Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.     

Histocytological characteristics of Eucalyptus urophylla?��?Eucalyptus grandis shoot apical meristems of different physiological ages

Histocytological characteristics of Eucalyptus urophylla × Eucalyptus grandis shoot apical meristems (SAMs) were described, comparing five outdoor and in vitro sources of akin genotypes differing in their physiological age. The size and the number of cells of the five zones identified within each SAM, i.e. the two tunica layers (L1 and L2), the central mother cells (CMC), the peripheral zone (PZ) and the combination of these four zones (4CZ) varied according to physiological age and plastochron phase. These five zones were significantly larger with higher numbers of cells for SAMs from mature and juvenile trees than for those from physiologically rejuvenated, in vitro mature and in vitro juvenile plants. However, these origin-related differences were not significant for SAMs in their early plastochron phase, to become obvious in a more advanced plastochron stage. Individual cell and nuclear measurements confirmed the rationale of distinguishing within SAM zones, characterized by specific cell and nuclear sizes liable to vary according to physiological age. The various histocytological investigations carried out established that SAM cell characteristics appeared to be the more reliable indicators of phase change. This was particularly true for the nucleoplasmic ratio and for more qualitative differences observed also at the nuclear level. SAM nuclei of the two in vitro origins were more evenly stained by naphtol blue-black, uniformly light for the juvenile source, whereas the mature source showed also darker nuclei. In contrast, SAM nuclei from outdoor origins had more chromocenters, darker and diffusely spread for the mature source than for the rejuvenated and the juvenile origins, where they were more peripherally distributed and where the nucleoli appeared more clearly. These results were discussed with respect to physiological ageing and in vitro culture influence, and suggest a determining influence of SAM cell nuclei on phase change phenomenon of arborescent species.     

In vitro rooting of genetically related Eucalyptus urophylla × Eucalyptus grandis clones in relation to the time spent in culture

In vitro responsiveness of microshoots derived from three genetically related and different age Eucalyptus urophylla × Eucalyptus grandis clones kept cultivated by monthly subcultures was assessed on two rooting media in relation to the time spent in culture. Significant differences of rooting capacity were found between the two 22-year-old half sibling genotypes 147 and 149 according to the concentration of BA added to the media and also to light intensity. The contrast was even more salient with the 1-year-old clone S, which displayed the poorest rooting ability despite being full sibling with clone 147 and much younger. The various criteria observed, i.e. rooting rate, number of roots produced, root length, microshoot height and shoot tip necrosis varied greatly and differently for the three origins according to the successive time replicates of the same rooting protocols, with rooting rates of more than 80% for clone 147 at certain dates. These results were discussed, suggesting the influence of endogenous rhythms as the most rationale cause of the time-related fluctuations of responses observed in steady in vitro environment, notwithstanding possible interferences of non-optimal tissue culture conditions. Such unexpected variations of rootability between closely related genotypes, and the notable interactions pointed out between the time spent in culture and the rooting conditions deserve to be taken into consideration before stating definitive judgment on the rooting capacities of a given genotype for arborescent species.     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

Community analysis of undergrowth vegetation in industrial plantation of Eucalyptus grandis×E.urophylla

基于群落调查方法,对巨尾桉林下植物进行群落学分析.结果表明:巨尾桉林下植物共有维管束植物57科、124属、154种及变种,以禾本科和菊科的种类占优势;林下植物物种组成较为分散,优势属不明显;种子植物53科划分为6个分布区类型和2个变型,以热带分布科为主;种子植物120个属有14个分布区类型和7个变型,以热带分布属为主,植物区系成分较为复杂.林下植被可分为灌木层和草本层,但是分层现象不明显,草本层植物占优势,偶见有少量的层间植物分布.林下物种丰富度表现为草本层>灌木层;而Simpson指数、Shannon-Wiener指数、Pielou均匀度指数则表现为灌木层>草本层,生境空间异质性及人为干扰活动影响了巨尾桉林下植物组成及分布.     

Hydrologic balance,net primary productivity and water use efficiency of the introduced exotic Eucalyptus grandis × Eucalyptus urophylla plantation in south-western China

Aims Land cover changes can disrupt water balance and alter the partitioning of precipitation into surface runoff, evapotranspiration and groundwater recharge. The widely plantedEucalyptustrees in south-western China have the potential to bring about hydrologic impacts. Our research aims to elucidate the hydrologic balance characteristics of the introduced exoticEucalyptus grandis×Eucalyptus urophyllaplantation and to assess whether its high productivity results from high water use efficiency (WUE) or large water consumption.     

Agrobacterium strain specificity and shooty tumour formation in eucalypt (Eucalyptus grandis ×E. urophylla)

Summary To develop a successful protocol forAgrobacterium-mediated transformation in plants it is essential to determine the most efficient bacterial strain/plant genotype interaction. In the present work, we evaluated the susceptibility ofEucalyptus grandis ×E. urophylla to fiveAgrobacterium rhizogenes and twelveA. tumefaciens wildtype strains. The results showed different degrees of virulence, according to the strain tested, indicating that transformation of this eucalypt hybrid by Agrobacterium-derived vectors is possible. All developed tumours showed an autonomous growth when transferred to a hormone-free medium. Some of these tumours formed shoots spontaneously, with a normal phenotype. Polymerase Chain Reaction (PCR) and Southern blot analyses were performed to confirm the absence of the oncogenic T-DNA in plants derived from these shooty tumours.     

A Newly Identified Passive Hyperaccumulator Eucalyptus grandis × E. urophylla under Manganese Stress

Manganese (Mn) is an essential micronutrient needed for plant growth and development, but can be toxic to plants in excess amounts. However, some plant species have detoxification mechanisms that allow them to accumulate Mn to levels that are normally toxic, a phenomenon known as hyperaccumulation. These species are excellent candidates for developing a cost-effective remediation strategy for Mn-polluted soils. In this study, we identified a new passive Mn-hyperaccumulator Eucalyptus grandis × E. urophylla during a field survey in southern China in July 2010. This hybrid can accumulate as much as 13,549 mg/kg DW Mn in its leaves. Our results from Scanning Electron Microscope (SEM) X-ray microanalysis indicate that Mn is distributed in the entire leaf and stem cross-section, especially in photosynthetic palisade, spongy mesophyll tissue, and stem xylem vessels. Results from size-exclusion chromatography coupled with ICP-MS (Inductively coupled plasma mass spectrometry) lead us to speculate that Mn associates with relatively high molecular weight proteins and low molecular weight organic acids, including tartaric acid, to avoid Mn toxicity. Our results provide experimental evidence that both proteins and organic acids play important roles in Mn detoxification in Eucalyptus grandis × E. urophylla. The key characteristics of Eucalyptus grandis × E. urophylla are an increased Mn translocation facilitated by transpiration through the xylem to the leaves and further distribution throughout the leaf tissues. Moreover, the Mn-speciation profile obtained for the first time in different cellular organelles of Eucalyptus grandis × E. urophylla suggested that different organelles have differential accumulating abilities and unique mechanisms for Mn-detoxification.     

An Efficient Procedure to Stably Introduce Genes into an Economically Important Pulp Tree (Eucalyptus grandis × Eucalyptus urophylla)

Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus. Seedlings from Eucalyptus grandis × Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity. After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens. A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype. According to kanamycin resistance and PCR verification, 120 transformants were generated. 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity). Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line. Northern analyses on the 5–16 and 3–23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition. With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses.     

Study on the adaptability of Eucalyptus grandis× E.urophylla in karst hills(Ⅰ):photosynthetic physiological ecology characteristics in spring

以岩溶石山生境的3年生巨尾桉人工林为研究对象,采用LI-6400型便携式光合仪测定巨尾桉在春季的叶片净光合速率(Pn)及其他生理生态因子日变化,同时测定巨尾桉光合-光响应曲线,并通过相关分析和通径分析探讨净光合速率与其他生理生态因子的关系.结果表明:巨尾桉光合-光响应曲线符合Walker的非直线双曲线模型.巨尾桉的光饱和点为1 340 μmolm-2s-1,光补偿点为14.68 μmolm-2s-1,表观量子效率(AQY)为0.06molmol-1,具有阳生植物的特点.净光合速率日变化呈现"单峰型"的特点.蒸腾速率(Tr)、气孔导度(Gs)和大气CO2浓度(Ca)是影响巨尾桉叶片净光合速率日变化的重要因子.巨尾桉有较高的水分利用效率,说明巨尾桉具有适应岩溶石山干旱的特征或避旱策略.     

Shooting Control in Eucalyptus Grandis × E. urophylla Hybrid: Comparative Effects of 28-Homocastasterone and a 5α-Monofluoro Derivative

28-Homocastasterone (28-HCTS), a brassinosteroid, was used to treat in vitro-grown shoots of a hybrid between Eucalyptus grandis and E. urophylla. Treated shoots showed enhanced elongation and formation of new main shoots (the shoots originating directly from the initial explant) at low doses. Coincidently, there was reduced elongation and formation of primary lateral shoots (shoots originating from the main shoot). However, a 5α-monofluoro derivative of 28-HCTS (5F-HCTS) was unable to either stimulate elongation or formation of new main shoots, although it did stimulate elongation of primary lateral shoots. In conclusion, it is quite apparent that exogenously supplied brassinosteroids are able to change shooting patterns in Eucalyptus. These findings have practical biotechnological applications, for example on the improvement of micropropagation techniques for clonal propagation of woody angiosperms.     

Adventitious bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Eucalyptus urophylla × E. tereticornis interspecific hybrid

In Vitro Cellular & Developmental Biology - Plant - A high-efficiency regeneration and genetic transformation system is indispensable for generating desirable traits in important trees such as...     

Targeted re-sequencing and genome-wide association analysis for wood property traits in breeding population of Eucalyptus tereticornis × E. grandis

Globally, Eucalyptus plantations occupy 22 million ha area and is one of the preferred hardwood species due to their short rotation, rapid growth, adaptability and wood properties. In this study, we present results of GWAS in parents and 100 hybrids of Eucalyptus tereticornis × E. grandis using 762 genes presumably involved in wood formation. Comparative analysis between parents predicted 32,202 polymorphic SNPs with high average read depth of 269-562× per individual per nucleotide. Seventeen wood related traits were phenotyped across three diverse environments and GWAS was conducted using 13,610 SNPs. A total of 45 SNP-trait associations were predicted across two locations. Seven large effect markers were identified which explained more than 80% of phenotypic variation for fibre area. This study has provided an array of candidate genes which may govern fibre morphology in this genus and has predicted potential SNPs which can guide future breeding programs in tropical Eucalyptus.     

Influences of medium consistency and shoot density on in vitro shoot proliferation of Populus alba × P. grandidentata

The in vitro shoot proliferation of Populus alba × P. grandidentata was affected by the medium consistency and shoot density, but not by three sizes of vessels. After 4 weeks of culture, the fresh weight and number of shoots per explant on liquid medium were significantly greater than those on agar-solidified medium. In particular, 3.2 shoots, 7 mm or longer per explant, were produced on liquid medium compared with 1.6 shoots per explant or agar-solidified medium. The fresh weight per explant after 4 weeks of culture on liquid medium and agar-solidified medium were 0.68 and 0.25 g, respectively. Increasing the number of shoots per vessel slowed the growth of the explants as measured by fresh weight and the number of shoots produced. There was little difference in the number of shoots produced between vessels with 1 or 2 shoots per vessel, but there were many fewer shoots produced when 3 shoots were placed in each vessel.Journal Paper No. J-11977 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 2210.     

Expression patterns of several floral genes during flower initiation in the apical buds of apple (Malus × domestica Borkh.) revealed by in situ hybridization

The apple (Malus?×?domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4?years) and a long reproductive period between the flower initiation and the mature fruit (14?C16?months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.     

Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique.     

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l⁻¹) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l⁻¹) than of zeatin, but was inhibited by a high (5 mg l⁻¹) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.