In vitro storage of cedar shoot cultures under minimal growth conditions

We developed procedures for slow-growth storage of Cedrus atlantica and Cedrus libani microcuttings of juvenile and adult origin, noting factors favouring the extension of subculture intervals. Microcuttings could be stored effectively up to 6 months at 4°C and reduced light intensity, provided that they were grown on a diluted modified MS medium. The addition of 6% mannitol to the storage media affected negatively survival and multiplication capacity of the cultures. The slow-growth storage conditions used in our experiments did not induce remarkable effects on both RAPD variability and average DNA methylation in the species.     

Long-termin vitro storage ofColocasia esculenta under minimal growth conditions

The storage of a clone ofColocasia esculenta at 28/24°C over a 12-h photoperiod in the absence of mannitol, was not feasible with transfer intervals of more than eight months. Mannitol had a positive effect on survival at this temperature, but caused abnormalities at high concentrations. At 9°C in total darkness,C. esculenta could be stored for more than eight years with transfer intervals of approximately three years. After this period, more than 90% of the cultures showed living shoots, but not all shoots per culture showed regrowth. Cultures which were transferred three times during the experimental period, had a significantly (=0.05) lower number of regrowing shoots than cultures which were transferred twice. This might be due to the fact that the former cultures were transferred at a less favourable developmental stage. The addition of mannitol to the storage medium did not improve survival and regrowth, nor did a more gradual lowering of the temperature to 9°C.     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

An in vitro method for the conservation and storage of garlic (Allium sativum) germplasm

Shoot cultures of three garlic (Allium sativum) cultivars were kept in various temperatures and media in order to maintain their viability without subculture. A high level of viability was recorded after 16 months of culture at 4°C with 100 gl^-1 sucrose in B-5 medium.     

In vitro storage ofXanthosoma spp. under minimal growth conditions

Xanthosoma germplasm requires regular subeulturing if stored in vitro under optimal culture conditions. An experiment was carried out to investigate whetherXanthosoma can be stored continuously under minimal growth conditions. Growth was reduced by incubation at low temperatures (9°C, 13°C and 17°C) and by adding mannitol (3.0% w/v) to the medium. Three investigatedXanthosoma species,Xanthosoma brasiliense, Xanthosoma robustum andXanthosoma sagittifolium could be stored in the dark for at least two years at 13°C. Addition of mannitol to the medium resulted in higher survival rates after three years of continuous storage. The storage period could be extended to at least 48 months forX. robustum andX. sagittifolium by annual subculture and regrowth under optimal culture conditions.     

In vivo growth of encapsulated axillary buds of mulberry (Morus indica L.)

Axillary buds excised from aseptic shoot cultures of mulberry were encapsulated in an alginate matrix under non-aseptic conditions. Addition of a fungicide to the alginate beads prevents contamination of the bud and increased survival of the buds when sown in soil.     

In vitro axillary shoot proliferation of apple rootstocks under different ethylene conditions

Summary Ethylene effect on in vitro shoot proliferation of two apple rootstocks, MM111 and M9, was studied. Ethylene biosynthesis was proportionally stimulated by increasing concentrations of the precursor 1-aminocyclopropane-1-carboxylic acid (ACC). When 25 μM or more ACC was applied without any control of the headspace of culture vessels, shoot proliferation of both rootstocks was negatively affected. However, when shoot cultures were transferred to ACC-supplemented medium after the second week of culture, ACC had no effect. Supplementing the medium with aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, together with the application of gas traps inside the flasks, significantly enhanced axillary shoot formation and elongation. Steady and high exogenous concentrations of ethylene in the culture flasks had negative effects on shoot proliferation. MM111 appeared to be more sensitive to ethylene than M9. For AVG a threshold dose was noticed, beyond which phytotoxic effects were induced.     

Spatial distributions of foliar nitrogen and phosphorus in crowns of Eucalyptus grandis

Summary Eucalyptus grandis trees were grown in plantations with and without added fertiliser to examine the effects of plant nutrition on photosynthesis and growth. Leaves were sampled from known locations within canopies of selected trees and leaf N and P concentrations were measured. Contour maps of N and P distributions were then produced for crowns of trees aged between 6 and 16 months. Gas exchange measurements on sample leaves were used to estimate parameters of a model of C₃ photosynthesis as a function of leaf N and P contentrations. Linear relationships were obtained between model parameters and leaf N concentration, but P appeared to be present in excess, since no correlation was found with P contentration. Photosynthetic light response curves were calculated for model leaves with differing N concentrations. The curves show that optimal concentrations of N in leaves depend on mean levels of irradiance during growth.     

In vitro culture of adult and juvenile bud explants of Passiflora species

Cultivar E23, an F1 hybrid of P. edulis and P. edulis f. flavicarpa is usually propagated by shoot-tip grafting. Various media were tested to evaluate the potential of E23 for in vitro propagation. Adult tissue was difficult to culture and did not respond to media containing low (<10 µM) concentrations of growth regulators. Growth of adult buds on intact stem sections was promoted by 1 week of dark incubation on MS basal medium plus 150 µM 2iP, 200 µM adenine sulphate and 17.1 µM IAA (3 mg l^–1), and further developed into shoots on MS medium plus 4.9 µM 2iP (1 mg l^–1) and 5.7 µM IAA (1 mg l^–1). By contrast, juvenile shoots of E23, and Passiflora species: edulis f. flavicarpa, edulis, alata, caerulea, mollissima, coccinea, herbertiana and suberosa grew rapidly on MS medium plus 10 µM kinetin and 5 µM IAA. Rapid multiplication was achieved on MS plus 20 µM BA, 10 µM kinetin, 5 µM IAA, and roots initiated on MS plus 5 µM IAA.Abbreviations IAA indole-3-acetic acid - 2iP N6-iso pentenyl adenine - BA N6-benzyl adenine     

In vitro plant regeneration in Holarrhena antidysenterica wall., through high-frequency axillary shoot prolieferation

Summary An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N⁶-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.     

Chloroplast DNA variation and population structure in the widespread forest tree, Eucalyptus grandis

Recognition of genetic structure of populations and the ability to identify vulnerable populations is useful for the formation of conservation management strategies for plants. Eucalyptus grandis is a tall forest tree that has a major area of occurrence in subtropical eastern Australia, with smaller populations located in the east coast tropics. Many widespread forest species exhibit population differentiation that corresponds to geographic regions. However, Eucalyptus grandis appears to be an exception based on isozyme and morphological data. This is intriguing given a large discontinuity between northern populations and those in the southern part of the species range. In this study, the distribution of a maternally inherited chloroplast locus was examined because it was more likely to reveal genetic structure due to the slower evolution of the chloroplast genome and limited dispersal of seed in eucalypts. As expected, the G _ST for chloroplast DNA was higher than that for nuclear DNA but indicated low population differentiation for a forest tree species. Phylogeographic analysis indicated that the 15 populations grouped into three broad geographical regions; however, overall population structure was weak suggesting that the large geographical disjunction in the distribution of E. grandis may be relatively recent. A paradigm for conservation management of E. grandis based on chloroplast DNA haplotype distribution would take into account the low differentiation among populations.     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

三种整地措施下尾巨桉人工林碳储量及其分配格局

以不炼山＋人工穴垦、不炼山＋机械带垦和炼山＋机械全垦3种不同整地组合下的2.5年生尾巨桉人工林为对象,对其碳储量及其分配格局进行研究。结果表明：(1)3种整地组合下尾巨桉各器官碳含量平均值为44.37％~57.42％,大小顺序为叶>干>枝>根>皮,带垦最大(51.21％),炼山全垦最小(49.95％);不同整地组合尾巨桉人工林林下地被物层的碳含量均无显著差异(P>0.05);土壤层(0~100 cm)碳含量均随土层深度的增大而减小,各层土壤平均碳含量总体趋势表现为带垦>炼山全垦>穴垦。(2)穴垦、带垦、炼山全垦措施下乔木层总碳储量依次为18.01、30.49和23.56 t.hm-2,各器官碳储量大小顺序为干>根>叶>枝>皮;除皮外,其余器官碳储量排序均为带垦>炼山全垦>穴垦。(3)尾巨桉人工林生态系统的总碳储量表现为带垦(197.03 t.hm-2)>炼山全垦(161.16t.hm-2)>穴垦(144.77 t.hm-2);不同整地措施碳储量分配格局均为土壤层>植被层>枯落物层。土壤层和乔木层碳储量均是带垦最大,在整个生态系统碳储量中处于主导地位,占整个系统碳储量在93％以上;不同整地组合措施对枯落物层的碳储量无显著影响。因此,从提高尾巨桉林分系统碳储量方面考虑,在雷州半岛及相似立地条件地区进行尾巨桉人工林造林时宜采取不炼山＋机械带垦的整地组合方式。     

In vitro production of microtubers for conservation of potato germplasm: Effect of genotype,abscisic acid,and sucrose

Summary With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 gl⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6±1°C.     

农杆菌介导巨桉Eg5高效遗传转化

以巨桉(Eucalyptus grandis)无性系Eg5叶片为外植体, 探讨了农杆菌(Agrobacterium tumefaciens)侵染时间、共培养pH值和共培养时间对瞬时转化效率的影响, 分析了不同筛选策略对遗传转化植株筛选效果的影响。结果表明, 外植体侵染45分钟, 共培养pH值为5.8, 共培养3天所得到的瞬时转化效率最高; 逐步提高卡那霉素(Km)浓度筛选转基因植株有效, 筛选率达到15%, 转化率达到0.26%。经过GUS染色分析和PCR检测, 证实为转基因植株。     

Model simulations of spatial distributions and daily totals of photosynthesis in Eucalyptus grandis canopies

Summary A simulation model for radiation absorption and photosynthesis was used to test the hypothesis that observed nonuniform distributions of nitrogen concentrations in young Eucalyptus grandis trees result in greater amounts of daily assimilation than in hypothetical trees with uniform N distributions. Simulations were performed for trees aged 6, 9, 12 and 16 months which had been grown in plantations under a factorial combination of two levels of fertilization and irrigation. Observed leaf N distribution patterns yielded daily assimilation rates which were only marginally greater (<5%) than for hypothetical trees with uniform distributions. Patterns of assimilation distribution in individual tree crowns closely resembled those for absorbed radiation, rather than for N. These conclusions were unaffected by three choices of alternative leaf area density distributions. The simulation model was also used to calculate hourly and daily rates of canopy assimilation to investigate the relative importance of radiation absorption and total canopy nitrogen on assimilation. Simulated hourly rates of carbon assimilation were often lightsaturated, whereas daily carbon gain was directly proportional to radiation absorbed by the tree crown and to total mass of N in the leaves. Leaf nitrogen concentrations determined photosynthetic capacity, whereas total leaf area determined the amount of radiation absorbed and thus the degree to which capacity was realized. Observed total leaf area and total crown N were closely correlated. The model predicted that nitrogen use efficiences (NUE, mol CO₂ mol^–1 N) were 60% higher for unfertilized than for fertilized trees at low levels of absorbed photosynthetically active radiation (PAR). Nitrogen use efficiency was dependent on fertilizer treatment and on the amount of absorbed PAR; NUE declined with increasing absorbed PAR, but decreased more rapidly for unfertilized than for fertilized trees. Annual primary productivity was linearly related to both radiation absorbed and to mass of N in the canopy.     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

In vitro growth reduction of tomato and carnation microplants

Shoots which proliferated from shoot tip explants of Colorado White Simm carnation and Fantastic tomato on MS medium containing 5 mgl^-1 benzyladenine were rooted and grown in vitro as microplants. Tomato microplants grown in medium with 5 gl^-1 sucrose had less overall shoot and root growth than those with 10,20, or 30 gl^-1 sucrose regardless of NAA level. Carnation shoot growth was reduced by 5 g l^-1 sucrose but root growth was not affected except when no sucrose was supplied. Microplant height and rooting of carnation were maximal when grown in 20 gl^-1 sucrose whereas tomato microplant growth was greatest with 30 gl^-1 sucrose. Microplants of both species had reduced height and root growth when the MS nutrient salts were lowered to 25%, 50%, or 75% compared to full strength when sucrose was supplied at 5 gl^-1.     

Application of gas-permeable bags for in vitro cold storage of strawberry germplasm

Summary This study reports the first use of gaspermeable, heat-sealable polyethylene bags for cold storage of plant tissue cultures. The bags were used to develop a new cold storage system for the in vitro strawberry collection at the National Clonal Germplasm Repository (NCGR), Corvallis. In vitro Fragaria plantlets of 96 different accessions (species and cultivars) were transferred to bags with basal medium without growth regulators, heat-sealed, grown for one week at 25°C, cold hardened for one week, and then stored in the dark at 4°C. These in vitro cultures were successfully stored for up to 24 months in polyethylene bags. Evaluations at three month intervals provided information on the condition of the diverse collection. Over 75% of the accessions originally stored remained in storage for 15 months and 47% remained for over 18 months. None of the 96 accessions studied was lost due to contamination or decline in vigor. Over 300 Fragaria accessions are currently stored using this system.Abbreviations BA N⁶-benzyladenine - IAA indole-3-acetic acid - GA₃ gibberellic acid     

In vitro shoot proliferation of 5-leaf aralia explants from field grown plants and forced dormant stems

A. Sieboldianus (5-leaf aralia) is recalcitrant for micropropagation, but has very good landscaping potential. This research was conducted with the following objectives: (1) to study effects of BA, TDZ, CPPU, 2iP, kinetin and zeatin in woody plant medium on the performance of softwood shoot nodal explants produced by field grown 5-leaf aralia plants; (2) to investigate influences of BA or TDZ in the forcing solution on subsequentin vitro shoot initiation of nodal explants taken from forced softwood growth. Shoot initiation of softwood nodal explants from field-grown plants was promoted by adding BA, TDZ or CPPU to the culture medium. Kinetin, zeatin and 2iP were ineffective for micropropagation ofA. Sieboldianus. The forced softwood growth for use as explants was “primed” by forcing dormant stems in solution containing 200 mg 8-HQC per liter plus 2% sucrose, 44.4, 222, or 444 μM BA, or 45.4, 227, or 454 μM TDZ. BA and TDZ in the forcing solution enhanced subsequentin vitro axillary shoot initiation of nodal explants taken from forced stems by doubling the number of shoots produced per explant to 3.3 from 1.65 shoots per explant taken from field grown plants. This forcing solution technique also reduced the time needed from culture initiation to potted plants to half of the time needed for the conventional micropropagation method (12 to 14 vs. 25 to 27 weeks), thus expediting the micropropagation ofA. Sieboldianus.