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1.
<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Perilla frutescens</Emphasis> 总被引:3,自引:0,他引:3
A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying -glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies. 相似文献
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Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in
thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 106/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l
α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis
of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l
zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS)
medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid.
Regenerated plants have normal morphology. 相似文献
4.
We developed an efficient gene transfer method mediated by Agrobacterium tumefaciens for introgression of new rice for Africa (NERICA) cultivars, which are derivatives of interspecific hybrids between Oryza glaberrima Steud. and O. sativa L. Freshly isolated immature embryos were inoculated with A. tumefaciens LBA4404 that harbored binary vector pBIG-ubi::GUS or pIG121Hm, which each carried a hygromycin-resistance gene and a GUS
gene. Growth medium supplemented with 500 mg/l cefotaxime and 20 mg/l hygromycin was suitable for elimination of bacteria
and selection of transformed cells. Shoots regenerated from the selected cells on MS medium containing 20 g/l sucrose, 30 g/l
sorbitol, 2 g/l casamino acids, 0.25 mg/l naphthaleneacetic acid, 2.5 mg/l kinetin, 250 mg/l cefotaxime, and 20 mg/l hygromycin.
The shoots developed roots on hormone-free MS medium containing 30 mg/l hygromycin. Integration and expression of the transgenes
were confirmed by PCR, Southern blot analysis, and histochemical GUS assay. Stable integration, expression, inheritance, and
segregation of the transgenes were demonstrated by molecular and genetic analyses in the T0 and T1 generations. Most plants were normal in morphology and fertile. The transformation protocol produced stable transformants
from 16 NERICA cultivars. We also obtained transformed plants by inoculation of calluses derived from mature seeds, but the
frequency of transformation was lower and sterility was more frequent. 相似文献
5.
The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments.
Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to
screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were
identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’
optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g
l−1 of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g
l−1), glucose (12.7 g l−1), soy peptone (10.9 g l−1), and ethanol (13.8 ml l−1) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA
concentration of 2.0 g l−1, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l
fermentor and 2 g l−1 PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum
strategy. 相似文献
6.
Hamedi J Malekzadeh F Saghafi-nia AE 《Journal of industrial microbiology & biotechnology》2004,31(10):447-456
The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed. 相似文献
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Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min–1) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l–1) was utilized to further investigate CA production. Medium containing 23 g l–1 led to the highest CA productivity (722 mg l–1 in 120 h) and that one containing 30 g l–1 gave the highest CA titre (753 mg l–1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism. 相似文献
8.
C. Douglas Boyette Mark A. Weaver Robert E. Hoagland Kenneth C. Stetina 《World journal of microbiology & biotechnology》2008,24(11):2721-2726
A mycelial formulation of the fungus Myrothecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive
weed kudzu. The mycelium can be rapidly (48–72 h) produced in several media, including an inexpensive soy flour–corn meal
medium. Mycelial yields were 2, 10, and 25 g dry weight l−1 in Czapek-Dox, Richard’s V-8, and soy flour–corn meal media, respectively. Scale-up production in soy flour–corn meal medium
using laboratory fermenters (10–25 l), resulted in a mycelial formulation that caused 90% mortality of naturally-occurring
mature (0.9–1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium
produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A
(limit of detection 2 μg ml−1). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the
probability of EPA registration and subsequent commercial development of this bioherbicide. 相似文献
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Acid phosphatase production by recombinant Arxula adeninivorans was carried out in submerged fermentation. Using the Plackett–Burman design, three fermentation variables (pH, sucrose concentration,
and peptone concentration) were identified to significantly affect acid phosphatase and biomass production, and these were
optimized using response surface methodology of central composite design. The highest enzyme yields were attained in the medium
with 3.9% sucrose and 1.6% peptone at pH 3.8. Because of optimization, 3.86- and 4.19-fold enhancement in enzyme production
was achieved in shake flasks (17,054 U g−1 DYB) and laboratory fermenter (18,465 U g−1 DYB), respectively. 相似文献
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S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing
the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads
to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp. 相似文献
11.
Qi Zhu Fengtao Wu Feng Ding Dong Ye Yongqin Chen Yi Li Yang Zhifan 《Plant Cell, Tissue and Organ Culture》2009,96(3):317-324
Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important
steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and
female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive
to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation
medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three
days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for
formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh
selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of
the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to
produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated
from initial infected callus explants. 相似文献
12.
Chu-Ting Liu I-Ting Hsu Cheng-Chun Chou Pei-Ren Lo Roch-Chui Yu 《World journal of microbiology & biotechnology》2009,25(5):883-890
In the present study, the production of exopolysaccharides (EPS) by 13 strains of Lactobacillus and 6 strains of Bifidobacterium in a chemical defined medium (CDM) supplemented with 30 g lactose/l was first compared. The highest EPS production of the
Lactobacillus strains was found in L. salivarius BCRC 14759 while among the Bifidobacterium strains examined, B. bifidum BCRC 14615 showed the highest EPS production. Analyzes of the effect of lactose concentration and cultivation temperature
on EPS production revealed that L. salivarius produced the highest amount of EPS (45.3 mg/l) in CDM supplemented with 5 g lactose/l at 40°C while B. bifidum produced the highest EPS (17.0 mg/l) in CDM supplemented with 40 g lactose/l at 35°C. α-Phosphoglucomutase, UDP-glucose pyrophosphorylase
and UDP-galactose-4-epimerase exhibited a markedly notable activity compared with other enzymes examined in the cell extract
of both test organisms. This indicates their possible involvement in the biosynthesis of EPS. 相似文献
13.
An efficient transformation protocol was developed for Eucalyptus tereticornis Sm. using cotyledon and hypocotyl explants. Precultured cotyledon and hypocotyl explants were cocultured with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pBI121 containing the uidA and neomycin phosphotransferase II genes for 2 d and transferred to selective regeneration medium containing 0.5 mg/l 6-benzylaminopurine
(BAP), 0.1 mg/l naphthalene acetic acid, 40 mg/l kanamycin, and 300 mg/l cefotaxime. After two passages in the selective regeneration
medium, the putatively transformed regenerants were transferred to Murashige and Skoog (MS) liquid medium containing 0.5 mg/l
BAP and 40 mg/l kanamycin on paper bridges for further development and elongation. The elongated kanamycin-resistant shoots
were subsequently rooted on the MS medium supplemented with 1.0 mg/l indole-3-butyric acid and 40 mg/l kanamycin. A strong
β-glucuronidase activity was detected in the transformed plants by histochemical assay. Integration of T-DNA into the nuclear
genome of transgenic plants was confirmed by polymerase chain reaction and southern hybridization. This protocol allows effective
transformation and direct regeneration of E. tereticornis Sm. 相似文献
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Attempts were made to optimize the cultural conditions for the production of L-asparaginase by Streptomyces albidoflavus under submerged fermentations. Enhanced level of L-asparaginase was found in culture medium supplemented with maltose as
carbon source. Yeast extract (2%) was served as good nitrogen source for the production of L-asparaginase. The optimum pH
for enzyme production was 7.5 and temperature was 35°C. The release of L-asparaginase from the cells of S. albidoflavus was high when strain was treated with cell disrupting agents like EDTA and lysozyme. The enzyme produced by the strain was
purifi ed by ammonium sulfate, Sephadex G-100 and CM-Sephadex C-50 gel fi ltration and the molecular weight was apparently
determined as 112 kDa. 相似文献
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Yan Zheng Jing Quan Xin Ning Li-Min Zhu Bo Jiang Zhi-Yan He 《World journal of microbiology & biotechnology》2009,25(1):41-46
Lipase-catalyzed transesterification of soybean oil and methanol for biodiesel production in tert-amyl alcohol was investigated. The effects of different organic medium, molar ratio of substrate, reaction temperature, agitation
speed, lipase dosage and water content on the total conversion were systematically analyzed. Under the optimal conditions
identified (6 mL tert-amyl alcohol, three molar ratio of methanol to oil, 2% Novozym 435 lipase based on the soybean oil weight, temperature 40°C,
2% water content based on soybean oil weight, 150 rpm and 15 h), the highest biodiesel conversion yield of 97% was obtained.
With tert-amyl alcohol as the reaction medium, the negative effects caused by excessive molar ratio of methanol to oil and the by-product
glycerol could be reduced. Furthermore, there was no evident loss in the lipase activity even after being repeatedly used
for more than 150 runs. 相似文献
18.
Ming Chang Shun-Gui Zhou Na Lu Jin-Ren Ni 《World journal of microbiology & biotechnology》2008,24(4):441-447
Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that
the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus
thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in
the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL−1 and higher entomotoxicity of 1,050 IU μL−1 were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations
also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for
safe SPW disposal and production of high potency and low cost bioinsecticide. 相似文献
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