Slash pine (Pinus elliottii Engelm.) somatic embryogenesis I. Initiation of embryogenic cultures from immature zygotic embryos

Immature female gametophytes and enclosed zygotic embryos of slash pine (Pinus elliottii Engelm.) collected at various developmental stages were cultured either on eight initiation media supplemented with different plant growth regulator (PGR) combinations or on LPM medium solidified with two brands of agar. Embryonal-suspensor cell masses extruded from gametophyte explants infrequently produced white or translucent mucilaginous embryogenic cultures. Variation in extrusion frequency was observed across seed sources (families), explant collection dates, agar types and culture media. Increased explant maturity (as related to collection dates) reduced cell extrusion rates. Four types of extrusions were grouped according to their morphology. One type of extrusion produced the greatest number of established and long-surviving embryogenic cultures. This extrusion type (type I) was associated with an extrusion condition termed empty in which the entirety of the embryonal-suspensor mass was extruded out of the gametophyte. Three other extrusion types (types II, III and IV) showed less culture establishment and survival.     

Provenance and family variability in slash pine (Pinus elliottii var. elliottii Engelm.) grown in southern Brazil and northeastern Argentina

A total of 172 seed orchard open-pollinated families of slash pine (Pinus elliottii var. elliottii Engelm.), generallysuperior for growth in the southeastern United States, wereevaluated in southern Brazil and northeastern Argentina. Mostfamilies were from selections originally made in four majorphysiographic regions within the species' natural distribution:Atlantic Coastal Flatwoods, East-Central Florida Flatwoods, GulfCoastal Flatwoods, and Southern Coastal Plain (withoutflatwoods). Ninety-one families were established in 1982 inthree tests in the Brazilian states of Santa Catarina and Parana. In 1986, 119 families, including 35 common to the Braziliantests, were planted in four tests in Corrientes and Misionesstates in Argentina. Variation among provenances for individualvolume growth was significant for both countries withEast-Central Florida Flatwoods consistently having the highestmean age-eight year volume and Southern Coastal Plainconsistently the lowest. Heritabilities were 0.20 in Argentinaand 0.44 in Brazil with within country type B geneticcorrelations of 0.66 and 1.00 for age-eight individual volume,respectively. The between country type B genetic correlation was0.63. Type B genetic correlations between United States breedingvalues for age-fifteen volume and Argentinian and Brazilianbreeding values for age-eight volume were 0.10 and 0.15,respectively. Families were generally superior to local checksin both South American countries.     

Somatic embryo production and plant regeneration of Japanese black pine (Pinus thunbergii)

Somatic embryogenesis in Pinus thunbergii was initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of somatic embryos were obtained on maturation media containing maltose, activated charcoal, abscisic acid, and polyethylene glycol as osmotic agent. The best result among the cell lines tested was achieved with the cell line T-205-3. More than 900 somatic embryos per petri dish, on average, were obtained after about 8 weeks of culture on maturation medium. Sixty percent of somatic embryos tested germinated after transfer to plant growth regulator-free medium and then 85% of them converted into plantlets.     

Efficient plant regeneration of Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis

This report describes the efficient plant regeneration of Chamaecyparis obtusa Sieb et Zucc. via somatic embryogenesis. Embryogenic cultures were initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated by 2–3-week interval subcultures in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of cotyledonary embryos were obtained on maturation medium containing maltose, polyethylene glycol, activated charcoal, and abscisic acid. Somatic embryos germinated readily after transfer to plant growth regulator-free medium. Growth of regenerated emblings has been monitored in a greenhouse.     

Scots pine (Pinus sylvestris L.) plantations contribute to the regeneration of holly (Ilex aquifolium L.) in mediterranean central Spain

The aim of this study is to evaluate the recruitment possibilities of holly (Ilex aquifolium L.) populations under Pinus sylvestris plantations in the mountains of Central Spain. Population recruitment of holly under P. sylvestris L. plantations was studied at two localities. We sampled holly seed rain, seed bank, and seedling emergence and survival (of both emerged and planted seedlings) in both localities. Seed germination was analysed by randomly sowing Ilex seeds and their subsequent recollection and analysis over the following 3 years (only in one locality). Seed rain, seed bank and emerged seedling densities did not differ between localities. Seed bank density was between 10 and 20 seeds m⁻², considerably higher than the observed seed rain (0.4–1.2 seeds m⁻²), and also higher than the emerged seedling density (0–4 seedlings m⁻²). But Ilex is not very efficient in germinating under pine forests (29.1% germination after 3 years). This may indicate a seed accumulation process of at least 5 years. Seedling survival for the first year differed among the studied sites (50–60 vs. 17%). Nevertheless these survival rates are higher than those found for the same seedling populations in different forest habitats (not Scots pine forests) from near locations. Differences in summer water stress between the study sites are proposed as the main cause of the observed differences in seedling survival. It is concluded that P. sylvestris forests may provide one of the best regeneration habitats for holly in the Mediterranean area, where this species is close to the Southern limit of its distribution.     

Influences of antibiotics on plantlet regeneration via organogenesis in Ioblolly pine （Pinus taeda L.）

Three antibiotics ampicillin, carbenicillin, and cefotaxime were evaluated for their effects on induction, growth, and differentiation of organogenic calli, as well as rooting of regenerated shoots of three Ioblolly pine (Pinus taeda L.) genotypes. Of the antibiotics administered, cefotaxime maximally increased the frequency of callus formation and growth rate of organogeni ccalli, carbenicillin maximally increased the frequency of shoot regeneration and the average number of adventitious shoots per piece of organogenic callus, ampicUlin maximally decreased the rooting frequency of regenerated shoots and mean number of roots per regenerated shoot, in comparison with antibiotic-free media. Compared with the control, ampicillin minimally increased the frequency of callus formation, cefotaxime minimally increased the frequency of shoot regeneration, and carbenicillin minimally decreased the rooting frequency of regenerated shoots in three Ioblolly pine genotypes tested. All three antibiotics increased the frequencies of callus formation and shoot regeneration, and reduced the rooting frequency ot regenerated shoots suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into Ioblolly pine need to select a suitable antibiotic. This investigation could be useful for optimizing genetic transformation of conifers.     

Experiments in rooting bishop pine (Pinus muricata D. Don) cuttings

Presented here are results of rooting studies using hedges established from juvenile seedlings of blue and green foliaged bishop pine (Pinus muricata D. Don) from Mendocino and Sonoma Counties, California. Rootability, averaged over all clones and all setting dates, was 88%. The average time for 50% of the cuttings to root was 6 months. In general, cuttings began to root rapidly in late winter/early spring. The time of year when cuttings were set determined how soon they began a phase of rapid rooting, with cuttings set in winter and early spring beginning faster than other setting dates. The period of rapid rooting lasted 2–3 months until mid/late summer, beyond which time, rooting was slow. Population and family differences in rooting were not significant; differences in rooting among clones, however, were large and significant. Analyses of clones in two experiments indicated that rooting was heritable.     

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that: 1) germination rate of grade 3 embryos in immature seeds with 0.6–0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4–0.6 cm diameter was 77.8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2) The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3) The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata var. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.     

First-year freeze hardiness of pure species and hybrid taxa of Pinus elliottii (Engelman) and Pinus caribaea (Morelet)

Six pine taxa were compared for freeze hardiness following 2 whole plant freeze tests carried out in early and late winter 1993–94. Pure species included slash pine (Pinus elliottii var. elliottii Engelman) (PEE), Caribbean pine (Pinus caribaea Morelet) var. bahamensis (PCB), and var. hondurensis (PCH). The same six slash pine parents served as the female parents of the pure slash pine taxon and were control pollinated to create the F₁ hybrids of slash pine × Caribbean pine var. bahamensis (F1B), the F₁ of slash pine × Caribbean pine var. hondurensis (F1H), and the backcross of slash pine × (slash pine × var. hondurensis (B1H). Four traits related to adaptation (bud set, foliage/stem damage, survival) were compared. Significant differences existing among the three species at all temperatures for both freeze tests. The 3 hybrid taxa were intermediate to parental taxa for all measured traits and only at extreme temperatures was there an indication of hybrid vigor. However, inspection of foliage damage curves to estimate temperatures at which 50 percent damage occurs indicated that the hybrids deviated from the mid parent value toward the more freeze susceptible parent. Discriminant functions which included foliage damage and survival traits provided the best segregation of taxa groupings and the combining of canonical variable 1 for these discriminant functions placed hybrids intermediate to parental taxa. Parent × taxa interaction was significant, suggesting that within these taxa, family to family variation occurred, but different maternal parents were associated with the freeze hardly families in different taxa.This is Florida Agricultural Experiment Station Journal Series No. R-04852 of the Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, 32611     

The variation of tree-ring widths of Scots pine (Pinus sylvestris L.) affected by air pollution

Using eight populations of Scots pine growing in foothills of the Sudetes (southern Poland) as an example, the character and causes of annual ring variation in 1900–1999 were studied. The climate had a short-term effect on radial growth during the whole period investigated. A long-term decrease of radial growth occurred after 1960, but only in some localities. It was probably caused by non-climatic factors, most likely by industrial pollution. The decline of trees during that period is mainly evidenced by a high variation of width of annual rings, a small similarity of chronologies of annual ring widths, and a lack of signal years. After 1990, the tree recovery process was evident. It was most intensive in localities where the earlier stress was the most severe. Then, trees started to produce wide annual rings, homogeneity of the growth reaction increased, and the signal years began to occur.     

Micropropagation of western hemlock (Tsuga heterophylla [Raf.] Sarg.) from embryonic explants

An in vitro propagation protocol is described for western hemlock, an important forestry species in Canada. For shoot bud induction, embryonic explants were placed initially on one-third strength Schenk and Hildebrandt medium containing 5 M N⁶-benzyladenine (BA) or 5 M BA in combination with either 5 M kinetin or 5 M 2-isopentenyl adenine for 14 days. The explants were transferred to basal medium without cytokinins for 3 weeks, and then to basal medium containing 0.05% activated charcoal. Elongating shoots were subcultured every 4 weeks on charcoal medium. Shoots, 10 mm in stem height, were rooted either in agar or sterilized peat/perlite (1:1). Up to 70% of the shoots formed roots when they were transferred to the latter, moistened with 1/2 strength Gresshoff and Doy medium containing 5 M -naphthaleneacetic acid. About 90% of the plantlets survived transfer to greenhouse conditions.     

In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP (2.0 mg·L^–1) and NAA (0.1 mg·L^–1). The optimal multiplication medium was a modified MS medium supplemented with BAP (1.0 mg·L^–1) and NAA (0.1 mg·L^–1). This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA (0.2 mg·L^–1) and acti-vated charcoal (0.5 mg·L^–1). Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproduci-ble procedure can be adopted for large-scale propagation of T. wilfordii.     

Modelling mortality of Scots pine (Pinus sylvestris L.) plantations in the northwest of Spain

Mortality is an important element of growth and yield models, especially if only low intensity silvicultural treatments are carried out. The objective of the present study was to develop a model for predicting tree number decline in planted even-aged stands of Scots pine (Pinus sylvestris L.) in Galicia (northwestern Spain). The model was constructed using data from two inventories of a trial network involving 68 permanent plots located in unthinned stands, or stands thinned lightly from below. Two alternatives were tested. In one alternative, a two-step modelling strategy was applied. First, a binary response function predicting the survival probability of all the trees in the stand was constructed, and an equation for reduction in tree number was developed, using only data where death had occurred over the period analyzed. Three different approaches were then used to compare the application of the above-mentioned functions together. In the other alternative, a mortality function for directly predicting the reduction in tree number was fitted, including all plots (with and without occurrence of mortality). Both alternatives provided similar results, showed logical behavior, and performed satisfactorily in evaluation tests. However, in choosing the best strategy for inclusion in a stand-level simulator, the use of the second alternative is recommended because it possesses the path invariance property required in a mortality model.     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

Peroxidase activity, isoenzymes and histological localisation in sapwood and heartwood of Scots pine (Pinus sylvestris L.)

Peroxidase activity and isoenzymes of fresh wood samples of the third shoot of 12-year old trees and from the sapwood, transition zone and heartwood of c. 60-year old stems of Scots pine (Pinus sylvestris L.) were investigated. Wood samples were ground at −30°C, extracted, and the extracts concentrated c. 20-fold for peroxidase activity assays (guaiacol method) and for IEF-PAGE. At least 11 major isoenzymes could be found in the gels. Even the heartwood contained some peroxidase isoenzymes. Isoenzyme patterns of the juvenile wood did not change with the season. However, juvenile wood showed the highest peroxidase activity at the end of the growing season. Peroxidase activity decreased from the outer sapwood towards the heartwood. Thin sections of different wood zones stained for peroxidase revealed activity in ray parenchyma and resin canal epithelial cells. Intensive staining was localised in the bordered pits of vertical and ray tracheids, and in the end walls of ray parenchyma cells.     

Influence of different forest conversion strategies on ground vegetation and tree regeneration in pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) stands: a case study in NE Germany

The conversion of anthropogenic into more natural, self-regenerating forests is one of the major objectives of forestry throughout Europe. In this study, we present investigations on permanent plots with different silvicultural treatment in NE German pine stands. Management of old-growth pine stands on acidic and nutrient-poor sandy sites differs in fencing, thinning, and planting of certain tree species. The investigations were carried out on the community, population, and individual level of the pine forest ecosystems. Thus, vegetation changes, size and height of tree populations, and height increment of tree individuals were observed over a time span of 6 years. Special attention was paid to short-lived tree species such as, e.g., Frangula alnus and Sorbus aucuparia, as well as to Fagus sylvatica as one of the most typical forest tree species of Central Europe. Vegetation changes are interpreted as a consequence of natural regeneration of formerly degraded forest sites, involving an increase in nutrient availability. High browsing pressure can be considered as a key factor for the inhibition of tree seedlings and growth of saplings. Some Sorbus aucuparia individuals, however, succeeded in growing out of the browsing height also in unfenced stands. Few found specimens of Fagus sylvatica proved that this species is able to establish spontaneously on these relatively dry, acidic sites under continental climate influence. Such natural regeneration processes, also including spontaneous rejuvenation of trees, can be integrated into silviculture as passive forest conversion management. An active management like thinning of stands, planting of trees, and fencing can accelerate forest conversion with regard to height growth and species number of trees.     

Lateral female strobili production in a Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone by exogenous cytokinin application

Application ofN ⁶-benzylaminopurine (BAP) and gibberellin (GA₃) to a grafted Japanese red pine (Pinus densiflora Sieb. et Zucc.) clone promoted flowering. BAP (250 mg/l) was sprayed five times to the top of branches in July or September 1992. GA₃ (100 or 500 mg/l) was applied five times to the whole plants by spray treatment during the above period, or GA₃ solution of the same concentration was injected once into a small incision made in the main stem of clone trees at the beginning of July or the middle of September 1992. In May 1993, no apical female strobili were observed at the top of the new shoots in any treatment. However, lateral female and bisexual strobili were produced in the lower part of new shoots by BAP application in September, regardless of GA₃ application. BAP applied in September without GA₃ application was sufficient to produce lateral female and bisexual strobili. In such treatment, production of female and bisexual strobili was 47 and 25, and they occurred in 16 out of 68 new shoots, 11 out of 15 treated branches and 5 out of 5 treated trees. The average number of female and bisexual strobili per new shoot was 1.06. BAP treatment either in July or in September significantly decreased (p<0.05) the production of male strobili. These results indicate that BAP application induces the development of lateral female strobili in Japanese red pine when applied during the period of floral differentiation. A part of this paper was presented at the 105th annual meeting of the Japanese Forestry Society.     

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.     

Bud development and dormancy in slash and loblolly pine. II. Effects of ethephon applications

Ethephon application on four dates did not induce budset in slash and loblolly pine seedlings. Ethephon promoted slash pine budbreak by 11 days when applied in mid-November and delayed loblolly pine budbreak by 8 days when applied in late November. Mid-November and early February applications of ethephon promoted height growth of both species.     

Environmental bioindication of sulfur in tree rings of Masson pine (Pinus massoniana L.) in the Pearl River Delta of China

In order to identify the potential of sulfur (S) content in the rings of Masson pine (Pinus massoniana) in the Pearl River Delta as a bio-indicator of regional history of atmospheric pollution, dendrochemistry was used to determine the temporal distribution of S content in the xylem of Masson pines from Zhaoqing Dinghushan and Nanhai Xiqiaoshan, Guangdong Province, southern China. The results indicated that contents of xylem S increased temporally and peaked in the rings formed in the most recent years at both sites. In the rings formed during the same period before the 1980s, S contents were not significantly different between the two sites, while in the rings formed at the same period after the 1980s, S content at Xiqiaoshan were significantly higher than those at Dinghushan. The chronosequences of the S indices at both sites could be easily marked as three periods: before 1970, during 1971–1985, and during 1986–2002. Based on the temporal changes of the xylem S contents and certain social-economic indices after the 1980s in the Delta, the history of atmospheric pollution at the study sites could be reconstructed as follows: 1) before 1970, a period in which the air was relatively clear, 2) 1971–1985, a period in which the air was gradually polluted, and 3) 1986–2002, a period in which the air was most severely polluted in the Delta. __________ Translated from Journal of Beijing Forestry University, 2008, 30(2): 1–7 [译自: 北京林业大学学报]