茎顶端分生组织在植物发育过程中的保持、转变和逆转

顶端分生组织(shoot apical meristems,SAM)为产生新的器官和组织而不断提供新的细胞,它的活性依赖于平衡分生组织细胞的增殖和器官发生之间关系的调控基因.来自不具备光合能力的顶端分生组织的细胞可形成具有光合能力的营养器官.在从营养生长到生殖发育的转变过程中,茎顶端分生组织,转变为花序分生组织,最终形成花分生组织.在进入开花决定状态以前,SAM的状态很大程度上受到环境信号和转录调控因子的影响.以模式植物拟南芥为主,对在顶端分生组织的保持和转变中复杂同时又有差异的基因调控网络进行讨论.在花和花序分生组织逆转过程中,SAM中的细胞也受到相关基因的调控,且表达方式存在明显的时空差异.因此,具有决定性的和未决定性双重特性的分生组织之间的转变和相互协调,对于器官发生和形态建成起到至关重要的作用.     

In vitro phytochrome dark reversion process

Thermal reversion of the far-red absorbing form of phytochrome to the red absorbing form in darkness has been investigated in crude and partially purified isolates from a number of etiolated and light grown higher plants. The influence of temperature, aging and urea on the rate of reversion was also determined.

Phytochrome isolated from all higher plants underwent reversion. The reversion proceeded in at least 2 distinct stages; a short rapid initial phase being followed a slow phase which continued for many hours. Reversion rate was highest in phytochrome isolated from green leaves of parsnip (Pastinacea sativa) and lowest in that isolated from etiolated oats (Avena sativa). Although the rate of reversion could be changed by modifying the tertiary structure of the protein component, the large differences in rate appeared to be characteristic of the plant source. Observed in vitro rates of reversion are slower than those occurring in vivo. Removal of other buffer solubilized material during purification had little effect on the rate of reversion of phytochrome isolated from etiolated material.

     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Putative dual pathway of auxin transport in organogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, damage to the superficial acropetal polar auxin transport (PAT) inhibits generative but not vegetative organ initiation. In order to verify whether in a vegetative phase auxin can be transported to the meristem in a different way, the research on wild-type and plants with defective PAT was performed. Distance from the differentiated vascular elements to the shoot apical meristem (SAM) was measured for Arabidopsis cultured in different experimental systems. The influence of this distance on the ability to induce organogenesis as well as transport of the fluorescent dye to the SAM, and the LEAFY gene expression were analyzed. The youngest protoxylem elements were used as a marker of the vascular tissues. The distance of protoxylem to the SAM and organogenesis were interrelated. Organ initiation occurred only when protoxylem was localized near to the SAM. Experimental elongation of internodes in a vegetative rosette caused an increase in the distance between protoxylem and the SAM organogenic zone. Thus, the inhibition of organ initiation took place already during the vegetative phase. The results suggest the presence of at least two pathways of acropetal transport of auxin inducing organogenesis: one superficial way through PAT, and the second, putative one, internal through the vascular system. Possibly, organogenesis is completely blocked only when both these pathways are dysfunctional.     

Immunohistochemical localisation of ubiquitin and the proteasome in sunflower (Helianthus annuus cv. Giganteus)

This study provides an immunohistochemical demonstration of the involvement of the ubiquitin- and proteasome-dependent pathway during differentiation and organogenesis in plants. The localisation of ubiquitin and the proteasome was studied in meristems, leaves, stems and roots of sunflower (Helianthus annuus L. cv. Giganteus). By using a new technique that enhances very low antigen signals, we obtained information on the structural distribution of the ubiquitin- and proteasome-dependent pathway, and of the importance of this pathway during organogenesis and plant development. Ubiquitin and the proteasome showed overall similarities in their cellular localisation. The highest antigenic signal was observed in the root and shoot apical meristems, in leaf primordia and vascular tissue. The cambium showed less expression than the apical meristems. During adventitious root formation in cuttings, no sign of increased expression was observed within dedifferentiating tissue, but as organogenesis progressed, the antigenic signal of ubiquitin and the proteasome gradually increased in the developing roots. Comparison of immunochemical results and Western blots demonstrated that important changes in the cellular antigen signal could only be detected by immunochemistry.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.     

拟南芥WUSCHEL基因在转基因烟草中的超表达

WUSCHEL(WUS)是近年报道的一个重要的干细胞调控基因.本实验用RT-PCR技术从拟南芥(Arabidopsisthaliana L.)中克隆到其cDNA并构建了双增强的CaMV3 5S启动子驱动的超表达载体pBKB.借助农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum L.),获得转基因植株.PCR和RT-PCR鉴定分别证明,外源WUS已整合到烟草基因组并已表达.转基因烟草地上部分出现大量异位增生的突起,扫描电镜观察表明:突起部分的细胞与分生组织细胞相似,部分突起能够发育为叶芽、花芽,表明WUS超表达引起烟草细胞异常分裂并在已分化组织中重新启动了器官形成.茎尖和花的内两轮器官没有上述变化.结合拟南芥的有关研究,推测烟草中可能也存在类似拟南芥WUS和其阻抑蛋白CLAVATA3、AGAMOUS间的反馈调节机制.转基因烟草叶发育表型变化明显,与生长素极性运输受抑制引起的表型相似,因此,作为生长点调控基因,WUS可能通过生长素对叶的发育进行调控.本研究为WUS基因的功能分析和有关生物技术应用提供了有意义的信息.     

拟南芥WUSCHEL基因在转基因烟草中的超表达(英文)

The Arabidopsis WUSCIHIEL (WUS） gene plays a key role in the specification of the stem cellsin the shoot apical meristem (SAM). A cDNA of WUShas been amplified with the RT-PCR approach fromArabidopsis. The plant overexpression vector was constructed. It was driven by a dual enhanced CaMV35Spromoter. The construct was transformed into tobacco (Nicotiana tabacum L.) via Agrobacterium mediation.Dramatic phenotypic changes appeared in the WUS overexpression transgenic plants. Aberrant celldivisions and ectopic organogenesis could be found in almost every aerial parts of the transgenic tobaccoexcept the meristems and the inner two floral whorls. The data showed a highly conserved function of WUSin tobacco, and suggested that WUS is involved in organogenesis. The leaves were malformed, whichstrongly matched those only described previously for plants grown in the presence of polar auxin transportinhibitors. It suggested a possible function of WUS in leaf development. These results provide usefulinformation for functional analysis of WUS and important biotechnological implication as well.     

Changes of axillary meristem localization in relation to flowering ofRaphanus sativus L. after GA treatment

Two phases of radish ontogenesis (I-when the plant had produced 3 –5 nodes and II-when the plant had produced 8 –10 nodes) were established on the basis of axillary, meristem localization. Flowering of the plants in response to GA treatment depends on the phases in which they were treated and on growth correlations in the apical meristem. The results obtained suggest that the reaction ofRaphanus sativus (LDP) to GA treatment is parallel to that ofChenopodium rubrum (SDP), and that the response of radish plants also depends on changes in growth correlations in the shoot apical meristem at the time of treatment.     

Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> roots to lipo-chitooligosaccharide from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and other chitin-like compounds

Lipo-chitooligosaccharides (LCOs) are bacteria-to-plant signals required for the establishment of rhizobia–legume nitrogen fixing symbioses. The ability of LCO [Nod Bj V (C_18:1, MeFuc)] isolated from B. japonicum (strain 532C), and of oligomers of chitosan (tetramer, pentamer) and chitin (pentamer) to affect the developmental morphology of roots in Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) was assessed using an interactive scanner-based image analysis system. LCOs have been shown to play a role in plant organogenesis at nanomolar concentrations. LCO and the chitin pentamer promoted root growth and development in Arabidopsis at concentrations of 10 nM and 100 μM, respectively. The LCO treated Arabidopsis plants had about 35% longer roots than untreated control plants. Similarly, treatment with 100 μM chitin pentamer (CHIT5) resulted in 26% longer roots than the untreated plants; however, chitosan oligomer (CH4 or CH5) treated plants did not differ from the control plants at either concentration (100 or 1 μM). Both LCOs and the chitin pentamer at higher concentrations increased root surface area, mean root diameter and number of root tips. However, leaf area increase was observed only in plants treated with LCO at 10 nM.     

Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in <Emphasis Type="Italic">Cedrela fissilis</Emphasis> Vellozo (Meliaceae)

Improvements to in vitro organogenesis are essential for optimizing shoot development and understanding basic physiological processes. The addition of polyamines (PAs) to the culture medium has been used to modulate organogenesis in plants, and this work evaluated the effects of exogenous PAs on direct organogenesis from apical and cotyledonary nodal Cedrela fissilis explants as well as the effects of putrescine (Put) on endogenous PA levels and variations in protein abundance. The effects of exogenous Put, spermidine, and spermine at 0, 0.5, 1, 2.5, or 5 mM on shoot development were tested. The comparison of the tested PAs to the control treatment revealed that 2.5 mM Put significantly increased the length of shoots from cotyledonary nodal explants, which are more sensitive than apical nodal explants, and treatment with 2.5 mM Put significantly increased the endogenous total free-PA and free-Put levels in shoots compared with the control (no Put). A comparative proteomic analysis of shoots indicated that 2.5 mM Put significantly changed the abundance of proteins, primarily metabolic and cellular proteins associated with stress and energy processes such as cell division. These results show that Put functions in endogenous PA metabolism and alters protein abundance, thereby contributing to shoot development in C. fissilis.     

Effects of apical damage on plant growth and male and female reproductive investments in <Emphasis Type="Italic">Ambrosia artemisiifolia</Emphasis>, a wind-pollinated plant

In wind-pollinated plants, apical damage may decrease male fitness by reducing height-dependent pollen dispersal distance, but may not affect female fitness because plant height is not always correlated with female fitness. We hypothesized that Ambrosia artemisiifolia responds to apical damage by (1) restoring plant height through compensatory growth from lateral buds, and/or (2) increasing the sex allocation to female function to compensate for the loss of male fitness. We tested these hypotheses by comparing a group of experimental removal of the apical meristem with three control groups and by field surveys on apically damaged plants. Experimental apical damage suppressed main stem growth, but promoted vertical secondary growth from lateral buds. These responses resulted in compensation of stem height in the apically damaged plants to the same height as one of three control groups. The numbers of male and female flowers and male racemes did not differ between damaged and undamaged plants, indicating that apically damaged plants did not change their sex allocation. Therefore, our results support our first hypothesis. The results of a field survey of naturalized populations also supported the first hypothesis in that plant height and the number of male racemes did not change in plants with apical damage. Consequently, our results suggest that A. artemisiifolia has a high ability of fitness compensation after apical damage by restoring height and male function. This ability may contribute to its invasiveness in disturbed habitats.     

大蒜花序轴离体培养器官发生途径的解剖学研究

以大蒜品种‘三月黄’(Allium sativum L.cv. Sanyuehuang)花序轴为外植体进行离体培养,对其器官发生过程进行了形态学和解剖学观察。结果显示:大蒜花序轴离体培养不经过愈伤组织,通过器官直接发生途径形成不定芽,其不定芽起源于大蒜花序轴维管组织韧皮部一侧周围的皮层薄壁细胞,属于外起源;皮层薄壁细胞经脱分化后,由最先形成的拟分生组织发育为茎尖分生组织,然后环绕其形成叶原基,茎尖和叶共同构成一个完整的不定芽;大蒜花序轴离体培养发生的不定芽与花苞中自然形成的营养芽发生部位一致。不定芽通过壮苗、生根培养可正常生根形成植株,如果继代培养周期超过21 d,鳞茎形成率可达90.56%。     

Effect of phyllotactic position and cultural treatments toward successful direct shoot organogenesis in dwarf ‘Pixie’ grapevine (Vitis vinifera L.)

In Vitis spp. where somatic embryogenesis-based regeneration predominates, an efficient, reproducible and robust method of direct shoot organogenesis from leaf explant material has been established in the dwarf wine grape ‘Pixie’ (Vitis vinifera). This regeneration system was achieved by testing the response of leaf material in two stages of development, and pre-conditioning the explant material in dark conditions and/or in liquid media prior to excising from the plant and placing it on solidified media. The pre-excision treatments included (1) a dark period of 24 h, with no regeneration medium; (2) soaking in regeneration medium followed by a dark period of 24 h; (3) a dark period of 24 h followed by soaking in liquid VRM (Vitis Regeneration Medium); (4) vacuum infiltration in liquid VRM followed by a dark period of 24 h; and (5) a control of no pre-conditioning treatment. Excised leaves from pre-treated intact plants in vitro significantly increased the frequency of shoot organogenesis. The most responsive explant material consisted of young semi-translucent apical leaves varying in size from 3 to 8 mm in length. The most successful combinations of factors contributing to shoot organogenesis involved the solely dark-exposed apical leaves or the soaking in VRM followed by a dark period. These results are expected to facilitate Vitis-related research in genetics, functional genomics, physiology, and other fields.     

Influence of type of explant,plant growth regulators,salt composition of basal medium,and light on callogenesis and regeneration in <Emphasis Type="Italic">Passiflora suberosa</Emphasis> L. (Passifloraceae)

Passiflora suberosa is used in popular medicine, improvement programs, and as an ornamental plant. The goal of this study was to establish efficient protocols for plant regeneration and callus induction from nodal, internodal and leaf segments excised from in vitro-grown plants. The different morphogenetic responses were modulated by the type and concentration of plant growth regulators, according to the basal medium and light conditions. Shoot formation occurred through three pathways: (1) development of preexisting meristems, (2) direct organogenesis, and (3) indirect organogenesis. Development of preexisting meristems was observed from nodal segments (1 shoot/explant) in response to α-naphthaleneacetic acid (NAA), picloram (PIC), and 2,4-dichlorophenoxyacetic acid (2,4-D), using two basal media (MS and MSM). Direct organogenesis in this species was obtained for the first time in this work, through shoot development from internodal segments in the presence of 6-benzyladenine (BA). The highest regeneration rates were achieved on MSM medium, regardless of the BA concentration. Indirect organogenesis was achieved from all explant types on media supplemented with BA, used alone or in combination with NAA. The highest regeneration efficiency was obtained from internodal segments cultured on MSM medium plus 44.4 μM BA. Compact, friable, or mucilaginous non-morphogenic calluses were induced by thidiazuron, PIC, 2,4-D, and NAA. High-yielding friable calluses obtained on MSM medium supplemented with 28.9 μM PIC are being used for the establishment of suspension cultures and further analysis of the production of bioactive compounds.     

The role of PENNYWISE and POUND-FOOLISH in the maintenance of the shoot apical meristem in Arabidopsis

Growth of the aerial part of the plant is dependent upon the maintenance of the shoot apical meristem (SAM). A balance between the self-renewing stem cells in the central zone (CZ) and organogenesis in the peripheral zone (PZ) is essential for the integrity, function, and maintenance of the SAM. Understanding how the SAM maintains a balance between stem cell perpetuation and organogenesis is a central question in plant biology. Two related BELL1-like homeodomain proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF), act to specify floral meristems during reproductive development. However, genetic studies also show that PNY and PNF regulate the maintenance of the SAM. To understand the role of PNY and PNF in meristem maintenance, the expression patterns for genes that specifically localize to the peripheral and central regions of the SAM were examined in Arabidopsis (Arabidopsis thaliana). Results from these experiments indicate that the integrity of the CZ is impaired in pny pnf plants, which alters the balance of stem cell renewal and organogenesis. As a result, pools of CZ cells may be allocated into initiating leaf primordia. Consistent with these results, the integrity of the central region of pny pnf SAMs can be partially restored by increasing the size of the CZ. Interestingly, flower specification is also reestablished by augmenting the size of the SAM in pny pnf plants. Taken together, we propose that PNY and PNF act to restrict organogenesis to the PZ by maintaining a boundary between the CZ and PZ.