Application of Ectomycorrhizal Fungi in Vegetative Propagation of Conifers

In forestry, vegetative propagation is important for the production of selected genotypes and shortening the selection cycles in genetic improvement programs. In vivo cutting production, in vitro organogenesis and somatic embryogenesis are applicable with conifers. However, with most coniferous species these methods are not yet suitable for commercial application. Large-scale production of clonal material using cuttings or organogenesis is hindered by rooting problems and difficulties in the maturation and conversion limit the use of somatic embryogenesis. Economically important conifers form symbiotic relationship mostly with ectomycorrhizal (ECM) fungi, which increase the fitness of the host tree. Several studies have shown the potential of using ECM fungi in conifer vegetative propagation. Inoculation with specific fungi can enhance root formation and/or subsequent root branching of in vivo cuttings and in vitro adventitious shoots. Germination of somatic embryos and subsequent root growth can also be improved by the use of ECM fungi. In addition, inoculation can increase the tree's ability to overcome the stress related to ex vitro transfer. A specific interaction between a fungal strain and tree clone occurs during root induction and germination of somatic embryos. Multiple rooting factors exist in this interaction that complicate the predictability of the response to inoculation. Fungal-specific factors that influence rooting responses to inoculation may include plant growth regulator production, modification of the rooting environment, and interactions with beneficial microbes. A combination of these factors may act synergistically to result in positive responses in tree genotypes that are compatible with the fungus.     

Somatic embryo and root regeneration from quince leaves cultured in ventilated vessels or under different oxygen and carbon dioxide levels

Summary The influence of the gaseous composition of the atmosphere inside culturing vessels on somatic embryogenesis and on adventitious root formation was investigated in the quince clone (Cydonia ablonga Mill.) BA29. Leaves taken from in vitro-grown shoots were cultured in glass Petri dishes and exposed to ventilation with atmospheric air (flow rate 25 ml min⁻¹) for 0, 5, 10, 20, and 40 d. Twenty days of ventilation reduced the frequency of embryogenic leaves and a further decrease was observed after 40d of treatment. Conversely, adventitious root formation in the ventilated dishes was never different from the untreated cultures. In a second test, leaves were incubated in atmospheres containing different levels of oxygen (0, 5.0, 10.0, and 21.0%) or carbon dioxide (0, 0.04, 0.15, 1.5, and 3.0%). Anoxia conditions almost completely inhibited somatic embryo and adventitious root formation, but without compromising callus formation and explant viability. In contrast, embryo and root regeneration occurred even in totally CO₂-free atmosphere. Oxygen seemed to influence somatic embryogenesis according to a quadratic response; a similar relationship was also observed for root regeneration. Instead, no clear trend could be inferred between embryo or root regeneration and CO₂ levels. Furthermore, in dishes flushed with gas mixtures containing oxygen or carbon dioxide somatic embryo formation was almost always lower than in confined dishes. A different result was observed for root regeneration, since the number of roots was never lower than in the control and increased appreciably with 3.0% CO₂. These results demonstrate that atmosphere composition of the culture head-space can influence somatic embryogenesis in quince. The finding that both vessel ventilation and atmosphere replacement with different gas mixtures reduced somatic embryo formation suggests that gaseous compounds, different from O₂ an CO₂, present in the gaseous environment may promote embryogenesis in this species.     

Cellular dynamics during maturation‐related decline of adventitious root formation in forest tree species

Adventitious root formation is a process in which roots are induced, from determined or differentiated cells that have not been specified to develop a root, at positions where they do not normally occur during development. In forest tree species, a decline in the capacity to form adventitious roots from similar cell types in stem cuttings is associated with tree age and maturity. This decline limits the success of vegetative propagation of selected adult trees. The joint action of local signals and a dynamic cascade of regulatory changes in gene expression, resulting in stereotypical cell division patterns, regulate cell fate changes that enable a somatic differentiated cell to reactivate meristem programs toward the induction of an adventitious root meristem.     

小麦根愈伤组织胚胎发育过程研究

实验通过对６个人工合成小麦品系和对照品种“中国春”种子根愈伤组织分化形成再生植株的过程进行形态和组织切片观察,发现分化初期有２种途径,一种是从愈伤组织先形成不定胚,然后再发育成不定芽和不定根,另一种途径是直接从愈伤组织中分化发育成不定根和不定芽;分化后期不定芽和不定根生长发育有３种类型：一种是不定芽发育先于不定根,一种是不定芽与不定期不定芽和不定根生长发育有种类型：一种是不一定芽发育先于不定根,一     

Production of ROL gene transformed plants of Rosa hybrida L. and characterization of their rooting ability

Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable.The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.     

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

The aim of this study has been to identify important processes that regulate early stages of embryo development in conifers. Somatic embryogenesis in Picea abies has become a model system for studying embryology in conifers, providing a well-characterized sequence of developmental stages, resembling zygotic embryogeny, which can be synchronized by specific treatments, making it possible to collect a large number of somatic embryos at specific developmental stages. We have used this model to analyze global changes in gene expression during early stages of embryo development by generating an expression profile of 12,536 complementary DNA clones. This has allowed us to identify molecular events regulating putative processes associated with pattern formation during the earliest stages of embryogenesis which have not been identified on the molecular level in conifers before. We recognize notable changes in the expression of genes involved in regulating auxin biosynthesis and auxin response, gibberellin-mediated signaling, signaling between the embryo and the female gametophyte, tissue specification including the formation of boundary regions, and the switch from embryonic to vegetative development. In addition, our results confirm the involvement of previously described processes, including stress, differentiation of a protoderm, and programmed cell death.     

Proteomic analysis of different mutant genotypes of Arabidopsis led to the identification of 11 proteins correlating with adventitious root development

A lack of competence to form adventitious roots by cuttings or explants in vitro occurs routinely and is an obstacle for the clonal propagation and rapid fixation of elite genotypes. Adventitious rooting is known to be a quantitative genetic trait. We performed a proteomic analysis of Arabidopsis (Arabidopsis thaliana) mutants affected in their ability to develop adventitious roots in order to identify associated molecular markers that could be used to select genotypes for their rooting ability and/or to get further insight into the molecular mechanisms controlling adventitious rooting. Comparison of two-dimensional gel electrophoresis protein profiles resulted in the identification of 11 proteins whose abundance could be either positively or negatively correlated with endogenous auxin content, the number of adventitious root primordia, and/or the number of mature adventitious roots. One protein was negatively correlated only to the number of root primordia and two were negatively correlated to the number of mature adventitious roots. Two putative chaperone proteins were positively correlated only to the number of primordia, and, interestingly, three auxin-inducible GH3-like proteins were positively correlated with the number of mature adventitious roots. The others were correlated with more than one parameter. The 11 proteins are predicted to be involved in different biological processes, including the regulation of auxin homeostasis and light-associated metabolic pathways. The results identify regulatory pathways associated with adventitious root formation and represent valuable markers that might be used for the future identification of genotypes with better rooting abilities.     

Root Formation in Ethylene-Insensitive Plants

Experiments with ethylene-insensitive tomato (Lycopersicon esculentum) and petunia (Petunia x hybrida) plants were conducted to determine if normal or adventitious root formation is affected by ethylene insensitivity. Ethylene-insensitive Never ripe (NR) tomato plants produced more below-ground root mass but fewer above-ground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on vegetative stem cuttings of wild-type plants but had little or no effect on rooting of NR plants. Reduced adventitious root formation was also observed in ethylene-insensitive transgenic petunia plants. Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious root formation on vegetative stem cuttings from NR and wild-type plants, but NR cuttings produced fewer adventitious roots than wild-type cuttings. These data suggest that the promotive effect of auxin on adventitious rooting is influenced by ethylene responsiveness. Seedling root growth of tomato in response to mechanical impedance was also influenced by ethylene sensitivity. Ninety-six percent of wild-type seedlings germinated and grown on sand for 7 d grew normal roots into the medium, whereas 47% of NR seedlings displayed elongated tap-roots, shortened hypocotyls, and did not penetrate the medium. These data indicate that ethylene has a critical role in various responses of roots to environmental stimuli.     

Adventitious root initiation in hypocotyl and epicotyl cuttings of eastern white pine (Pinus strobus) seedlings

The present paper reports results of experiments to develop a system for studying adventitious root initiation in cuttings derived from seedlings. Hypocotyl cuttings of 2-week-old eastern white pine (Pinus strobus L.) seedlings were treated for 5 min with 0, 100, 200, 300, 400, 500 or 600 mg l^?1 (0, 0.54, 1.07, 1.61, 2.15, 2.69 or 3.22 mM) 1-naphthaleneacetic acid (NAA) to determine the effect on root initiation. The number of root primordia per cutting was correlated with NAA concentration and the square of NAA concentration. Thus, the number increased from less than one per cutting in the 0 NAA treatment to approximately 40 per cutting at 300 mg l-1 NAA, above which no substantial further increase was observed. The larger number of root primordia formed in response to increasing concentrations of NAA was due to the formation of primordia over a larger proportion of the hypocotyls. Histological analysis of the timing of root primordium formation in hypocotyl cuttings revealed three discernible stages. Progression through these stages was relatively synchronous among NAA-treated hypocotyl cuttings and within a given cutting, but variation was observed in the portion of different cuttings undergoing root formation. Control-treated hypocotyl cuttings formed root primordia at lower frequencies and more slowly than NAA-treated cuttings, with fewer primordia per cutting. Epicotyl cuttings from 11-week-old seedlings also formed adventitious roots, but more slowly than hypocotyl cuttings. NAA treatment of epicotyl cuttings caused more rapid root initiation and also affected the origin of adventitious roots in comparison with nontreated cuttings. NAA-treated epicotyl cuttings formed roots in a manner analogous to that of the hypocotyl cuttings, directly from preformed vascular tissue, while control-treated epicotyl cuttings first formed a wound or callus tissue and subsequently differentiated root primordia within that tissue. This system of inducing adventitious roots in pine stem cuttings lends itself to studying the molecular and biochemical steps that occur during root initiation and development.     

Vegetative propagation of Cecropia obtusifolia (Cecropiaceae)

Cecropia is a relatively well-known and well-studied genus in the Neotropics. Methods for the successful propagation of C. obtusifolia Bertoloni, 1840 from cuttings and air layering are described, and the results of an experiment to test the effect of two auxins, naphthalene acetic acid (NAA) and indole butyric acid (IBA), on adventitious root production in cuttings are presented. In general, C. obtusifolia cuttings respond well to adventitious root production (58.3% of cuttings survived to root), but air layering was the better method (93% of cuttings survived to root). The concentration of auxins used resulted in an overall significantly lower quality of roots produced compared with cuttings without auxin treatment. Future experiments using Cecropia could benefit from the use of isogenic plants produced by vegetative propagation.     

EXPERIMENTS WITH ROOT CUTTINGS OF BRUSSELS SPROUT

A technique for the propagation of Brussels sprout by means of root cuttings is described. Adventitious shoots arise exogenously on callus tissue which develops around the base of side roots. Cuttings sometimes rot without forming adventitious shoots, and cuttings which remain sound do not all produce shoots. Rotting may largely be prevented by planting cuttings with the proximal end exposed above the surface of the medium, and by allowing the root portions to dry before planting. Surface sterilization with mercuric chloride controls rotting but reduces bud formation. Individual plants differ in their capacity to form buds on root cuttings, and this difference is carried by the clones derived from them. Portions of root form more buds if cut into several pieces than if planted intact.     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Explant preparation affects culture initiation and regeneration of Panax ginseng and Panax quinquefolius

The influence of explant preparation on culture initiation and regeneration was investigated. Explant preparation was defined as the application of surface disinfection and homogenization to ginseng roots and embryos. Surface disinfection significantly affected culture initiation and subsequent embryogenesis. A high success rate (80–97%) in culture initiation was associated with explants from non-surface disinfected root tissue. The cumulative contamination rate after 8 months was below 4%; in contrast, a contamination rate of 85% was observed in disinfected root explants. For non-disinfected explants, visible callus was induced in 1 week, adventitious root and somatic embryos were formed in 2 and 6 months, respectively. Explants from disinfected roots required 1 month to induce visible callus, 5–7 months for adventitious roots and 10 months for somatic embryogenesis. For germinating embryos, disinfected embryos required double the time for embryogenesis than non-disinfected ones. It was considered that surface disinfection imposed a stress and subsequently a carry-over effect on explants.     

Dormant Buds and Adventitious Root Formation by <Emphasis Type="Italic">Vitis</Emphasis> and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.