Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.     

Differential responses persist in shoot explants regenerated from callus of two mature black locust trees

Callus cultures were established from internodal segments of shoot cultures from two mature black locust Robinia pseudoacacia L. trees. Callus of both trees produced shoots on Murashige and Skoog (MS) medium supplemented with 10 microM 6-benzylaminopurine alone or in combination with 1 microM naphthaleneacetic acid. Regenerated shoots were successfully multiplied on MS medium containing 0.32 microM 6-benzylaminopurine, and produced roots on 0.1 strength MS medium containing 1 microM indole-3-butyric acid. One clone consistently outperformed the other with respect to shoot proliferation and proportion of shoots that produced roots. This distinction had previously been observed in shoots produced from bud explants obtained from the mature trees.     

Somatic embryogenesis from vegetative shoot apices of mature trees of Pinus patula

Embryogenic cultures were initiated and established from apical shoots of mature trees of three genotypes of Pinus patula Scheide et Deppe. Factors affecting initiation, including cold pretreatment, basal medium composition, growth regulators and gelling agent concentration, and the effect of partial desiccation on somatic embryo maturation were investigated. Cold pretreatment of thick sections (0.5-1.0 mm) of apical shoots at 2 degrees C for 3 days on 0.3% activated charcoal induced white mucilaginous embryogenic callus on initiation medium. Subculture of this embryogenic callus on maintenance medium resulted in the formation of embryonal suspensor masses with proembryos. Partial desiccation (12-90 h) of embryogenic tissue at the proembryo stage of development, prior to transfer to maturation medium containing 9 g l(-1) Gellan gum, enhanced somatic embryo maturation and germinability. The frequency of maturation increased from 5.3 to 16.5% after 12 h of desiccation and from 16.5 to 73.8% after 24 h of desiccation, but longer periods of desiccation were ineffective.     

Biochemical investigations during in vitro adventitious shoot regeneration in leaflet explants from nodal segments of a mature Albizia procera tree

The in vitro adventitious shoot differentiation in leaflet explants of an adult tree differed from that of leaflet explants of seedlings of Albizia procera(Roxb.)Benth. reported previously elsewhere. The leaflet explants from an adult tree passed through an initial callus phase for30 days on MS medium supplemented with 3 % sucrose,2.5 l M 2,4-D followed by a subsequent adventitious shoot differentiation phase for another 30 days on half MS medium supplemented with 0.25 l M each of BA and IBA.The regeneration rate of in vitro adventitious shoots in explants from the adult tree, i.e.1.66 shoots/callus, was lower than that from seedlings, i.e. [10 shoots/callus,which was reported elsewhere. Correspondingly, the activities of nitrate reductase and peroxidase, and endogenous phenol content remained very low during in vitro adventitious shoot differentiation in leaflet explants of an adult tree possibly due to lower availability of competent stem(juvenile) cells for the process.     

Polyphenols as potential markers to differentiate juvenile and mature chestnut shoot cultures

The phenolic contents of eight in-vitro-cultivated chestnut clones (Castanea sativa Mill. and C. sativa x C. crenata Siebold & Zucc. hybrids) were analyzed both qualitatively and quantitatively. The aim of the work was to identify potential phenolic markers of: (i) juvenile or mature state; (ii) topophysical origin; and (iii) rooting capacity. A condensed tannin was detected in mature material but not in juvenile material, indicating that it could be used as a qualitative marker. Other qualitative phenolic differences were found between basal shoots and crown shoots of some clones, but it was not possible to discriminate among these materials in a general way. Canonical discriminant analysis was used for the study of quantitative markers. Differentiation between mature and juvenile material, and between materials differing in in vitro rooting capacity was possible according to the results of the analysis. Nevertheless, no significant quantitative differences were found between the phenolic content of material of basal shoot origin and that of crown shoot origin, indicating that the greater juvenility of material of basal origin compared with that of crown origin was not reflected in differences in phenolic content.     

Micropropagation of Elaeagnus angustifolia from mature trees

Multiple shoots were obtained from nodal segments of mature trees of Elaeagnus angustifolia L. cultured on MS medium (Murashige and Skoog 1962) supplemented with 0, 0.88 or 2.22 micro M N(6)-benzyladenine. When nodal segments taken from the in vitro proliferated shoots were cultured under the same conditions, additional multiple shoots were obtained. Rooting of the in vitro propagated shoots was achieved on full strength MS medium or on MS supplemented with 2.46 micro M indole-3-butyric acid. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

彩色辣椒子叶离体培养研究初报

选用辣椒(CapsicumannuumL )杂交F1代2个彩色品种(橙色和紫色)的子叶进行诱导不定芽分化、生长及生根试验,建立了2个彩色辣椒品种的快速繁殖植株再生体系。每个外植体平均分化不定芽数8 9个,出苗6～8株。发现添加椰子水(CW)对不定芽的诱导分化及生长有较好的促进作用,外植体最高分化频率达100%。     

Tissue culture responses of explants taken from branch sources with different degrees of juvenility in mature black locust (Robinia pseudoacacia) trees

We studied the in vitro responses of cambial tissue and dormant vegetative buds obtained from top and epicormic branches of three mature black locust (Robinia pseudoacacia L.) trees. Cambial tissues isolated from epicormic branches produced more callus than cambial tissues isolated from top branches, whereas in vitro shoot cultures derived from buds excised from top branches grew faster than those derived from buds excised from epicormic branches. There were no significant differences between the two branch sources in in vitro bud break or shoot multiplication from bud explants or cambial-derived callus tissue, respectively. Furthermore, the top branches, generally considered to be the most mature in a tree, were not recalcitrant in terms of morphogenic capacity compared to epicormic branches.     

Changes in shoot properties in relation to vertical positions within the crown of mature canopy trees of Abies mariesii and Abies veitchii

We investigated current shoot properties in two contrasting vertical positions (leader crown; LC, and lower branch; LB) within the crowns of mature trees of two subalpine conifer species, Abies mariesii and A. veitchii. For both LCs and LBs, shoot length decreased with increasing branching order. However, shoot properties were different between LCs and LBs. Shoots in LCs had more needle biomass per unit of shoot length. Shoots in sunny conditions pack needles closer along the shoot and intercept incoming light more completely. This causes the shoots in the LCs to have more needles. In contrast, less needle packing per unit shoot length in LBs results in the avoidance of mutual shading among needles in order to intercept limited light more effectively. Because branch systems in lower layers tend to be more shaded, the quantity of irradiance received by the shoots in LBs is smaller. Thus, reduced needle amounts on the shoots in LBs reflect the needle arrangement acclimating to the lower light availability. This study suggests the importance of changes in the properties of individual shoot as a component of a branch system and accordingly a whole-crown system in mature canopy trees of A. mariesii and A. veitchii.     

In vitro shoot apex micrografting of mature Acacia mangium

Prospects for in vitro micrografting shoot apices of mature Acacia mangium trees were investigated with the use of 432 micrografts. Overall success rates of 51.5%s% were obtained for shoot apices ranging from 200 to 400 m in length, and a short basal wedge of underlying tissues top-grafted in aseptic conditions onto 2-to-3-month old in vitro grown Acacia mangium seedlings. The successfully established micrografts displayed, however, substantial variability in terms of further scion elongation as 41% of these micrografts, or 21.2% only of the total amount of the micrografts performed, had resumed growth two months after micrografting. The elongated scions exhibited different types of morphology, ranging from juvenile-like type composed leaves to the predominant mature-like phyllode morphology. Side-grafting, a more difficult procedure to perform than top-grafting, or placing the micrografts for 2 weeks in darkness after grafting, did not improve the scores. Moreover, attempts to micrograft meristems (150–200 m) resulted in 5% success only.     

Carbohydrate relations during propagation of cuttings from sexually mature Pinus banksiana trees

Concentrations of glucose, sucrose, soluble reducing sugars, starch and total non-structural carbohydrate were determined during propagation of cuttings from sexually mature Pinus banksiana Lamb. trees. Such cuttings rarely initiate adventitious roots whatever the method or duration of propagation. Terminals, needles, and upper and basal stem segments of cuttings were analyzed at Day 0 and every 2 days for 18 days. Comparison of the results with those of earlier studies with cuttings of P. banksiana seedlings, which root readily, indicated pronounced differences in carbohydrate concentrations and partitioning between the two types of cutting. Compared with those from seedlings, cuttings from sexually mature trees exhibited: (1) more total non-structural carbohydrate in each tissue at Day 0; (2) decreasing rather than increasing total carbohydrate (mainly starch) concentrations in each tissue during propagation; (3) different carbohydrate concentration ratios in each tissue during propagation; and (4) higher sucrose concentrations in terminals during propagation, relative to concentrations at Day 0. Cuttings from sexually mature trees also differed from cuttings of seedlings in having a much lower rate of dry matter accumulation during propagation. These findings suggest that the poor rooting ability of cuttings from sexually mature P. banksiana is not attributable to a lack of total carbohydrate, but that the rooting abilities of cuttings from seedlings and from sexually mature trees differ because of differences between the two types of cutting in rates of net photosynthesis and starch metabolism. The difference in starch metabolism becomes apparent during the first 2 days of propagation.     

利用重瓣大岩桐叶片诱导再生植株试验

利用重瓣大岩桐叶片诱导再生植株试验结果表明，采用MS＋BA1.5-2.00mg/L NAA0.15-0.20mg/L的培养基配比组合最适宜叶片再生小植株；在相同质量浓度下，用NAA处理好于用IBA处理。     

Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

Leaf-level acclimation to gap creation in mature Acer saccharum trees

Leaf-level morphological and physiological responses of mature, winter-deciduous, shade-tolerant Acer saccharum Marsh. trees to gap formation caused by selection harvest were studied experimentally over a 2-year period. We found no evidence for either physiological stress or positive acclimation following gap creation during the 1-2-week post-harvest period. Rather, lower-canopy leaves showed gradual increases in area-based maximum photosynthetic rates (Amax-area), stomatal conductance (gs), and leaf nitrogen concentration (Narea) over the entire 2-year study. These acclimation responses were directly related to changes in leaf mass per unit area (LMA) in the subsequent two leaf flushes. No change in Amax-area, gs, Narea, or photosynthetic nitrogen-use efficiency was observed that could not be accounted for by changes in LMA. The gradual acclimation responses in the lower canopy may account, in whole or in part, for the approximately 2-year lag in post-harvest growth response observed in Acer saccharum.     

Effect of auxins on axillary and de novo shoot regeneration from in vitro shoot cultures derived from forced epicormic buds of teak (Tectona grandis L.)

In this presentation,we report on de novo and axillary shoot regeneration and rooting of shoots maintained over a long term,from cultures of Tectona grandis L.Shoot-tips of teak shoots forced from epicormic buds were used as the starting material for axenic shoot-culture establishment.Long term maintenance of such axenic shoot cultures was carried out by regular sub-culturing on MS media supplemented with N 6-benzyleadenine (BA,8.8 μmol L-1) and indole-3-butyric acid (IBA,2 μmol L-1) for 24 months.Vigorously growing shoot tips (2 3 cm long) were inoculated on the MS basal medium supplemented with different concentrations (0,1,2,4,6,8 or 10 μmol L-1) of either IBA or α-naphthaleneacetic acid (NAA) for rooting.Axillary and de novo shoots were developed from axillary and cut basal ends of shoots,respectively.Shoots growing on auxins were further sub-cultured (every 15 days) and maintained for 45 days.The greatest number of de novo (5.06) as well as axillary shoots (2.85) was observed on the MS medium supplemented with 10 μmol L-1 NAA or 8 μmol L-1 IBA,respectively,after 45 days.The combinations of both IBA (μmol L-1) + NAA (μmol L-1) were tested at different concentrations (4 + 4,6 + 6,8 + 8) supplemented to a half strength MS basal medium with 0.1% activated charcoal for rooting of decapitated and non-decapitated de novo and axillary shoots.Rooting from non-decapitated de novo shoots was highest (93.33%) with a mean number of roots of 4.61 on this medium,supplemented with 6 μmol L-1 IBA + 6 μmol L-1 NAA,after 36 days of initial culture.Individual auxin,however,was not effective for root induction.Rooted shoots were acclimatized in a green house and after four weeks plantlets were transferred to the field.     

Field performance of Salix clones propagated via shoot cultures in vitro

Survival, growth, plant structure and insect damage of in vitro‐derived cuttings from two Salix clones (5. schwerinii and 5. viminalis) were compared in a field trial with performance of conventional cuttings of the same clones. No obvious morphological abnormalities were induced by in vitro treatment. The survival rate of in vitro‐derived cuttings of the S. schwerinii clone was much higher than that of conventional cuttings. In the 5. viminalis clone, height growth was slightly but significantly reduced for in vitro‐derived cuttings compared with conventional cuttings although stem basal area and estimated plant dry weight were unaffected. The number of stems per cutting, the number of side shoots per stem and gall midge damage did not appear to be affected by the type of cutting. Electrophoretic analysis of 12 enzyme systems revealed identical isozyme patterns for m vitro‐derived and conventionally propagated plants.     

Growth and soluble proteins of cell cultures derived from explants and protoplasts of Pinus pinaster cotyledons

Pinus pinaster Ait. cell suspension cultures were derived from chopped cotyledons and from cotyledon protoplasts. When transferred after 12 weeks in culture, growth of both cell types showed a lag of 5 days followed by an exponential phase of 14 days for the protoplast-derived cells and 23 days for the organ-derived cells. During the exponential growth phase, packed cell volume of protoplast-derived cultures increased 7-fold and that of organ-derived cultures 13-fold. During the stationary phase, the diameters of protoplast-derived cells averaged 80 microm and those of organ-derived cells 100 microm. After 30 days, the media containing protoplast-derived and organ-derived cells decreased in osmolarity by 50 and 120 mOs per kg water, respectively, and in pH by 1.4 and 2.0 units, respectively. Throughout the growth cycle, protein content per unit of packed cell volume was always at least 35% higher in the protoplast-derived cultures than in the organ-derived cultures. Two-dimensional electrophoretic separation of soluble proteins revealed three peptides present in protoplast-derived cells that were absent from organ-derived cells and two peptides present in organ-derived cells that were absent from protoplast-derived cells. Other peptides differed quantitatively between the cell types.     

Requirements for in vitro rooting of Quercus robur and Q. rubra shoots derived from mature trees

Stabilized shoot cultures initiated from crown material of six adult Quercus robur L. trees and from basal epicormic shoots of a Quercus rubra L. tree showed good in vitro rooting capacity. An initial five-day dark period generally improved the rooting response but was detrimental to plantlet quality. There were clonal differences in rooting capacity. The concentration and exposure time of the indolebutyric acid (IBA) treatment were critical for root induction. In both species, best rooting efficiency was achieved by culture in medium containing 25 mg l(-1) IBA for 24 h and subsequent transfer to an auxin-free medium containing 1% activated charcoal. For all clones tested, the charcoal benefited both shoot quality and root system development, the latter being enhanced by the formation of many lateral roots. Total root system area and length, measured with a digital image analyzer, were significantly greater in medium containing charcoal than in medium lacking charcoal. Because darkening the basal part of the shoots with aluminum foil during the rooting phase only caused a small increase in rooting, we conclude that the large effect of charcoal on rooting was the result of adsorption of inhibitory compounds from the medium or the explant or both, rather than of basal darkening. Other factors affecting the rooting response of Q. robur were: (a) the position on the tree of the material from which cultures were initiated (the topophysical effect); and (b) shoot quality. Recycling the same horizontally placed explant on multiplication medium allowed three successive crops of shoots to be obtained, and rootability was typically maintained from crop to crop.     

Genetic transformation of loblolly pine using mature zygotic embryo explants by Agrobacterium tumefaciens

Introduction1Genetictransformationinconifershasthepotentialtoallowtheselectiveimprovementofindividualtraitsinelitecloneswhilestillmaintainingtheexistingcombinationofgenesresponsibleforthesuperiorphenotype(Charestetal.1991;Jamesetal.1996;Walteretal.1999).Atpresent,althoughconsiderableresearchefforthasbeendevotedtothegeneticengineeringofconiferspecies(Sederoffetal.1986;Bekkauoietal.1988,1990;Robertsonetal.1992;Bomminenietal.1993;Shinetal.1994;Klimaszewskaetal.1997),ithaslaggedbehindadvancesma…