The mechanics of anchorage in seedlings of sunflower, Helianthus annuus L.

Forces applied to plants will subject many of the roots to tension, which must be transferred to the soil via shear if uprooting is to be prevented. The stress distribution will depend on the relative stiffnesses of the earth and root, and the mode of failure will depend on the relative strength of the soil and of the root soil bond. This study of the anchorage of sunflower radicles combined uprooting tests performed by a tensile testing machine with mechanical tests on the roots and soil.
The maximum extraction force increased with length to an asymptotic value and was reached at a very low displacement. Root hairs and soil particles covered the tapered top 20 mm of extracted root, but the lower cylindrical region was bare. The soil was stiffer than the root, so shear stress was initially concentrated at the top of the root, soil strength over the top 20 mm resisting uprooting. Lower regions of the root were stressed later, their sparser root hairs being sheared off, and resist uprooting only by friction. In a further lest upper and lower regions of radicles were uprooted separately. As predicted, the upper region generated much greater resistance to uprooting per unit length, and at much lower displacements than the lower region.
The top of the radicle is well adapted for anchorage, the profuse root hairs and mucigel it produces glueing the root to the soil. The lower regions are thus protected from damage.     

Agrobacterium tumefaciens-mediated transformation of hybrid larch (Larix kaempferi T L. decidua) and transgenic plant regenerationn

A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 μm coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3 months on selective medium, the transgenic tissue remained embryogenic, which allowed production of transgenic plants in the greenhouse. Stable integration of the transgene was confirmed by PCR and Southern hybridisation on transformed tissues and acclimatised plants. Received: 4 July 1996 / Revision received: 25 November 1996 / Accepted: 10 December 1996     

Pathogenicity of the pinewood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings

Pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus, to Japanese larch, Larix kaempferi, seedlings was tested with inoculation experiments under nursery conditions. Water suspensions of nematodes (mixed stages cultured on Botrytis cinerea or dispersal fourth-stage juveniles (DJ4) extracted from the adult Japanese pine sawyer, Monochamus alternatus) were injected into the stems of 2- and 3-year-old Japanese larch and Japanese black pine, Pinus thunbergii, seedlings growing in a nursery. In another treatment, Japanese pine sawyer adults holding DJ4 were released under a net that covered the upper half of the seedlings. Regardless of nematode inoculation method, Japanese larch seedlings were as susceptible as Japanese black pine seedlings to B. xylophilus under nursery conditions. The rate of disease development was similar on larch and pine seedlings. Nematode population densities were lower in the stems of dead larch seedlings than in the stems of dead pine seedlings. Histopathological observations revealed that the distribution of nematodes in the stems of dead larch seedlings was mostly limited to the cortex, phloem and cambial zone. Traumatic resin canal formation was one of the most characteristic symptoms in larch seedlings which was dissimilar to that in pine seedlings.     

Identification of larch species (Larix decidua, Larix kaempferi and Larix X eurolepis) and estimation of hybrid fraction in seed lots by RAPD fingerprints

Species-specific RAPD markers were used to identify the different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Although morphological differences between pure species and the hybrids exist, differentiation is not always possible, especially at an early stage (seed or plantlet). Eleven RAPD markers differentiated the two larch species, and 4 species-specific markers were sufficient to estimate the F₁ hybrid fraction in a seed lot. The species-specific markers were tested on individual trees of European and Japanese larches of diverse geographic origins and on several seed lots of different origins (F₁, F₂ hybrids and pure species). The 4 specific markers found for the European larch and the Japanese larch were monomorphic and present in all provenances and in all F₁ hybrid trees tested. Polymorphic SCAR fragments were obtained for 3 of the 11 fragments originally selected for the RAPD screening phase. For 2 of them, the sequence had some homology with the mitochondrial genome of other organisms and is thus mitochondrial. The two mitochondrial fragments and the OPF-13₁₀₀₀ fragment exhibited one polymorphic band, thereby maintaining its species-specific identity: OPF-13₁₀₀₀ is specific to the European larch. The 4 RAPD primers selected in this study offer a reliable, quick and cheap tool for the identification of different larch species (Larix decidua and Larix kaempferi) and their interspecific hybrid (Larix X eurolepis). Received: 28 February 1999 / Accepted: 29 April 1999     

Winter water relations of timberline larch (Larix leptolepis Gord.) on Mt. Fuji

 A krummholz mat of larch, Larix leptolepis, is the predominant growth form near the upper limit (near 2500 m) of the timberline ecotone on the south slope of Mt. Fuji, Japan. On the south-eastern slope, the tree line is lowered to 1600 m because of the last volcanic eruption. The extent and causes of winter desiccation were compared in timberline larch between 2500 m and 1600 m elevation over two winters. Bark abrasion due to wind-blown fine volcanic gravels caused a decrease in bark resistance to water loss and resulted in severe desiccation damage to current-year shoots of krummholz larches in the winter of 1986 – 87 at 2500 m, whereas abraded shoots at 1600 m maintained high water content during both winters. In the winter of 1985 – 86, shoots of krummholz larches at 2500 m did not experience bark abrasion and high water contents were maintained. Experimental abrasion of shoot surfaces resulted in similar results at each elevation. Thus, in timberline larch at 2500 m, abrasion by wind-blown fine volcanic gravels is the primary factor causing winter desiccation damage and krummholz formation. Based on field experiments, the estimated amount of water movement to non-abraded shoots was the same for the two elevations. At 2500 m, water movement to abraded shoots was less than to non-abraded shoots, but the reverse situation was noted at 1600 m. Water supply to abraded shoots at 2500 m was limited and insufficient to compensate for water loss. A cause of limited water supply at 2500 m may be xylem embolism. Received: 13 January 1995 / Accepted: 31 January 1996     

兴安、长白及华北落叶松RAPD分子标记的物种特异性鉴定

以中国北方地区主要乡土落叶松树种兴安落叶松（Larix gmelini）、长白落叶松（L.olgensis）和华北落叶松（L．principis—rupprechtii）的针叶及种子胚乳为研究材料,采用RAPD分子标记技术对3种落叶松进行不同物种的种间鉴别。结果表明,通过引物筛选得到了4个可以鉴别3种落叶松的RAPD引物,其中有2个引物在落叶松针叶和种子胚乳基因组中都扩增出相同的条带。引物OPB-11在兴安和长白落叶松基因组DNA中1500bp处扩增出特异条带,而在华北落叶松中没有：引物OPX-14在兴安落叶松基因组DNA中1200bp处扩增出特异条带,而在长白落叶松中没有：还有2个引物可分别作为3种落叶松苗木和种子鉴别的辅助标记。本研究从分子水平上为落叶松的种间鉴别提供了新的鉴定方法。     

Adventitious shoot formation in cultures of 30-year-old Larix decidua,L. leptolepis,L. eurolepis,and L. laricina trees

Primordial shoot explants excised from buds of one Larix decidua tree, about 30 years old, produced more adventitious buds, elongating into shoots, when grown on half strength Litvay medium than when grown on other basal media. Thidiazuron and N₆-benzyladenine (BA) were equally effective in adventitious bud induction. In a comparative study of 30-year-old L. decidua, L. leptolepis, L. eurolepis, and L. laricina trees, explants from L. eurolepis and L. decidua produced a high number of cultures with adventitious buds that elongated into shoots; those from L. leptolepis were less productive, and those from L. laricina failed to form adventitious buds. The highest response was obtained with material collected in August and September, and in March and April; the lowest response occurred in explants from the October collection.     

Nitrate uptake kinetics for rooted cuttings of Acer rubrum L.

Red maple (Acer rubrumL.) occurs in a broad spectrum of both woodland and managed landscapes in much of eastern North America. Earlier work has indicated considerable plasticity within the species in water use efficiency, as well as dry mass accumulation and tertiary root extension when grown in a solution in which all N was supplied as NO₃ ^-. Nitrogen uptake is largely a function of N supply, soil water availability and root surface area. Therefore, compensatory mechanisms could have evolved among cultivars to compensate for site differences. The objective of this study was to define NO₃ ^- uptake parameters for red maple in a hydroponic system through the use of two cultivars known to differ in root production and water use efficiency. Green wood stem cuttings of two red maple cultivars, `Autumn Flame' and `Franksred', were rooted and then transferred to solution culture. Using established techniques, three separate experiments were conducted to define ranges of I_max, K_m and C_min values and to determine root mass, length, mean radius and surface area. Estimates of I_max exhibited considerable variation between experiments with means ranging from 1.57E-5 to 5.91E-5 μmol cm^-2 s^-1. However, cultivar differences in I_max were statistically significant in only one of the three experiments. Means for K_m ranged from 2.04E-1 to 5.23E-1 μmol cm^-3, while C_min values were consistent at 0.001 μmol cm^-3 (the limit of analytical detection) across all experiments. While data on variation among red maple cultivars are not conclusive, some evidence exists for genotype differences that may relate to the broad range of site conditions under which red maple is successfully grown. This revised version was published online in June 2006 with corrections to the Cover Date.     

Gas-Exchange Properties of Salt-Stressed Olive (Olea europea L.) Leaves

The effects of two levels of salinity on photosynthetic properties of olive (Olea europea L.) leaves were observed either in low or in high H₂O vapor pressure deficit (vpd). Under moderate salt stress, stomata were found to be less open and responsive both to light and vpd, but the predominant limitation of photosynthesis was due to the mesophyll capacity of CO₂ fixation. We elaborate a procedure to correlate mesophyll capacity and liquid phase diffusive conductance. The estimated liquid phase diffusive conductance was reduced by salt and especially by high vpd; morphological and physiological changes could be responsible for this reduction. As a result, the chloroplast CO₂ partial pressure was found to decrease both under salt and vpd stress, thus resulting in a ribulose-1,5-bisphosphate carboxylase limitation of assimilation. However, under combined salt and vpd stress, O₂ sensitivity of assimilation increased, as would be expected under conditions of limiting ribulose 1,5-bisphosphate regeneration. Fluorescence induction measurements indicated that, under these conditions, energy supply may become limiting. When Cl⁻ concentration exceeded 80 millimolar in tissue water, zero growth and 50% leaf drop was observed. Fluorescence induction showed irreversible damage at Cl⁻ levels higher than 200 millimolar and basal leaves reached this concentration earlier than the apical ones.     

Ectomycorrhizal fungal community of naturally regenerated European larch (Larix decidua) seedlings

An investigation was undertaken to assess the community structure of ectomycorrhizal (ECM) fungi on naturally regenerating European larch (Larix decidua Mill.) seedlings grown under forest conditions. The sites examined were in two managed monoculture larch forests, differentiated by soil chemistry and mature tree density. Morphological and molecular analyses revealed a total of 22 fungal taxa. From detected ECM fungal taxa, 13 were noted at Site I and 13 at Site II. Only four taxa were found in both sites (Russula ochroleuca, Thelephora terrestris, Lactarius tabidus and Paxillus involutus). The most abundant species at Site I (lower mineral concentration, high tree density) was Hydnotrya tulasnei (25.7?%), followed by Pseudotomentella tristis, Tomentella sublilacina and Russula puelaris. At Site II (higher mineral concentration, low tree density) the dominant fungal symbiont of larch seedlings was clearly Wilcoxina mikolae, which accounted for 74?% of mycorrhizal tips. The less abundant species comprised T. terrestris, L. tabidus, Xerocomus pruinatus and R. ochroleuca. The analysis of similarity (ANOSIM) and non-metric multidimensional scaling (NMDS) ordination clearly separated the ECM fungal assemblages in the two sites tested. Because our study sites were differentiated by many factors, it is not easy to distinguish one factor in particular to explain the differences observed between the ECM communities at Sites I and II. The results obtained significantly increase our knowledge about the diversity of the ECM fungi hosted by L. decidua. The large number of ECM fungi detected was the first observation showing these fungi as symbiotic partners of European larch.     

Hydroponic versus rooted growth of Zostera marina L. (Eelgrass)

Seagrass fragments and seeds are important dispersal mechanisms by which individuals can be transported to new habitats. While dispersal distances of these free-floating stages have been recently investigated in some detail, almost nothing is known about how long fragments or seedlings may remain viable in the water-column. This study reports on the results of an experiment in which both mature and seedling life-history stages of the temperate seagrass, Zostera marina L. were successfully maintained hydroponically over a 1-month-period. It is suggested that a potential application of this hydroponic growth approach could be seedling culture for restoration activities.     

Development and characterization of microsatellite loci in western larch (Larix occidentalis Nutt.)

Western larch (Larix occidentalis Nutt.) is an important ecological and commercial species in the Pacific Northwest. We isolated nine microsatellite loci with variable polymorphism ranging from five to 19 alleles per locus. Observed and expected heterozygosities averaged 0.42 and 0.64 and ranged from 0.11 to 0.83 and from 0.48 to 0.80, respectively. These markers, along with those already existing, will be useful for the species' gene resource management activities.     

Root induction in pine (Pinus) and larch (Larix) spp. using Agrobacterium rhizogenes

Root induction on in vitro adventitious Pinus monticola Dougl. shoots from mature embryos was improved after 8 weeks co-cultivation with Agrobacterium rhizogenes Conn. strain A4 or the pRi transconjugant strain R1000 as compared to controls, and to naphthaleneacetic acid and indoleacetic acid treatments. An improvement in the number and quality of roots induced was observed on co-cultivated shoots as well as co-cultivated Pinus banksiana Lamb and Larix laricina (DuRoi) K. Koch de-rooted seedlings. Changes in substrate nutrient solution did not influence rooting as much as the presence of Agrobacterium in the rhizosphere. Rooting of cuttings from 3-year-old P. monticola trees was improved in two different seedlots with A. rhizogenes A4 strain.     

Phylogeography of Larix sukaczewii Dyl. and Larix sibirica L. inferred from nucleotide variation of nuclear genes

We investigated phylogeography of Larix sukaczewii and Larix sibirica using nucleotide variation at three following nuclear gene regions: 5.8 S rDNA including two internal transcribed spacers (ITS), cinnamyl alcohol dehydrogenase (CAD), and phytochrome-O (PHYO). We also included sequences of the 4-coumarate: coenzyme A ligase (4CL) gene region obtained in our recent study. CAD and PHYO showed very low nucleotide variation, but ITS and 4CL had levels of variation similar to those reported for other conifers. Pleistocene refugia have been hypothesized to exist in the Southern Urals and South Central Siberia, where four out of nine of the investigated populations occur. We found moderate to high levels of population differentiation (F _ST = 0.115 – 0.531) in some pairwise comparisons suggesting limited gene flow and independent evolution of some refugial populations. In L. sukaczewii, low levels of differentiation were found among populations from areas glaciated during the Pleistocene, indicating their recent origin. Our results also suggest these populations were created by migrants from multiple, genetically distinct refugia. Furthermore, some haplotypes observed in populations from previously glaciated areas were not found in putative refugial populations, suggesting these populations might have contributed little to the extant populations created after the Last Glacial Maximum. Some authors regard L. sukaczewii and L. sibirica as a single species, while others consider them as separate species. The observed conspicuous differences in haplotype composition and distribution between L. sukaczewii and L. sibirica, together with high values of F _ST between populations of the two species, appear to support the latter classification. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Ismael A. Khatab and Kariyawasam K.G.U. Hemamali contributed equally to this work.     

The role of larch budmoth (Zeiraphera diniana Gn.) on forest succession in a larch (Larix decidua Mill.) and Swiss stone pine (Pinus cembra L.) stand in the Susa Valley (Piedmont, Italy)

In the Alps, larch (Larix decidua Mill.) is severely affected by larch budmoth (Zeiraphera diniana Guénée) (LBM) attacks. The impact of these outbreaks on the Swiss stone pine (Pinus cembra L.) and on the dynamic processes acting in subalpine forest stands are still not well known. Dendroecological methods were used in this study to reconstruct past LBM outbreaks in Susa Valley, Piedmont, Italy. The analysis was carried out on 62 cores from larch and 101 cores from stone pine. The length and severity of each outbreak was quantified for both species and for each tree by means of the programme OUTBREAK. The frequency of the outbreaks was determined using singular spectral analysis and superposed epoch analysis was used to test the significance of the associations between outbreaks and tree-ring growth. In order to verify if trees belonging to different age classes are differently affected by LBM, the reconstructed outbreaks are then classified taking into account the cambial age of the tree at the moment of the outbreak. From 1760 to 1999, 19 outbreaks were recorded in the larch chronologies, while only three outbreaks in the stone pine chronologies. The larch growth is strongly influenced by LBM and the identified outbreaks are equally distributed in all the three age classes. On the stone pine the sporadic occurrence of the identified events made difficult any interpretation of the eventual effect of LBM. Our results lead us to argue that LBM has not played an important role both in determining the stone pine growth rate and in influencing the present observed succession from the stage dominated by larch, to a stage dominated by stone pine or by a mixed stone pine-larch forest.     

Isolation, characterization, and inheritance of microsatellite loci in alpine larch and western larch.

Microsatellite loci or simple sequence repeat loci (SSRs) were isolated in alpine larch (Larix lyallii Parl.) and western larch (Larix occidentalis Nutt.). In total, 14 SSR loci were characterized; two [(TCT)4, A7] came from published Larix DNA sequence data, one (CA)17 was obtained from a partial non-enriched alpine larch total genomic DNA library, and the remaining 11 loci were obtained from larch genomic DNAs enriched for (CA)n repeats. The SSR regions in these clones could be divided into three categories: perfect repeat sequences without interruption, imperfect repeat sequences with interruption(s), and compound repeat sequences with adjacent tandem simple dinucleotides. Eight of the 14 loci analyzed were found to be polymorphic and useful markers after silver-staining polyacrylamide gel electrophoresis. In addition, several SSR primers developed for alpine larch were able to successfully amplify polymorphic loci in its related species, western larch, and among other closely related taxa within the Larix genus. The inheritance of microsatellite loci was verified by analysis of haploid megagametophyte and diploid embryo tissues of progeny obtained from controlled crosses between western larch and alpine larch. All microsatellite loci analyzed had alleles that segregated according to expected Mendelian frequencies. Two species-specific markers (UAKLly10a and UAKLla1) allow easy and rapid identification of specific genetic entry of alpine larch and western larch at any stage in the sporophyte phase of the life cycle. Therefore, these markers are efficient in identifying the parental species and to validate controlled crosses between these two closely related species. These results are important in tree improvement programs of alpine larch and western larch aimed at producing genetically improved hybrid stock for reforestation in Western Canada and U.S.A.     

Genetic diversity of larch at the north of Primorskii Krai and limits of Larix olgensis A. Henry distribution

The genetic diversity of four mtDNA fragments and five microsatellite loci of cpDNA was examined in six larch population samples from the territory of the Sikhote-Alin Nature Reserve (northern Primor’e). In order to identify possible species-specific differences between the samples, the latter were collected at transects along the shore of the Sea of Japan and at different distances from the sea. Based on a number of morphological characters, some authors suggest that Olgan larch (Larix olgensis A. Henry) grows in the shoreline part of the reserve and, moving inland, it is replaced by Gmelin larch (Larix gmelinii (Rupr.) Rupr.). According to the other data, the northern border of the Olgan larch range does not reach the reserve territory. The data obtained were compared to those obtained previously for three samples from the south of Primor’e, including those for the locus classicus of Olgan larch. In the examined larch individuals (186, for mtDNA and 200, for cpDNA), a total of five mitotypes and 52 chlorotypes were recovered. According to the results of the AMOVA test, the proportion of variations accounted for the differences among all Larix population samples examined over both types of markers was higher (N _ST = 0.435, for mtDNA and R _ST = 0.041, for cpDNA) than that for the differences among the population samples within the reserve (N _ST = 0.079, for mtDNA and R _ST = 0.005, for cpDNA). No differences were detected between the groups of shoreline and continental populations.     

Mitochondrial DNA variation in Olga Bay larch (Larix olgensis A. Henry) from Primorsky Krai of Russia

Two mitochondrial DNA fragments, nad4(3c-4r) and nad5(1-2r), were sequenced in 58 larch accessions from the range of Larix olgensis A. Henry. Combinations of the nad4 polymorphic sites formed four haplotypes, two of which (H3 and H4) were unique and two (H1, H2) were common. Haplotype H1 was found only in pure L. olgensis from the vicinity of Olga Bay and in a number of accessions from the southern part of the range. Haplotype H2 was detected in the other samples from the range of Olga Bay larch, as well as in hybrid forms. Similarly to the nad4(3c-4r) fragment, the mtDNA fragment UBC460 was able to differentiate larch populations from the range of L. olgensis examined.