Further characterization of picloram-tolerant mutants of Nicotiana tabacum

Summary A genetic and preliminary biochemical analysis has been performed on four picloram-tolerant mutants of Nicotiana tabacum that were isolated from cell cultures. The four mutations define three distinct linkage groups. Mutant seedlings incorporate radioactively labeled picloram normally and do not modify or degrade the herbicide in a manner that alters its solubility characteristics.     

Changes in Glucose-6-Phosphate Dehydrogenase, Ribonucleases, Esterases and Contents of Viruses in Potato Virus Y Infected Tobacco Superinfected with Tobacco Mosaic Virus

Effects of the superinfection with tobacco mosaic virus (TMV) on susceptible tobacco plants infected with potato virus Y (PVY) were determined. Dynamic changes in the TMV and/or PVY contents, the ribonucleases (RNases), the phosphomonoesterase (PME), the phosphodiesterase (PDE) and the glucose-6-phosphate dehydrogenase (G6P DH) activities were studied. The PVY infection caused a substantial reduction in the multiplication of TMV. The content of TMV in the PVY inoculated leaves amounts to 6 and 9 % in the PVY systemically infected leaves when compared with single TMV. Surprisingly, the challenging virus (TMV) enhanced the content of inducing virus (PVY) in the locally inoculated leaves up to 130 – 141 %. In contrast, the reduction of PVY content down to 35 – 40 % by TMV was seen in the PVY systemically infected leaves. The activities of the RNase, the PME, the PDE and the G6P DH were increased (when compared with the healthy plants) during the acute phase of single virus multiplication (PVY or TMV). The increase in the activities of the enzymes in the leaves with mixed infection was at least as high as the sum of the increases of single infections. Moreover, a higher increase than the sum was seen for G6P DH and PDE (by about 20 – 35 %). This revised version was published online in July 2006 with corrections to the Cover Date.     

烟草类根瘤中ATPase的活性变化及分布特性

采用磷酸铅技术,对烟草类根瘤中ATPase的活性变化及分布特征进行了研究。分生细胞中没有磷酸铅颗粒,非含菌细胞的细胞质和细胞器中有少量的磷酸铅颗粒,但在年轻和成熟根瘤菌中却未见它们。相反,当非含菌细胞和根瘤菌开始衰老后,有大量的磷酸铅颗粒位于细胞的质膜和细胞壁上以及根瘤菌表面的内侧。随着它们进一步衰老,磷酸铅颗粒越来越多,广泛分布在细胞的液泡膜、质膜、细胞壁、胞间层、胞间隙及根瘤菌的表面、细胞质和拟核中。由于细胞的解体,磷酸铅颗粒明显减少,一般只位于质膜和由细胞器解体而来的膜泡状结构上。     

Does elevated atmospheric carbon dioxide affect internal nitrogen allocation in the temperate trees Alnus glutinosa and Pinus sylvestris?

Nitrogen‐fixing plant species growing in elevated atmospheric carbon dioxide concentration ([CO₂]) should be able to maintain a high nutrient supply and thus grow better than other species. This could in turn engender changes in internal storage of nitrogen (N) and remobilisation during periods of growth. In order to investigate this one‐year‐old‐seedlings of Alnus glutinosa (L.) Gaertn and Pinus sylvestris (L.) were exposed to ambient [CO₂] (350 µ mol mol ^{? 1}) and elevated [CO₂] (700 µ mol mol ^{? 1}) in open top chambers (OTCs). This constituted a main comparison between a nitrogen‐fixing tree and a nonfixer, but also between an evergreen and a deciduous species. The trees were supplied with a full nutrient solution and in July 1994, the trees were given a pulse of ¹⁵N‐labelled fertiliser. The allocation of labelled N to different tissues (root, leaves, shoots) was followed from September 1994 to June 1995. While N allocation in P. sylvestris (Scots pine) showed no response to elevated [CO₂], A. glutinosa (common alder) responded in several ways. During the main nutrient uptake period of June–August, trees grown in elevated [CO₂] had a higher percentage of N derived from labelled fertiliser than trees grown in ambient [CO₂]. Remobilisation of labelled N for spring growth was significantly higher in A. glutinosa grown in elevated [CO₂] (9.09% contribution in ambient vs. 29.93% in elevated [CO₂] leaves). Exposure to elevated [CO₂] increased N allocation to shoots in the winter of 1994–1995 (12.66 mg in ambient vs. 43.42 mg in elevated 1993 shoots; 4.81 mg in ambient vs. 40.00 mg in elevated 1994 shoots). Subsequently significantly more labelled N was found in new leaves in April 1995. These significant increases in movement of labelled N between tissues could not be explained by associated increases in tissue biomass, and there was a significant shift in C‐biomass allocation away from the leaves towards the shoots (all above‐ground material except leaves) in A. glutinosa. This experiment provides the first evidence that not only are shifts in C allocation affected by elevated [CO₂], but also internal N resource utilisation in an N₂‐fixing tree.     

Genetics of Burley and Flue-Cured Tobacco Resistance to Globodera tabacum tabacum

Genotypes of burley (cultivars B-21 and B-49), flue-cured (line VA-81 and cultivar PD-4), and Connecticut broadleaf (cultivar C9) tobacco (Nicotiana tabacum) resistant (R) or susceptible (S) to the tobacco cyst nematode Globodera tabacum tabacum were crossed. F1 progeny of burley and susceptible broadleaf were selfed and backcrossed to produce additional progeny for evaluation of resistance in greenhouse experiments. Plants without adult female nematodes visible (×10 magnification) on the root surface 6 weeks after inoculation were classified as resistant, whereas those plants in which one or more females were evident were classified as susceptible. Segregation ratios for progeny of resistant and susceptible plants were not different from 3:1 and 1:1 for F2 (F1 × F1) and BC1 (F1 × S) lines, respectively, indicating that resistance in burley to G. t. tabacum is conferred by a single, dominant gene. Segregation ratios for resistance in crosses between nematode-resistant burley and flue-cured tobacco (F1 and F2 progeny) and between burley-flue-cured hybrids and broadleaf BC1 (F1 × S) and BC2 (BC1 × S) progeny were consistent with the assumption that resistance to G. t. tabacum in burley and flue-cured tobacco is conferred by the same or closely linked single, dominant gene(s).     

重组色氨酸脱羧酶在烟草不同亚细胞区室的表达

将萜烯类吲哚生物碱代谢关键酶———色氨酸脱羧酶 (TDC)的编码基因转到烟草 (NicotianatabacumL .)植物体内 ,标定在不同的亚细胞区室表达。通过蛋白免疫印迹法和色胺在植物体内的累积量测定分析 ,对转基因植物进行筛选。结果表明 ,TDC在叶绿体和胞液中高效表达 ,TDC在叶绿体中的表达水平最高 ,高于在胞液中的表达 ,在内质网和液泡中表达水平很低 ,用蛋白免疫印迹法未检出。     

Stimulation of murine granulocyte macrophage-colony stimulating factor production by Pluronic F-68 and polyethylene glycol in transgenic Nicotiana tabacum cell culture

Pluronic F-68, PEG 8000, or PEG 20 000 added to cell suspension cultures of transgenic Nicotiana tabacum promoted cell growth and the production of the recombinant murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in a 5-l stirred tank bioreactor. The specific growth rates were enhanced from 0.27 d^–1 to 0.47 d^–1, 0.37 d^–1 and 0.4 d^–1 when Pluronic F-68, PEG 8000, or PEG 20 000 was added, respectively. The maximum cell density was also increased most to 13.6 g l^–1 when Pluronic F-68 was added (11.3 g l^–1 in the control culture). In terms of mGM-CSF production, PEG 8000 gave the greatest stimulation and with 2 g PEG 8000 l^–1, mGM-CSF increased from 1.6 to 6.6 ng ml^–1.     

Production of selectable marker-free transgenic tobacco plants using a non-selection approach: chimerism or escape,transgene inheritance,and efficiency

Public concern and metabolic drain were the main driving forces for the development of a selectable marker-free transformation system. We demonstrated here the production of transgenic tobacco plants using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. A. tumefaciens-infected leaf explants were allowed to produce shoots on a shoot induction medium (SIM) containing no selective compounds. Up to 35.1% of the A. tumefaciens-infected leaf explants produced histochemically GUS⁺ shoots, 3.1% of regenerated shoots were GUS⁺, and 72% of the GUS⁺ shoots were stably transformed by producing GUS⁺ T1 seedlings. When polymerase chain reaction (PCR) was used to screen the regenerated shoots, 4% of the shoots were found to be PCR⁺ for the transgene and 65% of the PCR⁺ shoots were stable transformants. Also, generation of PCR⁺ escapes decreased linearly as the number of subculture increased from one to three on SIM containing the antibiotic that kills the Agrobacterium. Twenty-five to 75% of the transformants were able to transmit transgene activity to the T1 generation in a Mendelian 3:1 ratio, and a transformation efficiency of 2.2–2.8% was achieved for the most effective binary vector. These results indicated that majority of the GUS⁺ or PCR⁺ shoots recovered under no selection were stable transformants, and only one-third of them were chimeric or escapes. Transgenes in these transgenic plants were able to transmit the transgene into progeny in a similar fashion as those recovered under selection.     

Identification and Quantification of Several Mammalian Steroid Hormones in Plants by UPLC-MS/MS

We have developed an effective method for the isolation, identification, and quantification of several mammalian steroid hormones and their metabolites in different plant tissues. The purification protocol was based on solid-phase extraction (SPE) combined with immunoaffinity chromatography (IAC) using immobilized generic polyclonal anti-Δ⁴-3-keto-steroid antibodies covalently bound to Affi-Gel 10 sorbent. The antibodies were characterized by means of enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA was 6.0 × 10⁻¹⁰ mol L⁻¹ and cross-reactivity with most Δ⁴-3-keto-steroids was very high as predicted (68–122%). The IAC allowed fast, single-step purification of different plant extracts prior to analysis by ultra-performance liquid chromatography-electrospray tandem mass spectrometry [UPLC-ESI(+)-MS/MS]. In multiple-reaction-monitoring (MRM) mode, the detection limit of the method for most of the steroids analyzed was close to 10 fmol and the response was linear up to 50 pmol injected. The analytical accuracy was validated using tobacco leaf samples spiked with known amounts of authentic and deuterium-labeled standards. The newly developed method was capable of detecting and quantifying at least 12 specified steroid compounds in plant extracts. In the analyzed extracts from three plant species, that is, common foxglove (Digitalis purpurea L.), tobacco (Nicotiana tabacum L.), and elecampane inula (Inula helenium L.), four endogenous steroids were detected, identified, and quantified. Progesterone was found in all three plants at concentrations comparable to those reported in previous studies. Three other steroids, androstendione, 17α-hydroxyprogesterone, and 16-dehydroprogesterone, were identified for the first time in plant extracts. 17α-Hydroxyprogesterone and 16-dehydroprogesterone occurred at significant concentrations in D. purpurea, whereas androstendione was found in N. tabacum and I. helenium but not in D. purpurea.