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1.
Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels
(200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing
15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal. 相似文献
2.
R. Prem Anand A. Ganapathi V. Ramesh Anbazhagan G. Vengadesan N. Selvaraj 《In vitro cellular & developmental biology. Plant》2000,36(6):475-480
Summary Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable
embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per
25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos
were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the
field was 8–10%. 相似文献
3.
《Plant Physiology and Biochemistry》2000,38(7-8):567-576
Long-term embryogenic lines were repeatedly obtained from nine asparagus (Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants. 相似文献
4.
Zhijian Li Abdoulaye Traore Siela Maximova Mark J. Guiltinan 《In vitro cellular & developmental biology. Plant》1998,34(4):293-299
Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing
staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth
medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development
medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the
frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of
22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using
this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types.
However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged
from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46.
Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of
cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological
and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use
of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of
a large number of plants from limited source materials. 相似文献
5.
G. Vengadesan N. Selvaraj R. Prem Anand V. Gaba A. Ganapathi 《In vitro cellular & developmental biology. Plant》2005,41(6):789-793
Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion.
Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid
medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM
l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of
heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium
containing 175.2 mM sucrose and 0.5 gl−1 activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6
mM sucrose and 1.4 μM gibberellic acid (GA3) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants. 相似文献
6.
High-efficiency somatic embryogenesis and plant regeneration from suspension cultures of grapevine 总被引:10,自引:0,他引:10
Embryogenic suspensions of grapevine (Vitis vinifera L.) were initiated from somatic embryos of `Thompson Seedless' and `Chardonnay'. Suspension cultures consisted of proembryonic
masses (PEM) that proliferated without differentiation in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). `Chardonnay'
somatic embryos developed fully from PEMs following subculture in medium without 2,4-D; however, somatic embryo development
did not advance beyond the heart stage in `Thompson Seedless' suspension cultures. Highly synchronized development of somatic
embryos was obtained by inoculating <960-μm PEMs into liquid medium without 2,4-D. Somatic embryos were also produced in large
numbers from suspension-derived PEMs of both cultivars on semisolid medium lacking 2,4-D. Somatic embryos matured and regenerated
into plants in MS basal medium containing 3% sucrose. Using this method more than 60% of the somatic embryos regenerated plants.
More than 90% of the regenerated plants were successfully transferred to the greenhouse.
Received: 27 July 1998 / Revision received: 15 October 1998 / Accepted: 27 October 1998 相似文献
7.
Summary
In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and
conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction,
efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants
grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at
13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced
concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being
transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency
of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed
into plantlets after being transferred to agar inedium with 0.44 μMN6-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant. 相似文献
8.
Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS
Murashige and Skoog (1962) medium
- B5
Gamborg et al. (1968) B5 medium
- 2,4-D
2,4-dichlorophenoxyacetic acid
- BAP
6benzylaminopurine
- NAA
1-naphthaleneacetic acid 相似文献
9.
Piedad Gallego Oscar Hita Nieves Villalobos Ana Dorado Luisa Martin Hilario Guerra 《In vitro cellular & developmental biology. Plant》2001,37(2):199-203
Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized
by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16
h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons
and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into
normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants
and N6-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration
of 2,4-D was decreased to 2.25 μM. 相似文献
10.
Summary A procedure for regenerating Zizyphus jujuba Mill. (Chinese date) plants through repetitive somatic embryogenesis (RSE) was developed. Primary somatic embryos were produced
from cotyledon-derived cultures of germinated plants in vitro. The highest induction frequency of primary somatic embryogenesis (PSE) was obtained with a combination of 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.49 μM indole-3-butyric acid (IBA), and 0.44 μM benzyladenine (BA) (17.4%). These primary somatic embryos were multiplied by RSE on media with different plant growth regulator
(PGR) combinations. The highest RSE frequency (51.3%), was obtained with 0.58 μM gibberellic acid (GA3). However, the highest number (4.4 per primary somatic embryo) of repetitive somatic embryos was obtained with 0.98 μM 6-r-r-dimethylallylaminopurine (2-iP). For germination of somatic embryos, different PGRs, cold and desiccation treatment were
tested. Desiccation of somatic embryos at 25±1°C for 2 wk was the best treatment for germination with epicotyl elongation
and root development. Of over 256 plants regenerated, 237 (92.6%) survived. 相似文献
11.
Sandra Aparecida de Oliveira Maria de Ftima Pires da Silva Machado Alberto Jos Prioli Claudete Aparecida Mangolin 《In vitro cellular & developmental biology. Plant》1995,31(1):47-50
Summary
Cereus peruvianus seedlings were used as a source of stem explants to determine the effective conditions for inducing and maintaining callus
tissues in a state of rapid growth, as well as to obtain plants regenerated from callus cultures. Factorial combinations of
2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin in MS medium were tested, and we concluded that the 18.1µM 2,4-D and 18.6 or 27.9µM kinetin combinations were suitable for callus induction. The cactus shoots were produced from the friable callus; root elongation
occurred within 2 wk in medium without 2,4-D and with 18.6µM kinetin. This method can be used to rapidly produce manyC. peruvianus plants. 相似文献
12.
A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l–1 2,4-D and 0.5 mg l–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l–12,4-D and 0.25 mg l–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked
cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo
formation was significantly influenced by the pH of the medium and the addition of AgNO3 and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented
with 0.01 mg l–1 BAP and 0.01 mg l–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and
chromosome number.
Received: 5 November 1997 / Revision received: 9 February 1998 / Accepted: 19 February 1998 相似文献
13.
Summary
In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses
were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred
to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented
with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation
and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened
and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction
and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l−1
L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development. 相似文献
14.
The study was carried out to establish in vitro culture conditions for plant regeneration of tef, Eragrostis tef (Zucc.) Trotter. Mature seeds of two Ethiopian varieties, DZ-01-354 and DZ-01-196, were used to initiate callus cultures
on Murashige and Skoog (MS) medium with different auxins. Four- and 8-week-old calli induced on a medium with 2.0 mg/l 2,4-dichlorophenoxyacetic
acid (2,4-D) were subcultured onto various media to induce somatic embryogenesis. Compact, nodulated, embryogenic callus was
observed after transfer onto MS-callus proliferating (CP) medium. Embryogenic tissue appeared on soft and amorphous callus
and developed into somatic embryos during a subsequent subculture to MS embryo-promoting (EP) media. Various growth regulator
combinations were tested in CP and EP media to obtain a high efficiency of somatic embryo formation. The highest frequency
of calli forming somatic embryos (56.1–68.3%) was observed when CP media with 2.0 or 4.0 mg/l 2,3,5-triiodobenzoic acid were
employed and then cultures were transferred to EP media with 0.5 mg/l 2,4-D and 0.5 mg/l kinetin followed by 0.5 mg/l indole-3-acetic
acid and 0.5 mg/l N6-benzyladenine. Plant development from somatic embryos was obtained on MS medium supplemented with 1.0 mg/l gibberellic acid.
On average, 71.2% of calli displaying somatic embryos converted into plants. Regenerated plants were successfully transferred
to soil. Neither chlorophyll-deficient plants nor morphological variants were found among regenerants. All regenerated plants
were fertile.
Received: 9 May 1997 / Revision received: 25 September 1997 / Accepted: 3 January 1998 相似文献
15.
Summary Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N6-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed
from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ.
However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis
that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared
to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers
of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following
transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several
roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is
suitable for further studies on embryo development and genetic transformation of Phalaenopsis. 相似文献
16.
Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal
plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus
onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture
was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension
cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown
up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos
to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated
cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions
survived, and were morphologically identical to the mother plant. 相似文献
17.
Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA3 and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar Klages × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA3 alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA3 produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid 相似文献
18.
Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N6 medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed. 相似文献
19.
Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine 总被引:2,自引:2,他引:0
Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature
zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm−3 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm−3 α-naphthalene acetic acid (NAA) + 0.5 mg dm−3 6-benzyladenine (BA) for embryos production and 0.03 mg dm−3 NAA + 0.5 mg dm−3 BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among
six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by
flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced
during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were
stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants
regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field
grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system. 相似文献
20.
Gumerova E. A. Galeeva E. I. Chuyenkova S. A. Rumyantseva N. I. 《Russian Journal of Plant Physiology》2003,50(5):640-645
An indirect somatic embryogenesis via the development of proembryogenic cell complexes (PECC) was observed in the in vitro cultured hypocotyl explants of 4–5-day-old buckwheat (Fagopyrum esculentum Moench.) seedlings. PECC development was shown to depend on culturing conditions, including 2,4-D concentration and the period of explant exposure to 2,4-D, sucrose concentration, and explant density. The culturing protocol was designed to ensure the development of buckwheat somatic embryos in two-month period. The cytogenetic analysis demonstrated that this protocol did not affect the chromosome numbers in the regenerated plants. 相似文献