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1.
根癌土壤杆菌介导转化诸葛菜子叶获得转基因植株 总被引:12,自引:0,他引:12
采用诸葛菜子叶为材料。在MS+BA 3mg/l,+NAA O.2mg/L培养基上诱导芽的再生,1/2 MS+IBA 0.03mg/L.上诱导生根,获得完整再生植株,建立了诸葛莱组织培养高频再生系统,其再生率可达100%。采用土壤杆菌介导转化诸葛菜子叶.在附加一定量的氨苄青霉索、头孢霉素和卡那霉素的相应培养基上进行筛选,进而培养出再生成苗。获得完整植株,经GUS和NPTI酶活测定,以及Southern blot分析.证实为转基因植株。转化子叶的芽再生率为51%.获得完整转基因植株的转化率为5.53%。在国内外首次建立了以根癌土壤杆菌(Agrobacterium tumefaciens)为载体,诸葛菜子叶为受体的遗传操作系统。 相似文献
2.
Maize embryogenic calli induced from pollen were subcultured for one and one half years on N, basic medium supplemented with 2 mg/1 kinetin, 1 mg/l 6-benzyl-aminopurine, 0.3 mg/l 2,4-D, 500 mg/l casein hydrolysate and 250 mg/l glutamine. These embryogenic calli were used for protoplast isolation. Protoplasts were cultured on Z2 medium (Table 1) which is composed of rice protoplast culture basic medium 1 supplemented with 0.2 mg/l kinetin, 0.1 mg/l 6-benzyl-aminopurine, 0.5 mg/l 2,4-D, 200 mg/l casein hydrolysate, 100 mg/l glutamine and 2% coconut milk. The first division of regenerated cell occurred after 4-6 days in culture. After 3 weeks later, small calli could be seen with naked eyes. At this moment, addition of the same Z2 medium with decreased osmoticum twice for the protoplast culture is necessary. Regenerated calli, 2–4 mm in diameter, were transferred in succession on differentiation medium Z3 and Z4 for organogenesis. Embryogenesis and plant regeneration could occur simultaneously on Z4 differentiation medium. It seems that except the cultural conditions genotype and using of embryogenic materials are the two key factors for plant regeneration of maize protoplast and the former may be the critical one. 相似文献
3.
H. -S. Lin C. van der Toorn K. J. J. M. Raemakers R. G. F. Visser M.J. De Jeu E. Jacobsen 《Plant cell reports》2000,19(5):529-534
Stem segments of seedlings from two Alstroemeria breeding lines, cultured on media supplemented with 4 mg/l 2,4-dichlorophenoxyacetic acid and 0.5–1.0 mg/l 6-benzylaminopurine
(BA), initiated soft callus, which became compact after subculture on a medium with only 0.5 mg/l BA. Friable embryogenic
calli were initiated from compact callus on a medium supplemented with 10 mg/l picloram. Proembryos developed from friable
embryogenic calli via embryos into plants after subculture on medium supplemented with 0.1 mg/l BA. The proembryos formed
friable embryogenic calli again after culture on medium supplemented with 10 mg/l picloram. The total time needed to regenerate
a complete plantlet from friable callus was approximately 6 months. This system for the production of embryogenic material
is considered to have valuable applications for genetic transformation in Alstroemeria.
Received: 22 April 1999 / Revision received: 16 July 1999 · Accepted: 20 July 1999 相似文献
4.
Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall. 总被引:1,自引:0,他引:1
A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified
Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 mg/l 2,4-D, 0.5 mg/l BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems
showing a plating efficiency of 0.8±0.5% and 6.5±0.7%, respectively. Upon transfer to Murashige and Skoog (1962) medium (MS)
with 1–2 mg/l BA, alone or in combination with NAA or 2,4-D at 0.1 mg/l, the protoplast-derived calli produced complete plantlets
through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5± 2.2%) occurred on MS medium
containing 0.1 mg/l NAA and 1 mg/l BA, whereas the maximum number of calli regenerating plantlets (64.7±6.2) was obtained
on MS medium with 0.1 mg/l NAA and 2 mg/l BA.
Received: 25 April 1997 / Revision received: August 1997 / Accepted: 2 September 1997 相似文献
5.
Indra Sandal Ajay Kumar Amita Bhattacharya Madhu Sharma Adarsh Shanker Paramvir Singh Ahuja 《Plant Growth Regulation》2005,47(2-3):121-127
Adventitious shoot regeneration via callus phase from in vitro leaf explants is reported for the first time in tea. Callus was obtained on Murashige and Skoog medium supplemented with
varied concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5, 5.0, 7.5 and 10.0 mg/l). Rhizogenesis was observed at
all concentrations of 2,4-D. Adventitious shoot buds developed indirectly on leaf explants after prolonged culture for 16 weeks
on medium supplemented with 10.0 mg/l 2,4-D. GC analysis of the medium and the tissues at different stages of development
showed that specific levels of 2,4-D in the tissue were responsible for morphogenesis. Shoot buds developed on rhizogenic
calli, only when 2,4-D declined to undetectable or negligible concentrations in the tissue probably due to detoxification
and metabolism. Alternatively, shoot buds could also be evoked when rhizogenic calli were transferred to medium supplemented
with low concentration of 2,4-D (1.5 mg/l). The adventitious nature of the shoots was confirmed through histological studies. 相似文献
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8.
A highly efficient tissue culture system and Agrobacterium-mediated transformation protocol for Chinese upland rice cultivar Handao297 has been established with mature embryos as explants.
Up to 81.2% of mature embryos were induced to regenerate good-quality calli on NB medium (a medium combining N6 macronutrient
components and B5 micronutrient and organic components) containing 3 mg/l 2,4-dichlorophenoxyacetic acid in 10 days. More
than 80% of the calli were morphogenic within 1 week and regenerated green plantlets within 1 month on Murashige and Skoog
medium supplemented with 0.5 mg/l 6-benzyladenine, 0.5 mg/l kinetin, 1 mg/l zeatin, 0.5 mg/l thidizazuron (TDZ), 0.5 mg/l
naphthaleneacetic acid, 0.15 mg/l indoleacetic acid, and 0.15 mg/l indolebutyric acid. This tissue culture system was suitable
for Agrobacterium-mediated transformation of upland rice Handao297. Furthermore, some important factors affecting transformation frequency
were investigated with Agrobacterium strain AGL1 containing the plasmid pCAMBIA1381. The addition of 30 mg/l hygromycin B followed by 60 mg/l hygromycin B to
the selection induction medium facilitated the revival of calli from selection and reduced false positive calli. Hygromycin
B at 10 mg/l was most effective in suppressing non-transgenic callus growth in the differentiation medium. The addition of
TDZ to the differentiation medium promoted the morphogenesis of calli and facilitated the generation of adventitious shoots
by five to tenfold in comparison to medium without TDZ. 相似文献
9.
TransformationofOrychophragmusviolaceusUsingAgrobacteriumtumefaciensAndRegenerationofTransgenicPlantsa¥ZHOUJi-ming(周冀明);WEIZh... 相似文献
10.
Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions. 相似文献
11.
Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x104 protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants. 相似文献
12.
Calll with many embryogenic cell colonies were produced from segments of seedlling of Peucedanum terebinthaceum (Fisch.) Fisch. ex Turcz. which were cultured on the 1/2MS agar medium (with half quantity of macronutrients) containing 1 mg/l 2,4-D. Cell suspension culture with high percentage of embryogenic cell colonies was established from the calli shaking in liquid medium. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CeCl2, 1 mmol/l KH2PO4, 0.6 mol/l mannital at pH 5.8 and 25℃. Cultured in a modified MS liquid medium containing 1 mg/l 2,4-D+ 0.5 mg/l zeatin, the protoplasts emered division after four days, and formed cell colonies of 0.5–1mm after about forty days. When transfered to 1/2 MS liquid medium supplemented with zeatin (0.5 mg/l), the cell colonies differentiated in to embryoids, then developed into plantlets with many green leaves and roots on the 1/2 MS agar medium devoid of phytohormones. 相似文献
13.
Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli. 相似文献
14.
Agrobacterium-mediated transformation of the wetland monocot Typha latifolia L. (Broadleaf cattail) 总被引:1,自引:0,他引:1
An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L. to achieve the long-term objective of introducing candidate genes for phytoremediation. Two binary plasmid vectors, pCAMBIA1301/EHA105 and pTOK233/LBA4404, both containing the gus (beta-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation. Fifty-day-old 5 mg/l picloram-derived calli were cocultivated and selected on medium containing 20 mg/l or 40 mg/l hygromycin. Resistant calli were regenerated on medium supplemented with 5 mg/l 6-benzylaminopurine, with or without 20 mg/l or 40 mg/l hygromycin and with or without charcoal (10 g/l). Transient GUS activity in explants ranged between 28% and 36%. Hygromycin-resistant calli, selected after 3 months, showed stable GUS expression. A total of 46 plants were regenerated and established in the greenhouse; 13 showed stable GUS expression. Cocultivation of dark culture-derived calli, directly selected on regeneration medium containing 20 mg/l hygromycin and rooted on medium with 20 mg/l hygromycin was the best protocol. The addition of charcoal did not have any effect on regeneration. PCR and Southern analyses of transgenic calli and transgenic plants confirmed the presence of the introduced genes. In conclusion, T. latifolia could be genetically transformed by Agrobacterium tumefaciens. 相似文献
15.
Protoplast of two mustard cultivars: Brassica juncea var. tsatsi cv. “Quxian Jiaoercai” and “Bangbangcai”, were isolated by enzymolysis from leaf grown in vitro. Protoplasts were suspended in liquid medium and semi-solidified medium with 0.35% low melting point agarose which formed a thin layer floating on the surface of the liquid medium. The first division appeared after 48h in the culture. One week after the original culture, a diluted medium with gradual dicrease of mannitol concentrations (6%→4%→zero) was then added to the culture three times respectively at one week's interval. In this culture method cell division and formation of microcalli were achieved. During the liquid culture of protoplasts, shaking at 20 rpm from time to time was beneficial in the formation of cell colonies and microcalli. Cell colonies developed into calli of approx 0.5—1mm in diameter one month after culture. The plating efficiency, which defined as the percentage of microcatli to numbers of protoplasts, was 0.2%—1%. Shoot regeneration occured when leaf protoplast-derived calli of “Quxian Jiaoercai” were transferred onto the modified MS medium supplemented with BAP 2.0mg/L, KT 1.0mg/L and NAA 0.2mg/L, and those of -'Bangbangcai" were transferred onto the modified MS medium supplemented with BAP 2.0mg/L. Individual shoot was rooted on a rooting medium supplemented with NAA 0.2 or 0.4 mg/L. 相似文献
16.
N. Bhagya K. R. Chandrashekar Anitha Karun U. Bhavyashree 《Journal of plant biochemistry and biotechnology.》2013,22(4):474-482
A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa. 相似文献
17.
枸杞髓组织培养中体细胞胚胎发生与过氧化物酶同工酶分析 总被引:8,自引:3,他引:5
枸杞髓组织在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L培养基上诱导愈伤组织发生。在 MS+6 - BA0 .1mg/ L+NAA0 .5mg/ L+CH50 0 mg/ L培养基上继代培养 ,再转入 MS+6 - BA2 mg/L +NAA 0 .5mg/ L的分化培养基上进行分化培养。显微观察表明 ,在培养过程中愈伤组织细胞由非胚性细胞转变为胚性细胞 ,直至发育成体细胞胚胎和完整植株 ;电泳结果显示 ,体细胞胚胎发生的各阶段 ,其过氧化物酶同工酶发生相应的变化。 相似文献
18.
Summary
Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring agropine-type Ri plasmid (pRi15834). A large number of transformed calli were obtained on Murashige and Shoog's (MS) medium lacking plant growth regulators. Frequency of transformation was about 3.5×10–3. In most of the calli, hairy roots appeared on MS medium without plant growth regulator. When the hairy roots were cut into segments and subcultured on MS medium lacking plant growth regulators, calli were readily formed. Plantlets were regenerated by transferring those calli to MS medium supplemented with 1 mg/l zeatin and 0.2 mg/l naphthaleneacetic acid. Frequency of plant regeneration was about 70%. 相似文献
19.
Callus cultures were raised from bulb scale segments ofOrinthogalum umbellatum L. (Liliaceae), on a Murashige and Skoog (1962) medium (MS) with 8 mg/l naphthaleneacetic acid (NAA). Bulbous shoots developed from calli after 2 months using MS medium with 2 mg/l NAA and 0.5 mg/l N6 - benzyladenine (BA). Shoots were also induced directly from scales of regenerated bulb used as secondary explants on MS medium supplemented with 0.5 mg/l BA. Shoots developed roots in 1/2 - strength MS medium. Regenerants multiplied rapidly in 1/2-MS liquid medium. Chromosome instability was reduced in callus grown on 2 mg/l NAA compared to callus grown on 8 mg/l NAA. Callus retained regeneration potential for 5 years in this modified MS medium. The chromosome analysis of regenerants dervied from callus, even from long term culture of 5 years, revealed only diploid cells with normal karyotype comprising 2n=46 chromosomes. Stable nature of callus and regenerants were further confirmed by cytophotometry. This procedure can be applied for securing stable regenerants on a mass scale inO. umbellatum. 相似文献
20.
Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS
Murashige and Skoog (1962)
- BAP
6-benzylaminopurine
- NAA
naphthaleneacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献