Elucidation of the role of activin in organogenesis using a multiple organ induction system with amphibian and mouse undifferentiated cells in vitro

Studies performed over the last century have clarified the mechanisms of organ and tissue formation. Mesoderm formation is one of the most important events in early body pattern determination during embryogenesis. In 1988, we found that activin A has mesoderm-inducing activity. As activin A could induce dorsal mesoderm formation, unlike fibroblast growth factor and bone morphogenetic protein, this factor was thought to be the molecular entity of the Spemann-Mangold organizer. Subsequently, the mechanisms of early embryogenesis have been clarified using molecular biological techniques, resulting in the identification of many genes that are involved in organ and tissue development. This finding that activin A could induce dorsal mesoderm formation spurred research into the application of agents that induce organs and tissues in vitro . In this regard, we have shown that many organ types can be induced by activin A in vitro . Moreover, we have found that other types of organs can be induced by changing the conditions of treatment. To date, more than 20 different types of tissues and organs have been successfully induced from Xenopus undifferentiated cells in vitro . In recent years, we have applied these protocols to mouse embryonic stem cells, and we have successfully induced several tissues, such as the pancreas and cardiomyocytes. We are also investigating how the pluripotency of undifferentiated stem cells is regulated. In this review, we summarize the current knowledge regarding activin as a mesoderm-inducing factor and its application for the induction of tissues and organs from undifferentiated cells. Moreover, we provide some examples of in vitro tissue differentiation from mouse embryonic stem cells, which may prove useful in regenerative medicine.     

Age and Physiological Condition of Donor Plants Affect in vitro Morphogenesis in Leaf Explants of Passiflora edulis f. flavicarpa

The regeneration potential of D.alata L. germplasm preserved in vitro was compared with the micropropagation of fresh material. Nodal cuttings were conserved for 9 months in different treatments based on D-571 culture medium modified, using several variable components (mannitol, benzylaminopurine and activated charcoal). Regeneration at 8 weeks, assessed by means of percentage of explant regenerating and the multiplication at 5 weeks through the shoot length and de novo bud count formation per explant were determined. The results showed high rates (100 and 98%) of explant regeneration and micropropagation from in vitro material maintained in D-571 medium with 1.5% of mannitol + 0.1 or 1 mg l^–1 of benzylaminopurine + 2 g l^–1 of activated charcoal, respectively.     

商陆属中五基数花起源于三基数花的新证据——非洲商陆的花器官发生

运用扫描电镜和石蜡切片方法对非洲商陆Phytolacca dodecandra L′Her.的花器官发生过程及花器官各部分的位置排列进行了观察。结果表明：非洲商陆萼片5枚,以螺旋向心方式发生;没有观察到花瓣原基的发生;雄蕊为螺旋离心方式发生,前4枚雄蕊分2对出现在对萼位置,但此后发生的雄蕊的发生顺序明显区别于其他种;心皮4-5枚,与内部雄蕊互生。本文探讨了时空因素对花器官发生的影响,基于对非洲商陆花器官发生的观察,否定了“商陆属源于二轮雄蕊样式”的假说,并为商陆属中“五基数花起源于三基数花”的观点提供了新的证据。     

提高榨菜离体培养植株再生频率

采用榨菜“浙桐1号”品种为材料,以MS为基本培养基,通过对不同植物生长调节剂的组合和不同外植体等主要因素的筛选,大幅度提高了榨菜离体培养植株再生频率。结果表明,2mg／L6．BA 0．2mg／L2,4-D的组合较为适宜,其不定芽再生频率可达50％,且外植体以下胚轴为好：而CPPU和2,4-D的适宜组合为1．5mg／L 0．2mg／L,其不定芽再生频率高达66．67％,最适外植体为带柄子叶。同时,研究结果显示,添加0．25～1mg／L的GA,对榨菜已分化的不定芽的伸长有抑制作用;子叶柄和下胚轴外植体的分化具有极性现象。     

Recent advances in Pelargonium in vitro regeneration systems

Recent advances in the development of protocols for in vitro culture and genetic manipulation have provided new avenues for the development of novel varieties of Pelargonium and for use as model systems for investigating the factors controlling plant morphogenesis. Optimized techniques of meristem culture have supplemented the culture indexing methods in commercial greenhouse production resulting in availability of large-scale pathogen indexed planting material. Currently, technologies are available for the mass in vitro propagation of F1 hybrid Pelargonium through both organogenesis and somatic embryogenesis. The somatic embryogenesis model system has allowed researchers to identify critical factors controlling plant morphogenesis in vitro such as regulation of regeneration by growth regulators, choice of explant and characterization of induction and expression phases of morphogenesis in Pelargonium. Also, optimization of technologies for genetic transformation of Pelargonium opened up the possibilities for developing genotypes with novel characters, including resistance to some of the major diseases. Finally, the development of regeneration systems for Pelargonium spp. has facilitated conventional crop improvement programs, thereby providing a valuable resource to the horticultural industry.     

In vitro regeneration of Sophora toromiro from seedling explants

Embryonic axes with cotyledons, shoot-tips of embryonic axes, isolated cotyledons, as well as axillary buds and leaves from 20-year-old trees of Sophora toromiro, were evaluated for their capacity to trigger organogenesis and to regenerate plantlets under in vitro conditions. Embryonic shoot-tips were the only explants capable of regenerating plants. They developed rapidly in vitro in the presence of NAA and BA while in subculture roots were induced at the proximal end in the presence of 0.49 μM IBA within 40–60 days. Development was completed with a subculture phase under non-sterile conditions using a mixture of equal parts of sterilized vermiculite/sand/soil in growth chambers, before final acclimation in the greenhouse. In the presence of NAA, BA and GA₃, whole embryonic axes formed multiple shoots that branched when grown in 2.27 or 11.35 μM TDZ in subculture. Similarly, callus was initiated at the embryo axis base, developing into several new shoots in the presence of TDZ. Because of the relatively high shoot induction rate along the embryonic axis, this axis presents a valuable source of new juvenile explants. Growth and rhizogenesis was satisfactory only when organs from seed pods of the year or from the previous season were used. Experiments with isolated cotyledons produced callus only, while axillary buds and leaves did not show any responses in the presence of several growth regulators assayed. Inoculation of seedlings with various strains of rhizobia under in vitro conditions resulted in root outgrowths, but not in nodules that are typical of rhizobia infection. This revised version was published online in June 2006 with corrections to the Cover Date.     

Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus

A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54 μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than 150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mannose: A Potential Selection System for Genetic Transformation of Annatto

The aim of the present work was to evaluate the feasibility of mannose as a selection system for the future genetic transformation of annatto (Bixa orellana L.). Hypocotyl segments, inverted hypocotyls and immature zygotic embryos were inoculated onto a Murashige and Skoog's medium supplemented with B5 vitamins, 87.6 mM sucrose and mannose in different combinations, 2.8 g dm^?3 Phytagel®, and 4.56 μM zeatin (organogenesis) or 2.26 μM 2,4-dichlorophenoxyacetic acid and 4.52 μM kinetin (somatic embryogenesis). Annatto explants did not regenerate on medium with mannose as the only carbon source when inverted hypocotyls and immature zygotic embryos were used. However, organogenesis (5 % frequency) occurred exclusively in hypocotyl-derived explants nearest to the crown (collar) of the seedlings. No further shoot development was achieved. Therefore the substitution of sucrose by mannose inhibited both organogenesis and embryogenesis, and thus the employment of mannose could constitute an alternative selective agent in protocols for genetic transformation of this species.     

台闽苣苔(苦苣苔科)花部器官的形态发生

在扫描电镜下对台闽苣苔 (T. oldhamii (Hemsl.) Solereder)进行了花部器官形态发生的观察,为探索该类群的个体发育、类群间的系统发育关系和进化趋势提供依据.研究发现该属植物萼片、花冠和雄蕊发生式样均为五数花类型,它们各自来源于花原基上分化出来的萼片原基、花冠原基和雄蕊原基;花冠与雄蕊的两侧对称性与花冠上唇生长稍快和退化雄蕊原基发育迟滞相关;萼片原基的发生和发育的顺序是不一致的:萼片原基发生的式样为近轴中原基-远轴2原基-2侧原基,发育式样则为近轴中萼片-2侧萼片-远轴2萼片,花蕾时为镊合状排列.花冠裂片原基的发生和发育式样是一致的,即远轴中裂原基(下唇中裂片)-远轴2侧裂原基(下唇2侧裂片)-近轴2裂原基(上唇2裂片).花蕾期卷迭式为覆瓦状排列,从外向内:下唇中裂片-下唇2侧裂片-上唇2裂片或下唇2侧裂片-上唇2裂片-下唇中裂片.雄蕊原基与花冠裂片原基互生,前方雄蕊原基在发生上稍迟于后方雄蕊原基,后者与退化雄蕊原基几乎同时发生,但较小,并与近轴心皮(或柱头上唇)对生.将该属与玄参科(Scrophulariaceae)的地黄属( Rehmannia )、苦苣苔科(Gesneriaceae)的异叶苣苔属( Whytockia)和尖舌苣苔属(Rhynchoglossum )的花部器官比较发现,这四个属在这方面呈现出多样性和交叉.过去一直按子房室数和胎座类型划分玄参科(子房2室、中轴胎座)和苦苣苔科(子房1室、侧膜胎座)这一做法受到了质疑.     

茄科马尿泡和天仙子的花器官发生

利用扫描电镜研究了茄科(Solanaceae)天仙子族(Hyoscyameae)中国特有属马尿泡属( Przewalskia Maxim.)马尿泡( Przewalskia tangutica Maxim.)和天仙子属( Hyoscyamus L.)天仙子( Hyoscyamus niger L.)的花器官发生和发育,研究结果表明:马尿泡和天仙子花器官的发生和发育具有以下3个共同特征:1)符合Hofmeister规律, 即新器官的发生首先出现在花顶已经存在的器官之间;2)花冠的发育模式符合茄科植物所具有的"后合瓣"("late sympetaly")现象,即花瓣单独发生但后来又通过它们基部分生组织的融合而连合起来;3)花被五基数且花器官原基发生顺序为向心发育.但是它们的花萼原基具有不同的发生方式.天仙子花萼裂片原基的发生方式为环状发生;马尿泡花萼裂片原基的发生方式为螺旋状发生,但5个花萼裂片原基在都出现后就连成了一个环.马尿泡是介于天仙子属和山莨菪属之间的类群,它比天仙子属原始但较山莨菪属进化.