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1.
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
2.
E. Carneros C. Celestino K. Klimaszewska Y.-S. Park M. Toribio J. M. Bonga 《Plant Cell, Tissue and Organ Culture》2009,98(2):165-178
Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes
containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates
of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families,
the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean
frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections)
were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only
seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the
families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured
after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This
effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were
tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However,
up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven
converted into somatic seedlings. 相似文献
3.
4.
Maggie Panaia Eric Bunn Jen McComb 《In vitro cellular & developmental biology. Plant》2011,47(3):379-386
Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity,
in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only
zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid
(2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic
embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic
embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto
fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture
periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per
culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all
previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent
experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for
efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion
to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis
and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii. 相似文献
5.
In Pinus spp., initiation of somatic embryogenesis (SE) is influenced by the developmental stage of immature embryos, the genotype
of the parent trees and the formulation of tissue culture medium. Optimizing all these factors can lead to improved initiation
and proliferation response; however, few studies have focused on improving these stages. For this reason, the objectives of
this research were to determine the best immature zygotic embryo developmental stage for initiation and to test the effect
of different sources of organic nitrogen in the initiation and proliferation steps in Pinus radiata SE. We have determined and verified the optimum zygotic embryo developmental stages 2–4 for embryogenic tissue (ET) initiation
and proliferation and identified the most responsive seed families in two consecutive years. Besides EDM (Walter et al. 1998), medium with high gellan gum content during ET proliferation maintained the embryogenic tissue in a better micro-morphological
arrangement for a longer time. 相似文献
6.
A method to produce transgenic plants of Vitis rotundifolia was developed. Embryogenic cultures were initiated from leaves of in vitro grown shoot cultures and used as target tissues
for Agrobacterium-mediated genetic transformation. A green fluorescent protein/neomycin phosphotransferase II (gfp/nptII) fusion gene that allowed for simultaneous selection of transgenic cells based on GFP fluorescence and kanamycin resistance
was used to optimize parameters influencing genetic transformation. It was determined that both proembryonal masses (PEM)
and mid-cotyledonary stage somatic embryos (SE) were suitable target tissues for co-cultivation with Agrobacterium as evidenced by transient GFP expression. Kanamycin at 100 mg l−1 in the culture medium was effective in suppression of non-transformed tissue and permitting the growth and development of
transgenic cells, compared to 50 or 75 mg l−1, which permitted the proliferation of more non-transformed cells. Transgenic plants of “Alachua” and “Carlos” were recovered
after secondary somatic embryogenesis from primary SE explants co-cultivated with Agrobacterium. The presence and stable integration of transgenes in transgenic plants was confirmed by PCR and Southern blot hybridization.
Transgenic plants exhibited uniform GFP expression in cells of all plant tissues and organs including leaves, stems, roots,
inflorescences and the embryo and endosperm of developing berries. 相似文献
7.
Luiz Ricardo Hanai Eny Iochevet Segal Floh Maria Helena Pelegrinelli Fungaro Claudete Santa-Catarina Francine Matias de Paula Ana Maria Viana Maria Lucia Carneiro Vieira 《In vitro cellular & developmental biology. Plant》2010,46(4):368-377
Ocotea catharinensis is a rare tree species indigenous to the Atlantic rainforest of South America. In spite of its value as a hardwood species,
it is in danger of extinction. The species erratically produces seeds showing irregular flowering and slow growth. Therefore,
plants are not easily replaced. Tissue culture-based techniques are commonly used for obtaining living material for tree propagation
and in vitro preservation. Therefore, a high-frequency somatic embryogenic system was developed for the species. In the present work,
the genetic fidelity of cell aggregates and somatic embryos at various stages of in vitro development of O. catharinensis was investigated using RAPD and AFLP markers. Both analyses confirmed the absence of genetic variation in all developmental
stages of O. catharinensis embryogenic cultures, verifying that the in vitro system is genetically stable. The cultures were also analyzed for their methylation profiles at 5′-CCGG-3′ sites by identifying
methylation-sensitive amplification polymorphisms. Some of these markers differentiated cell aggregates from embryo bodies.
The sequencing of ten MSAP markers revealed that four sequences showed significant similarity to genes encoding plant proteins.
Particularly, the predicted amino acid sequence of the fragment designated as OcEaggHMttc155 was similar to the enzyme 1–aminocyclopropane-1-carboxylate oxidase (ACO), which is involved in the biosynthesis of ethylene,
and its expression was reported to occur from the beginning to the intermediate stages of plant embryo development. Here,
we suggest that this enzyme is possibly involved in the control of the earliest stages of somatic embryogenesis of O. catharinensis, and an approach to study ACO expression during somatic embryogenesis is proposed. 相似文献
8.
Summary Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process
and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the
induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of
genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability
of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem
also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic
regulation of SE in the context of current molecular understanding of plant development. 相似文献
9.
10.
De Silva V Bostwick D Burns KL Oldham CD Skryabina A Sullards MC Wu D Zhang Y May SW Pullman GS 《Plant cell reports》2008,27(4):633-646
Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to
implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm
embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid
female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory
compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements.
We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth
and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated
this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and
mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at
P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65
nmoles per stage 2–3 FG) and the amount of citric acid that stimulates colony growth (25–50 nmoles) when applied topically
to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes
can provide valuable information leading to further applications of these molecules to improve LP SE protocols. 相似文献
11.
Małgorzata D. Gaj Aneta Trojanowska Agnieszka Ujczak Martyna Mędrek Aleksandra Kozioł Beata Garbaciak 《Plant Growth Regulation》2006,49(2-3):183-197
Plant hormones are considered to be the key factors involved in triggering in vitro induced plant morphogenesis, including somatic embryogenesis (SE). Mutants affected in SE and altered in hormonal response therefore provide valuable material for genetic research on in vitro induced plant embryogenesis. The capacity for SE was studied in 27 mutants with defects in response to different plant hormones: auxin, ABA, gibberellin and cytokinin, and evaluated in 2-week-old mutant and wild-type cultures in terms of their efficiency and productivity. SE was induced in vitro via a direct morphogenic pathway, through the culture of immature zygotic embryos on standard solid medium with 5 μM 2,4-D. The majority of the analyzed mutants displayed a significantly impaired capacity for SE; and those affected belonged to several different hormone-defective groups, including forms affected in auxin (axr4), gibberellin (ga) and ABA (abi, hyl1, cpb20, abh1) response. These mutants showed a significant decrease in embryogenic response as manifested by a low efficiency and/or productivity of SE. Additionally, SE efficiency was analyzed for axr4-1 mutant on media supplemented with different auxins while GA3 and inhibitors of gibberellins (uniconazol P and paclobutrazol), were applied for pkl1-1-mutant. The selected mutants provide a valuable research tool for studying the molecular mechanisms determining the induction of embryogenesis in cultures of somatic tissues. Their usefulness in further studies is discussed. 相似文献
12.
Summary
Baloskion tetraphyllum is a member of the Restionaceae and is an important species for the rehabilitation of disused mine sites and wetland areas, and is also highly prized as
a cut flower. Its use for restoration of disturbed land is, however, severely limited, due to very poor propagation success
by conventional methods. A study was conducted to evaluate the potential of somatic embryogenesis for the large-scale propagation
of this species. A variety of auxins (at different concentrations) were investigated for their efficacy in stimulating somatic
embryogenesis. Somatic embryos were successfully induced from excised coleoptiles of B. tetraphyllum on half-strength Murashige and Skoog (1/2MS) medium supplemented with 1 μM 2,4-dichlorophenoxyacetic (2,4-D). To scale up the production, proliferation of secondary somatic embryos was achieved using
primary somatic embryos as the tissue source, on 1/2MS+1 μM 2,4-D resulting in a 30-fold increase in somatic embryo, numbers. Almost all the somatic embryos developed into plants and
were established ex vitro. The other auxins investigated, including p-chlorophenoxyacetic acid, indole-3-acetic acid, α-naphthaleneacetic acid, and picloram, were not as effective as 2,4-D. The
age, of the explant material significantly influenced somatic embryogenesis with white, young coleoptiles (5–7 d) producing
50% more somatic embryos than green, more mature (8–14 d) coleoptiles. The protocol developed for B. tetraphyllum has the potential to be commercially viable, with an estimated 22 000 somatic embryos produced from 1 g of plant material.
This research may also have a positive impact on the propagation of other important Restionaceae species. 相似文献
13.
Summary We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic
embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid
(2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction
was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic
embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species. 相似文献
14.
Guohua Ma Jinfeng Lü Jaime A. Teixeira da Silva Xinhua Zhang Jietang Zhao 《Plant Cell, Tissue and Organ Culture》2011,104(2):157-162
Ochna integerrima is a medicinal and ornamental plant in Southeastern Asia. It has been listed as a rare and endangered species in China. Here
we studied the effects of plant growth regulators and their concentrations on the induction of somatic embryogenesis and shoot
organogenesis from leaf and shoot explants of O. integerrima for the first time. Cytokinins played a crucial role in somatic embryogenesis and shoot organogenesis. Among them, a higher
concentration of thidiazuron (10.0–15.0 μM TDZ) could induce both somatic embryogenesis and adventitious shoot formation whereas
low concentrations of TDZ (5.0 μM) could only induce adventitious shoots. However, 6-benzyladenine (BA at 5–15 μM) could only
induce adventitious shoots. Shoot explants induced more adventitious shoots and somatic embryos than leaf explants when cultured
on medium with the same concentration (5–15 μM) of TDZ or 15 μM BA. Medium containing 0.5 μM α-naphthaleneacetic acid and
8 μM indole-3-butyric acid and 0.1% activated charcoal could induce adventitious roots within 1 month. An efficient mass propagation
and regeneration system has been established. 相似文献
15.
Zhiyong Pan Shiping Zhu Rui Guan Xiuxin Deng 《Plant Cell, Tissue and Organ Culture》2010,103(2):145-153
The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic
cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant
tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development
of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic
changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l−1) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated.
Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic
stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated
callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and
an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D
could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells. 相似文献
16.
Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred
on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's
medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators.
The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D
(0.45 μM) and N6-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established
in the field. 相似文献
17.
18.
K. Balaraju S. Saravanan P. Agastian S. Ignacimuthu 《Acta Physiologiae Plantarum》2011,33(4):1123-1133
An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic
acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness
to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This
system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed
on Murashige and Skoog (MS) medium containing 1 mg L−1 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68%
of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L-1 GA3 respectively. The 2,4-D alone at 1.0 or 1.5 mg L−1 was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene
acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin
or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean
germination and percentage of survival were maximum in the medium containing 1.0 mg L−1 2,4-D in combination with 0.1 mg L−1 BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal
propagation of endangered plants. 相似文献
19.
Navtej Kaur Pratap Kumar Pati Madhu Sharma P. S. Ahuja 《In vitro cellular & developmental biology. Plant》2006,42(2):124-127
Summary A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS
medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of
collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy
acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l−1) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably
over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under
greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and
September.
IHBT Publication No: 0447 相似文献