Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine ( Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non-embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non-embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean ( Vicia faba ) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS-PAGE and non-denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light-yellow non-embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.     

Increases in cytokinin levels in Scots pine in relation to chilling and bud burst

Levels of isopentenyladenosine and zeatin riboside were monitored in buds and needles of Scots pine ( Pinus sylvestris L.) seedlings grown under controlled climatic conditions and in field-grown trees. Extracts were purified by immunoaffinity chromatography and high-performance liquid chromatography. Cytokinin levels were quantified with an enzyme-linked immunosorbent assay. The cytokinin content was low in buds and needles of dormant seedlings but increased during dormancy release, reaching peak values in buds just before resumption of shoot growth. Samples collected in the field also showed a marked increase in the levels of cytokinins just prior to bud burst. Further, the biological activity of applied cytokinins in activating the dormant buds of Scots pine is shown. The results indicate a probable role of cytokinins in seedlings during dormancy release.     

Detection of Intracellular Bacteria in the Buds of Scotch Pine (Pinus sylvestris L.) by In Situ Hybridization

Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.     

Field performance of micropropagated forestry species

Summary Micropropagated plants both from angiosperms (bamboo, birch, eucalyptus, tamarind, teak, willow) and gymnosperms (Douglas-fir, loblolly pine, Monterey pine, and redwood) have been established in the field. Plantlets were regenerated from juvenile explants (via adventitious or axillary buds) as well as explants from mature trees [apical and axillary buds (nodal segments)]. Plantlets regenerated from adventitious buds tend to show early maturation traits (Douglas fir, loblolly pine, Monterey pine). A population of selected clones showed superior performance and yield over seedlings derived from the same trees. Increased biomass production was obtained with plantlets derived from tissue culture ofEucalyptus spp. when compared to seedlings. No morphologic variation was observed in micropropagated plants. Plantlets derived from tissue culture grew very uniformly. Early flowering was observed with plantlets derived from tissue culture (tamarind, teak). Based on a presentation at an International Training Course on the Application of Biotechnology in Forest Trees held in Caracas, Venezuela, May 1991.     

Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana)

Opening of apical and axillary buds of mature Douglas-fir and sugar pine trees was obtained on a newly formulated basal medium (DCR) without growth regulators. Elongation of buds was observed on 1/2 strength DCR with 0.3% activated charcoal (DCR-1). In sugar pine, multiple shoots were obtained when explants on DCR with 0.5 mg/1 BAP for 5–6 weeks were transferred to DCR-1 medium. On subculture, axillary buds again developed when shoots were cultured on DCR with 0.2 mg/1 BAP for Douglas-fir and 0.5 mg/1 BAP for sugar pine. These buds were again elongated on DCR-1 medium. By subculturing 7–10 shoots of Douglas-fir and 2–3 shoots of sugar pine, over 100 shoots can be obtained in a year.Abbreviations BAP N⁶-benzylaminopurine - KN kinetin - NAA -naphthalene acetic acid - IAA Indole-3-acetic acid - MS Murashige-Skoog medium - WPM woody-plant medium     

Specific features of the development of Siberian stone pine megagametophytes and embryos in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitro cultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

Two Endophytic Fungi in Different Tissues of Scots Pine Buds (Pinus sylvestris L.)

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.     

Specific Features of Development of Megagametophytes and Embryos of the Siberian Stone Pine in vitro

Seedlings were grown in vitro from fertilized eggs and immature embryos of the Siberian stone pine. Cultivation of megagametophytes on a hormone-containing Murashige-Skoog medium from the egg formation until the globular embryo stage made it possible to manipulate fertilization and embryogenesis. Immature embryos are the most promising for in vitrocultivation. Their maturation and germination proceed within seven days of cultivation. When zygotic embryos were cultivated, adventitious buds were formed from cells at the cotyledon base and tips. When adventitious buds were subcultivated on a medium containing benzylaminopurine and naphthylacetic acid, organogenic callus and shoots were formed. Thus, cultivation of megagametophytes and embryos of the Siberian stone pine led to the completion of embryogenesis and formation of viable of seedlings.     

用RAPD法研究火炬松愈伤组织发生的遗传特性(英文)

体外培养对于植物的快速繁殖是非常有效的。和其它一些松果类硬木植物一样,火炬松的体外培养成功率却一直很低。本工作研究了不同的基本培养基和低温条件对于火炬松Ｊ－５６, Ｓ－１００３, ａｎｄ Ｅ－４４０等三个品系的成熟合子胚形成愈伤组织、分化出芽、成苗的影响。在不同的基本培养基条件下芽分化的程度差异很大。合子胚经过９－１２周培养,开始分化,形成具有器官发生的愈伤组织（Ｆｉｇ．２ａ）。分化后３周,开始诱导出芽（Ｆｉｇ．２ｂ）,芽的生长快慢不同（Ｆｉｇ．２ｃ,ｄ）。同一个愈伤组织上会生出几个芽来（Ｆｉｇ．２ｅ）。在添加有ＩＢＡ和ＢＡ的ＴＥ培养基上芽生长最快（Ｆｉｇ．１）。低温条件持续 １５天,能增加芽的数量和分化的程度（Ｔａｂｌｅ１）。上述培养基中增加ＧＡ３时表明,ＧＡ３对于根的诱导有决定性的作用。将９８株再生苗转移到特殊的混合土壤上;成活了７５株苗（Ｆｉｇ．２ｆ）。以这三种火炬松的再生苗尖为材料制备ＤＮＡ。用２０个引物进行ＲＡＰＤ分析,结果表明：这三种火炬松苗的扩增产物是相同的（Ｆｉｇ．２ｇ,ｈ＆ｉ）。这说明：用愈伤组织克隆植株的过程中没有引起植物遗传变异。     

Expression of beta-tubulin during dormancy induction and release in apical and axillary buds of five woody species

Cell cycle activity was studied in apical and axillary buds of Norway maple ( Acer platanoides L.), apple ( Malus ' M9 ') , pedunculate oak ( Quercus robur L.), Scots pine ( Pinus sylvestris L.) and rose ( Rosa corymbifera 'Laxa') during dormancy induction and release. Flow cytometric analyses revealed that in dormant buds, cells mainly were quiescent at the G₀/G₁ phase, while in non-dormant buds, a significantly higher frequency of G₂ cells was found in all species. In western blots accumulation of 55 kDa beta -tubulin was found in active growing plant material, whereas in dormant buds the accumulation was much lower or below detection level. It was observed for all species that during dormancy induction the amount of beta -tubulin decreased, while during dormancy release a fast accumulation of beta -tubulin occurred. The dynamics of the beta -tubulin accumulation reflected the dormancy status of tree buds of the five species studied suggesting that the beta -tubulin level might be useful as a marker for the dormancy status in buds of temperate woody species.     

适用于微卫星标记的湿地松、加勒比松DNA快速提取法

以湿地松(Pinuselliottii,PEE),包括古巴加勒比松(P.caribaea var.caribaea,PCC)、洪都拉斯加勒比松(P.caribaea var.hondurensis,PCH)、巴哈马加勒比松(P.caribaeavar.bahmaensis,PCB)3个变种在内的加勒比松,侧枝顶芽为试验材料,使用RETSCHMM400混合球磨仪破碎植物组织,然后利用改良CTAB法提取基因组DNA,完成48个样品仅用1h,1个工作日可提取300个样品以上,DNA分子量大于20kb,0.3g样品可提取DNA20-60μg,能够满足几百次PCR反应;利用所提DNA进行SSR分析,条带清晰,多态性好,说明该方法提取的DNA完全可以满足SSR反应的需要。本研究为SSR标记用于湿地松、加勒比松分子标记辅助选择育种提供了经济、高效、可靠的DNA提取方法。     

Host-plant influence on the population ecology of the jack pine budworm, Choristoneura pinus (Lepidoptera: Tortricidae)

Abstract.

• 1 Newly-emerged, second-instar jack pine budworm (Choristoneura pinus Freeman) establish spring feeding sites preferentially in the pollen cones of their host tree, Pinus banksiana Lamb.
• 2 Laboratory studies showed that the rate of establishment and survival of jack pine budworm on pollen cones was high throughout the entire spring emergence period of the insect.
• 3 In contrast, the rate of establishment and survival of jack pine budworm on vegetative buds was very poor early in the spring. Vegetative buds were only acceptable as feeding sites to the jack pine budworm for a relatively brief period in late spring.
• 4 Field studies showed that the change in population density of jack pine budworm during the spring emergence stage, as expressed by k-values, was a function of the abundance of pollen cones in the stand. Population reduction was greatest in those stands with the fewest pollen cones.
• 5 Direct measurement of spring dispersal by jack pine budworm showed that dispersal and consequent losses to the budworm population were greatest in stands with the fewest pollen cones.
• 6 We conclude that changes in the density of jack pine budworm are strongly influenced by production of pollen cones in the host stand. Because pollen cone production is related to previous years of defoliation by the jack pine budworm, we propose that pollen cones act as a density-dependent factor governing the density of early-stage jack pine budworm.
• 7 The resulting dynamics are compared to those of other budworm species and used to explain observed regional and temporal patterns of jack pine budworm outbreaks.

     

Changes in gene expression of Scots pine buds during the winter and under experimentally altered light and temperature conditions

Vegetative buds of Scots pine ( Pinus sylvestris L.) were collected during the apparently dormant phase in January and February and at the beginning of the growth phase in May. Wintering Scots pine plants were placed in climate chambers in which either the daily photoperiod or the temperature simulated the situation in early spring, whereas the other conditions were characteristic of midwinter.
The amount of total ribosome populations and their in vitro translation capacities were independent of the height of the tree or place of collection, but both were dependent on the season; the amount of ribosomes per fresh weight of buds was lower in spring than in winter, whereas the translation capacity in spring exceeded that in winter. Poly(A)⁺ RNA was purified from the ribosomes and translated in vitro and the translation products were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The polypeptide patterns proved that changes in gene expression can occur in plants growing outdoors even during the season of severe cold. In the climate chamber experiments, lengthening of the daily photoperiod increased the in vitro translation capacity of the buds within 7 days even at temperatures below 0°C, whereas a rise in temperature seemed to cause a more transient stress effect. Both treatments induced alterations in the pattern of in vitro polypeptide synthesis. It is suggested that while improvements in both light and temperature hastened the development of the buds under experimental growth conditions, the lengthening of the day may be the factor which induces a change in wintertime metabolism under natural conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of mature <Emphasis Type="Italic">Pinus sylvestris</Emphasis> buds stored at freezing temperatures

Changes of morphogenic competence in mature P. sylvestris L. buds due to frozen storage were investigated. The highest callus formation was registered on explants stored at –18°C for three months, but on explants stored for five months, it was also higher than in the control. Budding and development of needles in vitro was observed only for buds frozen three to five months. Peroxidase activity was lowest in these buds. In contrast, polyphenol oxidase activity in bud tissues continually increased during frozen storage. Within 10 months of frozen storage the content of starch and sugars in resting buds changed. It may be concluded that changes in composition of non-structural sugars in pine buds after five months of frozen storage are part of metabolic changes leading to loss of morphogenic capacity.     

Does timing of boron application affect needle and bud structure in Scots pine and Norway spruce seedlings?

Loss of apical dominance is a well-known boron (B) deficiency symptom in trees. Recent field studies indicate that B deficiency may cause irreversible damage in emerging leader buds leading to bushy growth, and changes in developing needles in mature Norway spruce trees. We experimentally studied if timing of B application affects needles and buds of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings with low initial B levels. The treatments were: no B (B0); B supply from the beginning of the simulated summer (B1); starting soon after bud burst (B2) and starting at the occurrence of first needle primordia in new spruce buds (B3). At the end of the experiment, B concentration in B1 was 23 mg kg⁻¹ (pine) or 17 mg kg⁻¹ (spruce) and lower in the later applications. In B0 it was at deficiency limit. In B0, B2 and B3, there were fewer sclerenchyma cells, and cavities occurred in vascular cylinders in pine needles, and in spruce buds there were more tanniferous cells in the primordial shoots compared to B1. Furthermore, in all but B1 there was cell collapse in the bud apex of some spruce seedlings. The experimentally induced changes were the same as earlier reported in B deficient conifers in the field, and indicate, similarly as in the field that adequate B is necessary throughout the growing season for healthy growth, particularly for spruce. The differences between spruce and pines are due, at least partly, to the differences in time frame of needle development and in the differences in development of conducting tissues in the buds.     

Effect of different nitrogen nutrients on the viability, protein synthesis and tannin production of Scots pine callus

The effect of two different media on the growth, metabolism and viability of Scots pine ( Pinus sylvestris ) callus cultures was studied. Inorganic nitrogen in the culture media (modified MS) was in the form of either KNO₃ or NH₄NO₃. The cultures were started from buds of mature Scots pine. Growth was poor on the medium with KNO₃, but this compound had a noticeable effect on the metabolism of the callus, which was reflected in alterations in protein and polyphenol synthesis and the pH of the culture medium. Although the fresh mass, water content and viability of the callus decreased when KNO₃ was the exclusive inorganic nitrogen nutrient, protein synthesis was more abundant. Electrophoretic analyses indicated alterations in the patterns of soluble proteins and purified glycoproteins. Phenylalanine ammonia-lyase (EC 4.3.1.5) activities were high in all the calluses, and concentrations of condensed tannins and their precursors, catechins, were higher than in intact buds. The role of inorganic nitrogen nutrition in the deterioration of tissues is discussed on the basis of the effect of ammonium on the metabolism of pine callus.     

Secondary buds in Scots pine trees infested with <Emphasis Type="Italic">Gremmeniella abietina</Emphasis>

In winter 2000–2001, there was a serious outbreak of Gremmeniella abietina Morelet in southeastern Norway. During the outbreak, we noted that injured Scots pine trees (Pinus sylvestris L.) developed secondary buds in response to the fungus attack, and we decided to study the relationship between injury, appearance of secondary buds and recovery of the trees thereafter. For this purpose, 143 trees from 10 to 50 years of age were chosen and grouped into crown density classes. Injury was assessed in detail, and buds were counted before bud burst in the spring of 2002. In addition, a subset of 15 trees was followed through the summer of 2002 to assess recovery. All injured trees developed secondary buds, with a clear overweight of dormant winter buds in proportion to interfoliar buds. Healthy control trees did not develop secondary buds at all. The secondary buds appeared predominantly on the injured parts of the tree; interfoliar buds in particular developed just beneath the damaged tissue. Most of the secondary buds died during the winter of 2001–2002, mainly because the fungus continued to spread after the first outbreak. Many of the remaining buds developed shoots with abnormal growth during the summer. Secondary buds may help trees to recover from Gremmeniella attacks, but this strategy may fail when the fungus continues to grow and injure the newly formed buds and shoots.     

马尾松高效再生体系的建立(简报)

利用组织培养技术快速繁殖针叶树,不仅为植树造林所需大量苗木开辟了一条快速高效的新途径,是生产优良无性系的捷径,而且也是植物基因工程技术应用于针叶树品种改良的前提条件。它将在林木种苗产业化和林木良种化进程中发挥重要作用。针叶树的离体培养和植株再生一直是植物组织培养研究中难度较大的一个领域。近几十年来,随着世界各国对发展无性系育林业的日益重视,针叶树组织培养研究取得了很大的进展[1-3]。马尾松(Pinus massoniana Lamb.)是我国南方主要的造林及绿化树种,其木材和松脂具有重要的经济价值。然而在生产实践中,种子园种子…     

Seasonal Variations in the Occurrence of Indole-3-Acetic-Acid in Buds of Pinus silvestris

Studies with a fluorescence spectrophotometer and gas chromatography confirm earlier results that IAA occurs in buds and sprouting shoots of Scots pine. IAA could be found during the whole vegetation period but not during winter dormancy. The largest amount was obtained during shoot growth in spring and early summer. During the most intensive elongation phase, a gradient could be found in the concentration of auxin with the highest amount in the basal part and a decreasing concentration upwards in the shoot.