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The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using
Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental
evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development. 相似文献
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The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient,
direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency,
ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants.
These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin
distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues
of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic
potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis. 相似文献
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The relationship between somatic embryogenesis (SE) and the expression of the BABY BOOM (BBM) gene was studied in cultured immature zygotic embryos (IZEs) using a transgenic line of Arabidopsis thaliana containing a BBMPro::GUS construct. Results showed spatio-temporal differences in BBM expression in explants during culture. BBM promoter activity was observed in freshly isolated IZEs except distal parts of cotyledons. At the beginning of culture, considerable
increase of GUS staining intensity was observed in all parts of explants, which maintained at high level over next few days
and coincide with cell divisions. Gradual decrease of GUS distribution in explants was observed at about the 5th day of culture.
BBM promoter activity became largely restricted to dividing cells, then to developing somatic embryos, shoot-like structures
and callus. In parts of explants not involved in morphogenesis BBM promoter activity was absent or hardly seen. Thus the in vitro expression of BBM coincides with cell proliferation and morphogenesis. 相似文献
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Iyyakkannu Sivanesan Mi Young Lim Byoung Ryong Jeong 《Plant Cell, Tissue and Organ Culture》2011,107(2):365-369
A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured
on Murashige and Skoog (MS) medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively.
Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under
light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3%
(w/v) activated charcoal (AC) and 1.0 mg L−1 GA3. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse
with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications
for genetic transformation, and mass clonal propagation. 相似文献
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Summary Somatic embryo (bipolar) or shoot (monopolar) morphogenesis in mesophyll cells of Euphorbia nivulia Buch.-Ham in vitro was dependent on the type of auxin supplementing Murashige and Skoog (MS) medium containing benzyladenine. Direct in vitro morphogenesis, i.e., organogenesis, and somatic embryogenesis were significantly influenced by seasonal growth of the donor
plant, explant position (proximal, mid, and distal), and light. Explants collected in march/April were superior to July/August
material. Proximal explants underwent morphogenesis more readily than mid- and tip-derived explants. Incubation in the light
favored morphogenesis while darkness was inhibitory. Kinetin (Kn) was also inhibitory to morphogenesis. MS medium enriched
with different levels of N6-benzyladenine (BA) alone, or in combination with α-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), induced adventitious
shoots directly. Explants collected in March/April cultured on medium with 13.3 μM BA and 2.69 μM NAA developed the highest number of shoots, a mean of 15.2 shoots per proximal explant. Developed shoots rooted the best
on half-strength MS medium with 2.46 μM indole-3-butyric acid, which developed a mean of 5.2 roots per shoot. Rooted healthy shoots could be transplanted to small
pots, with an 80% survival rate. Addition of 2,4-dichlorophenoxyacetic acid (2.4-D) to BA-supplemented medium was obligatory
to develop somatic embryos. MS medium containing 2.26 μM 2,4-D and 4.44 μM BA induced a mean of 44.8 somatic embryos per proximal explant. The embryos passed through distinct stages of embryogenesis,
namely globular, heart, torpedo, and early cotyledonary. The embryos (88%) underwent maturation on half-strength MS medium
with 2.89 μM gibberellic acid (GA3), and its subsequent transfer on half-strength MS basal medium in light conditions facilitated 80% conversion of embryos
to plantlets. Direct shoots or embryos were originated from the mesophyll cells. Somatic embryo development was concurrent
with the independent origin of vasculature in the bulbous basal portion. The survival rate of embryo-derived plants was 90%. 相似文献
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Summary Efficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of
neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from
leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal
and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf
explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free
medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately
15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets
thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived
from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic
embryos. 相似文献
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Ananya Paul Kalyan Mitter Sarmistha Sen Raychaudhuri 《Plant Cell, Tissue and Organ Culture》2009,97(3):303-311
The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica
charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction
of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell
division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos
of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli
(5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic
media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented
with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous
free PA levels. 相似文献
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Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were
excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto
MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic
embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D),
and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant
were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl−1 activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain
somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic
response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected
by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the
protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of
zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In
spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants
from somatic embryos was observed. 相似文献
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Sylvain Legrand Theo Hendriks Jean-Louis Hilbert Marie-Christine Quillet 《BMC plant biology》2007,7(1):27
Background
Somatic embryogenesis (SE) is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E) plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE) under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory. 相似文献12.
Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from
explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development
of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of
plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically
similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation
of the ‘paradise tree’. 相似文献
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In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic
auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence
that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and
somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic
embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth
day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos
auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly
displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal
cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged
in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected. 相似文献
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Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis
was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic
embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible
to obtain somatic embryogenesis in C. arabica and C. canephora. 相似文献
16.
Perera PI Hocher V Verdeil JL Doulbeau S Yakandawala DM Weerakoon LK 《Plant cell reports》2007,26(1):21-28
Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 μM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 μM abscisic acid, followed by plant regeneration medium (with 5 μM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos. 相似文献
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Summary The types of auxin in Murashige and Skoog (MS) medium containing N
6-benzyladenine (BA) determined indirect morphogenesis, i.e. development to bipolar somatic embryos or monopolar shoots in
Euphorbia nivulia Buch.-Ham. Indirect in vitro morphogenesis depended on growth regulators, explant excision period, and light. Calli induced from explants collected in
March–April were superior in the induction of indirect morphogenesis to those collected in July–August. Light enforced in vitro morphogenesis, while darkness was inhibitory. The presence of kinetin in the medium also inhibited morphogenesis. Calli developed
on explants collected in March–April grown on MS medium fortified with α-naphthaleneacetic acid (NAA) and BA facilitated indirect
organogenesis, while those developed on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and BA underwent somatic
embryogenesis. MS medium with 13.3 μM BA and 2.69 μM NAA was the best for induction of shoots from callus, which developed a mean of 15.7 shoots. Shoots were best rooted on half-strength
MS medium enriched with 2.46 μM indole-3-butyric acid with a mean of 5.1 roots per shoot. MS medium supplemented with 2.26 μM 2,4-D and 4.44 μM BA induced the highest number (mean of 13.4) of somatic embryos. Of the embryos transferred on half-strength MS medium containing
2.89 μM gibberellic acid, 78% of embryos developed to the cotyledonary stage. Most cotyledonary embryos (80%) underwent conversion
to plantlets upon being transferred to half-strength MS basal medium in light. The survival rate of organogenesis and embryo-derived
plants was 80 and 90%, respectively. Calli transformed with Agrobacterium tumefaciens showed expression of the gusA transgene and resistance to kanamycin, but did not undergo morphogenesis. 相似文献
18.
Summary
In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation.
Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and
Skoog (MS) medium with 8.9 μM N6-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis
as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial
side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared
to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with
BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage.
Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized
protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently
transferred to field conditions with a survival rate of 90%. 相似文献
19.
Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations
and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and
germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm−3 kinetin and 0.2, 0.5 mg dm−3 indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots
and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation
revealed that the somatic embryos were similar to those of zygotic embryos. 相似文献
20.
Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.This research was supported by the Consorcio Brasileiro de Pesquisa e Desenvolvimento do Cafe (CBP&D-Cafe). 相似文献