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1.
S. Cogbill T. Faulcon G. Jones M. McDaniel G. Harmon R. Blackmon M. Young 《Plant Cell, Tissue and Organ Culture》2010,101(2):127-133
Rapid-cycling fast plants (Brassica rapa; RCBr) is also known as Wisconsin Fast Plant and is widely used in K-12 and undergraduate studies. RCBr has a short generation
time (seed-to-seed in 30–60 days), which allows for the completion of experiments in a semester. Previous studies have shown
that cotyledonary explants with attached petioles are capable of generating shoots. However, there is no published adventitious
shoot regeneration protocol to date. Sterile cotyledonary explants were excised; all edges and petioles were removed. Five-day-old
cotyledonary explants produced shoots on a Murashige and Skoog medium containing 1.5 mg/L thiadiazuron (TDZ) and 0.5 mg/L
1-naphthaleneacetic acid (NAA) (FPM I) at a mean rate of 8.8%. This rate increased to 14.8% in explants placed on FPM I medium
supplemented with 5.0 mg/L silver nitrate (AgNO3) (SRM 2). The rate increased to 32.5% when 5-day-old explants, excised from the part of the cotyledon nearest to the petiole,
were placed adaxial side up on SRM 2 medium. The shoot regeneration rate increased to 44.5% using 4-day-old cotyledonary explants.
A shoot regeneration rate of 23% was observed among 9-day-old leaf explants. Shoots from cotyledonary explants were elongated
on basal medium with 0.5 mg/L NAA, rooted on basal medium, and later acclimatized. This is the first report of shoot regeneration
from cotyledonary explants of rapid-cycling Brassica rapa without pre-existing meristematic tissues. 相似文献
2.
Susceptibility to Agrobacterium tumefaciens and cotyledonary node transformation in short-season soybean 总被引:3,自引:0,他引:3
Short-season adapted soybean [Glycine max (L.) Merrill] genotypes (maturity group 0 and 00) were susceptible to Agrobacterium tumefaciens in tumor-formation assays with A. tumefaciens strains A281, C58 and ACH5. The response was bacterial-strain and plant-cultivar dependent. In vitro Agrobacterium-mediated transformation of cotyledonary node explants of these genotypes with A. tumefaciens EHA105/pBI121 was inefficient but resulted in a transgenic AC Colibri plant carrying a linked insertion of the neomycin phosphotransferase
and β-glucuronidase (gus) transgenes. The transgenes were transmitted to the progeny and stable gus expression was detected
in the T7 generation. The low rate of recovery of transgenic plants from the co-cultured cotyledonary explants was attributed to inefficient
transformation of regenerable cells, and/or poor selection or survival of such cells and not to poor susceptibility to Agrobacterium, since, depending on the cultivar, explants were transformed at a rate of 27–92%, but transformation events were usually
restricted to non-regenerable callus.
Received: 8 January 1998 / Revision received: 30 June 1999 / Accepted: 12 July 1999 相似文献
3.
In vitro plant regeneration from callus of niger (Guizotia abyssinica Cass.) cv. Sahyadri 总被引:1,自引:0,他引:1
Callus formation was achieved with root, hypocotyl, and cotyledon explants of niger (Guizotia abyssinica Cass.) cultivar Sahyadri on Murashige and Skoog medium containing 0.5 mg l–1
β-indoleacetic acid + 1.5 mg l–1 6-benzylaminopurine (BAP). Hypocotyl and cotyledon-derived calli when transferred onto a medium with 0.5 mg l–1 BAP produced an average of 12–32 shoots/ callus culture. The callus retained its potential for shoot regeneration for more
than 19 months. The shoots formed an extensive root system and were transferred to pots kept in a greenhouse, where the survival
rate was 98%. The plantlets flowered in vitro if transfer to fresh medium or to soil was delayed by 40–50 days. All regenerants
were diploid with 2n=30.
Received: 13 March 1997 / Revision received: 17 May 1997 / Accepted: 5 July 1997 相似文献
4.
Genetic transformation of European chestnut 总被引:3,自引:0,他引:3
Stable incorporation of the nptII gene into Castanea sativa Mill. has been achieved by Agrobacterium-mediated transformation. The transformation assays were performed by infecting wounded hypocotyls with a strain of Agrobacterium tumefaciens, LBA 4404 harbouring the plasmid p35SGUSINT. Although two schemes of selection were tested, many escapes were obtained. The
best strategy to avoid this problem is the introduction of higher concentrations of kanamycin in the culture medium, immediately
after coculture. PCR analysis showed of the selectable nptII gene integration in the plant genome. β-Glucuronidase histochemical assay revealed the expression of the uidA gene in shoots, regenerated from transformed explants.
Received: 3 December 1996 / Revision received: 4 February 1997 / Accepted: 1 March 1997 相似文献
5.
Greenhouse-grown plants of turnip rape Brassica rapa ssp. oleifera (syn. B. campestris) cv. Valtti and Sisu were transformed by Agrobacterium tumefaciens infection. Of the three A. tumefaciens strains tested (C58C1, EHA105 and LBA4404), LBA4404 gave the best results. Segments excised from one to two upper internodes
of an inflorescence-carrying stem served as explants for the Agrobacterium infection. Cultivation of the explants horizontally during the first 3 days of co-cultivation with A. tumefaciens following immediate selection of transformed tissue of the stem segments placed vertically basal side down were critical.
Use of silver nitrate (5–10 mg/l) in the culture medium and Micropore (3 M) paper tape for sealing plates was also beneficial.
Transgenic shoots were recovered using either hygromycin or kanamycin (20–25 mg/l) selection. Hygromycin was preferable, as
the proportion of `escapes' was 90% under kanamycin and 10% under hygromycin selection. Regeneration was achieved by culturing
the explants for 3–6 days on 0.5 mg/l of 2,4-di-chlorophenoxyacetic acid and 1–2 weeks on 2–3 mg/l of 6-benzyl aminopurine
with/without 0.05 mg/l α-naphthaleneacetic acid. Recovered shoots were then cultured on hormone-free MS medium. This culture program gave 60–80% shoot
regeneration. Regenerants were tested by histological β-glucuronidase staining and Southern blotting. The recovery rate of transgenic shoots was 4–9% of the number of explants used
in the experiments.
Received: 28 November 1997 / Revision received: 25 March 1998 / Accepted: 22 November 1998 相似文献
6.
Flower buds, cotyledons and hypocotyls of Pharbitis nil were used as plant material. Flower buds (1–2 mm long) were excised from 3-week-old plants, grown in soil. Cotyledons of
7-day-old sterile seedlings were cut into 25 mm2 squares cotyledons whereas hypocotyls were cut to 1 mm long fragments. Explants were transferred into Petri dishes containing
the Murashige and Skoog medium (MS), supplemented with either BA (11 μM·L−1) alone or BA (22 μM·L−1) and NAA (0.55 μM·L−1), and different sugars: sucrose, fructose, glucose, mannose or sorbitol (autoclaved or filter-sterilized). Addition of glucose
instead of sucrose to the medium stimulated the induction of callus on flower buds and cotyledonary explants, but inhibited
its growth on fragments of hypocotyls. The medium supplemented with fructose (especially filter-sterilized) stimulated the
development of flower elements. Organogenesis of shoots and roots on explants was also observed. Flower buds and hypocotyls
were able to regenerate both organs. Addition of fructose or glucose to the medium stimulated the organogenesis of shoots,
whereas root organogenesis was inhibited on all explants used. Sorbitol strongly inhibited both induction of callus and organogenesis
on all explants used. 相似文献
7.
This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium
supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing
percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained
on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin
(2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants
was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained
with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were
achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction
medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully
acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction. 相似文献
8.
A simple protocol for mass multiplication of Crataeva nurvala, a medicinal tree, from seedling-derived explants is described. Six different types of explants (cotyledonary nodes, epicotyl
nodes, hypocotyl segments, first pair of leaves, cotyledons, and root segments) developed shoots on Murashige and Skoog's
(MS) basal medium or the same supplemented with different concentrations of 6-benzylaminopurine (BAP). Among the explants
tested for caulogenic potential, only the epicotyl and cotyledonary nodal explants developed shoots on MS basal medium, while
on BAP (0 – 2.0 mg dm−3) adjuvated media all the explants exhibited caulogenesis. The optimum concentration of BAP varied for these explants. The
shoots could be rooted on half strength MS with 0.02 mg dm−3 α-naphthalene acetic acid to get plants, which have been transferred to soil. The explants from in vitro regenerated shoots also possessed a similar caulogenic potential.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
9.
A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige
and Skoog's medium containing either N
6-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth
regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with
BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A
proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication
medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem
node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the
shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil.
Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998 相似文献
10.
A procedure for producing transgenic Chinese cabbage plants by inoculating cotyledonary explants with Agrobacterium tumefaciens strain EHA101 carrying a binary vector pIG121Hm, which contains kanamycin-resistance and hygromycin-resistance genes and
the GUS reporter gene, is described. Infection was most effective (highest infection frequency) when explants were infected
with Agrobacterium for 15 min and co-cultivated for 3 days in co-cultivation medium at pH 5.2 supplemented with 10 mg/l acetosyringone. Transgenic
plants of all three cultivars used were obtained with frequencies of 1.6–2.7% when the explants were regenerated in shoot
regeneration medium solidified with 1.6% agar. A histochemical GUS assay and PCR and Southern blot analyses confirmed that
transformation had occurred. Genetic analysis of T1 progeny showed that the transgenes were inherited in a Mendelian fashion.
Received: 15 December 1998 / Revision received: 2 July 1999 · Accepted: 8 July 1999 相似文献
11.
The regeneration ability of primary explants derived from mericlones of two commercial Bohemian hops was investigated. It
was found that these hops are able to regenerate shoots by direct organogenesis on media containing BAP or zeatin at concentrations
0.5–2 mg dm−3. The highest regeneration of shoots was achieved from either petioles or internodes at frequencies 21% and 52%, respectively,
on the medium containing zeatin (2 mg dm−3), while relatively low amount of regenerated shoots (1.3%) was observed for leaf blade explants. On the other hand, more
efficient rooting occurred on the leaf blades then on other explants. A similar pattern of regeneration we observed for HLVd-infected
mericlones of clone Osvald 31 even though viroid concentration inin vitro cultures was about 8-fold higher than in field-grown plants and was 31.1 pg mg−1 of fresh mass in the average. These results suggest that HLVd infection did not impair organogenesis. We found that high
2,4-D concentration pretreatment (11 mg dm−3) did not promote somatic embryogenesis. Although this treatment suppressed direct organogenesis, the inhibition was not complete
and in low frequency the shoot regeneration was seen. Sensitivity of hop explants to antibiotics commonly used inAgrobacterium-mediated transformation was assayed. It was found that kanamycin (100–200 mg dm−3) suppressed efficiently callogenesis, root formation and shoot proliferation. An estimation of effect of kanamycin (200 mg
dm−3) and ticarcillin (500 mg dm−3) on morphogenesis was performed using regeneration medium. The inhibitory effects observed suggest that these conditions
could be used inAgrobacterium transformation/selection system.
Communicated by J. TUPY 相似文献
12.
Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an
efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits
due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium
tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess
the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured
cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 μM), infection of explants for 30–45 min and
3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation
of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l−1). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern
blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency
of 4% was achieved for CN explants using this protocol. 相似文献
13.
Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and
Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant
(9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary
nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being
transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss,
soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots. 相似文献
14.
Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants,
leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types
began first with culture on 50 mg l−1 of Km for 6 wk and then transfer to 30 mg l−1 of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl−1 cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m−2s−1. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%,
and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment
for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four. 相似文献
15.
High Frequency of Shoot Regeneration from Hypocotyls and Stem Segments of Antirrhinum majus (Snapdragon) 总被引:1,自引:0,他引:1
A broadly applicable direct shoot regeneration method from hypocotyls and stem explants has been developed for six cultivars
of Antirrhinum majus L. In order to establish a stable and high frequency of shoot regeneration system, leaves, hypocotyls and stem explants of
six cultivars were tested with 72 combinations of auxin (naphthaleneacetic acid (NAA) or 3-indoleacetic acid (IAA)) and cytokinin
(6-benzylaminopurine (BA) or zeatin (Z)). A few adventitious shoots were directly regenerated from hypocotyl segments of cv.
Orchid on MS medium with NAA + BA, IAA + BA, NAA + Z and IAA + Z. High frequency of direct shoot regeneration was obtained
from hypocotyl segments on MS medium with 0.05, 0.1 or 0.25 mg l−1 NAA + 2 mg l−1 Z and 0.5 mg l−1 IAA + 2 mg l−1 Z. Finally, stable and high frequency (92–100%) of shoot regeneration with more than 10 adventitious shoots per explant was
achieved from the hypocotyls and stem explants of all six cultivars on MS medium with 0.25 mg l−1 NAA + 2 mg l−1 Z. The shoots emerged directly from the hypocotyls and stem segments 4 weeks after culture initiation. 相似文献
16.
The epicotyl segments bearing scaly leave(s), excised from in vitro grown seedlings of Syzygium cuminii, produced multiple shoots when cultivated on Murashige and Skoog's (MS, 1962) medium supplemented with different concentrations
of BA (0–2 mg l–1). The optimum response was recorded on the medium containing 1 mg l–1 BA. An average of 8.6 shoots per explant were produced 60 days after inoculation, following transfer to fresh medium after
30 days. The shoots were excised, and the residual explants were transferred to fresh medium, where they again developed shoots.
Up to five such passages resulted in the production of shoots from the repeatedly subcultured original explants. However,
during the fifth passage, organogenic response was negligible and the explants turned brown thereafter. Following repeated
harvesting of shoots and subculture of the residual explants, an average of 29 shoots per explant was obtained. The in vitro
developed shoots produced roots when transferred to Knop's medium supplemented with 2% sucrose and 1 mg l–1 IAA. The developed plantlets were planted in soil and transferred to fields after an acclimatization period of 7–8 months.
These plants have been thriving well for more than 3 years. The nodal explants excised from in vitro developed shoots and
plants also exhibited a similar response when cultured on MS+1 mg l–1 BA. Thus, a protocol has been developed to raise plants of S. cuminii at any time of the year.
Received: 1 December 1998 / Revision received: 1 July 1999 · Accepted: 12 July 1999 相似文献
17.
Dormant axillary buds of nodal explants collected from a mature (35-year-old) tree of Ficus religiosa L. sprouted on MS medium supplemented with 6-benzyladenine (BA, 5 mg l–1) and indole-3-butyric acid (IBA, 0.2 mg l–1 ) within 4 days. Multiple shoots were obtained when these explants were transferred to MS medium containing 1.5 mg l–1 BA and 1.5 mg l–1 adenine sulphate (ADS). These multiple shoots (1 cm) were allowed to elongate to a height of 4–5 cm by maintaining them on
MS medium containing 2 mg l–1 BA, 0.5 mg l–1 1-naphthalene acetic acid (NAA), and activated charcoal (0.3% w/v). Nodal segments taken from these in vitro-proliferated
shoots yielded multiple shoots when cultured on the multiple shoot-inducing MS medium mentioned above. Root induction in these
shoots (4–5 cm in height) was achieved by transferring them onto MS medium supplemented with 2 mg l–1 IBA and 0.1 mg l–1 NAA for 1 week; upon transfer to half-strength MS basal medium these shoots exhibited root proliferation. These rooted plantlets
were successfully established in soil after a short period of acclimatization.
Received: 17 April 1997 / Revision received: 2 September 1997 / Accepted: 20 September 1997 相似文献
18.
Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase
and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants
were used. Transient expression of β-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be
the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation
efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This
system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering
with this crop.
Received: 8 October 1996 / Revision received: 2 January 1997 / Accepted: 2 February 1997 相似文献
19.
A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of
shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media
fortified with 22.2 μm N6-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations
of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS)
basal medium supplemented with 2.22 μm N6-benzylaminopurine and 0.54 μm
α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency
on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may
be useful for improving the crop through genetic manipulations.
Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998 相似文献
20.
Gerard C. Douglas John Mcnamara 《In vitro cellular & developmental biology. Plant》2000,36(5):412-415
Summary Regeneration of adventitious shoots was obtained in over 80% of explants, consisting of wounded cotyledonary nodes of Acacia mangium, by culturing germinated seedlings on DKW medium with combinations of N6-benzyladenine and either thidiazuron or N-(2-chloro-4-pyridyl)-N-phenylurea. Electron microscopy showed the presence of adventitious buds arising from wound tissue of the cotyledons and
cotyledonary nodes. Shoot regeneration was also obtained at lower frequency in isolated cotyledon explants cultured with 6%
sucrose alone (10%), or with 3% sucrose and 30.0 mg l−1 (0.1 μM) 2–4-dichlorophenoxyacetic acid (2,4-D; 16%). With 2,4-D,>60% of explants produced organized structures but these did not
develop into shoots or somatic embryos. Shoot formation was not induced in either hypocotyl or root explants. 相似文献