability of quercetin, kaempferol and isorhamnetin in rats was increased remarkably after oral administration of GBP and GBS comparing with GBE. The bioavailabilities of GBP increased more than that of GBS.     

Geographic variation pattern ofLarix olgensis

On the basis of the data of provenance test for 10 years in the total natural range ofLarix olgensis from 10 seeds collection sites, the geographic variation patterns have been made by means of canonical correlation analysis: (1) The basic variation patterns of growth characters is in vertical gradual change along the elevation gradient as principal, and gradual change along the latitude as supplement. (2) The Xiaobehu provenance with low elevation and low equivalent latitude is the good gene resource center ofL. olgensis. It has the genetic characters of rapid growth, high stability and fine timber quality. (3) The synthetical interaction between water and heat factors is the major factor to produce the variation ofL. olgensis and the temperature is the principal one. (4) Among the genetic variations of geographic population characters, the variation of growth is the most obvious one, and it could be taken as the main character for provenance division. (5) By allocating the seeds from the low equivalent latitude region to the northern suitable afforestation areas, the greater genetic gain could be obtained. (Responsible Editor: Chai Ruihai)     

Provenance trials ofJuglans mandshurica

The data of provenance trial onJuglans mandshurica young tree were investigated and studied from different provenance. Analysis methods of variance and correlation analysis were used to analyze these data. The following conclusons were obtained: (1) The difference of growth characters ofJuglans mandshurica is relatively significant among provenances: (2) The result of first trial has indicated thatJulans mandshurica growth trend expresses two-way gradual change trend with different longitude and latitude. but the second trial has no obvious gradual change trend. Height ofJuglans mandshurica is negatively correlated with elevation. (3) The provenance of Shulan. Mao’ershan and Baishishan were selected as superior provenance of afforestational seeds for Mao’ershan and its brink region. Responsible Editor: Zhu Hong