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1.
The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus
of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis
from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing
4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots
and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric
acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide
dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High
catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli
and regenerated roots. 相似文献
2.
Summary Tissue culture and plant regeneration protocols for the salt marsh plants Juncus roemerianus Scheele and Juncus gerardi Loisel, were developed. J. roemerianus callus was induced from mature seeds cultured on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BA), 5.37 μM α-naphthaleneacetic acid (NAA), 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 ml l−1 coconut water (callus induction medium). The callus was subcultured on MS medium containing 2.22 μM BA, 5.37 μM NAA, and 9.05 μM 2,4-D for callus maintenance. Shoot regeneration occurred 2 wk after transferring the callus onto shoot regeneration medium,
which consisted of MS medium containing BA or thidiazuron. A high frequency of shoot regeneration was obtained when the medium
contained 13.3 μM BA. Regenerated shoots were transferred to MS medium supplemented with 10.7 μM NAA for root production. Rooting did not occur in the shoots regenerated on the thidiazuron-containing media. The callus
induction medium for J. roemerianus was also effective in inducing callus of J. gerardi from young inflorescences. The same medium was also used for callus maintenance. Shoot regeneration occurred 10 d after transferring
the callus onto MS medium supplemented with 0.44 μM BA and 0.57 μM indole-3-acetic acid. Root regeneration occurred after transferring the shoots onto MS medium plus 0.44 μM BA and 14.8 μM indole-3-butyric acid. The regenerated plants of both J. roemerianus and J. gerardi grew vigorously in potting soil in the greenhouse. J. roemerianus regenerants also grew well in a saltwater-irrigated field plot. Tissue culture-produced plants of J. roemerianus and J. gerardi can be used for planting in created or restored wetlands. 相似文献
3.
Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants
placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum
shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented
with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and
without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants
in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important
medicinal plant. 相似文献
4.
Hai Ping Hong Hongyi Zhang Paula Olhoft Steve Hill Hunt Wiley Effie Toren Helke Hillebrand Todd Jones Ming Cheng 《In vitro cellular & developmental biology. Plant》2007,43(6):558-568
A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes
as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced
on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with
various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect
on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a
low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside,
and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction
medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected
calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been
regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including
leaf, stem, and roots. Southern and T1 plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy
number ranging from 1–5 copies. 相似文献
5.
Summary Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production;
however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential
of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in
calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival
rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium+8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf dise calluses, and
cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf dise calluses
regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a
higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2±0.5 versus 2.0±0.3, than did leaf dise calluses
on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin
concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original
seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata. 相似文献
6.
Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented
with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot
induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot
developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil
and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age. 相似文献
7.
Summary Development of an efficient transformation method for recalcitrant crops such as sugar beet (Beta vulgaris L.) depends on identification of germplasm with relatively high regeneration potential. Individual plants of seven sugar
beet breeding lines were screened for their ability to form adventitious shoots on leaf disk callus. Disks were excised from
the first pair of true leaves of 3-wk-old seedlings or from partially expanded leaves of 8-mo.-old plants and cultured on
medium with 4.4 μM 6-benzylaminopurine for 10 wk. At 5 wk of culture, friable calluses and adventitious shoots began to develop. Rates of callus
and shoot formation varied between breeding lines and between individual plants of the same line. Line FC607 exhibited the
highest percentage (61%) of plants that regenerated shoots on explants. Among the plants with a positive shoot regeneration
response, line FC607 also had the highest mean number (8.3±1.1) of shoots per explant. Individual plants within each line
exhibited a wide range of percentages of explants that regenerated shoots. A similar variation was observed in the number
of shoots that regenerated per explant of an individual plant. No loss of regeneration potential was observed on selected
plants maintained in the greenhouse for 3 yr. Regenerated plants exhibited normal phenotypes and regeneration abilities comparable
to the respective source plants. Based on our results, it is imperative to screen a large number of individual plants within
sugar beet breeding lines in order to identify the high regenerators for use in molecular breeding and improvement programs. 相似文献
8.
Quan-le Xu Zhe Hu Chun-yuan Li Xin-yu Wang Chong-ying Wang 《In vitro cellular & developmental biology. Plant》2009,45(5):583-590
Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent
root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l−1 6-benzylaminopurine (BA) and 0.2 mg l−1 α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and
roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l−1 NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in
a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated
plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted
to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance
liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA)
were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning. 相似文献
9.
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
10.
Junli Wang Jue Wang Kun Liu Xuan Xiao Weizhen Gong Yuan Lu Mingfei Liu Dongting Xu 《In vitro cellular & developmental biology. Plant》2010,46(5):445-450
An efficient micropropagation system for Hylotelephium tatarinowii (Maxim.) H. Ohba, a rare medicinal plant, has been developed. Callus induced from leaf explants placed onto Murashige and
Skoog (MS) medium with supplementation of plant growth regulators. When the concentration of 2,4-dicholorophenoxy acetic acid
was as high as 2.0 mg l−1 in combination with 0.5 mg l−1 6-benzylaminopurine (6-BAP), the callus induction rate reached 92.1%. Adventitious shoots were observed on callus exposed
to 1.0 mg l−1 6-BAP, with 81.5% frequency of shoot regeneration after 30 d. Flower buds appeared after subculture. Regenerated shoots could
flower normally in vitro. Up to 100% of the regenerated shoots formed complete plantlets on half-strength MS medium without any growth regulator, with
an average of 5.9 roots per shoot explant. Quantitative analysis of flavonoids and rutin showed that the phytochemical profile
of callus and regenerated plants was similar to that of wild plants. 相似文献
11.
C. A. Martínez A. M. Giulietti J. Rodríguez Talou 《Plant Cell, Tissue and Organ Culture》2011,104(1):91-100
Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative
properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can
be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from
petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various
ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined
after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic
acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced
only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration
frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with
IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation
at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived
shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants
were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor
for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin. 相似文献
12.
Summary An improved protocol for shoot regeneration from hypocotyl segments of seedlings from open-pollinated seeds of lingonberry
(Vaccinium vitis-idaea L.) cultivars, ‘Ida’, ‘Splendor’, and ‘Erntesegen’, and a native clone from Newfoundland was developed. The effect of thidiazuron
(TDZ) on adventitious bud and shoot formation from apical, central, and basal segments of the hypocotyl was tested. Highly
regenerative callus was obtained from hypocotyl segments on modified Murashige and Skoog (MMS) medium containing 5–10 μM TDZ. A maximum of 10 buds and 12 shoots per apical segment for seedlings of cultivar ‘Ida’ regenerated on MMS containing
10 μM TDZ. Callus and bud regeneration frequency, callus growth, and number of buds and shoots per regenerating explant depended
not only on the specific segment of the hypocotyl, but also on parental genotype. Inhibition of shoot elongation by TDZ was
overcome by transferring shoot cultures to a shoot proliferation medium containing 1–2 μM zeatin. The optimal concentration of sucrose for shoot elongation was 20 gl−1. Shoots were rooted ex vitro on a 2 peat: 1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid, and rooted plants acclimatized readily under
greenhouse conditions. 相似文献
13.
R. Dibax C. Deschamps J. C. Bespalhok Filho L. G. E. Vieira H. B. C. Molinari M. K. F. De Campos M. Quoirin 《Biologia Plantarum》2010,54(1):6-12
The purpose of this research was Eucalyptus saligna in vitro regeneration and transformation with P5CSF129A gene, which encodes Δ1-pyrroline-5-carboxylate synthetase (P5CS), the key enzyme in proline biosynthesis. After selection of the most responsive genotype, shoot organogenesis was induced
on leaf explants cultured on a callus induction medium (CI) followed by subculture on a shoot induction medium (SI). Shoots
were subsequently cultured on an elongation medium (BE), then transferred to a rooting medium and finally transplanted to
pots and acclimatized in a greenhouse. For genetic transformation, a binary vector carrying P5CSF129A and uidA genes, both under control of the 35SCaMV promoter, was used. Leaves were co-cultured with Agrobacterium tumefaciens in the dark on CI medium for 5 d. The explants were transferred to the selective callogenesis inducing medium (SCI) containing
kanamycin and cefotaxime. Calli developed shoots that were cultured on an elongation medium for 14 d and finally multiplied.
The presence of the transgene in the plant genome was demonstrated by PCR and confirmed by Southern blot analysis. Proline
content in the leaves was four times higher in transformed than in untransformed plants while the proline content in the roots
was similar in both types of plants. 相似文献
14.
Y. Bao P. Dharmawardhana R. Arias M. B. Allen C. Ma Steven H. Strauss 《Plant cell reports》2009,28(6):947-962
We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs
to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common
in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems
as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were
placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary
growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response
regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events
recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended
to stimulate meristem development to promote clonal propagation and genetic transformation.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
15.
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
16.
17.
Summary Shoot tips and leaves excised from in vitro shoot cultures of Salvia nemorosa were evaluated for their organogenic capacity under in vitro conditions. The best shoot proliferation from shoot tips was obtained on Murashige and Skoog (MS) medium supplemented with
8.9 μM 6-benzylaminopurine (BA) and 2.9 μM indole-3-acetic acid (IAA). Leaf lamina and petiole explants formed shoots through organogenesis via callus stage and/or
directly from explant tissue. The highest values for shoot regeneration were obtained with 0.9 μM BA and 2.9 μM IAA for lamina explants. No shoot organogenesis was obtained on leaf explants cultured on MS medium supplemented with α-naphthaleneacetic
acid (NAA). The regenerated shoots rooted the best on MS medium containing 0.6 μM IAA or 0.5 μM NAA. In vitro-propagated plants were transferred to soil with a survival rate of 85% after 3 mo. 相似文献
18.
Sushma Tamta Lok Man S. Palni Vijay K. Purohit Shyamal K. Nandi 《In vitro cellular & developmental biology. Plant》2008,44(2):136-141
An efficient and reproducible method for the regeneration of multiple shoots of brown oak (Quercus semecarpifolia Sm.) has been developed in which a part of the petiolar tube containing a primary shoot is used as the explant. Explants
derived from in vitro grown seedlings were cultured either on Murashige and Skoog or Woody Plant medium (WPM) containing different concentrations
of benzyladenine (BAP) throughout the range of 1–20 μM. WPM supplemented with 20 μM BAP was found to be best for adventitious
shoot induction and for the multiplication of individual shoots. In-vitro-produced shoots were rooted using a two-step method. Firstly, shoots were cultured on WPM containing indolebutyric acid (IBA)
at either 50 or 100 μM for 24 or 48 h. Secondly, the shoots were transferred to plant-growth-regulator-free half-strength
WPM. The second step not only considerably improved the rooting percentage but also minimized the formation of basal callus.
The most effective first-step treatment was found to be 100 μM IBA for 24 h, which initiated rooting at a frequency of 100%.
Well-rooted plants were transferred to plastic cups containing nonsterile, sieved soil and farmyard manure, hardened under
greenhouse conditions, and then successfully established in pots. This procedure is suitable for use in large-scale production
of plants and may have potential application in additional oak species. 相似文献
19.
The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm−3 1-naphthaleneacetic acid (NAA) + 1.0 mg dm−3 thidiazuron and 1.0 mg dm−3 NAA + 2.0 mg dm−3 kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm−3 NAA + 1.0 mg dm−3 benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and
increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during
organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high
POX activity was observed in regenerated shoot. 相似文献
20.
Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot
regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm−3 kanamycin and 300 mg dm−3 cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm−3 kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl
explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival
rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on
transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved. 相似文献