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1.
Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and
hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos
of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass;
however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo
cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture
where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from
SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with
both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
2.
Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials. 相似文献
3.
Kee-Yoeup Paek Eun-Joo Hahn Sung-Ho Son 《In vitro cellular & developmental biology. Plant》2001,37(2):149-157
Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production
cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate
the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors
equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for
micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation
via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were
cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction
medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted
in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of
virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of
culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation
and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in
Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different
stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos
in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost
for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation
industries. 相似文献
4.
Schlatmann JE Nuutila AM Van Gulik WM Ten Hoopen HJ Verpoorte R J Heijnen J 《Biotechnology and bioengineering》1993,41(2):253-262
The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc. 相似文献
5.
Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth
regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and
Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior
and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous
with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength
MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets
acclimatized under field conditions with 90% survival. 相似文献
6.
In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]
We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l–1 2,4-D, 20 mg l–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l–1 potassium nitrate, and 0.05 mg l–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion. 相似文献
7.
Chris Selby W. Colin McRoberts John T. G. Hamilton Barbara M. R. Harvey 《Plant Growth Regulation》1996,20(1):37-42
The maturation of somatic embryos of Sitka spruce [Picea sitchensis (Bong.) Carr.] was found to be highly dependent on the method used to seal plastic Petri dishes. Large numbers of well-formed mature embryos developed if dishes were sealed with PVC cling-film (CF) whilst sealing with Parafilm M (PF) greatly reduced the numbers of embryos forming. Inclusion of potassium permanganate oxidation traps, normally used to deplete the atmospheric ethylene, greatly stimulated somatic embryo maturation under PF sealing. Similarly, traps of adsorption agents (Tenax, activated charcoal or soft white paraffin), capable of removing volatiles from the culture vessel head-space, stimulated somatic embryo maturation under PF sealing although to a lesser extent than the oxidation traps. Incorporation of silver nitrate or 2-chloroethylphosphonic acid (ethephon) in the culture medium indicated that ethylene was not the agent supressing somatic embryo maturation under PF sealing.Abbreviations ABA
abscisic acid
- CF
PVC cling-film
- PF
Parafilm M 相似文献
8.
Explants of germinating zygotic embryos of Eleutherococcus sessiliflorus,an important medicinal plant, produced somatic embryos directly on Murashige and Skoog (MS) medium with 4.5 M 2,4-D. In addition, embryogenic callus formed at a low frequency (less than 7%) from hypocotyl segments after prolonged culture. High frequency somatic embryogenesis was obtained through cell suspension culture after the cells were transferred to medium lacking 2,4-D. Maturation and germination of embryos was influenced by the sucrose concentration of the medium. At a low concentration of sucrose (1%), maturation and germination of embryos occurred readily. At over 6% sucrose, somatic embryos did not germinate although this could be overcome by GA3 treatment. Cold treatment during acclimatization after transfer to soil enhanced survival. Surviving plantlets produced new sprouts after overwintering in the field. 相似文献
9.
Jingli Yang Chenguang Zhou Lihui Liu Du Jia Zhiyang Yin Myongjo Kim Changyeon Yu Chenghao Li 《Plant Cell, Tissue and Organ Culture》2012,110(2):289-298
Embryogenic callus was obtained from young petiole, stem, and root explants of 4-year-old Siberian ginseng plants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Embryogenic callus differentiated into somatic embryos (SEs), most of which could germinate but developed abnormally. Friable embryogenic callus was induced mainly from the root regions of germinated primary SEs or regenerated plantlets on plant growth regulator-free medium. Histological studies showed that the embryogenic callus initiated from the subepidermal cells of young roots. The bioreactor system was more efficient than suspension culture regarding the number and growth of SEs, although a similar amount of embryonic tissue was used. An average of 115,370 germinated SEs developed from an initial 400?mg of embryogenic callus, and 64.7?% of germinated SEs converted into plantlets after a 4-weeks culture on agar medium. During the bioreactor culture process, secondary SEs were induced directly from SEs at various stages, a phenomenon that rarely occurred in suspension culture. These secondary SEs developed quickly and germinated during the bioreactor culture process. Proline content and peroxidase and catalase activities of SEs cultured in bioreactors were higher than in SEs cultured in suspension culture. 相似文献
10.
An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM
callus maintenance medium
- 2,4D
2,4-dichlorophenoxy acetic acid
- PCV
packed cell volume
- MS
Murashige and Skoog medium 相似文献
11.
Summary
In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses
were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred
to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented
with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation
and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened
and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction
and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l−1
L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development. 相似文献
12.
Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA
fluorescein diacetate
- ABA
abscisic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
13.
Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured
on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation
decreased with an increasing concentration of 2,4-D up to 10 mg l−1, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses
using half-strength SH medium supplemented with 0.1 mg l−1 of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos
and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at
a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to
mass propagation and conservation of this species. 相似文献
14.
Anne Katharina Jäger Brigitte Schottländer Ulla Wagner Smitt Ulf Nyman 《Plant cell reports》1993,12(9):517-520
Cell cultures from different species of the genus Thapsia (Apiaceae) have been investigated. In one 4-yearold line of T. garganica L. spontaneous somatic embryogenesis up to the globular stage occurred in a suspension culture containing 1 mg l–12,4-dichlorophenoxyacetic acid (2,4-D). Also callus cultures of this line, previously maintained on a medium containing 1 mg l–1 2,4-D, when transferred to various media deprived of 2,4-D, produced somatic embryos that developed into plantlets. Cell culture, embryos and regenerated organs were analysed for their content of thapsigargins. The undifferentiated cell culture did not synthezise thapsigargins, but was found to produce a yet unidentified compound not present in planta. White embryos in the pre-cotyledonary stage did not synthezise thapsigargins either, but when the embryos developed to the cotyledonary stage and became green, the synthesis started. Regenerated roots and shoots also contained thapsigargins.Abbreviations BAP
Benzylaminopurine
- 2,4-D
2,4-Dichlorophenoxyacetic acid
- EtOAc
ethyl acetate
- FDA
fluorescein diacetate
- IAA
Indole-3-acetic acid
- IBA
indole-3-butyric acid
- 2-iP
2-isopentenyladenine
- NAA
1-Napthaleneacetic acid 相似文献
15.
Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid. 相似文献
16.
Myung Jin Oh Hye Ryun Na Hong-Keun Choi Jang Ryol Liu Suk Weon Kim 《Plant biotechnology reports》2008,2(1):87-92
An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension
cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when
cultured on half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly
with increasing concentrations of 2,4-D up to 3 mg l−1, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus
were established using half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos
and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by
up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting
soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high
frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield,
which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could
be useful for the mass propagation and transformation of selected elite lines. 相似文献
17.
Summary Embryogenic cell suspension cultures and somatic embryos of five genotypes of beech, were obtained from aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2, 4-dichlorophenoxyacetic acid and N6-benzyladenine. The origin of somatic embryos was traced from single cells. Embryos remained arrested at the globular stage on liquid media, further development was achieved after plating embryogenic aggregates on Murashige and Skoog's medium with half strength major salts supplemented with glutamine and low levels of growth regulators. Cultures of different genotypes showed significant differences in maturation frequency which was not affected by the hormone treatments assayed. The frequency of conversion of embryos into plantlets was low. This frequency increased after cold storage of embryos for up to 7 months.Abbreviations BA
N6-benzyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- EtOH
ethanol
- GA3
giberrellic acid
- IAA
indole-3-acetic acid
- IBA
indole-3-butyric acid
- MS
Murashige and Skoog (1962)
- NAA
naphthalene acetic acid
- WPM
woody plant medium (Lloyd and McCown, 1980)
- Z
zeatin 相似文献
18.
H. Brittain-Loucas S. R. Bowley B. D. McKersie 《In vitro cellular & developmental biology. Plant》1998,34(4):281-284
Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in
liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0);
and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was
greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium),
but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%).
In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture
callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical
factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures. 相似文献
19.
Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators. 相似文献
20.
Alexander I. Kuklin Plamen D. Denchev Atanas I. Atanassov Alan H. Scragg 《Plant Cell, Tissue and Organ Culture》1994,38(1):19-23
A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA
abscisic acid
- B5
Gamborgs B5 medium (Gamborg et al. 1968)
- COT
cotyledon embryo state
- 2,4-d
2,4-dichlorophenoxyacetic acid
- FW
fresh weight
- ID
internal diameter
- MS
Murashige and Skoog medium (Murashige & Skoog 1962)
- PEG
polyethylene glycol
- POLY
polyembryos
- VVM
volume of gas/volume of bioreactor 相似文献