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1.
In order to develop a more efficient genetic transformation system for cacao somatic embryos, the effects of polyamines and
β-lactam antibiotics on somatic embryogenesis, hygromycin as selective agent, and different factors affecting uidA gene transfer have been evaluated. The polyamines putrescine, spermidine, and spermine significantly improved secondary somatic
embryogenesis in cacao. Spermine at 1,000 μM provided the best responses, increasing 6.7× the percentage of embryogenic callus
and 2.5× the average number of embryos per embryogenic callus. The β-lactam antibiotics timentin and meropenem, used for Agrobacterium tumefaciens counter-selection, had a non-detrimental effect on secondary somatic embryogenesis, depending on their concentration, whereas
the commonly used β-lactam cefotaxime inhibited it, irrespective of the tested concentration. Hygromycin showed a strong inhibitory
effect on secondary somatic embryogenesis of cacao, impairing completely the embryo production at 20 mg l −1. Following the criterion of GUS activity, the best conditions for T-DNA transfer into cotyledon explants from primary somatic
embryos of cacao were a sonication of the explants for 100 s, a 20-min incubation period in Agrobacterium solution, an Agrobacterium concentration of 1.0 (OD 600), and cocultivation of the explants on tobacco feeder layers. These findings will have important implications for studies
on functional genomics of cacao. 相似文献
2.
Summary A procedure for the regeneration of cacao ( Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing
staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μ M 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth
medium supplemented with 20 g glucose per L, 9 μ M 2,4-D, and 1.4 n M kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development
medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the
frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of
22.7 n M was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using
this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types.
However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged
from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46.
Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of
cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological
and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use
of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of
a large number of plants from limited source materials. 相似文献
3.
Levels of wheat germ agglutinin have been determined by radioimmunoassay in tissues of immature wheat embryos cultured under different conditions in order to determine the suitability of the lectin as a marker for somatic embryogenesis. Embryos cultured on media favouring continued embryo development accumulated lectin in a similar manner to zygotic embryos in planta unless precocious germination occurred. Embryos cultured on media containing 2,4-D produced callus, and some of this developed somatic embryos. Both embryogenic and non-embryogenic callus contained WGA, that in non-embryogenic callus possibly arising from developmentally arrested root primordia.Abbreviations ABA
abscisic acid
- dpa
days post anthesis
- PBS
phosphate buffered saline, (10 mM KH 2PO 4 K 2HPO 4, 145 mM NaCl, pH 7.4)
- RIA
radioimmunoassay
- WGA
wheat germ agglutinin
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
4.
Mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were germinated on a Murashige and Skoog medium lacking growth
regulators. However, when the zygotic embryos were cultured on MS medium containing increased levels of macrosalts (NH 4NO 3, KNO 3, KH 2PO 4, MgSO 4, or CaCl 2) to result is a mild salt stress, growth of zygotic embryos was strongly suppressed and eventually browning occurred. Somatic
embryos or embryogenic calli were formed directly from these abnormal stressed zygotic embryos. Cotyledons were the most competent
tissue for somatic embryo production. The highest frequency of somatic embryo formation (56.3%) was observed on medium containing
61.8 mM of NH 4NO 3. The highest frequency of somatic embryo formation by five different macrosalt treatments occurred in the following order:
NH 4NO 3> KNO 3> KH 2PO 4> MgSO 4> CaCl 2. Somatic embryos were regenerated into plants with a shoot and root, and the plants survived on soil in the greenhouse.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
5.
橡胶树的花药愈伤组织在长期继代过程中,胚性易下降甚至丧失;而AgNO3作为乙烯活性抑制剂,被广泛应用于植物组织培养中.该研究以继代培养4 a以上的热研7-33-97花药愈伤组织为材料,在继代培养基中添加2.5 mg·L-1 AgNO3预培养35 d后,观察预培养前后愈伤组织表形及其细胞形态的变化,并设计不同浓度AgNO3及不同处理时间对其进行体胚诱导,90 d后分别统计胚状体总数和正常胚数.结果表明:浅黄色质地柔软的愈伤组织在含AgNO3的培养基上预培养后能转变成鲜黄色易碎愈伤组织,在倒置显微镜下前者大多表现为不规则多边形,细胞内含物较稀薄;而后者则呈圆形或椭圆形,细胞内含物丰富,属于典型的胚性细胞.在体胚诱导的第1个月添加5 mg·L-1 AgNO3能显著促进体胚的发生,AgNO3浓度升至10 mg·L-1时体胚发生受到抑制,且畸形胚的形成率显著增加;在含5 mg·L-1 AgNO3的培养基中培养2个月以上,体胚发育明显受阻,大部分形成畸形胚.该研究结果在一定程度上恢复了橡胶树长期继代花药愈伤组织的胚性能力,并提高了其体胚发生频率,为橡胶树花药胚性愈伤组织长期继代培养过程中胚性的保持提供了参考. 相似文献
6.
Summary The development of efficient tissue culture systems for cacao holds the potential to contribute to the improvement of this
tropical erop by providing a rapid and efficient vegetative propagation system for multiplication of elite genotypes. It may
also find application in facilitation of germplasm movement across quarantine borders, enhancement of germplasm conservation
via cryo-preservation, and development of genetic transformation systems. Somatic embryogenesis using floral tissue explants
was previously the only tissue culture procedure for regeneration of cacao. We report the development of a secondary embryogenesis
system utilizing primary somatic embryo cotyledon explants, which results in up to a 30-fold increase in somatic embryo production
compared to primary somatic embryogenesis. The influence of genotype on the efficiency of the system was evaluated. To understand
the cellular origins and developmental pathways operative in this system, we investigated the morphological changes occurring
over time using light and scanning electron microscopy. While primary embryos arise from clusters of cells forming embryonic
nodules, secondary embryos arise predominantly from the division of single cells, in a pathway reminiscent of zygotic embryogenesis.
These results have important significance to the application of tissue culture to cacao improvement programs. 相似文献
7.
Somatic embryogenesis was induced in immature zygotic embryos of pea ( Pisum sativum L.), synthetic auxins α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) being used. Only one (line HM-6) of 46 genotypes tested exhibited good potential for somatic embryogenesis. 2,4-D was found as the best somatic embryo inductor. Three different ways of somatic embryo conversion have been described. Plantlets from individual somatic embryos were micropropagated as somaclones and subsequently rooted. A sterile morphological mutant has been found within a group of fertile plants of T 0-generation. Sufficient amount of T 1-seeds is available for somaclonal variation studies. 相似文献
8.
Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA
benzyladenine
- NAA
naphthaleneacetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BA
indolebutyric acid
- MS
Murashige & Skoog (1962) medium
- SH
Schenk & Hildebrandt (1972) medium
- G
Gamborg (1966, PRL-4-C) medium (macronutrients in mg l –1: NaH 2PO 4·H 2O, 90; Na 2HPO 4, 30; KCl, 300; (NH 4) 2SO 4, 200; MgSO 4·7H 2O, 250; KNO 3, 1000, CaCl 2·2H 2O, 150)
- PGR
plant growth regulator 相似文献
9.
Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH 4
+ and NO 3
– ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 M) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH 4
+ and NO 3
– concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 M) enhanced the conversion of somatic embryos to plantlets. 相似文献
10.
Plant regeneration via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and increased osmolarity of the medium were necessary for embryo induction. Relatively high amounts of embryogenic calli could be obtained in 2 genotypes. After a period on hormone-free medium, a second induction of somatic embryos was possible. Further development of somatic embryos was accomplished on GA 3 — containing medium.Abbreviations ABA
abscisic acid
- BA
6-benzylaminopurine
- 2.4-D
2.4-dichlorophenoxyacetic acid
- GA 3
gibberellic acid
- Kin
kinetin
- NAA
naphthaleneacetic acid
- Pic
Picloram, 4-amino-3,5,6-trichloropicolinic acid 相似文献
11.
A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt
to improve somatic embryo production in zonal geranium ( Pelargonium × hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced
by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the
morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With
1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage
embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher
ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply
of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine
(BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no
interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos
can be achieved in zonal geranium. 相似文献
12.
A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium ( Pelargonium × hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 µM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 µM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium. 相似文献
13.
Five genotypes of chickpea ( Cicer arietinum L.) PG1, PG5, PG12, N59 and C235 were evaluated for induction of somatic embryogenesis. Somatic embryogenesis was induced from immature cotyledons of genotypes PG12 and C235 and immature embryo axes of genotypes PG5, PG12 and C235. Genotypes N59 and PG1 showed no response. The maximum frequency of globular embryo formation occurred in cotyledonary segments on MS medium with 3.0 mg/l 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Further embryo development was achieved only in somatic embryos derived from cotyledonary segments of genotype PG12. Globular-stage embryos derived from immature embryo axes of PG5, C235, PG12, and cotyledonary segments of C235 dedifferentiated and formed callus. The cotyledonary stage embryos of genotype PG12 germinated on half-strength MS medium supplemented with 1 mg/l zeatin. The regenerated plants were transferred to soil and grown to maturity.Abbreviations ABA
abscisic acid
- BAP
6-benzylamino purine
- CM
coconut milk
- Dicamba
3,6-Dichloro-2-methoxybenzoic acid
- 2,4-D
2,4-dichlorophenoxy-acetic acid
- GA 3
gibberellic acid
- IAA
indoleacetic acid
- MS
Murashige and Skoog medium (1962)
- NAA
1-napthaleneacetic acid
- Picloram
4 amino-3,5,6-trichloropicolinic acid
- 2,4,5-T
2,4,5-trichlorophenoxy-acetic acid
- zeatin
(6-[4-Hydroxy-3-methyl-2-butenylamino] purine) 相似文献
14.
Arbutus unedo L. is a perennial tree, native in the Mediterranean area, and tolerant to stress conditions. Due to its economic potential, there is an increasing demanding for plants by producers and farmers. In order to offer cloned material with assured quality, several micropropagation protocols have been developed, including somatic embryogenesis induction. However, little is known about this process on strawberry tree and a great deal of work is still necessary to successfully clone in A. unedo through somatic embryogenesis. Thus, the main goals of this work were: (i) to test the effect of the genotype on somatic embryogenesis induction, (ii) to analyse the role of adult and young materials on induction, and (iii) to perform a comparative histological analysis between somatic embryos and their zygotic counterparts. Somatic embryogenesis was induced on apical expanded leaves from in vitro shoots of several genotypes in Andersson medium with 3% sucrose and different concentrations of BAP (2.0 mg L?1) and NAA (0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0 mg L?1). Embryogenesis induction rates ranged from 0 to 94.5%. Higher induction rates were achieved on the medium with 2 mg L?1 BAP and 2 mg L?1 NAA, and are genotype dependent. After a 3-month induction period, the highest somatic embryogenesis induction rate was 94.5% on genotype AU4. Embryos at different developmental stages were found, as well as abnormal somatic embryos. SEM images showed different anomalies being the most common embryos displaying more than two cotyledons or fused embryos. Embryo germination was not genotype dependent and the maximum embryo conversion rate achieved was 73.5%. However, only 39.21% of the embryos were able to grow into plantlets which displayed a normal chromosome number (2n?=?26). Histological analysis showed differences in the cell organization between somatic and zygotic embryos, as well as several morphological anomalies. Overall, the developed somatic induction protocol proved to be very efficient, with high induction rates achieved, both from seedling and adult material, but its genotype dependent. However, embryo conversion still needs to be improved, in order to fully seize the potential of this micropropagation technique. 相似文献
15.
The native gibberellin A 4 (GA 4) was fed as [1, 2- 3H]GA 4 (1.3 Ci/mmol) to anise somatic cultures maintained either at a proembryo-like stage with 2,4-dichlorophenoxyacetic acid (2,4-D), or allowed to undergo embryogenic development on a - 2,4-D medium. Proembryos, although only 20% of the dry wt of embryos, absorbed 1.4-times more [ 3H]GA 4/g dry wt than embryos. The [ 3H]GA 4 was metabolized to GA 1 and GA 8, and at least six conjugates [GA 4-glucoside (GA 4-G), GA 4 glucosyl ester (GA 4-GE), GA 1-0(3)-G, GA 1-0(13)-G, GA 1-GE and a GA 8-glucosyl conjugate]. The major metabolite was GA 4-G at each of two, 204 and 348 hr harvests (56–71 %), with GA 8-G increasing from < 1 % to 13 % with harvest time. The percentage and amount of GA 4-GE was highest at 204 hr (2% and 8 %, for embryos and proembryos, respectively), dropping to < 1 % at 348 hr, thereby indicating hydrolysis (e.g. reversible conjugation). Embryos had reduced amounts and percentages of biologically active GA 4 and GA 1, and most of their conjugates, but increased amounts and percentages of GA 8 and its conjugate(s). This finding is consistent with the hypothesis (based on present and past work) that high levels of biologically active GAs, especially GA 1, inhibit somatic embryogenesis in anise and carrot. The auxin, 2,4-D, may thus derive, at least in part, its ability to maintain the proembryo-like stage by inhibiting oxidative metabolism and conjugation of biologically active GAs. 相似文献
16.
The aim of this study was to improve the direct somatic embryogenesis and initiate embryogenic callus formation in camphor
tree ( Cinnamomum camphora L.) on hormone-free medium. The influence of osmotic stress pretreatment of immature zygotic embryos (0.5 and 1.0 M solution
of sucrose for 12, 24, 48, 72, 96, 120, and 144 h at 4 or 25°C) before cultured on hormone-free medium, on embryogenesis efficiency
was assessed. The embryogenesis frequency was improved from 16.29 to 93.27%, while the average number of somatic embryos per
explant increased from 3 to 12.57. Activated charcoal (AC), medium renewal, basal medium, light conditions and sucrose concentration
in culture medium were also evaluated for their effect on somatic embryogenesis. AC addition and 10-day medium renewal did
not increase embryogenesis efficiency significantly, and Murashige and Skoog (MS) medium proved to be more beneficial for
somatic embryo formation than others. No differences were found between embryogenesis frequencies when cultured in darkness
or under light, but culturing under light yielded more embryos. After the sucrose solution pretreatment, high level concentration
of sucrose in induction medium was not needed for somatic embryogenesis, for it had a negative effect on somatic embryo formation
when the concentration of sucrose was higher than 50 g l −1. The derived embryogenic lines were maintained via repetitive embryogenesis on hormone-free medium. Low ratio formation of
embryogenic callus was observed on the surface of somatic embryos both on induction and proliferation medium. Plantlets derived
from somatic embryos grew vigorously with normal appearance similar to germinated zygotic embryos. 相似文献
17.
Summary A plant regeneration method via somatic embryogenesis of several Helianthus annuus L. genotypes was developed. Starting from cotyledonary explants high frequency embryo induction was obtained following several subcultures on defined media. An appropriate cotyledon developmental stage was identified. Etiolated explants and darkness treatment were necessary to obtain somatic embryos in all tested genotypes. After 20–25 days on somatic induction medium containing an auxin:cytokinin ratio of 1:1, the germination of embryos was induced by a reduction of the hormonal ratio (1:2). Shoots were excised from callus and transferred onto a medium containing various vitamins. The range of embryogenesis frequency was 33–72%, depending on the genotype. High frequency of rooting (49–82%) was obtained using a medium supplemented with 0.5 mg/L of ancymidol and by a reduction of photoperiod. A large percentage of somatic embryos developed into normal regenerated plants producing viable seeds.Abbreviations MS
Murashige and Skoog (1962)
- NAA
naphthaleneacetic acid
- BAP
benzylaminopurine
- GA 3
gibberellic acid
- EIM
embryo induction medium
- GM
germination medium
- VM
vitamins medium
- RA2
ancymidol rooting medium
- EtOH
ethanol 相似文献
18.
An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred
to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and
Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency
(100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated
on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10 4) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without
plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary
embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However,
when they were treated with gibberellic acid (GA 3), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots
and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully
transferred to forest mountain soil. Following overwintering, these plants produced new growth. 相似文献
19.
Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised
unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three
to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were
low, but increased in the presence of gibberellic acid (GA 3). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs).
The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher
than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced
embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had
two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia,
but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological
and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of
somatic embryo origin. 相似文献
20.
Fifty genotypes of each of three cultivars of alfalfa ( Medicago spp.) were tested in three medium protocols for their capacity to produce somatic embryos and plantlets from callus cultures. Highly productive genotypes produced somatic embryos regardless of medium protocol or explant source, while other genotypes produced somatic embryos in a medium-specific or explant-specific fashion. The results showed that embryogenesis in mature leaf-derived calli could be predicted from the frequency of embryo formation in cotyledon-derived calli of the same genotype. The results also indicated that highly productive genotypes can be selected from cultivars with a low frequency of regeneration. 相似文献
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