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1.
Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue 总被引:12,自引:0,他引:12
Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue.
Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension
cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were
inoculated with 1 ml of diluted (OD600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT.
When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic
tissue confirmed T-DNA integration.
Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997 相似文献
2.
John J. Finer Raymond W. Saxton Barbara L. Norris Jeffrey A. Steele Sha Rahnema 《Plant cell reports》1991,10(8):380-383
Summary Growth of 6 different common laboratory bacteria (Escherichia coli, Flavobacterium balustrum, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas fluorescens, and Agrobacterium tumefaciens) in a bacterial medium, fresh plant medium, and spent plant media was initially measured. In all cases, bacteria grew best in the bacterial medium followed by the fresh plant medium. The spent plant medium did not support growth of the bacteria and apparently was actively toxic to bacterial cells. Proliferating, embryogenic suspension cultures of cotton (Gossypium hirsutum) were then inoculated with these 6 different bacteria. Two to three d following bacterial inoculation, embryogenic tissues were placed in various concentrations of bleach for various amounts of time, rinsed with sterile water, and placed on a bacterial culture medium. Clumps of embryogenic tissue which showed no visible bacterial growth after 3 d of culture were then transferred to an agar-solidified plant tissue culture medium to determine viability of bleachdisinfested tissues. Viable, single pieces of the disinfested embryogenic tissue were then used to reinitiate embryogenic suspension cultures. Treatment of contaminated tissue with a 1% bleach solution for 1–5 min resulted in the highest recovery of viable, disinfested tissues using 5 of the 6 bacteria. It was not possible to remove F. balustrum from clumps of embryogenic tissue without also killing the plant tissue.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
Salaries and research support were provided by an Ohio Academy of Sciences/National Science Foundation summer internship award to JAS and by State and Federal funds appropriated to OSU-OARDC. OARDC Journal Article No. 391-89 相似文献
3.
John J. Finer Philippe Vain Mark W. Jones Michael D. McMullen 《Plant cell reports》1992,11(7):323-328
Summary A simple and inexpensive particle bombardment device was constructed for delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles using pressurized helium in combination with a partial vacuum. The particles are accelerated directly in a helium stream rather than being supported by a macrocarrier. Bombardment parameters were partially optimized using transient expression assays of a ß-glucuronidase gene in maize embryogenic suspension culture and cowpea leaf tissues. High levels of transient expression of the ß-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea. Stable transformation of embryogenic tissue of soybean has also been obtained using this bombardment apparatus.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- PCV
packed cell volume
- GUS
ß-glucuronidase
- NOS
nopaline synthase
Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark of proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 34-92 相似文献
4.
Development of the Particle Inflow Gun 总被引:7,自引:0,他引:7
Philippe Vain Noel Keen Jesus Murillo Carl Rathus Cheri Nemes John J. Finer 《Plant Cell, Tissue and Organ Culture》1993,33(3):237-246
A simple and inexpensive particle acceleration apparatus was designed for direct delivery of DNA to plant cells. The Particle
Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles directly in a helium steam. High levels of transient
expression of theβ-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of maize and soybean, and leaf
tissue of cowpea. Stable transformation of soybean and maize has also been obtained using this bombardment apparatus. 相似文献
5.
High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda) 总被引:5,自引:0,他引:5
Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large- scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene -glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co- cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species. 相似文献
6.
Detailed analyses of the physical parameters inherent in the microprojectile bombardment technology necessary to produce optimum transient -glucuronidase (GUS) expression were undertaken in pollen and embryogenic tissues of white spruce. Higher helium pressure used for microprojectile bombardment resulted in lower GUS expression in pollen, but in higher GUS expression in embryogenic tissues. Modification of the osmoticum of the culture medium had a limited effect on GUS transient expression in pollen but substantially increased the transient expression in embryogenic tissues. The viability of transformed pollen was not affected by the bombardment procedure. This is the first detailed analysis of microprojectile bombardment technology reporting the conditions needed for optimum transient transformation of pollen and embryogenic tissues of white spruce. 相似文献
7.
Montoro P Rattana W Pujade-Renaud V Michaux-Ferrière N Monkolsook Y Kanthapura R Adunsadthapong S 《Plant cell reports》2003,21(11):1095-1102
A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees. 相似文献
8.
已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体.另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染.本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响 DNA 转化效率的因素.研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1~2周,诱导产生直径为2~3 mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于 2 mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率.此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响 T-DNA 插入基因组的效率.用 1 ml A600值为 0.5 浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3 d或直到可见农杆菌菌落,此方法可以得到较高转化效率.将再生的潮霉素抗性的转化植株在含有 50 mg/L 潮霉素的分化和生根培养基中筛选得到,并对转化植株 gus 基因的表达进行 PCR 检测.结果显示,用 A600值为 0.5 浓度的农杆菌浸泡侵染 20 min和滴加浸染法,分别得到PCR阳性植株率为 70% 和92%. 相似文献
9.
Agrobacterium‐mediated transformation of Vitis Cv. Monastrell suspension‐cultured cells: Determination of critical parameters
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Mingyu Chu Carmen Quiñonero Hülya Akdemir Nuria Alburquerque María Ángeles Pedreño Lorenzo Burgos 《Biotechnology progress》2016,32(3):725-734
Although some works have explored the transformation of differentiated, embryogenic suspension‐cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication‐assisted Agrobacterium‐mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp‐expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725–734, 2016 相似文献
10.
Cerda Francisca Aquea Felipe Gebauer Marlene Medina Consuelo Arce-Johnson Patricio 《Plant Cell, Tissue and Organ Culture》2002,70(3):251-257
Embryogenic cultures from immature zygotic embryos of Pinus radiata seeds were established on semisolid proliferation medium with 2,4-D and BAP. Growing embryogenic masses containing embryonal cells and suspensor cells were subcultured on this media every 2 weeks. After 10 weeks, embryogenic masses (1.5 cm diameter) were transferred to a maturation medium containing ABA. Fully developed somatic embryos were obtained in this medium after 12 weeks. Embryogenic masses were genetically transformed using Agrobacterium tumefaciens. The pBI121 vector containing -glucuronidase (uidA) and the neomycin phosphotransferase (nptll) genes was introduced into this tissue. After co-cultivation with Agrobacterium, the embryogenic tissues were transferred to a selection media containing geneticin and carbenicillin. After 1 month of selection, histochemical assays showed extensive GUS positive activity zones in the transformed embryogenic tissues. Under light microscope, blue crystals were seen inside the embryogenic and suspensor cells, and also completely blue somatic embryos were obtained. The uidA gene was also detected by PCR analysis in genomic DNA isolated from transformed embryogenic tissues. These results indicate stable transformation of P. radiata somatic embryogenic tissues using Agrobacterium-mediated transformation. 相似文献
11.
Borys Chong-Pérez Maritza Reyes Luis Rojas Bárbara Ocaña Blanca Pérez Rafael G. Kosky Geert Angenon 《Plant Cell, Tissue and Organ Culture》2012,111(1):79-90
An efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of the important banana cultivar ‘Dwarf Cavendish’ (Musa AAA). A high embryogenic response was obtained in 1.36 % of immature male flower explants. Once embryogenic structures were transferred to liquid medium, embryogenic cell suspensions (ECSs) with high regeneration capacity were obtained. ECSs were incubated under different conditions with Agrobacterium tumefaciens strain EHA101 harboring vector pFAJ3000 that contains pNos-nptII-tOcs and p35S-uidAintron-t35S expression cassettes. The effect of spermidine and infection time on transformation efficiency was examined. The highest efficiency was obtained when ECSs were infected for 6 h, in medium supplemented with 200 μM acetosyringone and 1.0 mM spermidine, with more than 600 independent lines/~50 mg FW of settled cells. Spermidine showed an enhancing effect, increasing significantly the transient Gus expression and the number of transformed embryo colonies and regenerated plants in comparison with the same treatments without this polyamine. This is the first report showing efficient Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in the ‘Dwarf Cavendish’ banana cultivar. 相似文献
12.
Summary A characteristic phenotype of highly embryogenic explants along with the location of embryogenesis- and transformation-competent
cells/tissues on immature cotyledons of soybean [Glycine max (L.) Merrill.] under hygromycin selection was identified. This highly embryogenic immature cotyledon was characterized with
emergence of somatic embryos and incidence of browning/necrotic tissues along the margins and collapsed tissues in the mid-region
of an explant incubated upwards on the selection medium. The influences of various parameters on induction of somatic embryogenesis
on immature cotyledons following Agrobacterium tumefaciens-mediated transformation and selection were investigated. Using cotyledon explants derived from immature embryos of 5–8 mm
in length, a 1∶1 (v/v; bacterial cells to liquid D40 medium) concentration of bacterial suspension and 4-wk cocultivation
period significantly increased the frequency of transgenic somatic embryos. Whereas, increasing the infection period of explants
or subjecting explants to either wounding or acetosyringone treatments did not increase the frequency of transformation. An
optimal selection regime was identified when inoculated immature cotyledons were incubated on either 10 or 25 mgl−1 hygromycin for a 2-wk period, and then maintained on selection media containing 25 mgl−1 hygromycin in subsequent selection periods. However, somatic embryogenesis was completely inhibited when inoculated immature
cotyledons were incubated on a kanamycin selection medium. These findings clearly demonstrated that the tissue culture protocols
for transformation of soybean should be established under both Agrobacterium and selection conditions. 相似文献
13.
A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 μm coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3
months on selective medium, the transgenic tissue remained embryogenic, which allowed production of transgenic plants in the
greenhouse. Stable integration of the transgene was confirmed by PCR and Southern hybridisation on transformed tissues and
acclimatised plants.
Received: 4 July 1996 / Revision received: 25 November 1996 / Accepted: 10 December 1996 相似文献
14.
Transformation of rice mediated by Agrobacterium tumefaciens 总被引:48,自引:0,他引:48
Agrobacterium tumefaciens has been routinely utilized in gene transfer to dicotyledonous plants, but monocotyledonous plants including important cereals were thought to be recalcitrant to this technology as they were outside the host range of crown gall. Various challenges to infect monocotyledons including rice with Agrobacterium had been made in many laboratories, but the results were not conclusive until recently. Efficient transformation protocols mediated by Agrobacterium were reported for rice in 1994 and 1996. A key point in the protocols was the fact that tissues consisting of actively dividing, embryonic cells, such as immature embryos and calli induced from scutella, were co-cultivated with Agrobacterium in the presence of acetosyringonc, which is a potent inducer of the virulence genes. It is now clear that Agrobacterium is capable of transferring DNA to monocotyledons if tissues containing competent cells are infected. The studies of transformation of rice suggested that numerous factors including genotype of plants, types and ages of tissues inoculated, kind of vectors, strains of Agrobacterium, selection marker genes and selective agents, and various conditions of tissue culture, are of critical importance. Advantages of the Agrobacterium-mediated transformation in rice, like on dicotyledons, include the transfer of pieces of DNA with defined ends with minimal rearrangements, the transfer of relatively large segments of DNA, the integration of small numbers of copies of genes into plant chromosomes, and high quality and fertility of transgenic plants. Delivery of foreign DNA to rice plants via A. tumefaciens is a routine technique in a growing number of laboratories. This technique will allow the genetic improvement of diverse varieties of rice, as well as studies of many aspects of the molecular biology of rice. 相似文献
15.
The photosynthetic properties of two commonly used suspension cultured lines, embryogenic and photoautotrophic (PA, SB-1 line) cells of soybean [Glycine max (L.) Merr.] were characterized. We found that compared to the dark green PA cells, the light green embryogenic cells contained fewer and smaller plastids with less-developed thylakoid membranes. The embryogenic cells also contained much lower contents of both chlorophyll and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) protein, an undetectable level of Rubisco small subunit protein, and a very low rate of photosynthesis. While the DNA contents of the nuclear genomes were similar in these two types of cultured cells, the embryogenic cells possessed a markedly lower content of plastid DNA. The 18-year-old PA suspension culture, SB-1, continues to evolve with higher Rubisco and plastid DNA contents than leaves, and with small decreases in nuclear DNA content that appears to mimic changes in chromosome numbers. These findings may prove useful in the application of plastid transformation, particularly when non-leaf or non-green tissues must be used as targets for transformation and plant regeneration. 相似文献
16.
Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens 总被引:4,自引:1,他引:3
Levée V. Garin E. Klimaszewska K. Séguin A. 《Molecular breeding : new strategies in plant improvement》1999,5(5):429-440
A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and -glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized. 相似文献
17.
Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants 总被引:4,自引:0,他引:4
We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The -glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration. 相似文献
18.
Antara Ghosh T. R. Ganapathi Pravendra Nath V. A. Bapat 《Plant Cell, Tissue and Organ Culture》2009,97(2):131-139
Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation
of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation
of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male
flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained
was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV)
was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried
out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression
of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration
of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation
with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The
present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of
disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist
in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana. 相似文献
19.
Eliane R. Santarém John J. Finer 《In vitro cellular & developmental biology. Plant》1999,35(6):451-455
Summary Transgenic soybean can be efficiently produced by particle bombardment of embryogenic suspension culture material. Unfortunately,
the time required to obtain a transformation-competent soybean suspension culture line is often lengthy and can result in
reduced fertility of regenerated plants. In addition, establishment and maintenance of embryogenic suspension cultures can
be very difficult. The objective of this work was to minimize the time required to obtain transformation-competent embryogenic
tissue and optimize DNA delivery into that tissue. Somatic embryos were induced from immature cotyledons of soybean [Glycine max (L.) Merrill cv ‘Jack’] by placement of cotyledons, adaxial side up, on a MS-based induction medium containing 40 mg (181
μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1 and 6% sucrose. Embryogenic tissues, which formed from the surface of the cotyledons
within 2–4 wk, were transferred to an embryo proliferation medium containing 20 mg (90 μM) 2,4-D per 1 and 3% sucrose. After 4 wk, proliferative embryogenic tissue could be used for transformation via particle bombardment.
Desiccation of target tissue, period of subculture prior to bombardment, and the number of bombardments per target tissue
were evaluated for enhancement of transient β-glucuronidase (GUS) expression. The highest number of blue foci was observed
when the target tissue was desiccated for 10 min in an uncovered Petri plate containing proliferation medium, subcultured
on the same day of bombardment, and bombarded three times on a single day. For stable transformation, selection was started
20 d after bombardment using 9 mg hygromycin per 1 for 4 wk, and 18 mg per 1 thereafter. Stably transformed clones were obtained
from tissue bombarded once and twice on a single day. GUS assays and Southern hybridization analysis of DNA from putative
clones confirmed stable integration of the introduced genes. Fertile transgenic plants were obtained in 11–12 mo following
culture initiation. 相似文献
20.
A rapidly growing, maintainable, embryogenic suspension culture of Glycine max L. Merrill. has been generated. The culture consisted almost entirely of clumps of proliferating globular embryos with very little nonembryogenic tissues. The number and size of somatic embryo clumps were used to quantify growth of embryogenic tissues under various conditions. Initiation and proliferation of this embryogenic suspension culture were dependent on the inoculum, method of subculture, and composition of the subculture medium. Twenty to 50 mg of highly embryogenic, early-staged soybean tissue were inoculated into 35 ml of liquid culture medium containing 5 mg 1–1 2,4-D and either 15 mM glutamine or preferably 5 mM asparagine. Suspension cultures were subcultured at the same inoculum density every 4 weeks. The embryos matured and germinated following placement on solid media, resulting in consistent plant regeneration. 相似文献