Botanical and chemical evaluation of Atractylodes lancea plants propagated in vitro and by division of the rhizome

Summary A single plant of Atractylodes lancea, a perennial herb of Asteraceae, was propagated in vitro by shoot culture from floral buds and shoot tips. The reproduced plants were then grown under field conditions for up to four years and their botanical and chemical characteristics were compared to those of control plants that were propagated by dividing the rhizome of the parent plant. The first-year-plants of the micropropagated plants were scanty, but in the second year they were comparable to the control in most botanical features. On the other hand, the contents of two sesquiterpenes (hinesol and -eudesmol) and a polyacetylenic compound (atractylodin) in the rhizome of the micropropagated plants remained low for the first two or three years of cultivation and at the third or fourth year they reached to the same level as that of the control. Accordingly micropropagation yields clonal plants of A. lancea comparable to those obtained by conventional propagation methods within three or four years of cultivation.     

Comparison of foliar terpenes between native and invasive Solidago gigantea

To test a defensive chemistry prediction of the Evolution of Increased Competitive Ability (EICA) hypothesis, Solidago gigantea plants from North American and European (invasive) populations were grown in a screen-enclosed garden. Terpenes from 80 seed grown (dried leaves) and 320 rhizome propagated (moist leaves) individuals were confirmed by GC/MS and quantified by GC-FID. Native seed grown plants were found to have significantly greater diterpene concentrations than their European counterparts; foliar sesquiterpenes did not differ. The occurrence of specific sesquiterpenes and diterpenes was homogeneous across the two seed sources suggesting these biochemical pathways remain unchanged. Leaves from native rhizome propagated plants also had significantly greater monoterpene and diterpene concentrations; again sesquiterpene levels did not differ. Rhizome propagated plants exhibited significant population differences in monoterpene and diterpene concentrations. These data support the defensive chemistry predictions of the EICA hypotheses but cannot discount the role of possible founder effects in the invasive range.     

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1^–1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1^–1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.Abbreviations MS Murashige and Skoog basal medium - IAA Indol-3-acetic acid - NAA naphthaleneacetic acid - 2,4-D dichlorophenoxyacetic acid - Mes 2-(N-morpholino)-ethanesulfonic acid - Cpw cell and protoplast washing solution     

Characteristics of Bupleurum falcatum plants propagated through somatic embryogenesis of callus cultures

Various characteristics including the saponin content in the root of Bupleurum falcatum plants propagated in vitro through somatic embryogenesis of callus cultures were compared with those of the plants propagated by seeds. The asexually propagated plants had an aerial part of more uniform characteristics than those of sexually propagated ones. However, both the mean and variance of root weight of the former were significantly larger than those of the latter. As for the saponin content of the root on a dry weight basis, there was little difference between the two groups. The amounts of saikosaponins c and d in a root were significantly larger in the asexually propagated plants than in the sexually propagated ones.     

Ent-labdane-type diterpene glucosides from leaves of Rubus chingii

Previously, a sweet steviol bisglucoside named rubusoside was isolated from leaves of a Chinese Rubus spp. which was tentatively assigned as R. chingii. From leaves of Japanese Rubus chingii (Japanese name Gosho-Ichigo) which are not sweet, five ent-labdane-type diterpene glucosides named goshonosidies F1-5 were isolated instead of rubusoside and their structures were elucidated. The name ‘R. suavissimus’ has been proposed for the Chinese plant.     

Field performance of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. plants propagated via somatic embryogenesis

Somatic embryogenesis is an in vitro clonal propagation method with potential to contribute to the improvement of cacao varieties. Before using this technology for commercial production, it is essential that somatic embryogenesis-derived plants be tested in field conditions. Therefore, we established a field test at Union Vale Estate, Saint Lucia. Thirty- to 50-yr-old trees were selected for clonal propagation as potentially high yielding based on local farmers observations. Clonal plants were propagated in vitro from immature flowers by embryogenesis and micropropagation. Multiple plants from nine genotypes were acclimated to greenhouse conditions then returned to Saint Lucia and planted in a field. Orthotropic rooted cuttings and locally propagated open pollinated seedlings were also planted for a total of 214 trees. Growth data were collected every 4–6 mo. including: stem diameter, stem height, length of the longest jorquette branch, number of jorquette branches, and dates of first flowering and fruiting. At 4.5 yr after planting in the field there were no major differences in all growth parameters among the propagation methods evaluated with exception of the orthotropic rooted cuttings. Trees grown from seeds were slightly taller then trees propagated by the other methods. Trees propagated as orthotropic rooted cuttings exhibited smaller average stem diameters, shorter stem heights to the jorquette, and shorter jorquette branches. We concluded that somatic embryo-derived plants demonstrated normal phenotypes in field conditions and have growth parameters similar to plants propagated by traditional methods.     

High rate of virus-free plantlet regeneration via garlic scape-tip culture

Shoot cultures of Allium sativum L. were established from stem-tip and scape-tip. Various combinations of media, growth regulators were compared. The best establishment and proliferation of stem-tip and scape-tip explants were obtained on MS medium containing 0.5 mg l^–1 NAA and 0.5 mg l^–1 KT. The roots were produced and the bulblets were formed after the micro-shoots were transferred to the hormone-free MS medium. The virus of each tube-plant was detected by ELISA. The virus elimination rates of plantlets from stem-tip with 3 leaf primordia, with 1–2 leaf primordia, and scape-up were 26.5%, 45.4%, and 77.6% respectively. The procedure of scape-tip culture would be applied for virus elimination to the species propagated vegetatively.     

Domestication of micropropagated plants of the spice damiana (Turnera diffusa)

Tissue culture propagation was performed on the spice shrub damiana (Turnera diffusa. Willd.) using MS medium (Murashige and Skoog 1962) supplemented with different combinations of the plant growth regulators, 6-benzyl adenine (BA) and indole-3-butyric acid (IBA). Organogenesis of leaf explants from wild plants and explants from propagated cuttings was compared; only the former regenerated complete plants. The highest shooting rate (92%) occurred at a concentration of 10^–7 M BA plus 10^–6 M IBA. Regenerated shoots were rooted in MS medium without any plant growth regulators. Foliage productivity of the micropropagated plants under field cultivation was determined yearly over 3 years. The yield increased annually for the first two years. The quantity of essential oils in propagated plants was similar to that of wild plants growing nearby. We propose tissue culture propagation of damiana as a viable means of domestication of this wild plant for semi-arid agriculture in Mexico. Commercial propagation would help to conserve wild populations of damiana that are currently threatened by overharvesting.Abbreviations BA 6-benzyl adenine - IBA indole-3-butyric acid     

6. Plant growth regulator effects in the in vitro propagation of three hardwood tree genera: Castanea,Juglans, and Quercus

Castanea, Juglans, and Quercus are genera containing hardwood forest species that are difficult to propagate asexually by conventional means. Their in vitro clonal propagation would assist forest tree improvement and silvacultural research. The research efforts in clonal propagation of these three genera are reviewed with respect to plant growth regulator effects as they are related to primary explant source, multiple shoot formation, somatic embryogenesis, rooting, and transfer to soil.     

Micropropagation and field performance of Yucca valida

Yucca valida is an important potential source of steroidal saponins closely related to Yucca schidigera, the species that is commercially exploited from the wild as a source of steroidal extracts. Neither of the species has been domesticated mainly because of their slow growth and long life span before harvesting. Here, we report a micropropagation method to generate isogenic or clonal lines for plantation purposes. Seventeen clonal lines were propagated and evaluated over a period of 26 months in an experimental plantation and compared with the performance of plants from seeds. The large variability found between the plants derived from seeds is manifested in the differences observed between the different clonal lines; however, these present a much smaller internal coefficient of variation than the one observed in the population of plants derived from seeds. Some clonal lines perform in a superior manner indicating that a process of selection and cloning can generate lines of fast growing individuals for plantations that can satisfy the demand for these materials without putting a natural resource at risk.     

RAPD Assessment for Identification of Clonal Identity and Genetic Stability of in vitro Propagated Chestnut Hybrids

Randomly amplified polymorphic DNA (RAPD) was used as a tool to assess the clonal identity of four in vitro propagated chestnut rootstock hybrids (Castanea sativa × C. crenata) described as originally isolated from the same mother tree. To confirm genetic stability after in vitro multiplication for more than 4 years, RAPD patterns of in vitro and donor plants were compared. From 40 arbitrary 10-mer primers used to amplify DNA, 21 provided patterns and were chosen for comparisons. Although significant differences were found in growth parameters between in vitro material of the putative clones, RAPD profiling showed polymorphism in none but one. This accession may then be withdrawn from the same clonal origin as the other three. As expected, no polymorphism was detected between the material propagated in vitro and the donor plants they originated from.     

Effects of nutritional and hormonal factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were found to produce anthraquinone glucosides. The effects of nutritional and hormonal factors on growth and anthraquinone production were investigated in order to study the enzymecatalyzed glucosylation of these metabolites.Abbreviations AQ Anthraquinone - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA 3-indole-acetic acid - HPLC High Performance Liquid Chromatography - TLC Thin Layer Chromatography     

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.     

Opposing effects of plant growth regulators via clonal integration on apical and basal performance in alligator weed

植物生长调节剂通过克隆整合对空心莲子草顶端和基部生长的不同作用 入侵植物不仅对全球生物多样性造成了巨大的威胁,同时也严重影响了农业生产与粮食安全。克隆整合使得相连植株进行资源共享,能促进入侵植物的生长从而获得优势。然而,入侵杂草 在植物调节剂(plant growth regulators, PGRs)影响下的克隆整合作用则很少有报道。PGRs被广泛应用于 农作物生产上,并能通过土壤淋溶、侵蚀和径流作用,影响分布在作物附近的农田杂草的生长。本 研究采用两种PGRs赤霉素(gibberellins, GA)和多效唑(paclobutrazol,PAC)处理恶性入侵杂草空心莲子草 (Alternanthera philoxeroides)基端,并保持或者通过剪切达到控制基端与顶端的连通,从而探究克隆整合作用在空心莲子草响应两种农业常用PGRs中的作用。研究结果表明,GA和PAC对空心莲子草生长的作用相反。GA通过克隆整合作用显著促进顶端植株的地上生长。相反地,PAC显著抑制基端和顶端的地 上生长,但是能够通过克隆整合作用显著促进基端和顶端的地下生长。这些研究结果解释了克隆整合作用能促进PGRs对空心莲子草生长的促进作用,这很可能是外来杂草能够成功入侵人为干扰较多的农业生态系统的重要原因之一。     

Growth and hare, Lepus timidus, resistance of white birch, Betula pendula, clones grown in different soil types

Many plant species have the ability to expand laterally through space by clonal growth. Plant pathogens can affect clonal growth characteristics thereby altering the success of host plants within populations and of clonal species within communities. We conducted a greenhouse experiment to determine the effects of the systemic fungal pathogen, Epichloë glyceriae , on clonal growth patterns of its host grass, Glyceria striata . We found that infected and uninfected plants produced similar total biomass and numbers of tillers plus primary stolons per mother ramet. However, biomass allocation to tillering (vegetative growth) vs stolon production (clonal growth) was significantly affected by pathogen infection. Infected plants produced more stolons and clonal growth biomass than uninfected plants while mother ramets of uninfected plants produced more tillers and biomass than infected plants. Stolon architecture of infected and uninfected plants also differed. In two of three populations, infected plants produced stolons with greater biomass and shorter spacer lengths, even though mean stolon lengths were similar for infected and uninfected plants. These results contrast strongly with most other clonal plant-pathogen systems where infected plants are less vigorous and have reduced clonal growth compared to uninfected plants. Greater clonal growth may be an effective mechanism for host genotypes to persist and spread when seed production is prevented, as is the case with castrating pathogens like Epichloë glyceriae .     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Brassica grown gall tumourigenesis and in vitro of transformed tissue

A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8–10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.     

Influence of plant development and environment on transgene expression in potato and consequences for insect resistance

Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance.     

Amritosides A, B, C and D: clerodane furano diterpene glucosides from Tinospora cordifolia

Four new clerodane furano diterpene glucosides (amritosides A, B, C and D) were isolated as their acetates from Tinospora cordifolia stems. The structures of these compounds were established on the basis of spectroscopic studies.