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1.
The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.Abbreviations BAP 6-Benzyl-aminopurine - IAA Indole-3-acetic acid - MS Murashige and Skoog medium - NAA -Naphthalene-acetic acidCommunicated by H. Lörz 相似文献
2.
M. Muñoz-Amatriaín A. M. Castillo X. W. Chen L. Cistué M. P. Vallés 《Molecular breeding : new strategies in plant improvement》2008,22(1):119-129
In cereals, albinism is a major obstacle to produce doubled haploids (DH) for breeding programs. In order to identify QTLs
for green plant percentage in barley anther culture, a specific population was developed. This population, consisting of 100
DH lines, was generated by crossing the model cultivar for anther culture “Igri” with an albino-producing DH line (DH46) selected
from Igri × Dobla, in search of a maximum segregation for the trait and minimum for the other anther culture variables. A
combination of bulked segregant analysis and AFLP methodology was used to identify markers linked to the trait. A linkage
map was constructed using these AFLPs, together with RAPD, STS and SSR markers. This study identified a new QTL for green
plant percentage on chromosome 3H and confirmed the previously reported one on chromosome 5H. Up to 65.2% of the phenotypic
variance for this trait was explained by the additive effects of these two QTLs. Thirty elite cultivars of barley from different
origin, row type, growth habit and end use, were selected to validate these QTLs. Since two of the markers linked to the QTLs
were AFLPs, we successfully converted them into simple PCR-based SCAR markers. Only the SSR HVM60, on chromosome 3H, was significantly
associated with the trait, explaining near 20% of the phenotypic variance. Among the allelic variants identified for this
marker, HVM60-120bp was associated with the highest values of green plant percentage. 相似文献
3.
An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.Abbreviations BAP 6-Benzylaminopurine - GA3 Gibberellic acid - GUS -Glucuronidase - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - TDZ 1-Phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron)Communicated by H. Lörz 相似文献
4.
Monika Höfer 《Acta Physiologiae Plantarum》2005,27(4):709-716
Based on optimized protocols for anther and microspore culture in apple (Malus x domestica Borkh.), the regeneration phase and the efficiency of the processes in general were compared by using the same androgenic material of two experimental years. Microspore culture resulted in an increase in embryo induction depending on the genotype (Höfer 2004), however anther culture was superior to microspore culture in the total number of regenerated plants. The regeneration process in anther and microspore culture is similar. Two developmental pathways were observed: 1) secondary embryogenesis followed by adventitious shoot formation and 2) direct adventitious shoot formation from primary embryos. Induction and regeneration processes are delayed in microspore culture as compared with anther culture. The reasons for the reduced regeneration efficiency in microspore culture are discussed. 相似文献
5.
The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis.
The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars.
Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were
carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on
a C17 induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was
incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements.
With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000
anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection
of ten F1 crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested
from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant
was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis,
named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol
can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain
DHs for most crosses involving the durum wheat cultivars grown in Spain. 相似文献
6.
Leaves of Solanum virginianum plants were used for protoplast isolation. To support cell wall formation and cell division, protoplasts were cultured in
thin alginate layers floated in liquid medium. When protoplasts were plated at a density of 1.0 × 106/ml in Kao and Michyaluk (KMp8) medium supplemented with 0.5 mg/l zeatin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, and 1.0 mg/l
α-naphthaleneacetic acid, 42.3% of the dividing cells developed microcalli in 3–4 weeks. Shoot formation via organogenesis
of protoplast-derived calli was achieved for 28% of calli transferred to solidified KMp8 medium supplemented with 2.0 g/l
zeatin and 0.1 mg/l 3-indol acetic acid in about 2 weeks. Further shoot development was observed in Murashige and Skoog (MS)
medium without growth regulators and roots were induced after transfer to MS medium containing 1.0 mg/l 3-indol butyric acid.
Regenerated plants have normal morphology. 相似文献
7.
The effects of growth regulators, cold-pretreatment of flower buds, ovule (embryo sac) developmental stage and genotype on
induction of gynogenesis in unpollinated ovule cultures were assessed in niger (Guizotia abyssinica (L. f.) Cass.). Indirect callus-mediated gynogenesis occurred in cvs JNC-6 and Ootacamund when the ovules were cultured on
MS medium supplemented with 30 g l−1 sucrose and 2,4-D either alone (0.5–2.0 μM) or in combination (2.0 μM) with different cytokinins, such as adenine, BA, 2iP
and kinetin (0.5–2.0 μM). An optimum induction of gynogenesis was fostered on medium supplemented with 2.0 μM 2,4-D and 1.0 μM
kinetin. Cold-pretreatment of flower buds had no stimulatory effect, but ovules collected one day before anthesis were most
responsive to gynogenesis. The results showed significant variations in genotypic competence for gynogenesis with cv. Ootacamund
being the most responsive (12.5%) and cv. IGP-76 the least (2.5%). Gynogenic embryos differentiated and matured on media (30 g l−1 sucrose) supplemented with 0.5 μM NAA plus 1.0 μM kinetin, and 0.5 μM ABA, respectively. The haploidy (2n = 1x = 15) of gynogenic plants was confirmed by cytological analysis. 相似文献
8.
Csaba Lantos Anikó Gémes Juhász György Somogyi Krisztina Ötvös Pál Vági Róbert Mihály Zoltán Kristóf Norbert Somogyi János Pauk 《Plant Cell, Tissue and Organ Culture》2009,97(3):285-293
The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi
80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated.
Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore
cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with
pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different
pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth
habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’,
three ‘Jaranda’, and a single ‘Szegedi 80’ plant. 相似文献
9.
The aim of the research was to make a preliminary determination of the effectiveness of the induction of haploids in Capsicum frutescens L. In order to induce androgenesis red and yellow fruit forms of species were used, each bred by the researchers on their
own. The experiment was performed in October. Anther cultures were conducted according to a modified method developed by Dumas
et al. (1981) for C. annuum L. The anthers were laid on CP medium containing 0.01 mg dm−3 2.4-D and 0.01 mg dm−3 kinetin, with the addition of 0.5 g dm−3 of activated carbon and 5 mg×dm−3 of silver nitrate, solidified with 8 g dm−3 of agar. The cultures were incubated in the dark at 35 deg C for 8 days. Next they were transferred to 25 deg C under a 12-hour
photoperiod. After 14 days of induction, anthers were transferred to R1 medium supplemented with 0.1 mg dm−3 kinetin. Obtained embryos were subsequently transplanted onto V3 hormone-free medium and well growing plants were planted in greenhouses. The efficiency of androgenesis for both C. frutescens L. forms was relatively low and it did not exceed 5%. The ploidy level of the resulting plants was determined by flow-cytometric
analysis. The regenerants consisted of about equal numbers of haploids and diploids. Additionally, among plants regenerated
from anthers of yellow fruit forms, two mixoploids were observed. 相似文献
10.
Tanoh Hilaire Kouakou Pierre Waffo-Téguo Yatty Justin Kouadio Josep Valls Tristan Richard Alain Decendit Jean-Michel Mérillon 《Plant Cell, Tissue and Organ Culture》2007,90(1):25-29
Studies of phenolic compounds were performed during cell suspension cultures in relation with the induction of embryogenic
structures in two cultivars of cotton. Coker 312 produced embryogenic structures, unlike R405-2000 which was found to be a
non-embryogenic cultivar. Embryogenesis induction in Coker 312 was strongly linked to a higher content of caffeic, ferulic
and salicylic acids and to the appearance of p-coumaric acid, benzoic acid, trans-resveratrol, catechin and naringenin. 相似文献
11.
Marchand S Fonquerne G Clermont I Laroche L Huynh TT Belzile FJ 《Plant cell reports》2008,27(3):443-451
Most Fusarium head blight (FHB) resistant barley (Hordeum vulgare L.) accessions perform relatively poorly from an agronomic point of view. Due to the polygenic inheritance of FHB resistance,
introgression of this complex trait into well-adapted elite germplasm will likely require multiple cycles of hybridization
and selection to combine resistance and agronomic performance. The use of anther culture to produce doubled haploids would
seem well justified to reduce the time required to achieve this goal. Unfortunately, little is known concerning the androgenic
response of the small number of genotypes with known partial FHB resistance. To make the best use of such FHB resistance donors
in a barley improvement program, we first characterized the FHB resistance of eight reported FHB resistance sources (Chevron,
Gobernadora, Seijo II, Shyri, Svanhals, Zhedar I, F104-250-9 and C97-21-38-3) in our own FHB nursery in Quebec City (QC, Canada).
In parallel, we assessed the androgenic response of these same eight lines with that of three cultivars (ACCA, Léger and Cadette)
of known androgenic response. Finally, the androgenic response of F1 hybrids involving some of these genotypes used as parents was measured and compared to that of the parental genotypes. Very
large and significant differences were observed in the number of green plants produced by the different accessions and F1s. Although anther culture seemed very promising for some accessions, for others, the androgenic response was so low that
a conventional approach would seem more appropriate. 相似文献
12.
Summary Hemp (Cannabis sativa L.) is cultivated in many parts of the world for ils fiber, oil, and seed. The development of new hemp cultivars with improved
traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate
the propagation of hemp in tissue culture and to establish a protocol for Agrobacterium-mediated transformation for foreign gene introduction. Stem and leaf segments from seedlings of four hemp varieties were
placed on Murashige and Skoog medium with Gamborg B5 vitamins (MB) supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM kinetin, 3% sucrose, and 8 gl−1 agar. Large masses of callus were produced within 4 wk for all cultivars. Suspension cultures were established in MB medium
containing 2.5 μM 2,4-D. To promote embryogenesis or organogenesis, explants, callus, and suspension cultures derived from a range of explant
sources and seedling ages were exposed to variations in the culture medium and changes to the culture environment. None of
the treatments tested were successful in promoting plantlet regeneration. Suspension cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary vector pNOV3635 with a gene encoding phosphomannose isomerase (PMI). Transformed callus
was selected on medium containing 1–2% mannose. A chlorophenol red assay was used to confirm that the PMI gene was expressed.
Polymerase chain reaction and Southern hybridization detected the presence of the PMI gene. Copy number in different lines
ranged from one to four. 相似文献
13.
Jesús Arellano Sara Isabel Fuentes Patricia Castillo-España Georgina Hernández 《Plant Cell, Tissue and Organ Culture》2009,96(1):11-18
A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic
axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production
was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred
to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots
per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of
the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development
and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure. 相似文献
14.
In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM
6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l−1 casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots)
to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop
into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented
with 4.4 μM BA, 0.29 μM gibberellic acid (GA3), and 500 mg l−1 CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration
(16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with
0.44 μM BA and 0.29 μM GA3. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response
and shoot numbers per explant were recorded on WPM supplemented with BA and GA3, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA3 were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20
shoots within 12 wk. Rooted plantlets were successfully acclimatized. 相似文献
15.
Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle 相似文献
16.
AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at
27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L
and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic
embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR
amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus
far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants. 相似文献
17.
Karin Sonntag Brigitte Ruge-Wehling Peter Wehling 《Plant Cell, Tissue and Organ Culture》2009,96(3):297-305
A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 105 protoplasts g−1 fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all
tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks)
and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown
pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids.
However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast
fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration. 相似文献
18.
Centaurea ragusina L. (Asteraceae) is an endemic Croatian plant species, which developed xeromorphic characteristics as a consequence of its natural environment – vertical limestone cliffs above the Adriatic sea. Cytogenetic status of C. ragusina long-term culture (94th subculture) and C. ragusina seedlings was analysed and compared after 4 weeks of growth. Cytogenetic stability was investigated in root meristem cells of C. ragusina cultured plants originated from Pen đa (cliffs near Dubrovnik) and seedlings originated from three different localities in south Adriatic (Penđa, Pasjača – cliffs near Dubrovnik and island of Komiža) using mitotic index and mitotic and chromosomal abnormalities as parameters. Mitotic indices of cultured plants and ‘Penđa’ seedlings were similar and showed significant increase compared to mitotic indices of ‘Komiža’ and ‘Pasjača’ seedlings. Although the highest number of mitotic abnormalities was recorded in root meristem cells of cultured plants, it was only a bit higher than in root tips of ‘Pasjača’ and ‘Penđa’ seedlings, while that of ‘Komiža’ was two times lower compared to cultured plants. Pattern of analysed mitotic abnormalities was very similar in root tips of cultured plants and ‘Pasjača’ and ‘Penđa’ seedlings, with exception of ‘Komiža’ seedlings. Presented results suggest that long-term cultivation of C. ragusina has almost no effect on culture ageing considering similar distribution of scored mitotic abnormalities as in ‘Penđa’ and ‘Pasjača’ seedlings. 相似文献
19.
Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N6-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation
frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium
augmented with 2.0 mgl−1 (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each
harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8%
of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl−1 (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil. 相似文献
20.
Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis. 相似文献