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1.
Lourdes Castelblanque Begoña García-Sogo Benito Pineda Vicente Moreno 《Plant Cell, Tissue and Organ Culture》2010,100(1):107-112
A simple and efficient protocol for plant regeneration from protoplasts of the potted plant Kalanchoe blossfeldiana Poelln. is reported. Mesophyll protoplasts were isolated from axenic leaves after a preculture. The enzymatic digestion of
the tissue with a solution containing 0.4% Cellulase Onozuka R-10 and 0.2% Driselase yielded 6.0 × 105 protoplasts per gram fresh weight after density gradient purification. Protoplasts were cultured in the dark at an initial
density of 1 × 105 protoplasts per milliliter in a liquid medium with 320 mM mannitol, 130 mM sucrose, 2.3 μM 2,4-dichlorophenoxy acetic acid
(2,4-D), 5.4 μM 1-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzyladenine (BA). Cell wall regeneration was observed within
4 days of culture and cell division began after 5–7 days. When cultured in a liquid medium with 5.4 μM NAA and 8.9 μM BA,
protoplast-derived colonies proliferated until small visible calli, and adventitious buds appeared after transfer to photoperiod
conditions. Developed shoots were rooted on a solid medium supplemented with 0.6 μM indole-3-acetic acid (IAA) and successfully
established under greenhouse conditions. The process required 4 months from isolation to rooted plants and the best conditions
found gave a plant regeneration efficiency of 6.4 plants per 1 × 105 protoplasts. This is the first protocol reported for plant regeneration from protoplasts for a Crassulaceae family species. 相似文献
2.
A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for
a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 105 protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in
Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of
culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material,
a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 105 protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first
cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h)
of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days
on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with
5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and
1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM
IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand
were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well. 相似文献
3.
T. Dennis Thomas 《In vitro cellular & developmental biology. Plant》2009,45(5):591-598
Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density
of 5 × 105 protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating
efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists
of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about
100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm
in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with
200 μmol m−2 S−1 of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM)
and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies
of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ
and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered
following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid. 相似文献
4.
Mamta Singhvi Dipti Joshi Shalaka Gaikaiwari Digambar V. Gokhale 《Indian journal of microbiology》2010,50(1):97-100
Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast
buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with
various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer
using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS
medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli
as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii. 相似文献
5.
The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min,
1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO2 overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on
media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to
the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea
(CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated
on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses
were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic
acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 106 protoplasts per gram of fresh weight. 相似文献
6.
Yuichi Uno Shigeyuki Nakao Yumiko Yamai Ryohei Koyama Michio Kanechi Noboru Inagaki 《Plant Cell, Tissue and Organ Culture》2009,98(3):303-309
An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured
cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l−1 casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing
the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield
of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells,
and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was
obtained with 1 × 105 protoplasts ml−1, 25 μg batch−1 of plasmid vector, and 30% polyethylene glycol 4,000. 相似文献
7.
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
8.
High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 °C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 °C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 °C, 23.2± 0.24× 106 protoplasts g−1 fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures. 相似文献
9.
Wang Xiao Xue-Lin Huang Xia Huang Ya-Ping Chen Xue-Mei Dai Jie-Tang Zhao 《Plant Cell, Tissue and Organ Culture》2007,90(2):191-200
A protocol for plant regeneration from protoplasts of Musa acuminata cv. Mas (AA) via somatic embryogenesis was developed. Viable protoplasts were isolated from embryogenic cell suspensions
at a yield of 1.2 × 107 protoplasts/ml packed cell volume (PCV). Liquid and feeder layer culture systems with medium-A and medium-B were used for
protoplast culture. In liquid culture system, medium-B was more efficient for inducing cell division (17.5% at 14 days) and
colony formation (6.7% at 28 days) than medium-A. However, all protoplast-derived cell colonies (PDCC) obtained from liquid
culture system could not develop further. In feeder layer culture system, there was no significant difference between medium-A
and medium-B on cell division and colony formation of the cultured protoplasts, and the cell division frequency at 14 days
and colony formation frequency at 28 days were 24.5% and 11.2%, respectively, in medium-B. Comparative study on the effects
of BAP (2.2 μM, 4.4 μM, 8.8 μM), zeatin (0.4 μM, 0.8 μM, 1.2 μM) and TDZ (0.2 μM, 0.4 μM, 0.6 μM) on embryo formation of PDCC
from feeder-layer culture indicated that TDZ was best. TDZ at 0.4 μM induced 7906 mature embryos per ml PCV PDCC, which was
4-fold the frequency as with BAP at 4.4 μM, 7.5-fold as with zeatin at 0.8 μM and 150-fold as control medium (no mentioned
cytokinins) after 45 days on M3 medium. About 44% of the mature embryos were converted into plantlets with poor root system
after subculture on M4 medium. Root further development of regenerated plantlets was promoted by addition of activated charcoal
(AC) to MS basal medium. 相似文献
10.
The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×108/ml fromT. koningii and 6×107/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells. 相似文献
11.
Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 106 / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl2 and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.Abbreviations BAP
benzylaminopurine
- NAA
naphthaleneacetic acid
- PCR
Polymerase Chain Reaction
- fw
fresh weight 相似文献
12.
Qin-Mei Wang Feng-Zhan Gao Xiang Gao Fan-Yu Zou Xin Sui Meng Wang Yue-Jun Hui Li Wang 《Plant Cell, Tissue and Organ Culture》2012,109(2):191-200
An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were
incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic
acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young
ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently,
callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration
from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM
KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing
9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals
had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when
shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted
to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological
changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats
were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic
similarities with mother plants and 89.0–100.0% similarities with each other. 相似文献
13.
A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 105 protoplasts g−1 fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible. 相似文献
14.
The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to
its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following
the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation
period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol.
The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher
yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white
M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell
division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained. 相似文献
15.
A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn. 相似文献
16.
S. V. Raikar R. H. Braun C. Bryant A. J. Conner M. C. Christey 《Plant biotechnology reports》2008,2(3):171-177
This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme
combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS,
1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation
proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a
nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating
efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived
cell colonies. 相似文献
17.
Xiaohuan Wang Zhenhua Gao Yunzhen Wang Ray A. Bressan Stephen C. Weller Xia Li 《In vitro cellular & developmental biology. Plant》2009,45(4):435-440
An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on
stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and
regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut
water (CW), 20 g l−1 sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots
per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically
similar to Black Mitcham parents. 相似文献
18.
Anber Hassanein Latifa Hamama Karine Loridon Noëlle Dorion 《Plant cell reports》2009,28(10):1521-1530
Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses
from a 33-μF capacitor in a 250-V cm−1 electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient
expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast
suspension conductivity of >1,500 μS cm−1 allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing
uidA and nptII genes. When selection (50 mg l−1 kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l−1 kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant
rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated
plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones. 相似文献
19.
《Journal of Fermentation and Bioengineering》1993,75(1):65-67
Over 2 × 107/ml protoplasts were obtained from mycelia of hyper lignolytic fungus (nomenclatured as strain IZU-154) by treatment with the lytic enzyme NovoZym 234 in the presence of 0.05 M maleic acid buffer (pH 5.6) containing 0.6 M MgSO4. The protoplasts regenerated at more than 10% of frequency on solid 2% agar medium containing 0.6 M sucrose as an osmotic stabilizer overlaid with 0.5% agar containing the stabilizer. In the determination of the lignolytic activities of 50 regenerants from protoplasts, 2 strains which degraded more than 56% of the lignin during incubation for 30 d and showed activity higher than the parent were found. The regeneration from protoplasts of this fungus was suggested to be useful for the breeding of strains having higher lignolytic activity than this fungus. 相似文献
20.
Kaitlin J. Palla Paula M. Pijut 《In vitro cellular & developmental biology. Plant》2011,47(2):250-256
A plant regeneration protocol was developed for white ash (Fraxinus americana L.). Hypocotyls and cotyledons excised from embryos were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine
(BA) plus thidiazuron (TDZ), and compared for organogenic potential. Sixty-six percent of hypocotyl segments and 10.4% of
cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 3.5 ± 0.9 and 2.5 ± 1.5,
respectively. The best regeneration medium (52% shoot formation; 47% shoot elongation) for hypocotyls was MS basal medium
containing 22.2 μM BA plus 0.5 μM TDZ, producing a mean of 3.9 ± 0.4 adventitious shoots. Adventitious shoots were established
as proliferating shoot cultures following transfer to MS medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM
TDZ. For in vitro rooting, woody plant medium with indole-3-acetic acid (IAA) at 0, 2.9, 5.7, or 8.6 μM in combination with 4.9 μM indole-3-butyric
acid (IBA) was tested for a 5- or 10-d dark culture period, followed by culture under a 16-h photoperiod. The best rooting
(78% to 81%) of in vitro shoots was obtained with a 5 d dark culture treatment on medium containing 2.9 or 5.7 μM IAA plus 4.9 μM IBA, with an average
of 2.6 ± 0.4 roots per shoot. Rooted plants were successfully acclimatized to the greenhouse. This adventitious shoot regeneration
and rooting protocol will be used as the basis for experimental studies to produce transgenic white ash with resistance to
the emerald ash borer. 相似文献