Changes in polyamine concentration associated with aging in Pinus radiata and Prunus persica

Endogenous polyamine concentrations in apical buds of peach (Prunus persica (L.) Batsch cv. 'Hakuho') and monterey pine (Pinus radiata D. Don) seedlings, saplings and mature trees were analyzed by high performance liquid chromatography coupled with fluorescence detection to establish physiological correspondences and disparities between the species. Although there were many differences in polyamine metabolism between peach and monterey pine, several similarities were found. Specifically, in both species, the ratio between concentrations of free polyamines and low molecular-weight polyamine conjugates was greater than and less than unity in juvenile and adult plants, respectively. Free putrescine concentration decreased with age in both species.     

Reinvigoration of Pinus radiata is associated with partial recovery of juvenile-like polyamine concentrations

Polyamine concentrations in consecutive radiata pine (Pinus radiata D. Don) grafts of 30-year-old trees on 1-year-old seedling rootstocks were analyzed by high performance liquid chromatography coupled with fluorescence detection to determine whether reinvigoration is associated with the recovery of polyamine concentrations typical of those in juvenile tissues. Reinvigoration of radiata pine was correlated with the attainment of some, but not all, polyamines characteristic of juvenile trees. In response to reinvigoration, free putrescine, the ratio of free polyamines to low molecular weight polyamine conjugates, and the relative content of putrescine versus spermidine plus spermine increased to values approaching those characteristic of juvenile tissue. In contrast, there was no noticeable change in the concentrations of low molecular weight polyamine conjugates during reinvigoration even though these conjugates increased greatly during tree maturation. We conclude that certain polyamines defined as reinvigoration markers can be used in forestry upgrading programs to assess the morphogenic ability of reinvigorated trees.     

Reinvigoration of mature chestnut (Castanea sativa) by repeated graftings and micropropagation

Gradual reinvigoration of adult chestnut (Castanea sativa M. cv. Montemarano) shoots was obtained by serial grafting onto juvenile rootstocks. The phenomenon was evaluated on the basis of percentage of primary nodes regenerating axillary shoots and length and number of shoots (> 10 mm) per primary node. In vitro growth of explants from serially grafted shoots was significantly lower than that of explants from seedlings at the end of the establishment phase. Only microshoots from seedlings and plants that had been serially grafted four times could be subcultured on proliferation medium. Repeated subculture on medium containing a low cytokinin concentration induced progressive reinvigoration of microshoots derived from plants that had been serially grafted four times. The number of axillary shoots per explant increased significantly after six subcultures. After 12 subcultures, microshoots from serially grafted plants showed an increase in stem elongation, rooting and plantlet survival. After in vitro stabilization, there was no difference in in vitro performance between microshoots derived from seedlings and serially grafted plants. Microshoots multiplied from serially grafted plants displayed only a transitory appearance of juvenile traits.     

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.     

Different culture conditions applied to in vitro shoot multiplication of two Eucalyptus benthamii explant sources

Eucalyptus adult material requires more successive subcultures in the in vitro multiplication phase for increased vigor and cellular activity. This study evaluated the endophytic manifestation and shoot multiplication of one 13-year-old Eucalyptus benthamii clone under different culture conditions and used canopy branches(CB) and trunk base material as explant sources. The culture media were wood plant medium(WPM), Murashige and Skoog medium(MS) and JADS(Correia and co-authors medium).Based on the results of the initial multiplication experiment, further tests examined sucrose concentrations and p H. Morphophysiology, dry mass production, endophyticmanifestation and histochemical were determined. Explant sources responded differently to MS and JADS media, but the WPM medium promoted homogeneous development.The responses were similar for both explant sources when sucrose concentrations varied. Shoots died in the absence of sucrose, showed high oxidation at 60 g L-1 and optimal development at 30 g L-1. Endophytes were more evident for shoots from the CB origin. Explant sources responded distinctively to treatment due to physiological and intrinsic genetic factors. Therefore, explant sources, different culture media, sucrose concentration and p H may determine micropropagation success and influence the presence and/or intensity of endophytic manifestation.     

Photosynthesis of oak leaves under water stress: maintenance of high photochemical efficiency of photosystem II and occurrence of non-uniform CO(2) assimilation

Shoot cultures of Quercus rubra (L.) were established from both juvenile and adult plant material. Initial explants from epicormic shoots formed on the basal zone of the trunks had a greater capacity for in vitro establishment than explants from crown branches. The growth of vigorous axillary shoots was obtained by culturing decapitated shoots horizontally on Woody Plant Medium supplemented with 0.2 mg l(-1) of 6-benzylaminopurine. After 3 weeks of culture the shoots were transferred to fresh medium for two more weeks, giving a 5-week multiplication cycle. Efficient shoot production was achieved by combining three treatments favoring the growth of lateral buds: excision of the apex, horizontal culture and cytokinin treatment. The addition of indoleacetic acid or indolebutyric acid to the multiplication medium did not improve shoot proliferation rates, and naphthaleneacetic acid was detrimental. Recycling the same explant for several successive subcultures improved the efficiency of the propagation procedure. Using the optimal multiplication procedures, nine clones (six of juvenile origin and three from adult trees) were tested in vitro and it was found that genotype and age affected performance.     

Micropropagation of Vitex negundo L. and Random Amplified Polymorphic DNA Analysis of Micropropagated Plants

A micropropagation protocol was established for a medicinal plant Vitex negundo. Genetic stability of micropropagated plants was investigated. Multiple shoots were induced from nodal explants cultured on Murashige and Skoog (MS) medium with 0.53 μM naphthalene acetic acid (NAA) and 11.0 μM benzyl aminopurine (BAP) along with additives (ascorbic acid, 283.9 μM; citric acid, 130.1 μM; and arginine, 143.6 μM). Shoots were further multiplied by repeated transfer of the mother explant. The shoots were further multiplied on MS medium + 0.57 μM indole-3-acetic acid (IAA) and 6.6 μM BAP. The micropropagated shoots were pulse treated with 122.5 μM indole-3-butyric acid (IBA), in liquid MS medium and then transferred to autoclaved soilrite. These rooted ex vitro. Shoots were also rooted in vitro on a half-strength MS medium + 2.45 μM IBA. The survival rate of in vitro rooted plantlets was poor during hardening compared to ex vitro rooted plantlets. About 95% of the ex vitro rooted, hardened plantlets survived in the field. Genetic stability of micropropagated plants was tested by using 25 random amplified polymorphic DNA primers. The cloned plants exhibited no variation in banding pattern in comparison with the mother plant.     

Polyamines in embryogenic cultures of Norway spruce (Picea abies) and red spruce (Picea rubens)

Embryogenic cultures of red spruce (Picea rubens Sarg.) and Norway spruce (Picea abies (L.) Karst.) were initiated from dissected mature zygotic embryos. The tissues were grown on either proliferation medium or maturation medium. On proliferation medium, the embryogenic tissue continued to produce early stage somatic embryos (organized meristems attached to elongated, suspensor-like cells), whereas on maturation medium fully mature embryos developed from the embryonic tissue. Analysis of polyamines in tissues grown on these two media showed that: (1) both putrescine and spermidine concentrations were always higher in cultures grown on proliferation medium than in cultures grown on maturation medium; (2) in both species, spermidine concentrations declined with time in the tissues grown on maturation medium; and (3) spermine was present in only minute quantities and showed only a small change with time. The presence of difluoromethylornithine in the culture medium had little effect on polyamine concentration, whereas the presence of difluoromethylarginine caused a decrease in putrescine concentrations in both red spruce and Norway spruce tissues grown on proliferation medium or maturation medium.     

Optimization of rhizogenesis for in vitro shoot culture of Pinus massoniana Lamb.

The rooting capacity of Pinus massoniana is poor,especially for mature trees,and has prevented the development of clonal forestry for P.massoniana.In this study,we varied explant types,subculture times and exogenous hormones for plantlet regeneration and assessed shoots for rooting rate and root number for P.massoniana.Following five repetitive grafts,new shoots from grafts used as explant sources were rejuvenated as observed from juvenile shoot morphology and anatomy,leading to greatly enhanced plant regeneration in comparison to that of mature materials from 26-year-old P.massoniana trees.The rooting capacity of subcultured shoots increased with successive subcultures,reaching a peak at 20 subcultures with 35-40 days per subculture.However,rooting performance was significantly reduced after 30 subcultures.The addition of naphthaleneacetic acid(NAA) plus indoleacetic acid in the medium improved the root number,but the combination of exogenous NAA with paclobutrazol(PBZ) increased rooting rate and root number.We thus greatly improved the rooting capacity of mature P.massoniana trees by optimizing explant types(rejuvenated),subculture times(20 subcultures,35-40 days per subculture) and addition of NAA+PBZ to the rooting medium.The conditions can be used for efficient plantlet regeneration of P.massoniana.     

共生微生物在植物离体快繁中的应用潜力

获得品质优良的种苗是植物离体快繁中的技术关键, 但在生产环节中出现的组培苗玻璃化现象、移栽后的脱水萎蔫伤亡、放养群体低抗逆性等共性问题仍具挑战性.无茵条件下体外再生植株导致与之自然共生的微生物的缺失是其中的重要原因, 而共生菌与宿主植物存在自然的共生关系并对宿主发挥独特的生理功能, 能有效提高存活率, 帮助幼苗抵御移栽后的不良环境; 对某些药用植物而言, 还能促进其次生物质的积累.文中综述了国内外学者将共生菌引入植物快繁技术所取得的成果, 并分析了其应用潜力.     

Propagation method influences first year field survival and growth of Paulownia

Field trials at three locations were used to evaluate survival and early growth of Paulownia elongata, P. fortunei, and P. × 'Henan 1' plants produced from seeds, shoot cuttings, and micropropagated shoots. One year after field establishment, trees produced by either vegetative propagation method survived with higher frequency and were taller and of greater diameter breast height than trees grown from seed. The influence of propagation method was more pronounced in P. elongata and P. fortunei than in P. × 'Henan 1'. Survival frequency was similar for each species/hybrid, but both species were taller than P. × 'Henan 1' across propagation method and site. Field site had a pronounced influence on tree survival and height. Species/hybrid differences were not detected at the site that promoted the greatest tree growth, but P. elongata outperformed P. fortunei and P. × 'Henan 1' at the two sub-optimal sites. Clones within species/hybrid produced through micropropagation differed in survival frequency, height, and diameter breast height. Under the conditions used for this study, Paulownia field survival and early growth in central North Carolina was improved by using P. elongata or P. fortunei produced by rooting shoot cuttings or micropropagated shoots. Significant variation among clones within these species demonstrated the potential benefit of identifying superior clones to improve first year field survival and growth.     

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^-1 6-benzyladenine and 0.2 mg L^-1 a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^-1 NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.     

银杏雄花芽分化期间内源激素、碳水化合物和矿质营养含量的变化

银杏雄花芽生理分化期间花芽中赤霉素 (GA1 3)、脱落酸 (ABA)含量下降 ,玉米素 (ZRs)、异戊烯基腺嘌呤类 (iPAs)的含量以及ZRs GA1 3、iPAs GA1 3、ZRs ABA、iPAs ABA上升且高于叶芽 ;形态分化开始后 ,花芽内ZRs、iPAs的含量以及ZRs GA1 3、iPAs GA1 3、ZRs ABA、iPAs ABA下降且维持低水平 ,花芽内ABA含量和ABA GA1 3在高于叶芽的水平上波动。在银杏雄花芽分化过程中 ,雄花芽叶中钾和总糖含量均高于同期雄叶芽叶 ,而全氮的含量基本低于同期雄叶芽叶 ;生理分化期雄花芽叶内磷含量高于同期雄叶芽叶。     

Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium

We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4+ and NO3- were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein.     

Rooting of Hybrid Clones of Populus tremula L. × P. tremuloides Michx. by Stem Cuttings Derived from Micropropagated Plants

Propagation costs could be cut by replacing part of the micropropagation process with steps involving more traditional techniques. This study explored possibilities for improving existing vegetative propagation techniques for aspen using stem cuttings obtained from micropropagated plants. Vegetative propagation through stem cuttings was studied in 10 micropropagated hybrid aspen clones (Populus tremula L.     

Selection gains for essential oil traits using micropropagation of Eucalyptus polybractea

The value of eucalyptus oil for medicinal purposes is based largely on its cineole content, and the prime commercial species for pharmaceutical grade eucalyptus oil is Eucalyptus polybractea (blue mallee). This study applies a recently developed micropropagation protocol to selected elite oil yielding blue mallee seedlings and investigates the potential of a trial plantation based on micropropagated clones to increase cineole yields. The study aims to assess two micropropagated clones of blue mallee for growth and key essential oil traits in comparison to two related half-sibling families, and to assess consistency for oil traits within clones. After 23 months growth in a plantation, all ramets from the micropropagated clones appeared healthy and showed similar growth forms to the half-sibling saplings at the time of harvest. Total above ground biomass was significantly greater in the half-sibling families compared to their relative clones. Nevertheless, the foliar essential oil concentration and the proportion of the total oil as cineole was greatest in the clones compared to their related half-sibling families, reflecting the oil traits of the ortets. The oil composition profiles were very consistent within clones. This was particularly evident for cineole which had the remarkably narrow ranges of 90–92% in one clone and 92–94% in the other, suggesting strong genetic control of essential oil composition. Overall, the results suggest that micropropagation has great potential for the establishment of blue mallee plantations using genotypes selected for key oil traits. The application of the micropropagation protocol to older plants of blue mallee (for which both key oil and biomass traits are known) is likely to produce greater selection gains for cineole yield.     

32种经济竹种的组培及苗木培育技术研究

选用了32个竹种作为微体快速繁殖材料,采用以芽繁芽的途径实现快繁。当选用竹子种子作为外植体时得到的组培效果最好,其次是幼年竹节芽和小枝芽,成年竹节芽最差。同等条件下,毛环刺竹、实心瓜多竹、孝顺竹、黄金碧玉竹、非洲锐药竹、麻竹和大佛肚竹更易成活和发笋,人面竹的成活率最低。选用蛭石作为育苗基质取得了较为理想的试验结果。用麻竹组培苗(1 a生苗)作为试验材料,得出腐殖土可作为优良的育苗基质,30%锯末+70%红土次之。组培苗移栽后施以NPK复合肥作为叶面肥,配以微量元素的追肥可提高成活率。对竹苗进行封顶处理可促进发笋。播种苗和组培苗便于运输,且成本低,但因竹子种子不易获得,所以组培苗是作为生产竹苗的最佳选择。     

Developmental stages during the rooting of in-vitro-cultured Quercus robur shoots from material of juvenile and mature origin

In-vitro-cultured shoots of clones initiated from shoots of the basal parts (BS) and the crown (C) of two mature Quercus robur L. trees were subjected to rooting experiments to relate rooting with shoot topophysical origin. The BS shoots exhibited morphologically juvenile characteristics and rooted more easily after indole-3-butyric acid (IBA) treatment than C shoots. When naphthylphthalamic acid (NPA) was applied to BS shoots, rooting capacity decreased and root emergence was delayed at least 2 days compared with shoots treated with IBA only. During the first days of the rooting process, endogenous indole-3-acetic acid (IAA) concentration was higher in C shoots than in BS shoots, regardless of whether the shoots were treated with NPA. Mitotic figures were observed in cells from the basal part of both BS and C shoots 24 h after IBA treatment. After 4 days of IBA treatment, the occurrence of histological events differed between BS shoots and C shoots. Cells of BS shoots became meristematic, giving rise to meristemoids and root primordia, whereas no differentiation of root meristemoids occurred in cells of C shoots. Thus, although adult oak material (C shoots) is capable of responding to the initial stimulus of auxin during the adventitious rooting process, the endogenous IAA concentration is not the factor limiting rooting in adult material.     

Influence of environment, fertilizer and genotype on shoot morphology and subsequent rooting of birch cuttings

Differences in rooting ability of birch (Betula pubescens J.F. Ehrh.) cuttings were observed as a result of differences in genotype and physiology of the stock plants. The uniformity in response among cuttings from micropropagated plants compared with cuttings from seed plants confirmed the advantage of using micropropagated plants to study environmental effects. Shoot morphology of the seed stock plants was influenced by both photoperiod and thermoperiod. A day/night temperature of 15/25 degrees C reduced stem elongation compared with a day/night temperature of 25/15 degrees C regardless of photoperiod, and a continuous light regime resulted in more shoots per plant in both temperature regimes than a 16-h photoperiod. A reduction in the supply of macronutrients did not influence shoot morphology, but increased rooting substantially and seemed to override the effects of environmental factors. In cuttings of seed plants, the highest rooting percentage and number of roots were obtained in a 16-h photoperiod with a day/night temperature of 15/25 degrees C. In micropropagated stock plants, there was a positive correlation between shoot length and number of leaves per shoot and topographical distribution of light within the plants, but there was no correlation between these parameters and rooting ability of the cuttings. A rooting temperature of 16 degrees C delayed the rate of root production compared with the rate at higher temperatures, but the final rooting percentage was the same over the range from 16 to 28 degrees C. Root branching increased with temperature. At all temperatures, there was a large increase in sucrose content at the base of the cuttings during rooting, whereas the concentration of nontranslocated sugars remained constant. The carbohydrate content at the base of cuttings from micropropagated stock plants was three times higher than at the base of cuttings from seed stock plants, but the higher carbohydrate content was not correlated with a higher rooting potential.