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151.
152.
153.
Estelle Villemont Frédéric Dubois Rajbir S. Sangwan Gérard Vasseur Yvan Bourgeois Brigitte S. Sangwan-Norreel 《Planta》1997,201(2):160-172
Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP
6-benzylaminopurine
- BM
basal medium
- BrdUrd
bromodeoxyuridine
- GUS
-glucuronidase
- KmR
kanamycin resistant
- T-DNA
transferred DNA 相似文献
154.
Summary Microscopy techniques were used to identify the pathway of transport of soybean leaf vegetative storage proteins (VSP/ and VSP94) to the vacuoles of a specialized cell type, the paraveinal mesophyll (PVM), where they accumulate. PVM cells are enriched in endoplasmic reticulum and Golgi bodies relative to surrounding mesophyll cells. The margins of medial and trans Golgi cisternae had attached or closely associated noncoated vesicles with densely staining membranes and lumenal contents of the same appearance as material that accumulated in the vacuole. These vesicles appeared to be transported preferentially to the tonoplast, where fusion with the membrane released the granular contents into the vacuole. Cytochemical staining with phosphotungstic acid and silver methenamine supported this interpretation as both the Golgi vesicles and the tonoplast stained intensely with these reagents, unlike the tonoplast of mesophyll cells which do not accumulate VSP. Immunocytochemical localization for VSP/ labeled the Golgi bodies and associated vesicles, and vacuolar material in PVM cells, but not in mesophyll. Similar labeling was seen in PVM of another legume species previously found to accumulate antigenically similar VSPs. Immunolocalization for VSP94, a lipoxygenase, labeled the PVM cytosol and material in the PVM vacuole, but not the Golgi or vesicles. The results of this study demonstrate that the Golgi pathway is utilized for transport of VSP/ in the PVM, which follows the mechanism of deposition demonstrated for certain seed storage proteins. VSP94 appeared to follow a separate path for accumulation in PVM vacuoles.Abbreviations LOX
lipoxygenase
- PVM
paraveinal mesophyll
- RER
rough endoplasmic reticulum
- TEM
transmission electron 相似文献
155.
Tissue culture techniques involving cell suspension, protoplast fusion and culture in the genus Allium are different as Allium
is recalcitrant due to the biological peculiarity of the genus. A procedure is described for the establishment of a regenerable
suspension culture and for the isolation and culture of protoplasts of Allium cepa.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
156.
本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。 相似文献
157.
Protoplasts of Boergesenia forbesii (Harvey) were treated with inhibitors of protein synthesis in order to investigate their effects on cellulose synthesis. Cellulose synthesis was reversibly inhibited by 10 μM cycloheximide as assayed by fluorescence microscopy of Tinopal binding to cellulose. Freeze fracture and image analysis of cycloheximide- treated cells indicated a reduction in the number of intramembrane particles; however, the terminal synthesizing complexes remained at all times. Treatment with 10 μM actinomycin D, when applied during the first hour of protoplast formation, irreversibly inhibits cellulose synthesis and terminal complex formation. De novo protein synthesis is required for cell wall regeneration by protoplasts. The data suggest that the structural subunits visualized in the terminal complex do not undergo significant turnover, but that there may exist an essential proteinaceous component of cellulose synthesis which must be continually renewed. 相似文献
158.
Chlorella pyrenoidosa (UTEX 1230) cells in late log phase of growth were induced to form viable protoplasts by enzymatic digestion only when incubated in 2-deoxy-d-glucose (2DG) for 24 h. The combination of hemicellulase (4% w/v), Cellulysin (4% w/v), and glucuronidase (5% v/v) with 0.8 M mannitol and 8 mM CaCl2 in modified Bristol's solution, was most effective for obtaining viable protoplasts as determined by light and electron microscopy, and vital staining with primuline (0.01% w/v). Resistance of cell walls to extensive extraction (acetolysis), and infrared analysis indicated that sporopollenin is a component of the cell wall. Transmission electron miscroscopy of acetolysed cell walls also allowed visualization of the laminate nature of the wall. This is the first report of successful induction of protoplasts from algae which contain sporopollenin in their cell walls. 相似文献
159.
New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli 总被引:4,自引:0,他引:4
A new type of microfluorometer was applied to assess photosynthesis at the single-cell level by chlorophyll fluorescence using the saturation pulse method. A microscopy–pulse amplitude modulation (PAM) chlorophyll fluorometer was combined with a Zeiss Axiovert 25 inverted epifluorescence microscope for high-resolution measurements on single mesophyll and guard cells and the respective protoplasts. Available information includes effective quantum yield of photosystem II, relative electron transport rate and energization of the thylakoid membrane due to the transthylakoidal proton gradient. Dark–light induction curves of guard cell (GCPs) and mesophyll cell protoplasts (MCPs) displayed very similar characteristics, indicating similar functional organization of thylakoid membranes in both types of chloroplasts. Light response curves, however, revealed much earlier saturation of photosynthetic electron flow in GCPs than in MCPs. Under anaerobiosis, photosynthetic electron flow and membrane energization were severely suppressed. A similar effect was observed in guard cells when epidermal peels were incubated with the fungal toxin fusicoccin which activates the plasma membrane H+-ATPase and causes irreversible opening of stomata. The drop in electron transport rate was prevented by blocking ATP consumption of the H+ pump or by glucose addition. These results show that chlorophyll fluorescence quenching analysis allows profound insights into stomatal physiology. 相似文献
160.
Chen Zhi-xian Li Shu-jun Yue Jian-xiong Jiao Gai-li Liu Shao-xiang She Jian-ming Wu Jing-yin Wang Hai-bo 《植物学报(英文版)》1989,31(12)
Protoplasts were isolated from an embryogenic suspension culture of commercial cotton cv. The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium with K3 mineral salts and modified Km8p organic compositions, supplemented with 0.05–0.1 mg/l 2,4-D, 0.2–0.5 mg/l 2ip in the dark. The regenerated plantlets from protoplasts of coker312 and coker 201 cv. were obtained. Embryogenesis from protoplast of Jin4 cv. and microcolonies form protoplasts of JiHe321 and Lul cv. were observed. 相似文献