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Sánchez MC Martínez MT Valladares S Ferro E Viéitez AM 《Journal of plant physiology》2003,160(6):699-707
Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis. 相似文献
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Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration
from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic
embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected
developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical
seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development
(since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical
for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the
biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary
somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher
than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein
content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive
storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis
pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea. 相似文献
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V. Šunderlíková J. Salaj I. Matušíková E. Wilhelm 《Trees - Structure and Function》2009,23(1):135-144
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Interactions of growth regulators and polyethylene glycol on maturation of geranium somatic embryos were investigated. Somatic embryos were induced on medium with 20 M thidiazuron for 3 days. The growth regulators used were 1 µM abscisic acid, jasmonic acid, napthaleneacetic acid and benzylaminopurine at 21 days from the start of induction. Benzylaminopurine and napthaleneacetic acid did not enhance abscisic acid effects on maturation frequency but only improved maturation frequency in the presence of polyethylene glycol. Abscisic acid significantly improved protein content in the presence of polyethylene glycol. Benzylaminopurine and napthalene acetic acid in combination with abscisic acid and jasmonic acid improved protein types in somatic embryos only in the absence of polyethylene glycol. Osmoticum effected by polyethylene glycol seems the main component required for protein synthesis. This study showed significant improvement of somatic embryo quality for artificial seed production. 相似文献
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Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification 总被引:2,自引:0,他引:2
Cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (Asparagus officinalis L.) were cryopreserved by vitrification. The vitrification solution (PVS) contains (w/v) 22% glycerol, 15% ethylene glycol, 15% propylene glycol and 7% DMSO in Murashige-Skoog medium enriched with 0.5M sorbitol. After initial cryoprotection with sorbitol supplemented MS medium containing 12% ethylene glycol, cells or embryos were exposed stepwise to 85% PVS at 0°C. They were loaded into 0.5 ml transparent straws, and were then plunged directly into liquid nitrogen. After rapid warming, PVS was removed and diluted stepwise. The highest survivals of vitrified cells and embryos were about 65 and 50%, respectively. Surviving embryos developed into plantlets.Abbreviations DMSO
dimetyl sulfoxide
- PVS
vitrification solution
- LN
liquid nitrogen
- DSC
differential scanning calorimeter
- MS
Murashige-Skoog salt medium
- NAA
naphthalene acetic acid
- BA
6-benzyladenine 相似文献
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Xin Y. Li Feng H. Huang J. Brad Murphy Edward E. Gbur 《In vitro cellular & developmental biology. Plant》1998,34(1):22-26
Summary A culture medium that can efficiently produce mature somatic embryos was developed for loblolly pine (Pinus taeda L.). The medium contained maltose as a carbohydrate source and polyethylene glycol as an osmoticum. This medium formulation
significantly enhanced embryo maturation efficiency compared to a medium with only maltose, or with sucrose combined with
polyethylene glycol. Maltose at 4% and polyethylene glycol at 6% resulted in the highest embryo maturation efficiency; an
average of around 100 cotyledonary embryos were produced from 1 g of embryogenic tissue. These results suggested that previous
ineffective embryo maturation in loblolly pine may be due to the lack of the proper combination of osmoticum and carbohydrate
source. This embryo maturation method also improved morphology of cotyledonary embryos of loblolly pine. 相似文献
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Randall P. Niedz Scott E. Hyndman Eldridge T. Wynn Michael G. Bausher 《In vitro cellular & developmental biology. Plant》2002,38(6):552-557
Summary Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphogenesis of somatic
embryos. To normalize development, glycerol-induced globular-stage somatic embryos of sweet orange [C. sinensis (L.) Osbeck cv. ‘Hamlin’] were cultured on 6000–8000 MW cutoff cellulose acetate, >400 000 MW cutoff cellulose acetate, nitrocellulose,
polyvinylidene fluoride (PVDF), cellulose filter paper, or positive or neutral charged nylon membranes. Only the two cellulose
acetate membranes resulted in the development of normal, two-cotyledon, bipolar, heart-shaped embryos, and no aberrant teratoma-like
structures. Heart-shaped embryos developed and germinated normally on Murashige and Tucker basal medium with 0.5% sucrose
and 1 μM gibberellic acid. Culture of embryogenic callus directly onto cellulose membranes also resulted in the development of normal
heart-shaped embryos, indicating that glycerol induction of globular-stage embryos is not necessary. Heart-shaped embryos
were not observed when the osmotic potential of the medium was increased by the addition of 2.5–15% polyethylene glycol; neither
were they observed when the matric potential of the medium was increased by increasing the gelling agent concentrations of
agar and Gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively. 相似文献
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Summary Cotyledonary Quercus robur L. somatic embryos from two cell lines were encapsulated in 4% (w/v) sodium alginate. An artificial endosperm was provided
by the addition of P24 medium plus 3% (w/v) sucrose. Oak somatic embryos and oak synthetic seeds were germinated on P24 medium plus 0.1 μM indole-3-butyric acid and 0.9 μM 6-benzylaminopurine or were dehydrated prior to germination. The highest conversion rates (26%) were obtained with encapsulated
somatic embryos as well as artificial endosperm-coated somatic embryos. Encapsulation improved the regeneration into oak plantlets
in one of the two cell lines tested. The artificial endosperm had no additional beneficial effect on conversion frequency,
but increased germination rate in one cell line tested. Significant higher conversion could be attributed to slow desiccation
compared to the non-encapsulated control. Cold storage as a post-maturation treatment had no influence on the germination
ability of oak synthetic seeds. Differences in the response of the cell lines with respect to conversion frequencies and timing
of germination were observed. Fifty-six well-developed plantlets regenerated 12 wk after germination, and 29 plants were transferred
to the greenhouse, where they have been successfully established in substrate. 相似文献
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Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation. 相似文献
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Veronique Troch Stefaan Werbrouck Danny Geelen Marie-Christine Van Labeke 《Plant Cell, Tissue and Organ Culture》2009,98(1):115-123
Factors affecting conversion of horse chestnut (A. hippocastanum L.) somatic embryos into plantlets were evaluated. Anther filament derived embryogenic tissue developed bipolar structures
with two cotyledons and a well-developed shoot and root apical meristem upon auxin omittance from the culturing medium. The
impact of carbohydrate type (glucose, fructose, sucrose and maltose) and concentration (3 and 6%) on somatic embryo maturation
and conversion were evaluated. Although conversion frequencies were high for all treatments, overall quality of regenerated
plantlets was poor. Increasing the carbohydrate concentration in the maturation medium did not increase conversion of somatic
embryos or quality of regenerated plantlets in terms of shoot height. On the contrary, addition of PEG (polyethylene glycol)
in maturation media had a beneficial effect on shoot quality of regenerated plantlets. Sucrose was a superior carbon source
when PEG was included in the maturation medium, in terms of conversion rate (65.7%) as well as of shoot quality of plantlets
(43.8% of plantlets had shoots >2 cm). Clonal fidelity of the different development stages of somatic embryogenesis and of
converted plantlets was assessed by flow cytometry and no major ploidy changes were found. 相似文献
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《Plant Physiology and Biochemistry》1999,37(2):145-154
High concentrations of sucrose are often used to cryopreserve regenerable plant cell cultures in liquid nitrogen. A 21-h pretreatment of carrot somatic embryos in medium containing 0.4 M sucrose allows 80 % of them to germinate after freezing. Substitution of sucrose by polyethylene glycol 6000 led to lower germination rates. However, a high level of freezing tolerance was restored by addition of 1 μM abscisic acid in the pretreatment medium. Using these different media, both total water soluble protein, using SDS-PAGE, and boiling-stable protein, using 2-D electrophoresis, were studied in relation to acquisition of cryopreservation tolerance. Only boiling-stable protein patterns showed some changes: five polypeptides accumulated in 0.4 M sucrose-pretreated embryos or in embryos pretreated by media containing abscisic acid. This accumulation was not detected with polyethylene glycol 6000 used as sole cryoprotectant. Although over-accumulation of polypeptides was highest with media containing ABA, the best germination rates were linked to pretreatment with 0.4 M sucrose. The addition of okadaic acid in 0.4 M sucrose medium led to embryo death after freezing, confirming the existence of a message leading to metabolic changes and acquisition of cryotolerance. Water-soluble proteins obtained from 0.4 M sucrose-pretreated embryos appeared more active than those extracted from control embryos in protecting in vitro a freeze-labile enzyme. Boiling-stable proteins, corresponding to a part of total proteins, were more active than total proteins. These results suggest that these polypeptides may be involved in a mechanism of protection needed for cell survival during freezing stress. 相似文献
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A. Ramarosandratana L. Harvengt A. Bouvet R. Calvayrac M. Pâques 《In vitro cellular & developmental biology. Plant》2001,37(1):29-34
Summary The influence of carbon sources and polyethylene glycol combined with 0.45 and 0.9% (w/v) of gellan gum on the maturation
of maritime pine somatic embryos was tested. The effect of the carbon source and polyethylene glycol varied widely between
lines. One out of the five lines tested showed a striking response to polyethylene glycol (PEG) treatment; the addition of
this osmoticum limited the embryonal-suspensor mass (ESM) proliferation while it enhanced the maturation rate. Conversely,
the ESM proliferation was stimulated by PEG in the other lines without subsequent improvement of the maturation rate. The
use of a high concentration of gellan gum (0.9%) improved the maturation of the five ESM lines. It was concluded that the
most efficient culture medium to recover cotyledonary embryos from all lines is one supplemented with sucrose at 6% (w/v)
and gellan gum at 0.9% (w/v) without PEG. The determining factor in the maturation of maritime pine somatic embryos is the
genotype and/or the quality of ESM. The possible relationship between maturation performances and ESM morphology, particularly
the suspensor organization, is discussed. 相似文献
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Soybean (Glycine max L. Merrill) somatic embryos have been useful for assaying seed-specific traits prior to plant recovery. Such traits could be assessed more accurately if somatic embryos more closely mimicked seed development. Amino acid supplements, carbon source, and abscisic acid and basal salt formulations were tested in an effort to modify existing soybean embryogenesis histodifferentiation/maturation media to further normalize the development of soybean somatic embryos. The resultant liquid medium, referred to as soybean histodifferentiation and maturation medium (SHaM), consists of FNL basal salts, 3% sucrose, 3% sorbitol, filter-sterilized 30 mM glutamine and 1 mM methionine. SHaM-derived somatic embryos are more similar to seed in terms of protein and fatty acid/lipid composition, and conversion ability, than somatic embryos obtained from traditional soybean histodifferentiation and maturation media. 相似文献
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Márquez-Martín B Sesmero R Quesada MA Pliego-Alfaro F Sánchez-Romero C 《Journal of plant physiology》2011,168(17):2028-2034
Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a medium gelled with 10 g l−1 agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos. 相似文献
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Emilio Maruyama Yoshihisa Hosoi Katsuaki Ishii 《In vitro cellular & developmental biology. Plant》2007,43(1):28-34
Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both
media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to
20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic
acid (3 μM) and 6-benzylaminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l−1), activated charcoal (2 g l−1), abscisic acid (100 μM), and polyethylene glycol (100 g l−1). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to
51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field. 相似文献