In Vitro Screening and Characterization of Selected Elite Clones of Eucalyptus tereticornis Sm. for Salt Stress

Journal of Plant Growth Regulation - The present investigation aimed to determine the salt tolerance level of six already selected elite clones of Eucalyptus tereticornis. The response of...     

Factors affecting micropropagation and acclimatization of an elite clone of Eucalyptus tereticornis Sm.

Several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm. were investigated. Amongst different cytokinins tested, 6-benzyleadenine proved to be the most effective cytokinin for shoot multiplication and elongation. The initial size of the shoot clump (inoculum) also influenced shoot multiplication and elongation. The number of shoots proliferated per culture vessel were significantly higher (342 shoots per culture vessel) when larger shoot clumps (15?C20 shoots) were inoculated, compared to smaller shoot clumps (4?C5 shoots), which resulted in a reduced shoot proliferation rates (245 shoots per culture vessel). However, the number of elongated shoots (65 per culture vessel) and shoot length (5.23?cm) were higher in cultures which were inoculated with smaller shoot clumps in comparison to those cultures which were inoculated with larger shoot clumps (54 shoots per culture vessel with shoot length of 4.17?cm). The maximum number of rooted shoots (80.7?%) was obtained on one fourth-strength MS medium supplemented with 5.0???M indolebutyric acid. The number of shoots proliferated, elongated, rooting frequency, and subsequent survival of plants after acclimatization were higher in cultures incubated under photosynthetically active radiation (PAR) compared to those incubated under cool fluorescent lights (CFL). Osmotic potential of the sap and chlorophyll content of cultures incubated under PAR were also higher than those incubated under CFL. Following transfer of plants to soil, inoculation with a suspension of Bacillus subtilis (plant growth-promoting bacterium) increased the survival rate of plants by 10?%, yielding successful transfer of 84?% of plants. Random amplified polymorphic DNA and inter simple sequence repeat analyses indicated a high level of clonal uniformity amongst regenerated plants and also with that of the mother plant.     

Micropropagation of Eucalyptus tereticornis Smith.

Axillary shoot bud multiplication has been achieved in Eucalyptus tereticornis Smith. using explants from different regions of 8–10 years old elite trees, growing in the field. Results showed that addition of NAA at 0.1 mgl^-1 and BAP at 1.0 mgl^-1 to modified MS medium induced maximum number of shoot buds. For inducing axial growth in regenerated bud promordia, the hormone concentration of the medium was lowered. The addition of charcoal and gibberellic acid to the medium were beneficial. Rooting was best in Knop's medium containing 1.0 mgl^-1 IBA. The key factor in root induction was primarily a dark incubation for a short period. The percentage of both rooting of shoots and survival of the rooted shoots was 60–80.Continuous trials using explants from the elite trees throughout the year showed that the period between July–September was the best season for the explant source for rapid and increased multiplication of axillary buds. Phenolic exudation was also minimum at this period. The experiments were repeated using 50 populations from different plantations. It was observed that during culture, genotypically different populations responded differently in spite of optimal growth conditions.     

Nest boxes in planted and regrowth Forest Red‐gum (Eucalyptus tereticornis Sm.) ecosystems

Nest boxes were deployed in planted and regrowth areas in association with a revegetation project to restore Forest Red‐gum (Eucalyptus tereticornis) ecosystems on abandoned former agricultural land. A year after revegetation began, 36 boxes were installed in each of the planted and regrowth areas in 2003, and these were monitored to 2013. Sixteen vertebrate species utilised boxes, which included breeding by four species and two species that were not detected by other survey methods. More boxes were used by fauna in the planting compared to regrowth in all but one audit. Significantly, more boxes were used by reptiles in regrowth than planting, but significantly more by birds in planting than regrowth. Nearly 90 per cent of boxes remained intact over the 10‐year period. While the study's capacity to attribute results to habitat types was limited, the results do add weight to the possibility that nest boxes made of good quality materials can provide valuable habitat for a wide range of species during the early recovery phase of reforestation projects.     

AFLP genetic maps of Eucalyptus globulus and E. tereticornis

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient technique for detecting large numbers of DNA markers in eucalypts. We have used AFLP markers in a two-way pseudo-testcross strategy to generate genetic maps of two clones of different Eucalyptus species (E. tereticornis and E. globulus). Of 606 polymorphic fragments scored, 487 segregated in a 1 : 1 ratio, corresponding to DNA polymorphisms heterozygous in one parent and null in the other. In the maternal E. tereticornis map, 268 markers were ordered in 14 linkage groups (919 cM); the paternal E. globulus map had 200 markers in 16 linkage groups (967 cM). Results from PGRI software were compared with MAPMAKER. The average density of markers was approximately 1 per 3.9 cM. Framework markers were ordered with an average confidence level of 90%, covering 80–100% of the estimated Eucalyptus genome size. In order to investigate the homologies between the E. tereticornis and the E. globulus genetic linkage maps, we included 19 markers segregating 3 : 1 in the analysis. Some homeologous linkage groups were recognized. The linkage data developed in these maps will be used to detect loci controlling commercially important traits. Received: 17 July 1997 / Accepted: 13 October 1997     

Micropropagation of 4-year-old elite Eucalyptus tereticornis trees

 A protocol for the micropropagation of mature Eucalyptus tereticornis Smith has been developed using regenerated shoots from axillary bud explants. The trees were selected on the basis of their better growth rate, physical and phenotypic characteristics and freedom from disease. Regeneration was obtained in modified Murashige and Skoog (1962) medium. Evaluation of explant regeneration throughout the year indicated that the incidence of browning of explants was maximum during the month of February, while dominance of the microbes in endogenously infected explants peaked in August–September. Regeneration from primary explants was maximum during the months of March–April. Subcultures were carried out every 4 weeks. Effects of hormones and media composition on regeneration and growth were studied. Phytagel induced vitrification, while calcium chloride dihydrate reduced vitrification and induced the elongation of shoots. Best rooting was obtained with half-strength, modified MS medium supplemented with 1.0 mg/l indolebutyric acid. Plantlets were hardened in a nonsterile potting mix at high humidity and gradually exposed to the ambient environment over a period of 6 weeks, and upon transfer to field conditions the survival rate varied from 84% to 100%. Received: 15 October 1998 / Revision received: 18 June 1999 · Accepted: 12 July 1999     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

Phytochemical constituents of cultured cells of Eucalyptus tereticornis SM

Callus induced from shoot explants of Eucalyptus tereticornis was maintained for eight months on a defined MS medium. The lipid composition of the callus of E. tereticornis were -sitosterol, stigmasterol and cholesterol. In addition, we report the presence of a flavanoidal glycoside, aglycon identified as Kaempferol. Further, the presence of 2,3-dihydroxybenzaldehyde and 3,4-dihydroxyphenyl acetic acid was established from the methanol fraction.List of Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - Kn Kinetin - NAA 1-Naphthaleneacetic acid - IAA Indole-3-acetic acid - BA 6-Benzylaminopurine     

不同林龄尾细桉人工林的生物量和能量分配

对广东省遂溪县北坡林场1～4年生尾细桉人工林的生物量和能量进行研究.结果表明:林龄对林分现存生物量影响极显著(P0.01),1～4年生林分生物量在10.61～147.28t.hm-2,随林龄增加,各组分和林分的生物量均增加,叶片、枝、树皮生物量占林分总生物量的比例逐年减小,而树干则呈逐年升高趋势.4个林龄阶段各组分生物量的分布规律,1～2年生为树干枝树皮根叶片,3～4年生为树干根枝树皮叶片.不同林龄各组分的平均灰分含量在0.47%～5.91%,以树皮的灰分含量最高、树干最低.各组分的平均干质量热值和去灰分热值分别为17.33～20.60kJ.g-1和18.42～21.59kJ.g-1,均以叶片数值最高、树皮最低.林龄对枝、树干、树皮的干质量热值及对叶片、树干、树皮的去灰分热值有显著影响(P0.05),对叶片和根的干质量热值、枝和根的去灰分热值及植物体热值的影响不显著(P0.05).1～4年生尾细桉的林分能量现存量在199.98～2837.20GJ.hm-2,林龄对其的影响达极显著水平(P0.01),随林龄增长,各组分和林分能量现存量增加,且各组分能量分配比例的变化趋势与生物量相同.     

Genetic dissection of vegetative propagation traits in Eucalyptus tereticornis and E. globulus

We have detected quantitative trait loci (QTLs) affecting vegetative propagation traits in Eucalyptus tereticornis and Eucalyptus globulus. Using amplified fragment length polymorphism (AFLP) genetic linkage maps, the inheritance of 199 markers was assessed in 94 F₁ individuals with extreme adventitious rooting response, and in 221 randomly chosen F₁ individuals. Phenotypes were scored in 1995 and 1996. QTL analyses were performed using chi-square tests (χ²), single-marker analysis (SMA), interval mapping (IM) and composite interval mapping (CIM). All approaches yielded similar QTL detection results. Three QTLs are hypothesized for mortality (MORT=% dead cuttings), nine for adventitious rooting (ROOT, RCT=% rooted cuttings relative to the surviving or total cuttings, respectively), four for petrification (PETR=% surviving unrooted cuttings), one for sprouting ability (SPR=number of stump sprout cuttings harvested in 1995) and four for the stability of adventitious rooting (STAB=absolute value of the difference ROOT95-ROOT96). All putative QTLs for MORT and PETR were located on the E. tereticornis map, and for SPR and STAB on the E. globulus map. We found different QTLs for MORT, ROOT, RCT, SPR and STAB. Putative QTLs accounted for 2.6–17.0% of the phenotypic variance of a trait (R²). Estimated standardized gene substitution effects varied between 0.13 and 0.49 phenotypic standard deviations (σ_p). These results indicate that the phenotypic variation in these traits has a meaningful genetic component and that stable QTLs can be found in a family of reasonable size where no previous knowledge of the trait was available. Received: 1 September 1998 / Accepted: 24 February 1999     

Genetic analysis of enhanced-axillary-branching-derived Eucalyptus tereticornis Smith and E. camaldulensis Dehn. plants

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. Received: 8 October 1996 / Revision received: 22 July 1997 / Accepted: 30 July 1997     

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis

Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/l benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/l BA, 500 mg/l polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4–6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - mWP modified Woody Plant medium Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station.     

In vitro cloning of Helleborus niger

. This study shows that it is possible to propagate Helleborus niger by means of in vitro cloning. Universal (U) medium has been used for the in vitro culture. This medium was created by Haensch as a universal medium to meet the average nutritional requirements of many different plants. Apical buds of H. niger seedlings were established on U-medium supplemented with 8.9 µmol/l 6-benzylaminopurine (BAP) and 2.7 µmol/l !-naphthaleneacetic acid. Propagation was carried out on U-medium supplemented with 2.2 µmol/l BAP and 2.9 µmol/l gibberellic acid. The highest rooting success rate of 96.7% was achieved by adding 4.9 µmol/l indole-3-butyric acid to the medium. Shooting and rooting were dependent on the seedling cloned. More than 80% of the in vitro plants survived and thrived in the greenhouse.     

In vitro propagation of Eucalyptus grandis L. by tissue culture

Multiple shoots were induced from nodal segments of five year old trees of Eucalyptus grandis L. on solid medium containing Murashige and Skoog's (MS) Basal medium supplemented with additional thiamine, BAP and NAA. Rooting could be achieved from shoot culture on half strength MS salts or white's medium supplemented with low auxins like IAA, IBA and NAA.Abbreviations Kn Kinetin - BA 6-Benzyl adenine - 2iP Isopentyl adenine - NAA -Naphthalene acetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - GA Gibberellic acid - PVP Polyvinyl pyrrolidone     

In vitro studies on Eucalyptus globulus rooting ability

Summary Micropropagation has the potential to quickly introduce selected genotypes of adult Eucalyptus globulus clones and it is now widely used in Portugal as a part of genetic improvement programs. Several clones have been established and multiplied in vitro. The different clones have individual requirements for successful rooting. Rejuvenation was achieved at different periods after culture initiation for the different clones. Subculturing preceding rooting in multiplication medium supplemented with riboflavin and cholene chloride allowed the increase of rooting ability for several clones tested. Removal of boron from the rooting medium increased rooting by 10%. Indolebutyric acid (IBA) dipping before transfer to the rooting medium resulted in a rooting percentage of 80–95% for the best clones tested. Acclimatization was performed without difficulties (90–95% success) and the rooted plants were either planted directly or used as mother plants for further cutting production, depending on the needs. The results described in this paper increase the commercial feasibility of the micropropagation system for E. globulus.     

In vitro field collection techniques for Eucalyptus micropropagation

A simple method was established for the collection and short-term storage of shoot material from field-grown Eucalyptus grandis and E. grandis hybrids for micropropagation. Initial studies were undertaken with plants grown outside a greenhouse, which were neither fertilised nor treated with fungicides. The method was then tested and adapted for field-grown clones. It involved collecting 35–50 mm long stems with three nodes and no leaves, spraying them with 70% (v/v) ethanol and storing them in glass bottles containing moist sterile vermiculite for 48 h. Addition of 1 g l^–1 calcium hypochlorite to the first culture medium (bud break) inhibited endogenous contamination. Multiplication yields after storage of field-grown explants were 160–264 shoots/100 explants, depending on clone. This offers an alternative, improved means for explant collection over present standard procedures of maintaining parent plants in hedges or transporting shoots to the micropropagation laboratory in buckets.     

巨桉的组织培养与植株再生

1　植物名称　巨桉 (Eucalyptusgrandis)。2　材料类别　三～五年生优树萌芽枝带芽茎段。3　培养条件　 ( 1 )启动培养基 :S + 6 BA 0 .5mg·L- 1 (单位下同 ) +NAA 0 .0 5 + 3%蔗糖 ;( 2 )丛生芽诱导培养基 :S + 6 BA 1 .0 +NAA 0 .0 5 + 3%蔗糖 ;( 3)增殖培养基 :S + 6 BA 0 .5 +NAA 0 .1 +生物素2 + 4 %蔗糖 ;( 4 )壮苗培养基 :MS(铁盐和有机物 1 .5倍 ,其余全量 ) + 4 %蔗糖 ;( 5 )生根培养基 :1 /2MS+ABT生根粉 1号 1 .0 + 2 %蔗糖。S培养基是对MS培养基下列组分作了调整的培养基 :KNO311 40 ,NH4NO391 0 ,KH2 PO42 1…     

Species discrimination, population structure and linkage disequilibrium in Eucalyptus camaldulensis and Eucalyptus tereticornis using SSR markers

Eucalyptus camaldulensis and E. tereticornis are closely related species commonly cultivated for pulp wood in many tropical countries including India. Understanding the genetic structure and linkage disequilibrium (LD) existing in these species is essential for the improvement of industrially important traits. Our goal was to evaluate the use of simple sequence repeat (SSR) loci for species discrimination, population structure and LD analysis in these species. Investigations were carried out with the most common alleles in 93 accessions belonging to these two species using 62 SSR markers through cross amplification. The polymorphic information content (PIC) ranged from 0.44 to 0.93 and 0.36 to 0.93 in E. camaldulensis and E. tereticornis respectively. A clear delineation between the two species was evident based on the analysis of population structure and species-specific alleles. Significant genotypic LD was found in E. camaldulensis, wherein out of 135 significant pairs, 17 pairs showed r(2)≥0.1. Similarly, in E. tereticornis, out of 136 significant pairs, 18 pairs showed r(2)≥0.1. The extent of LD decayed rapidly showing the significance of association analyses in eucalypts with higher resolution markers. The availability of whole genome sequence for E. grandis and the synteny and co-linearity in the genome of eucalypts, will allow genome-wide genotyping using microsatellites or single nucleotide polymorphims.     

EST—CAPS标记在尾叶桉×细叶桉F1中分离的研究

对7个EST-CAPS标记在尾叶桉×细叶桉F1中分离的研究表明：5个EST-CAPS标记呈1∶1的分离,但其中1个偏离孟德尔分离（α=0.05）;另外2个EST-CAPS标记呈1∶1∶1∶1的分离,且均符合孟德尔分离。孟德尔正态分离的EST-CAPS标记比例达85.7%,偏分离标记比例为14.3%,表明EST-CAPS标记是一种可靠的遗传标记,可用于桉属树种遗传图谱构建和相关研究。另外,探讨了偏分离的可能原因。     

Changes in growth performance and fecundity of Eucalyptus camaldulensis and E. tereticornis during domestication in southern India

Bulk seedlots of two unpedigreed multiprovenance seed production areas (SPAs) each of Eucalyptus camaldulensis and Eucalyptus tereticornis and one pedigreed seedling seed orchard (SSO) of E. tereticornis were planted in genetic gain trials at three southern Indian trial sites. At the time of seed collection, fewer than 30% trees flowered in these orchards, except in one E. camaldulensis SPA where 73% of the trees flowered, which had an estimated outcrossing rate of 86%. The E. tereticornis SSO was dominated by pollen from five highly fecund families of the Indian Mysore gum land race, which contributed 59% of the fruits produced. The SPA and SSO seedlots were compared with a bulked natural-provenance seedlot of E. camaldulensis (Morehead, Laura, and Kennedy Rivers, Queensland), another natural-provenance seedlot (Petford, Queensland), commercial eucalypt clones at two sites, and a Mysore gum seedlot at one site. At 3 years, progeny from all the four SPAs displayed good survival (79–93%) and performance similar to that of the natural provenances and the commercial clones. Progeny from the E. tereticornis SSO had significantly lower growth (at two sites) and lower survival at all three test sites. The Mysore gum seedlot displayed high fecundity and lower growth but better survival than the SSO progeny. Seed orchard genetic composition and flowering contributions thus affected progeny performance and the extent to which orchard genetic diversity was captured in the progeny. SPA progeny displayed greater fecundity than the natural provenances, indicating a response to selection for fertility.