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101.
102.
Matthias Huber Ralph Hahn Dieter Hess 《Molecular breeding : new strategies in plant improvement》2002,10(1-2):19-30
Improved gene transfer techniques are necessary to obtain adequatenumbers of stable transgenic wheat plants needed for practical purposes.Considering that wheat transformation is genotype-dependent, we used cv. Combiin all experiments, which had been selected from agronomically important Germanspring wheat cultivars because of its high transformation ability. In mostwheatgene transfer attempts, immature embryos or embryogenic scutellar calli weremicrobombarded. We compared both methods under optimised conditions, usingbar, uidA, andgfp as markers in co-transformation attempts. Integrationof the genes mentioned above was proven by Southern blotting, expression levelswere measured by assays on phosphinothricin acetyltransferase and-glucuronidase activities, and by monitoring for green fluorescentproteinin most developmental stages. Following bombardment of scutellar calli, anaverage transformation frequency of 0.13% was attained. Using immature embryos,mean transformation frequency (1.06%) was 8-fold higher. In addition, embryotechniques were over 2 weeks faster than scutellar callus procedures.Introducing gfp as a vital marker led to an improvement ofembryo-based techniques. In a first screening, transientgfp-expressing embryos were transferred tophosphinothricincontaining callus medium. Only gfp-expressing calli whichdeveloped on it were cultured further on phosphinothricin containingregeneration medium. Shoots obtained from gfp-expressingcalli were rooted on phosphinothricin-free medium, and cultured exvitro. Average transformation frequency (4.93%) was 38-fold higherthan with scutellar callus techniques. Differences between the transformationstrategies used were of high statistical significance. Combining greenfluorescent protein screening with phosphinothricin selection in embryo-basedtechniques offers a promising system to obtain high wheat transformationfrequencies. 相似文献
103.
Siela?N.?Maximova Larence?Alemanno Ann?Young Nicole?Ferriere Abdoulaye?Traore Mark?J.?GuiltinanEmail author 《In vitro cellular & developmental biology. Plant》2002,38(3):252-259
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
104.
The influence of exogenous gibberellic acid (GA3) andpaclobutrazol, an inhibitor of gibberellin biosynthesis, on growth of callusandsomatic embryogenesis in petiole-derived tissue cultures of Medicagosativa L. has been investigated. GA3 (0.5–500M) or paclobutrazol(5–100 M) were added to either an induction (with 2,4 Dand kinetin) or a differentiation medium (without plant growth regulators).Gibberellin A3, applied during the induction as well as thedifferentiation stage, reduced the weight of callus and increased the number ofsomatic embryos in Medicago sativa L. tissue cultures.Somatic embryo production was increased more by the presence of exogenousGA3 in the differentiation than induction medium. The inclusion ofpaclobutrazol in the induction or differentiation medium caused the inhibitionof callus growth and embryo production. Callus growth was much less affectedthan embryogenesis. These results indicate that gibberellins are beneficial forboth embryoinduction and formation. The level of endogenous gibberellins is presumablysufficient for callus induction and growth. However, it seems not optimal forthe induction and particularly for the differentiation of embryos. 相似文献
105.
Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N6) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N6 medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively
more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic
calluses to hormone-free MS or N6 regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets
on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N6 medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis
and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’
explants such as immature embryos and unemerged inflorescences. 相似文献
106.
Regeneration and transformation of Egyptian maize inbred lines via immature embryo culture and a biolistic particle delivery system 总被引:2,自引:0,他引:2
Hanaiya?A.?El-ItribyEmail author Shireen?K.?Assem Ebtissam?H.?A.?Hussein Fathy?M.?Abdel-Calil Magdy?A.?Madkour 《In vitro cellular & developmental biology. Plant》2003,39(5):524-531
Summary A regeneration system was developed for elite Egyptain maize inbred lines using immature embryos as explants. This system
proved to be highly genotype-dependent. Line Gz 643 was identified as the best line, revealing the highest regeneration frequency
(42.2%). Addition of l-proline and silver nitrate to culture media greatly enhanced the formation of embryogenic type II callus and the regenerability
of some of the tested lines. Transformation of the scutellar tissue of immature embryos from inbred line Gz643 was performed
with the particle delivery system using a single plasmid carrying both the GUS and Bar genes (pAB-6) or by co-transformation with two plasmids, pAct1-F (GUS) and pTW-a(Bar). Different transformation parameters were evaluated, i.e. ostomic treatment, acceleration pressure, and number of shots.
Osmotic treatment (0.25 M sorbitol + 0.25 M mannitol) along with the use of either acceleration pressure 1300 psi and one shot per plate (for co-transformation with
pAB-6) or 1100 psi and two shots per plate (for transformation with pAct1-F and pTW-a) gave the best results, as expressed
by the number of blue spots in the β-glucuronidase (GUS) assay. Stable transformation was confirmed in Ro transformed plants
by means of histochemical GUS assay and herbicide application. PCR and Southern blot analysis proved the integration of the
full-length genes in some of the transgenics. 相似文献
107.
R.?S.?Nirwan S.?L.?KothariEmail author 《In vitro cellular & developmental biology. Plant》2003,39(2):161-164
Summary A highly efficient protocol for callus induction and plant regeneration in Sorghum bicolor was developed by varying the concentrations of copper (0.1, 0.3, 0.5, 0.7, 1, 1.5, 2.5 μM) in Murashige and Skoog (MS) medium. The mature embryos of Sorghum bicolor were cultured on MS medium containing 2,4-dichlorophenoxyacetic acid (9μM), kinetin (2.3 μM), and 3% (w/v) sucrose for embryogenic callus induction. Plant regeneration from this callus occurred on MS medium containing
kinetin (9.2 μM) and indole-3-acetic acid (2.85 μM). A much greater response was noted on these media with higher levels of copper. Frequency of plant regeneration and number
of regenerants dramatically increased with an optimal amount of copper (2 μM) in the MS medium. Rooting of the regenerated shoots readily occurred on half-strength MS medium supplemented with α-naphthaleneacetic
acid (10.7 μM) and 3% (w/v) sucrose. Well-developed plantlets were transferred to the field where 100% survival and normal seed setting
was noted. 相似文献
108.
Bueno MA Gomez A Sepulveda F Seguí JM Testillano PS Manzanera JA Risueño MC 《Journal of plant physiology》2003,160(8):953-960
Microspore-derived embryos produced from cork oak anther cultures after long-term incubations (up to 10-12 months) were analysed in order to determine the genetic variability and ploidy level stability, as well as morphology, developmental pattern and cellular organisation. Most of the embryos from long-term anther cultures were haploid (90.7%), corresponding to their microspore origin. The presence of a low percentage of diploid embryos (7.4%) was observed. Microsatellite analysis of haploid embryos, indicated different microspores origins of the same anther. In the diploid embryos, homozygosity for different alleles was detected from anther wall tissues, excluding the possibility of clonal origin. The maintenance of a high proportion of haploid embryos, in long-term anther cultures, is similar in percentage to that reported in embryos originating after 20 days of plating (Bueno et al. 1997). This suggests that no significant alterations in the ploidy level occurred during long incubations (up to 12 months). These results suggest that ploidy changes are rare in this in vitro system, and do not significantly increase during long-term cultures. Microscopical studies of the microspore embryos in various stages revealed a healthy and well developed anatomy with no aberrant or chimeric structures. The general morphology of embryos appearing at different times after plating, looked similar to that of earlier embryos, as well as the zygotic embryos, indicating that they represent high quality material for cork oak breeding. 相似文献
109.
Imai KS 《Differentiation; research in biological diversity》2003,71(6):346-360
Nuclear localization of beta-catenin is most likely the first step of embryonic axis formation or embryonic cell specification in a wide variety of animal groups. Therefore, the elucidation of beta-catenin target genes is a key research subject in understanding the molecular mechanisms of the early embryogenesis of animals. In Ciona savignyi embryos, nuclear accumulation of beta-catenin is the first step of endodermal cell specification. Previous subtractive hybridization screens of mRNAs between beta-catenin-overexpressed embryos and nuclear beta-catenin-depleted embryos have resulted in the identification of beta-catenin downstream genes in Ciona embryos. In the present study, I characterize seven additional beta-catenin downstream genes, Cs-cadherinII, Cs-protocadherin, Cs-Eph, Cs-betaCD1, Cs-netrin, Cs-frizzled3/6, and Cs-lefty/antivin. All of these genes were expressed in vegetal blastomeres between the 16-cell and 110-cell stages, although their spatial and temporal expression patterns were different from one another. In situ hybridizations and real-time PCR revealed that the expression of all of these genes was up-regulated in beta-catenin-overexpressed embryos, and down-regulated in beta-catenin-suppressed embryos. Therefore, the accumulation of beta-catenin in the nuclei of vegetal blastomeres activates various vegetally expressed genes with potentially important functions in the specification of these cells. 相似文献
110.