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1.
Jing Li Yang Yu Da Niu Chuan Ping Yang Gui Feng Liu Cheng Hao Li 《Plant Cell, Tissue and Organ Culture》2011,106(3):391-399
Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised
unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three
to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were
low, but increased in the presence of gibberellic acid (GA3). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs).
The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher
than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced
embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had
two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia,
but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological
and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of
somatic embryo origin. 相似文献
2.
D. A. Steinmacher G. C. Cangahuala-Inocente C. R. Clement M. P. Guerra 《In vitro cellular & developmental biology. Plant》2007,43(2):124-132
The factors affecting the induction and development of somatic embryos and plantlet acclimatization of peach palm (Bactris gasipaes Kunth) were evaluated to establish an efficient regenerative protocol based on somatic embryogenesis. Mature zygotic embryos
were cultured in Murashige and Skoog (MS) medium supplemented with 0–40 μM of picloram (4-amino-3,5,6-trichloropicolinic acid)
and 0 or 5 μM of 2-isopentyladenine (6-dimethylaminopurine) (2-iP). After 5 mo. in culture embryogenic callus arose from primary
calli. Picloram (10 μM) was effective in inducing embryogenic calli in 9.8% of the explants. The use of 1 μM of AgNO3 enhanced embryogenic competence. Embryogenic calli showed an organized structure, a globular aspect, and were white to yellowish
in color. Histological analyses showed that cell proliferation arose from subepidermal cells adjacent to vascular bundles,
resulting in primary callus formed by a meristematic zone from which somatic embryos arose. Protein profile analyses revealed
two high molecular mass bands in these embryogenic calli, but not in other tissues. Embryogenic calli were transferred to
a culture medium containing 40 μM of 2,4-dichlorophenoxyacetic acid, 10 μM of 2-iP, plus 1 g l−1 of glutamine, hydrolyzed 0.5 g l−1 casein, and activated 1.5 g l−1 of charcoal. Morphogenetic responses achieved in this medium were the development of somatic embryos, rooting, and loss of
embryogenic capacity. Somatic embryos were converted to plantlets on MS medium plus 24.6 μM of 2-iP and 0.44 μM of naphthalene
acetic acid. Plantlets were maintained in MS medium with activated charcoal (1.5 g l−1) until they were 6 cm tall, and then acclimatized. After 16 wk, 84.2 ± 6.4% survival was observed.
M. P. Guerra and C. R. Clement are Fellows of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília,
DF. 相似文献
3.
A. Othmani C. Bayoudh N. Drira M. Marrakchi M. Trifi 《Plant Cell, Tissue and Organ Culture》2009,97(1):71-79
Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year
old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth
regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months
on MS medium supplemented with 10 mg l−1 2,4-D and 0.3 mg l−1 activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping
and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with
1 mg l−1 ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus
was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and
306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments
was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l−1 NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots.
The growth of regenerated somatic plants was also monitored in the field. 相似文献
4.
Chun-Lai Zhang Dong-Fang Chen Marie Kubalakova Jian Zhang Nigel W. Scott Malcolm C. Elliott Adrian Slater 《Plant Cell, Tissue and Organ Culture》2008,93(2):209-221
Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation
of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the
starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings
of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic
embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic
calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this
method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic
embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the
induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs).
Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration
of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic
transformation of sugar beet. 相似文献
5.
Friable embryogenic callus and somatic embryo formation from cotyledon explants of African marigold (Tagetes erecta L.) 总被引:3,自引:0,他引:3
Embryogenic callus and somatic embryos were induced from cotyledonary explants of African marigold (Tagetes erecta L.). Cotyledons were first cultured on MS medium supplemented with 2.0 mg l–1 2,4-D and 0.2 mg l–1 kinetin. After 5 weeks, calli were transferred to MS medium supplemented with 0.02 mg l–1 thidiazuron where compact embryogenic callus developed. Friable embryogenic callus developed when the compact embryogenic
callus was transferred to medium containing 2,4-D and subcultured every 2 weeks. Friable embryogenic callus has been maintained
for more than 2 years without losing the capacity to generate embryos. Embryo development was obtained when friable embryogenic
callus was transferred to MS medium supplemented with 3 mg l–1 ABA and 60 g l–1 sucrose. The addition of 10–30 mM
l-glutamine improved embryo development.
Received: 13 May 1997 / Revision received: 24 February 1998 / Accepted: 28 March 1998 相似文献
6.
Daniela Lopes Paim Pinto Ana Maria Rocha de Almeida Mailson Monteiro Rêgo Maurecilne Lemes da Silva Evelyn Jardim de Oliveira Wagner Campos Otoni 《Plant Cell, Tissue and Organ Culture》2011,107(3):521-530
Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of
6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented
with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological
and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells
with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small
nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated
charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed. 相似文献
7.
Yong Wook Kim So Young Park In Sun Park Heung Kyu Moon 《Plant biotechnology reports》2007,1(4):237-242
We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3–4 weeks postanthesis.
The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized
culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium.
The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos
were converted into normal plantlets in 1/2 MS medium containing gibberellic acid (GA3). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or
radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture,
were transferred to a nursery, and have grown normally. 相似文献
8.
Q. C. Liu H. Zhai Y. Wang D. P. Zhang 《In vitro cellular & developmental biology. Plant》2001,37(5):564-567
Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic
callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and
Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established.
Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with
9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred
to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of
cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred
to soil, showed 100% survival. No morphological variations were observed. 相似文献
9.
Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower 总被引:2,自引:1,他引:1
Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s
germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary
and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however,
100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos
matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non
root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized
and successfully transferred to the field. 相似文献
10.
We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin
for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic
embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7
d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in
regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L
giberellic acid (GA3). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern
hybridization to confirm gene integration. 相似文献
11.
An efficient protocol was established for regeneration of Desmodium motorium via somatic embryogenesis. Embryogenic calli were induced from cotyledon segments (6 mm, 16 days old) lacking embryo axis,
excised from seedlings grown in vitro on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) (2.9 μM)
in combination with 6-benzyladenine (BA) (4.44 and 8.88 μM). Differentiation of embryogenic calli into globular and heart-shaped
somatic embryos was achieved on transfer to hormone-free MS medium. When incubated for 4 days on MS medium supplemented with
BA (8.88 μM), 95% of the globular and heart-shaped somatic embryos matured into torpedo and cotyledonary stages with minimum
(10%) abnormalities. Modified MS basal medium without hormones and containing half-strength macronutrients and 0.88 M sucrose
was suitable for germination of mature somatic embryos. Regenerated plantlets were successfully transferred to earthen pots
with survival rate of 50%. Secondary embryogenesis was observed when pre-existing somatic embryos at globular and heart-shaped
stages were cultured on MS medium supplemented with various concentrations of BA, adenine sulphate (AdS) and abscisic acid
(ABA) individually. 相似文献
12.
Nasser J. Y. Sholi Anjana Chaurasia Anuradha Agrawal Neera Bhalla Sarin 《Plant Cell, Tissue and Organ Culture》2009,99(2):133-140
Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 μM
2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of
scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid
MS medium supplemented with 4.5 μM 2,4-D and 1.0 μM zeatin. Upon transfer to semi-solid MS medium of the same composition
as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 μM abscisic acid (ABA), maturation
of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in
the medium. Upon transfer to MS medium supplemented with 1.25 μM 6-benzyladenine (BA), 80% of germinated embryos developed
into plantlets. 相似文献
13.
R. Scorza P. H. Morgens J. M. Cordts S. Mante A. M. Callahan 《In vitro cellular & developmental biology. Plant》1990,26(8):829-834
Summary Peach leaf segments, immature embryos, and long-term embryogenic calli have been transformedin vitro with the engineeredAgrobacterium tumefaciens strain A281 containing pGA472. All three tissue sources proliferated callus which grew on a medium containing 100–200 mg/l
kanamycin or 10–20 mg/l G-418 as selective agents. These calli were shown to produce neomycin phosphotransferase. The results
of Southern analyses were consistent with the incorporation of foreign DNA into the genome of leaf, embryo and embryogenic
peach callus. 相似文献
14.
Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA3treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis. 相似文献
15.
Regeneration of Acacia mangium through somatic embryogenesis 总被引:2,自引:0,他引:2
Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos
of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of
amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic
cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first
stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA3 followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic
embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy
showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could
be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic
structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation
layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important
tropical forest species.
Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September 相似文献
16.
M. Muruganantham S. Amutha A. Ganapathi 《In vitro cellular & developmental biology. Plant》2010,46(1):34-40
The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred
to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped
embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared
to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred
to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage
embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed
into plants. 相似文献
17.
Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos.
The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM
2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred
to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells
destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular,
heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects
of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium
supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas
cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
18.
Somatic embryos were obtained from immature zygotic embryos of Cedrela fissilis Well. (Meliaceae), after a culture period of 12 months, with regular subcultures every 6–8 weeks. Callus was developed on explants in 2 months
on Murashige and Skoog (MS) medium containing 2,4 dichlorophenoxyacetic acid (2,4-D) or picloram (PIC). When the calli were
transferred to fresh medium, embryogenic tissue appeared on MS + 45 μM 2,4-D, or 22.5 μM 2,4-D + 0.4 μM 6-benzyladenine (BA),
or 20.7 μM PIC after 6 months. Sub-culture of embryogenic tissue in MS medium supplemented with 4.5 μM 2,4-D resulted in the
differentiation into somatic embryos after further 4 months. Repeated secondary somatic embryogenesis was achieved by regular
subculture on this medium. Maturation and conversion of somatic embryos into plantlets was achieved on MS medium without plant
growth regulators and the conversion frequency was approximately 12.5 %. The plantlets were successfully acclimatized in pots
with soil. Histological studies showed that somatic embryos had no detectable connection with the mother explants and that
somatic embryos in advanced stages were bipolar with shoot and root apical meristems, they contained vascular system and showed
typical characteristics of a somatic dicotyledonous embryo. 相似文献
19.
Itziar Lanas Piedad Gallego Luisa Martin Javier Fernandez Angel Alonso Juana Elena-Rosello Antonio Blazquez Nieves Villalobos Hilario Guerra 《Plant Growth Regulation》2006,49(1):49-60
High production of viable somatic embryos was obtained from cultured anthers in the second phase of meiosis, using microscopic
level observations of tetrads. The medium with the greatest embryogenic efficiency was H6, composed of Murashige and Skoog
(MS) medium with 2 mg l−1 of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l−1 of kinetin. All (100%) of the somatic embryos obtained germinated and produced 63% green and 37% albino seedlings. In general,
embryogenic calli had a higher ion concentration than non-embryogenic calli, with the exception of calcium whose concentration
was higher in non-embryogenic calli. The calli induced in the different media differed in their sucrose and starch compositions.
The most embryogenic medium H6-induced calli with the highest sucrose concentration and the lowest starch concentration, before
visible embryos were observed. In the leaves of the albino seedlings, sucrose concentrations were very high while those of
starch were very low. Ion concentrations were also lower in albino plants than in the leaves of green seedlings, with the
exception of calcium, whose concentration was higher. Most of the albino individuals were homozygous, even when their progenitors
were heterozygous, thereby confirming their haploid nature. 相似文献
20.
Maurecilne Lemes da Silva Daniela Lopes Paim Pinto Miguel Pedro Guerra Eny Iochevet Segal Floh Cláudio Horst Bruckner Wagner Campos Otoni 《Plant Cell, Tissue and Organ Culture》2009,99(1):47-54
The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and
4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number
of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically,
embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with
small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated
charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the
production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos,
including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the
first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary
embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a
vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible
protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation
protocol. 相似文献