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1.
Jin Cui Jianjun Chen Richard J. Henny 《In vitro cellular & developmental biology. Plant》2009,45(1):34-43
Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and
cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D),
or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71%
of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium
containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction
medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants
cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto
MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth
regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously
after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after
their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from
which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry
analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines,
like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg
2C−1, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy
level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans. 相似文献
2.
Kottackal Poulose Martin A. K. Pradeep Joseph Madassery 《Acta Physiologiae Plantarum》2011,33(4):1141-1148
Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source
of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from
the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture
to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot
morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower
concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA)
or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of
shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation
as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived
whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole
branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with
BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing
2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix:
a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants
regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar
to that of the source plants flowered normally and set fruits. 相似文献
3.
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
4.
S. Antony Ceasar S. Ignacimuthu 《In vitro cellular & developmental biology. Plant》2008,44(5):427-435
An efficient somatic embryogenesis and plant regeneration system was developed from shoot apex explants of finger millet,
Eleusine coracana. Eight genotypes, CO 7, CO 9, CO 13, CO 14, GPU 26, GPU 28, GPU 45, and GPU 48, were assessed in this study. The maximum
somatic embryogenic induction, at 98.6%, was obtained from explants cultured on Murashige and Skoog medium supplemented with
18.0 μM dichlorophenoxyacetic acid and 2.3 μM kinetin. The highest number of shoot induction (26) was observed after transfer
of embryonic callus to regeneration medium supplemented with 4.5 μM thidiazuran and 4.6 μM kinetin. Significant differences
were observed between genotypes for somatic embryogenesis and plant regeneration. GPU 45 gave the best response, while CO
7 was the least responsive under the culture conditions tested in this study. Regenerated plants were successfully rooted
and grown to maturity after hardening in soil. 相似文献
5.
Muthu Thiruvengadam K. T. Rekha Chang-Hsien Yang Narayanasamypillai Jayabalan Ill-Min Chung 《Plant biotechnology reports》2010,4(4):321-328
An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature leaf explants of Momordica charantia, a very important vegetable and medicinal plant. Calluses were induced from immature leaf explants excised from in vitro
(15-day-old seedlings) mature leaf explants of vivo plants (45 days old). The explants were grown on Murashige and Skoog (MS)
medium with Gamborg (B5) vitamins containing 30 g l−1 sucrose, 2.2 g l−1 Gelrite, and 7.7 μM naphthalene acetic acid (NAA) with 2.2 μM thidiazuron (TDZ). Regeneration of adventitious shoots from
callus (30–40 shoots per explant) was achieved on MS medium containing 5.5 μM TDZ, 2.2 μM NAA, and 3.3 μM silver nitrate (AgNO3). The shoots (1.0 cm length) were excised from callus and elongated in MS medium fortified with 3.5 μM gibberellic acid (GA3). The elongated shoots were rooted in MS medium supplemented with 4.0 μM indole 3-butyric acid (IBA). Rooted plants were
acclimatized in the greenhouse and subsequently established in soil with a survival rate of 90%. This protocol yielded an
average of 40 plants per leaf explant with a culture period of 98 days. 相似文献
6.
K. Samuel D. Debashish B. Madhumita G. Padmaja Siva Ram Prasad V. Bhaskara Ramana Murthy P. S. Rao 《In vitro cellular & developmental biology. Plant》2009,45(4):466-473
The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken
to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for
micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds
when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA3). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts)
supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds
decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which
germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction
was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary
medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after
two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively. 相似文献
7.
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
8.
Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine
(BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number
of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators,
and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration
was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots
per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing
the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same
medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants
survived and became established. 相似文献
9.
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
10.
Callus cultures from nodal and leaf explants of Phyllanthus amarus were established on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. The leaf-derived
callus induced on 2.26 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) + 2.32 μM Kinetin (Kin) upon transfer to medium containing
thidiazuron (TDZ) exhibited higher shoot regeneration (32.4 ± 1.3 shoots per culture). Four-week-old shoots rooted readily
on 1.5 μM Indol acetic acid (IAA)-containing medium and were successfully acclimatized with 98% survival. The lignans, Phyllanthin
(PH) and Hypohyllanthin (HPH), of leaf extracts from naturally grown plants were identified by using TLC, HPLC and H1-NMR.
The PH and HPH production in the regenerated shoots was compared to their production in callus cultures, plants under field
conditions and in naturally grown plants. The regenerated shoots on MS + 2.27 μM TDZ produced about two times higher PH and
HPH than the leaves of naturally grown plant. The present study provides a useful system for further studies on in vitro morphogenesis,
elicitor-assisted production of PH and HPH and A. rhizogenes-mediated genetic transformation in Phyllanthus amarus. 相似文献
11.
Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome
doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants.
Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring
the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO3). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip
explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production
of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot
tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The
fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture
of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from
parthenogenetic melons. 相似文献
12.
S. Paul A. Dam A. Bhattacharyya T. K. Bandyopadhyay 《Plant Cell, Tissue and Organ Culture》2011,105(2):271-283
A reproducible protocol for direct and indirect somatic embryogenesis was established in a small aromatic tree, Murraya koenigii. Embryogenic callus was obtained from 90% zygotic embryonic axis (ZE) and 70% cotyledon (COT) explants in Murashige and Skoog
(MS) basal medium supplemented with 8.88 μM 6-benzyladenine (BA) and 2.675 μM α-naphthaleneacetic acid (NAA). Globular somatic
embryos were induced and further matured from such embryogenic callus by subsequent culture on the same basal media containing
thidiazuron (TDZ) (2.27–9.08 μM). The highest frequency of somatic embryos (14.58 ± 0.42) was recovered from ZE-derived callus
after 6 weeks. The age and type of explant and concentration of TDZ played an important role in the development of somatic
embryos. Explants excised from 60-day-old seed differentiated from 96.67% of ZE explants and 86.67% from COT explants when
cultured on MS basal medium supplemented with 4.54 and 9.08 μM TDZ, respectively, after 4 weeks. The best result obtained
for the average frequency of somatic embryos (11.28 ± 0.32) was from ZE explants, which was significantly higher than COT
explants (7.34 ± 0.97). Most of the somatic embryos (above 95%), irrespective of their origin, germinated after 4 weeks in
1/2 MS basal media containing 2.32 μM kinetin (KN) and 1.07 μM NAA. Well-rooted plantlets were successfully acclimatized.
Histological analysis and scanning electron micrographs confirmed the initiation, development, and germination of somatic
embryos from both explants. 相似文献
13.
The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old
white oak (Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo
induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular
structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis
process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage.
Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain
cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces
and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7–8 weeks after culture
initiation, although most emerged after 9–12 weeks in culture. The sequence of application of media for somatic embryo induction
was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and
2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic
lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%)
were achieved after cold storage for 2 months. 相似文献
14.
Phillip A. Wadl Adam J. Dattilo Lisa M. Vito Robert N. Trigiano 《Plant Cell, Tissue and Organ Culture》2011,106(3):513-516
Pityopsis ruthii is an endangered herbaceous perennial species from the United States. In vitro multiplication of this species can be valuable
for germplasm conservation. Flower receptacles of P. ruthii were cultured on Murashige and Skoog medium (MS) supplemented with 11.4 μM indole-3-acetic acid (IAA) in combination with
2.2, 4.4 or 8.8 μM 6-benzyladenine (BA). Shoots were visible within 14–28 days and three plants were successfully rooted on
MS medium supplemented with 5.7 μM IAA. A two tailed t-test for paired-variates revealed that shoot regeneration on MS medium
amended with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than on other treatments. Leaf explants were also cultured on MS not supplemented with growth regulators or supplemented
with 11.4 μM IAA in combination with 0, 2.2, 4.4 or 8.8 μM BA. Shoots were visible within 21–35 days and one plant was successfully
rooted on MS medium supplemented with 5.4 μM NAA. Shoot regeneration on MS medium augmented with 11.4 μM IAA and 2.2 μM BA
was significantly higher (P < 0.05) than the other treatments according to analysis of variance (ANOVA) with a rank transformation. Hyperhydricity and
rooting of shoots was problematic for explants derived from flower receptacles and leaf tissue, but viable plants were regenerated
using both explants sources indicating the potential role for micropropagation in the ex situ conservation of the species. 相似文献
15.
Rishi K. Tyagi Rajni Goswami Rajkumari Sanayaima Rakesh Singh Rajesh Tandon Anuradha Agrawal 《In vitro cellular & developmental biology. Plant》2009,45(6):721-729
Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture
in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously
on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation
medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about
70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained
by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment
and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at
least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium
for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of
20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation
was detected among the in vitro-conserved plants compared with the mother plants. 相似文献
16.
María Laura Vidoz Pablo Klusacek Hebe Yolanda Rey Luis Amado Mroginski 《Plant Cell, Tissue and Organ Culture》2006,86(1):111-115
In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and organogenesis were developed using immature leaves as explants. Morphologically normal somatic embryos were obtained on culture media composed of 20.70 or 41.41 μM picloram (PIC) with the addition of 0.044 μM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively. The source of explants and the developmental stage of the leaves had a marked effect on somatic embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive producing up to 30% embryogenesis in MS+41.41 μM PIC. Embryos converted into plants after transfer to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) with or without 0.044 μM BA, achieving plant regeneration in the induction media. The highest percentage of bud formation was obtained on culture medium composed of␣MS+10.74 μM NAA+0.044 μM BA (12.5%). Roots were formed on all culture media tested. Regenerated plants were transferred to pots and grew well under greenhouse conditions. 相似文献
17.
Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and
nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured
on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP).
Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior
growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the
proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with
an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength
MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening,
with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be
produced within eight subcultures from callus obtained from leaf explant through the methods described here. 相似文献
18.
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
19.
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
20.
A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing
Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with
a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation
medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid
proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were
obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for
10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival
(40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf
index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy
of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating
plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings. 相似文献