Efficient embryogenic callus induction and plant regeneration from embryonic axis explants inQuercus acutissima

Embryogenic callus ofQuercus acutissima was successfully induced from embryogenic cultures, and plants were regenerated from the callus. The development of the techniques involved will allow mass propagation and gene transformation in this species. Embryogenic cultures were formed from embryonic axis explants (i.e., embryos without cotyledons) excised from immature embryos, after culture on Murashige and Skoog (MS) medium containing indolebutyric acid and benzyladenine. Attempts to induce embryogenic cultures from cotyledon explants were unsuccessful. Embryogenic calli were induced at high frequency from embryogenic cultures on MS medium containing 2,4-dichlorophenoxyacetic acid. However, benzyladenine inhibited embryogenic callus formation. Somatic embryo development from embryogenic calli occurred on MS medium in all of the seven cell lines tested. Germination of somatic embryos was induced on half strength MS medium without plant growth regulators. Finally, acclimated plants growing in soil were obtained.     

Aboveground biomass and nitrogen in four short-rotation woody crop species growing with different water and nutrient availabilities

We quantified the effect of water and nutrient availability on aboveground biomass and nitrogen accumulation and partitioning in four species from the southeastern United States, loblolly pine (Pinus taeda), slash pine (Pinus elliottii), sweetgum (Liquidambar styraciflua), and sycamore (Platanus occidentalis). The 6-year-old stands received five levels of resource input (control, irrigation with 3.05 cm water week⁻¹, irrigation + 57 kg N ha⁻¹ year⁻¹, irrigation + 85 kg N ha⁻¹ year⁻¹, and irrigation + 114 kg N ha⁻¹ year⁻¹). Irrigation significantly increased foliage, stem, and branch biomass for sweetgum and sycamore, culminating in 103% and 238% increases in total aboveground biomass. Fertilization significantly increased aboveground components for all species resulting in 49, 58, 281, and 132% increases in total aboveground biomass for loblolly pine, slash pine, sweetgum, and sycamore, respectively. Standing total aboveground biomass of the fertilized treatments reached 79, 59, 48, and 54 Mg ha⁻¹ for loblolly pine, slash pine, sweetgum, and sycamore, respectively. Fertilization increased foliar nitrogen concentration for loblolly pine, sweetgum, and sycamore foliage. Irrigation increased total stand nitrogen content by 6, 14, 93, and 161% for loblolly pine, slash pine, sweetgum, and sycamore, respectively. Fertilization increased total nitrogen content by 62, 53, 172, and 69% with maximum nitrogen contents of 267, 212, 237, and 203 kg ha⁻¹ for loblolly pine, slash pine, sweetgum, and sycamore, respectively. Growth efficiency (stem growth per unit of leaf biomass) and nitrogen use efficiency (stem growth per unit of foliar nitrogen content) increased for the sycamore and sweetgum, but not the loblolly or slash pine.     

Diversity and abundance of carabid beetles in short-rotation plantings of sweetgum, maize and switchgrass in Alabama

This study compares assemblages of carabid beetles, potentially important regulators of herbivorous insect pests, in short-rotation plantings of sweetgum (Liquidambar styraciflua L.) with and without grass cover crops, maize (Zea mays L.) and switchgrass (Panicum virgatum L.). A total of 38 species were trapped during the summers of 1996 and 1997. The dominant carabid encountered was Harpalus pensylvanicus Latreille, a widespread omnivorous species; H. pensylvanicus was particularly prevalent in maize plots. Several other commonly encountered species were primarily associated with one plot type. Overall carabid activity was found to be similar in maize and switchgrass plots and much greater than that in sweetgum plots of both cover types. This may be related to the presence of more consistently available dense ground cover in these plots compared to sweetgum plots, a situation favored by most carabids, although both abundance and diversity were similar in sweetgum plots with and without cover. Diversity, as indicated by species richness and dominance levels, was greater in switchgrass than in maize or sweetgum plots, which were similar. This revised version was published online in June 2006 with corrections to the Cover Date.     

Prescribed burning and hexazinone herbicide as release treatments in a sapling hardwood-loblolly pine stand

To compare release treatments, a randomized complete block study was established in a 7-year-old hardwood-loblolly pine (Pinus taeda L.) stand in central Louisiana established using chopping and burning. There were 5 blocks of 3 treatments each: (1) check, (2) hexazinone applied once, and (3) prescribed backfiring applied twice. The first burn in December 1985 (7 years after site preparation) had a fire intensity of 90 kJ/s/m. The hexazinone herbicide was applied in April 1986 (the 8th year after site preparation) with a metered spotgun applicator at a rate of 3.0 kg active ingredient/ha. The second burn in March 1989 (the 11th year after site preparation) had a fire intensity of 106 kJ/s/m.The two prescribed burns increased the number of stems less than 1.5 m tall from 1,380 to 2,960/ha red maple (Acer rubrum L.), blackgum (Nyssa sylvatica var. bifora), and sweetgum (Liquidambar styraciflua L.). Burning reduced the number of loblolly pines less than 2.0 m tall, which resulted in a significant increase in average loblolly pine height and diameter. Loblolly pine comprised 62 and 59% of the basal area on the check and burn treatments, respectively, 11 years after site preparation. Hexazinone reduced the number of blackgum, sweetgum, and oak (Quercus spp.) from 6,100 to 4,560 stems/ha and resulted in significantly less hardwood tree basal area than found on the check or burn treatments. Therefore, the herbicide treatment resulted in principally a loblolly pine stand (over 80% of the total tree basal area was pine) four years after hexazinone application.     

Ectomycorrhizal inoculation of Douglas-fir transplanted container seedlings with commercially produced inoculum

Commercially produced vegetative inocula of Laccaria laccata and Hebeloma crustuliniforme successfully formed ectomycorrhizae with Douglas-fir transplanted container (plug+1) seedlings. After 4.5 months in containers, 83% and 90%, respectively, of short roots were mycorrhizal. L. laccata- or H. crustuliniforme-inoculated seedlings had significantly more mycorrhizal and total short roots than Pisolithus tinctorius-inoculated (4% mycorrhizal root tips) or uninoculated control seedlings. No significant differences were detected in seedling growth at the end of the container phase.After transplantation and growth in nursery beds for 17 months, mean new short root colonization of all seedlings was 80%. H. crustuliniforme persisted as a dominant mycorrhizal fungus on seedlings initially inoculated with this fungus. L. laccata-inoculated seedlings had 40% of their short roots colonized by L. laccata and another 40% by native fungi Rhizopogon and Thelephora spp. All mycorrhizae of control seedlings and those inoculated with P. tinctorius were formed by fungi native to the nusery beds. A significant fungal treatment effect was detected for shoot height only. Control seedlings were significantly taller than L. laccata-inoculated seedlings after transplanting.This article is part of senior author's thesis in partial fulfillment of the Ph.D. degree in the Department of Forest Science at Oregon State University, Corvallis.     

Somatic embryogenesis inCephalotaxus harringtonia embryo-megagametophyte co-culture

Somatic embryogenesis was initiated fromCephalotaxus harringtonia (Forbes) K. Koch embryo culture. Explants consisted of embryo and megagametophyte halves both cut longitudinally. They were removed aseptically from mature seeds and grown together on a solid Murashige and Skoog modified medium supplemented with 5 mg·l ⁻¹ 2,4-dichlorophenoxyacetic acid. Embryogenic cultures started from callus after three or more months on the primary medium. The embryogenic callus originated from the suspensor region of the embryo. All chromosome counts made in the cells of the embryonic structures demonstrated a diploid stage, which suggest that they originated from zygotic embryo tissue. The early stages of somatic embryogenic development were achieved,i.e., formation of small clusters consisting of an embryonal region made up of isodiametric meristematic cells. A more advanced stage was reached in some cultures in which the distal embryonal end of the embryo appeared smooth and opaque. The ultrastructural characteristics of the embryos, the two types of embryo cells, embryonal and suspensor cells, as well as their contents were similar to those already reported in the case of somatic embryogenesis of other conifers.     

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.     

Somatic embryogenesis of Pinus sylvestris

Embryogenic cultures (EC) of Scots pine (Pinus sylvestris L.) were initiated from immature embryos. Whole ovules were used as expiants. The responsive period for initiation began just after fertilization and remained throughout the development of first stages of early embryos. The main part of the embryogenic cultures were initiated by the time of cleavage polyembryony. Strong correlation was obtained between degree days and the responsive period. During subsequent years the experiments were repeated in Finland and Sweden. In all cases the responsive period for initiating embryogenic cultures was the same, about two weeks after fertilization.

In 1991–1993, a total of 138 clones of elite pine trees were tested for their ability to initiate embryogenic cultures. Of these, 33% were responsive under our experimental conditions. Based on about 300 ovules per clone the number of embryogenic lines induced by the responding clones varied from 0.2% to 9.0% of the expiants.

Several nutrient media were found to be suitable for initiation and proliferation of ECs. About half of the cell lines responded to abscisic acid by producing maturing embryos. The embryos reached full maturity in cultures of only a few cell lines. Some of the embryos that produced roots were planted in soil and transferred to a greenhouse.     

Plant recovery from seedling-derived shoot tips ofFaidherbia albida grown in vitro

In vitro cultures were initiated from shoots taken from seedlings ofFaidherbia albida on Murashige and Skoog based medium supplemented with different combinations of 6-benzylaminopurine (BA) and -naphthaleneacetic acid (NAA). The shoots grew and rooted on all media. Rooting and vigorous growth were most successful on medium supplemented with 10^–7M NAA alone on which 87% of the shoots formed roots. Seventy-one percent of plantlets which were transferred to soil were successfully established and nodulated with the nativeRhizobium. The procedures provide a basis for the development of in vitro techniques for rapid multiplication and physiological studies of the species.     

Somatic embryogenesis in sawara cypress (<Emphasis Type="Italic">Chamaecyparis pisifera</Emphasis> Sieb. et Zucc.) for stable and efficient plant regeneration,propagation and protoplast culture

Somatic embryogenesis inChamaecyparis pisifera was initiated from immature seeds collected from the end of June to early July. We obtained initiation frequencies ranging from 12.5 to 33.3% using whole seed explants in liquid media. Embryogenic cultures were maintained and proliferated for more than a year in solid and liquid media. High maturation frequencies of ‘high quality’ embryos were obtained on maturation media containing abscisic acid (ABA), activated charcoal (AC), and polyethylene glycol (PEG) as osmotic agent. More than one thousand cotyledonary embryos on average per 100 mg initial fresh weight of embryogenic cells were attained on medium containing 100μM ABA, 2 gL⁻¹ AC, and 150 gL⁻¹ PEG. About 97% germination frequencies and 92% plant conversion rates were achieved without any pretreatment. Growing of plants regenerated from somatic embryos has been monitored in the field. Furthermore, a procedure for culture of protoplasts isolated from embryonal masses was also described. This work was supported in part by the Japan Science and Technology Corporation and in part by a Grant for Research for the Future Program from the Japan Society for Promotion of Science.     

Notes on beech (Fagus crenata Blume) seed and seedling mortality due to rodent herbivory in a northernmost beech forest, Utasai, Hokkaido

We investigated the mortality of post-dispersal seeds and current-year seedlings of a beech species (Fagus crenata Blume) at the northernmost extent of its geographical range at Utasai (Kuromatsunai), Hokkaido, Japan. Introduced seeds that were protected from rodent predation by wire cages had a higher survival rate from winter to the following spring (mean ± SD: 84 ± 16.7%,n = 10) than controls without cages (mean ± SD: 12 ± 17.9%,n = 10). Unprotected transplanted current-year seedlings with almost fully opened primary leaves never survived more than three days (0%,n = 30), while more than 80% (n = 30) of seedlings within cages survived to the end of the growing season. These results indicate that rodent herbivory is a major mortality factor that strongly prevents the establishment of beech seedlings. In a natural population, we observed that 90% of seedlings (n = 197) disappeared within 10 days after their emergence and rodent herbivory caused this heavy mortality. A herbivorous rodent, the gray-backed vole,Clethrionomys rufocanus bedfordiae, was remarkably abundant in late June (101.5 voles/ha), suggesting that this species strongly affects the formation of the seedling bank. The presence ofC. rufocanus bedfordiae may be one of the reasons for the scarcity of beech seedlings and saplings and the rarity of recruitment in this northernmost beech forest.     

Growth and Mycorrhizal Dependency of Acacia mangium Willd. Inoculated with Three Vesicular Arbuscular Mycorrhizal Fungi in Lateritic Soil

Three VA-mycorrhizal fungi; Glomus occultum, Glomus aggregatum (local isolates) and G. mosseae (strain from Bangalore, India) were inoculated to assess their effect on growth of Acacia mangium in lateritic soil. All inoculations enhanced growth with respect to shoot height, root diameter, leaf area, chlorophyll content and biomass of A. mangium significantly compared to uninoculated control seedlings. G. occultum proved most efficient among the three. The mycorrhizal dependency factor indicated that the growth of A. mangium was 57% dependent on G. occultum, 47% on G. mosseae and 46% on Glomus aggregatum.     

Prevalence and incidence of the root-inhabiting fungi, Fusarium, Cylindrocarpon and Pythium, on container-grown Douglas-fir and spruce seedlings in British Columbia

Surveys were made at the end of the 1990 and 1991 growing seasons for root-inhabiting fungi in the genera Fusarium, Cylindrocarpon and Pythium from the roots of one year-old container-grown Douglas-fir and spruce seedlings grown under greenhouse conditions. In the 1990 survey of four nurseries, it was found that 61–97% of both Douglas-fir and spruce roots were colonized with Fusarium, Cylindrocarpon or Pythium. There were significantly (p0.05) more Douglas-fir roots than spruce roots colonized by Fusarium at all nurseries, however, there were significantly (p0.05) more spruce roots than Douglas-fir roots colonized by Cylindrocarpon and Pythium. Root colonization of Douglas-fir and spruce by the three fungal genera during 1991 varied from 0–82% at three nurseries, however, only at a south coastal nursery was there significantly (p0.05) more spruce than Douglas-fir roots colonized by Cylindrocarpon. Significantly more seedlings were infected in 1990 than in 1991. In 1991, there were few significant differences between Douglas-fir and spruce, in the percentage of seedlings with colonized roots and in the percentage of growth medium colonized by the fungi. However, there were significant differences between nurseries.     

Effect of Frankia inoculation on the growth of Alnus sieboldiana on unsterilized soil

The effect of inoculation with Frankia, a N-fixing actinomycete, on the growth of Alnus sieboldiana seedlings was studied on unsterilized soil from a nursery and an alder stand (forest of Alnus firma). The seedlings of A. sieboldiana were inoculated with Frankia before or after a 2-month culture on sterilized vermiculite, during which they nodulated, and transplanted to unsterilized soil from the nursery and the alder stand. The control seedlings were also cultured on sterilized vermiculite for about 2 months and transplanted to unsterilized soil without Frankia inoculation. The seedling growth, nodulation and N-fixing activity were measured 3, 10 and 16 weeks after the transplantation. Growth and nodule biomass of the seedlings inoculated with Frankia and those grown on the alder soil were better than those without inoculation with Frankia and grown on the nursery soil, respectively. The seedlings inoculated before spontaneous nodulation grew better than those inoculated at the transplantation. Nitrogen-fixing activity measured by acetylene reduction assay at 16 weeks after the transplantation was higher in the seedlings grown on the soil from the nursery than on the soil from the alder stand.     

Dry matter production and allocation in Eucalyptus cloeziana and Eucalyptus argophloia seedlings in response to soil water deficits

Effects of soil water availability on seedling growth, dry matter production and allocation were determined for Gympie (humid coastal) and Hungry Hills (dry inland) provenances of Eucalyptus cloeziana F. Muell. and for E. argophloia Blakely (dry inland) species. Seven-month-old seedlings were subjected to well-watered (100% field capacity, FC), moderate (70% FC) and severe (50% FC) soil water regimes in a glasshouse environment for 14 wk. There were significant differences in seedling growth, biomass production and allocation patterns between species. E. argophloia produced twice as much biomass at 100% FC, and more than three times as much at 70% and 50% FC than did either E. cloeziana provenance. Although the humid provenance of E. cloeziana had a greater leaf area at 100% FC conditions than did the dry provenance, total biomass production did not differ significantly. Both E. cloeziana provenances were highly sensitive to water deficits. E. argophloia allocated 10% more biomass to roots than did E. cloeziana. Allometric analyses indicated that relative biomass allocation patterns were significantly affected by genotype but not by soil water availability. These results have implications for taxon selection for cultivation in humid and subhumid regions.     

Growth reactions of a Gremmeniella abietina isolate and Scots pine embryogenic tissue cultures differ in a host–parasite in vitro system

Summary The aim of this study was to screen Scots pine embryogenic tissue culture material to Gremmeniella abietina. According to the results Scots pine (Pinus sylvestris) embryogenic tissue lines (27 lines) could be ranked based on significant differences in G. abietina effects estimated by changes in glucosamine concentrations, growth, and dry matter contents of inoculated tissue pieces. The ranking order of the extreme tissue lines stayed basically similar, regardless of the variable measured. These findings imply a possibility to use the ranked embryogenic tissue lines in more detailed investigations of interactions between G. abietina and its host. This is the first report of using embryogenic tissue cultures in studying host–parasite interaction between Scots pine and G. abietina.     

Cryopreservation of an embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration

Embryogenic suspension cultures of sitka spruce (Picea sitchensis) were successfully cryopreserved. The best method for cyropreservation as demonstrated by regrowth tests was: 1) preculture in medium containing 0.4 M sorbitol and pretreatment with 5% (v/v) DMSO (dimethylsulfoxide), 2) freezing the cultures at a cooling rate of 0.5°Cmin^‐1 to —40°C followed by transfer to LN (liquid nitrogen). After thawing in a waterbath at 45°C the growth rates of cell cultures cryopreserved by this method were equal to growth rates of untreated unfrozen cultures. The cryopreserved cultures gave rise to mature somatic embryos which developed cotyledons, roots and shoots 3–4 months after thawing.     

Selection of efficient VA mycorrhizal fungi for Casuarina equisetifolia—second screening

An investigation was carried out to screen and select efficient vesicular arbuscular mycorrhizal (VAM) fungi for inoculating the forest tree species, Casuarina equisetifolia. The seedlings were inoculated with 10 different VAM fungi, obtained from various sources. Inoculated seedlings generally had greater plant height, stem girth, biomass and P content than uninoculated plants. They also had more mycorrhizal root colonization and spore numbers in root zone soil. C. equisetifolia seedlings responded best (in biomass) to inoculation with Glomus mosseae (Nicolson and Gerdemann) Gerdemann and Trappe, closely followed by Acaulospora laevis Gerdemann and Trappe and G. fasciculatum (Thaxter Sensu Gerdemann) Gerdemann and Trappe; all the three being statistically on par with each other.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

Cross breeding of Populus and its hybrids for cold resistance

Populus tomentosa was crossed with P. tremuloidis, P. grandidentata, P. alba × P. grandidentata and P. alba × Ulmus pumila in order to maintain its rapid growth and high wood quality and improve its resistance to cold. Two methods were used to increase the germination rate from 1.5% to 41.1% and the remaining rate from 1.7% to 44.2%. Forty crossing combinations were conducted and 2 744 hybrid seedlings were obtained. MX₄ × P. grandidentata (G-1-58), MX₃ × P. tremuloidis (T-44-60), MX₂ × P. tremuloidis (1-13-87-37) and MX₂ × (P. alba × P. grandidentata) were regarded as superior combinations after analysis and selection. Thirty seedlings of these combinations and 11 triploid seedlings identified by counting their chromosomes were selected as super plants. [Supported by the Doctoral Foundation of Ministry of Education (Grant No. 200220022004) and the National “863” Project (Grant No. 2002AA241071)]