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1.
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
2.
A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics
of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog
(MS) medium supplemented with 3 μM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated
clearly that the first sign of meristematic cell division could be seen after 1–2 days of pre-culture, and meristematic tissues
multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained
from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 μM
thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for
organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle
bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene. 相似文献
3.
F. D. Espasandin M. M. Collavino C. V. Luna R. C. Paz J. R. Tarragó O. A. Ruiz L. A. Mroginski P. A. Sansberro 《Plant Cell, Tissue and Organ Culture》2010,102(2):181-189
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which
carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented
with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during
the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots
per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS
strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and
Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines
were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90%
of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase
from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter
showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the
same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the
cloned materials was established. 相似文献
4.
Eline Kirk Mørk Karin Henriksen Henrik Brinch-Pedersen Kell Kristiansen Karen Koefoed Petersen 《Plant Cell, Tissue and Organ Culture》2012,108(3):501-512
A protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. A Murashige and Skoog medium with 1.0 mg l−1 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. Addition of 0.1 mg l−1 indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots
longer than 1 cm. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation.
A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants
with Agrobacterium tumefaciens. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for β-glucuronidase (GUS) were used. Regeneration of transgenic plants after co-cultivation with A281 was independent
of cultivar, and all explants produced callus followed by indirect shoot formation. In ‘Victoria Fanny’ shoots were formed
faster and without a callus phase after co-cultivation with LBA 4404 or C58. The highest number of potentially transformed
shoots was regenerated after co-cultivation of ‘Victoria Fanny’ leaf explants with LBA 4404. Integration of the transgenes
in the plant genome was confirmed using PCR and Southern blot hybridisation. To verify that the transgenes could be transferred
to offspring, crosses were conducted between three transgenic lines of ‘Victoria Fanny’ and two wild type pollen donors. It
was demonstrated that viable seeds were produced and that the uidA gene was inherited. 相似文献
5.
Claudia Simões Norma Albarello Cátia Henriques Callado Tatiana Carvalho de Castro Elisabeth Mansur 《Plant Cell, Tissue and Organ Culture》2009,98(1):79-86
Alternative methods for in vitro shoot culture of Cleome rosea, a Brazilian herbaceous species with ornamental value and medicinal potential, were evaluated. A protocol for rapid in vitro
multiplication of roots, a valuable source of medicinal compounds, was also developed. Stem explants were cultured in liquid
media (continuous immersion and paper bridge), while root explants were cultivated in continuous immersion and on solidified
media. The highest numbers of shoots, 20 ± 4.6 shoots/explant, were obtained from stem explants incubated in a continuous
immersion system in a liquid medium supplemented with 2.2 μM BA. Root explants cultivated in liquid media produced only hyperhydrous
adventitious shoots. However, these explants generated 5.8 ± 0.8 shoots/explant by indirect organogenesis when cultivated
on solidified medium supplemented with 2.2 μM BA. In addition, root multiplication was achieved in liquid medium in the presence
of α-naphthaleneacetic acid. Adventitious shoots developed on newly formed roots when inoculated on solidified medium supplemented
with 2.2 μM BA. Shoot microcuttings developed roots when transferred onto solidified MS medium without growth regulators.
Rooted microcuttings were efficiently acclimatized when transferred ex vitro. 相似文献
6.
M. Arshad J. Silvestre G. Merlina C. Dumat E. Pinelli J. Kallerhoff 《Plant Cell, Tissue and Organ Culture》2012,108(2):315-322
Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron
(TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM
TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture
of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for
both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength
MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars
developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions.
When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of
Roses’ and ‘Atomic Snowflake’, respectively. 相似文献
7.
Valentine Otang Ntui Gunaratnam Thirukkumaran Pejman Azadi Raham Sher Khan Ikuo Nakamura Masahiro Mii 《Plant cell reports》2010,29(9):943-954
Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi”
resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with
Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing
100 mgl−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges
of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’,
respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype
NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the
wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same
selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies
of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic
lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants. 相似文献
8.
S. B. Nandeshwar Sandhya Moghe P. K. Chakrabarty M. K. Deshattiwar Keshav Kranthi P. Anandkumar C. D. Mayee B. M. Khadi 《Plant Molecular Biology Reporter》2009,27(4):549-557
An Agrobacterium-mediated gene transfer protocol was developed for the diploid cotton Gossypium arboreum using meristematic cells of shoot tips, followed by direct shoot organogenesis or multiple shoot induction of putative transformants.
Seven-day- old shoot tips of in vitro-germinated seedlings of G. arboreum cv. RG8 were excised by removing cotyledonary leaves and providing “V”-shaped oblique cuts on either side of explants. Excised
explants were inoculated with an overnight-grown culture of Agrobacterium tumefaciens carrying a plant cloning vector harboring the cry1Ac gene. The explants were co-cultivated in Murashige and Skoog (MS) medium supplemented with 30 mg/L acetosyringone, 100 mg/L
myoinositol, 10 mg/L thiamine, and 30 g/L glucose for three days in the dark. Following co-cultivation, explants were incubated
on the same medium supplemented with 20 mg/L kanamycin, for first three passages of 10–12 days each and subsequently on 50 mg/L
kanamycin to facilitate stable expression of transgene. Explants were then transferred to a fresh MS medium supplemented with
either kinetin (0.1 mg/L), myoinositol (100 mg/L), thiamine (10 mg/L) and glucose (30 g/L) or benzyl adenine, BA (2 mg/L),
kinetin (1 mg/L), myoinositol (100 mg/L), thiamine (10 mg/L), and glucose (30 g/L) to induce either single or multiple putative
transformant shoots, respectively. Following 6 weeks, shoots were transferred to a rooting medium consisting of liquid MS
supplemented with 0.05–0.1 mg/L NAA and glucose (15 g/L). Rooted plantlets were first acclimatized in liquid MS with 0.05 mg/L
NAA and 15 g/L glucose, transferred to plastic pots containing soilrite Mix-TC (a mixture of Irish peat moss and horticultural
grade expanded perlite, 75:25), and grown under controlled temperature and humidity conditions in a growth chamber. Acclimatized
plants were then transferred to clay pots and grown in the greenhouse. These plants were confirmed as transgenic for cry1Ac gene using polymerase chain reaction, enzyme linked imunosorbent assay, and Southern blot analyses. 相似文献
9.
Guang-Zhe Lin Xiao-Mei Zhao Soon-Kwan Hong Yu-Ji Lian 《Plant Cell, Tissue and Organ Culture》2011,106(1):93-103
We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple
shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l−1 indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot
induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l−1 Zn, and 0.5 mg l−1 IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l−1 Zn and 0.5 mg l−1 IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of
development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses
were transferred to MS medium containing either (1) 1.5 mg l−1 Zn and 0.05 mg l−1 IAA or (2) 1.0 mg l−1 BA and 0.05 mg l−1 IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic
embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l−1 α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture
of mountain soil and perlite. 相似文献
10.
When cotyledonary explants, excised from in vitro germinated seedlings, of pomegranate (Punica granatum L.) were incubated on solid Murashige and Skoog (1962) medium supplemented with 21 μM naptheleneacetic acid (NAA) and 9 μM 6-benzyladenine (BA), 80% of explants developed callus.
A high frequency of shoot organogensis was obtained when explants were incubated on MS medium supplemented with 8 μM BA, 6 μM
NAA, and 6 μM giberrellic acid (GA3). However, adding 24 μM silver nitrate (AgNO3) to this medium markedly enhanced shoot regeneration frequency (63%) and mean number of shoots per explant (11.26) and length
of shoots (2.22 cm). Highest frequency of in vitro rooting, mean number of roots/shoot (4.32), and mean root length (2.71 cm)
were obtained when regenerated shoots were transferred to half-strength MS medium supplemented with 0.02% activated charcoal.
Well-rooted plantlets were acclimatized, and then transferred to soil medium. Moreover, when zygotic embryos of P. granatum, excised from seeds collected at 16 weeks following full bloom, were incubated on MS medium containing 30 g l−1 sucrose, 15% coconut water, 21 μM NAA, and 9 μM BA, they developed the highest frequency of embryogenic callus, clumps with
globular embryos, and mean number of both globular and heart-shaped embryos per callus clump. Subjecting zygotic embryo explants
to six-week dark incubation period was essential for embryogenic callus induction, and these were subsequently transferred
to 16 h photoperiod for further growth and development of somatic embryos. Germination of somatic embryos was observed when
these were transferred to MS medium was supplemented with 60 g l−1 sucrose. 相似文献
11.
Tantravahi Srinivasan Koppolu Raja Rajesh Kumar Pulugurtha Bharadwaja Kirti 《Plant Cell, Tissue and Organ Culture》2010,101(3):303-309
Though peanut tissue culture has advanced to a considerable extent using a number of explants with the subsequent production
of transgenic plants, wild Arachis species appeared to be recalcitrant to using similar explants. In this study, the use of cotyledonary nodes as explants prepared
from 7-day old seedlings resulted in the development of a simple and rapid regeneration protocol for five diploid wild species
including A. diogoi, A. stenosperma, A. duranensis, A. cardenasii and A. correntina belonging to the genus Arachis for producing multiple shoots. Shoot bud initiation was observed 10 to 15 days after culture initiation. Responding cotyledonary
nodes with shoot buds were subcultured to lower levels of cytokinin and finally to MS basal medium for further shoot development
and elongation. Production of multiple shoots was observed in all the five diploid species with a maximum of 9 to 16 shoots
were obtained per explant in the primary cultures. The number of shoot buds increased significantly with repeated explant
subculturing with recovery up to 45 shoots from responding explants. These shoots were rooted efficiently on MS medium supplemented
with 1 mg l−1 naphthalene acetic acid and the time taken from explanting to the transfer of shoots to potting mixture was about 12 weeks.
All rooted shoots were successfully established in soil in glass house and further transferred to field. These plants survived
to maturity and set seed. 相似文献
12.
Ganeshan Sivanandhan Thankaraj Salammal Mariashibu Muthukrishnan Arun Manoharan Rajesh Sampath Kasthurirengan Natesan Selvaraj Andy Ganapathi 《Acta Physiologiae Plantarum》2011,33(6):2279-2288
An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine
(BA) [1.5 mg L−l], indole-3-acetic acid (IAA) [0.3 mg L−l] and with the addition of polyamine, spermidine (20 mg L−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same
medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L−l) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average
of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were
cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from
all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities
of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants
compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated
plants. This protocol will be useful for scale-up production of withanolides on commercial scale. 相似文献
13.
An efficient micropropagation system via direct shoot organogenesis from hypocotyl segments of Embelia ribes Burm F. was developed. A high frequency (84%) of adventitious shoot induction was obtained on Murashige and Skoog (MS) medium
supplemented with additives (283.85 μM ascorbic acid [AA], 118.96 μM citric acid [CA], 142.33 μM cysteine, and 684.22 μM glutamine)
and 1.13 μM of thidiazuron (TDZ) after 4 weeks following culture. Further development of shoot primordia into well-grown shoots
of 4–5 cm in length was achieved by sub-culturing explants along with shoot primordia on MS medium supplemented with 0.44 μM
benzyl adenine (BA) and 0.49 μM indole butyric acid (IBA) for three sub-culture periods with an interval of 15 days between
them. The highest shoot multiplication was obtained when explants were incubated on MS medium supplemented with 2.2 μM BA
and 0.49 μM IBA in 4 weeks. All in vitro developed shoots, 3–4 cm in length, rooted when grown on half-strength MS basal medium
along with 2.47 μM IBA within 4 weeks. Moreover, 100% of shoots developed roots when these were treated with 4.93 μM IBA for
20 min and then transferred to pots containing soilrite mix and grown in the greenhouse. In vitro and ex vitro rooted plants
showed a survival of 85 and 95% respectively, during hardening in the greenhouse for a 6-week period. 相似文献
14.
15.
An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium
supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled
as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number
of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after
4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks)
also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots
after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the
species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll
cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities. 相似文献
16.
Patricia Corral Rubén Mallón Juan Rodríguez-Oubiña María Luz González 《Plant Cell, Tissue and Organ Culture》2011,105(2):211-217
In vitro culture is a useful tool in the ex situ conservation of rare, endemic, and threatened plant species. Crepis novoana (Compositae) is an endangered endemic in northwestern Spain. Use of in vitro culture tools is necessary due to the poor conservation
status of populations of the species. The systems of in vitro propagation developed for this species in the present study
were caulogenesis from leaf explants and growth of axillary buds from shoots. Explants were produced by placing fragments
of leaves on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzyladenine (BA) and 2.69 μM naphthaleneacetic
acid (NAA); caulogenesis was induced in 80% of explants, with development of a mean number of 2.48 shoots per explant. Axillary
bud development from shoots was highest with MS supplemented with 4.44 μM BA and 0.54 μM NAA, resulting in production of a
mean number of 49.77 shoots per explant. Immersion of the basal side of shoots in a solution of 5.37 mM NAA for 30 s yielded
90% success in the production of rooted shoots. Plantlets were well acclimatized, and almost 100% of plants transferred to
soil recovered successfully. 相似文献
17.
Bilal Haider Abbasi Mubarak Ali Khan Tariq Mahmood Mushtaq Ahmad Muhammad Fayyaz Chaudhary Mir Ajab Khan 《Plant Cell, Tissue and Organ Culture》2010,101(3):371-376
The morphogenic potential and free-radical scavenging activity of the medicinal plant, Silybum marianum L. (milk thistle) were investigated. Callus development and shoot organogenesis were induced from leaf explants of wild-grown
plants incubated on media supplemented with different plant growth regulators (PGRs). The highest frequency of callus induction
was observed on explants incubated on Murashige and Skoog (MS) medium supplemented with 5.0 mg l−1 6-benzyladenine (BA) after 20 days of culture. Subsequent transfer of callogenic explants onto MS medium supplemented with
2.0 mg l−1 gibberellic acid (GA3) and 1.0 mg l−1 α-naphthaleneacetic acid (NAA) resulted in 25.5 ± 2.0 shoots per culture flask after 30 days following culture. Moreover,
when shoots were transferred to an elongation medium, the longest shoots were observed on MS medium supplemented with 0.5 mg l−1 BA and 1.0 mg l−1 NAA, and these shoots were rooted on a PGR-free MS basal medium. Assay of antioxidant activity of in vitro and in vivo grown
tissues revealed that significantly higher antioxidant activity was observed in callus than all other regenerated tissues
and wild-grown plants. 相似文献
18.
Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring
conservation. In this study, fresh shoot sprouts of P. tuberosa, produced by tubers, were used as explants for in vitro micropropagation. Surface-sterilized nodal shoots were incubated
on Murashige and Skoog (MS) medium supplemented with 8.88 μM benzyladenine (BA), 50 mg l−1 ascorbic acid, and 25 mg l−1 of each of citric acid and adenine sulphate. Cut ends of nodal stem segments rapidly turned brown, and cultures failed to
establish. When 100 mg l−1 ascorbic acid (ABA) and 25.0 mg l−1 polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established. Bud-breaking
of nodal stem explants resulted in multiple shoot formation. Shoots proliferated (35–40 shoots per culture vessel) on MS medium
as described above, but supplemented with 4.44 μM BA and 0.57 μM indole acetic acid (IAA) and additives. After 4–5 passages,
proliferating shoots exhibited tip-browning and decline in growth and multiplication. However, when shoots were transferred
to fresh shoot proliferation medium supplemented with 2.32 μM kinetin (Kn), sustained growth and high rate of shoot proliferation
(50–60 shoots per culture vessel) was observed. Shoots rooted when transferred to medium consisting of half- strength MS medium
with 9.84 μM indole butyric acid (IBA) and 0.02% activated charcoal. Alternatively, individual shoots were pulsed with 984.0 μM
IBA and transferred to glass bottles containing sterile and moistened soilrite. These shoots rooted ex-vitro and were acclimatized
in the greenhouse. Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin. 相似文献
19.
Xingyu Yang Jinfeng Lü Jaime A. Teixeira da Silva Guohua Ma 《Plant Cell, Tissue and Organ Culture》2012,109(2):213-221
Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for
its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China
are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium
containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated
on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced.
This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins
supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days
following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic
embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants
from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium
containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three
ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce
secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious
roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and
transfer to a potting mixture (1:1, sand:vermiculite). 相似文献
20.
In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated
using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations
of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured
in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%)
formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented
with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was
observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage
of GUS+ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium
containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated
shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l
kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated
shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR+ plants. 相似文献