Embryogenic Cell Suspension Cultures Research Articles

Defence responses induced in embryogenic cultures of Norway spruce by two fractions of Gremmeniella abietina mycelia

SummaryWe have examined defence responses in embryogenic cell suspension cultures of Norway spruce (Picea abies L.) elicited by intracellular protein and wall fractions (PF and WF, respectively) prepared from mycelia of the fungus Gremmeniella abietina (Lagerberg), focusing on changes in levels of phenolics (soluble and cell wall‐bound), polyamines (free and soluble conjugates) and activities of enzymes that catalyse important steps in their metabolism: phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) and diamine oxidase (DAO, EC 1.4.3.6), respectively. Treatment with both preparations induced increases in the spruce cells total contents of phenolic acids and polyamines but they responded more rapidly to WF than to PF. The contents of soluble (especially benzoic acid derivatives) and cell wall‐bound (especially ferulic acid) phenolic acids and soluble conjugates of putrescine (mainly amides of hydroxycinnamic acids), started to increase relative to controls (C) within 4 h of the addition of the WF preparation and, more weakly, within 8 h of exposure to the PF preparation. These changes correlated with the induction of PAL activity in spruce cells at 4 and 8 h following the addition of WF and PF preparations, respectively. Increases in the spruce cell’s DAO activity were also detected after just 4 h exposure to WF, and after 24 h exposure to PF. These results demonstrate that both intracellular protein and cell wall fractions prepared from A. abietina mycelium elicit changes in the metabolism of phenylpropanoids and polyamines, substances that are known to be heavily involved in the defence responses of plants to pathogens.

Expression of hepatitis B small surface antigen in Santalum album embryogenic cell suspension cultures

Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated from the colony with expression of 11.09 μg(HBsAg) g−1(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed the expression of 19.95 μg(HBsAg) g−1(f.m.).

An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus

A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100μM acetosyringone for 5days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6M BH3 and 0.15M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

Improvement of bananas for black sigatoka and panama disease resistance through genetic manipulation

Bananas are a staple food in Eastern Africa, with 25.3% of the total world production. The production is, however, threatened by the presence of several diseases, of which the fungal diseases black sigatoka and Panama disease are the most important. With the development of embryogenic cell suspension cultures, the isolation of protoplasts therefrom and their successful regeneration, an invaluable vegetative material for genetic manipulation of bananas became available. The discovery of new types of antifungal proteins (AFP’s) and the cloning of their encoding genes provide a source of resistance to fungal diseases that can be introduced into plant cells by genetic engineering. Transformation techniques are currently under investigation. Electroporation of banana protoplasts have resulted in transient expression frequencies of more than 1% as visualized by the action of the β-glucuronidase (GUS) marker gene. Experiments with particle bombardment on cell suspensions have resulted in transient expression frequencies up to 3300 blue spots/0.5 ml cells, and stable transformants have also been selected. Optimisation of the cell suspension culture technique and transformation methodology is in progress.

High frequency plant regeneration system for Nymphoides coreana via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l−1 of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l−1 with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l−1 of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.

Cryopreservation of embryogenic cell suspensions of Catharanthus roseus L. (G) Don.

An efficient cryopreservation protocol was established for embryogenic cell suspension cultures of Catharanthus roseus. This involved a vitrification-based cryopreservation method wherein embryogenic cells were exposed to a preculture/pretreatment medium prior to their immersion in liquid nitrogen. These cell suspension cultures were first initiated from friable embryogenic callus derived from hypocotyls of C. roseus on a medium containing 4.52 μM 2,4-Dichlorophenoxy acetic acid (2, 4-D). Among different sucrose (0.09–0.6 M) and sorbitol (0.2–0.6 M) levels evaluated during preculture, 0.4 M sucrose promoted highest cellular regrowth. Whereas, among pretreatments Dimethyl sulphoxide (DMSO) (5 or 10%) and glycerol (5 or 10%) at six different levels either alone or in combinations (PT 1–PT 6), the cryopreservation treatment combinations of either 0.4 M sucrose, 5% DMSO, and 5% glycerol (PT-5) or 0.4 M sucrose and 5% DMSO (PT-1) resulted in the highest frequency of viability of embryogenic cultures. However, the PT-1 treatment produced highest number of cell colonies (10.06 ± 0.55) following reculture of cryopreseved cultures. All calluses regrown in an optimized medium, containing 6.62 μM 6-benzyladenine (BA) and 5.37 μM α-naphthaleneacetic acid (NAA), resumed normal growth, and produced somatic embryos similar to those from non-frozen embryogenic cultures. These somatic embryos were converted into regenerated plantlets, and all plantlets exhibited normal morphology.

Long-term suspension cultures of cucumber (Cucumis sativus L.) with high embryogenic potential

Cucumber (Cucumis sativus L.) cytokinin-independent embryogenic cell suspension cultures were derived and maintained for more than 3.5 years without losing the embryogenic potential. The preparation and the characteristics of the cucumber embryogenic cell suspension possess many similarities to that of carrot. The cultures were induced from hypocotyl explants of in vitro grown cucumber plants in liquid MS media containing 2,4-dichlorophenoxyacetic acid as the sole growth regulator during 6 weeks and they contained a heterogeneous array of several different types of single cells and cell clusters (PEMs). The established cell suspensions were subcultured in 1-week interval, while the inoculation density was optimized to 2.0 × 105 cells ml−1 using cell viability as a marker. Somatic embryos were obtained after the transfer of the proembryogenic masses to a hormone-free semisolid MS medium with a frequency of 388 ± 57 somatic embryos per 1 ml of packed cell volume of the established cucumber embryogenic culture within 7 days. The frequency of normal somatic embryos with two cotyledons was found to be 78%. Such embryos possessed the potential of spontaneous maturation and the embryo conversion rates were 87%. The yield of normally growing plants was much higher compared with that previously described for cucumber systems. Somatic embryo-derived plants were successfully transferred to the greenhouse where they flowered and fruited.

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV) was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.

In vitro plant regeneration from embryogenic cell suspension culture of Astragalus chrysochlorus (Leguminoseae)

In vitro plant regeneration was achieved from embryogenic cell suspension culture of Astragalus chrysochlorus. When 30-day-old aseptically grown seedlings were cultured on Murashige and Skoog (MS) medium containing 0.1 mg/l -naphthaleneacetic acid (NAA) plus 1.0 mg/l 6-benzyladenine (BA), friable callus was formed within two weeks from the mesocotyl of the seedling. After three weeks, proliferated actively growing calli were transferred to MS liquid medium containing 2,4- dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA) or NAA and subcultured at two week intervals. After two weeks, induction of somatic embryos up to the torpedo stage occured at all tested concentrations of 2,4-D, IAA or NAA. Somatic embryos developed only in MS medium containing 0.5 mg/l IAA within two weeks and 2% of globular embryos were developed into the cotyledonary stage embryos. Eighty one percent of somatic embryos cultured in MS medium supplemented with 0.5 mg/l IAA were found to be diploid by flow cytometric analysis. Plantlet propagation was achieved on half strength MS liquid medium supplemented with 3% (w/v) sucrose after four weeks of culture. After a month on half strength MS medium [1.5% (w/v) sucrose and 0.8% (w/v) agar] 29 of 71 shoots developed into rooted plantlets.

Plant regeneration from suspension cells induced from hypocotyls derived from interspecific cross Alstroemeria pelegrina × A. magenta and transformation with Agrobacterium tumefaciens

Embryogenic cell suspension cultures were established using the ovule culture of an interspecific cross, Alstroemeria pelegrina var. rosea × A. magenta. Ovules harvested 14 days after pollination were cultured on Murashige and Skoog (MS) medium without plant growth regulators (PGRs); calli were produced on the hypocotyl surface in germinating zygotic embryos. Suspension cells were induced from the calli by using liquid MS media containing 2,4-dichlorophenoxyacetic acid or 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (picloram). Adventitious embryos developed from the suspension cells on half-strength MS medium supplemented with 0.5 mg l−1 of both α-naphthaleneacetic acid and N6-benzylaminopurine; they grew into plantlets on the same medium. The plantlets formed rhizomes following transfer to half-strength MS medium without PGRs, and acclimatized plants were easily established. Subsequently, Agrobacterium-mediated transformation system was applied. The suspension cells were co-cultivated with A. tumefaciens strain EHA101/pIG121Hm or LBA4404/pTOK233, both of which contain neomycin phosphotransferase II, hygromycin phosphotransferase and intron-containing ß-glucuronidase (intron-GUS) genes. Seven days after co-cultivation, the cells were subjected to GUS assay; staining was most pronounced in the cells subcultured in a picloram-containing liquid medium and co-cultivated with EHA101/pIG121Hm. The co-cultivated cells were transferred to the MS medium containing picloram and 20 mg l−1 hygromycin; 1 month later, several hygromycin-resistant callus lines showing GUS activity were obtained. Transgenic plants were obtained through our plant regeneration system, and foreign gene insertion into the regenerated plants was confirmed by polymerase chain reaction.

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l−1, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l−1 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.

Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album.

Methyl jasmonate induced overproduction of eleutherosides in somatic embryos of Eleutherococcus senticosus cultured in bioreactors

This study was concentrated on the production of eleutherosides and chlorogenic acid in embryogenic suspension cultures of Eleutherococcus senticosus by exposing them to different concentrations (50-400 µM) of methyl jasmonate (MJ) during the culture period. In the bioreactor cultures, eleutheroside content increased significantly by elicitation of MJ, however, the fresh weight, dry weight and growth ratio of embryos was strongly inhibited by increasing MJ concentrations. The highest total eleutheroside (7.3 fold increment) and chlorogenic acid (3.9 fold increment) yield was obtained with 200 µM MJ treatment. There was 1.4, 3.4 and 14.9 fold increase in the eleutheroside B, E, and E1 production respectively with such elicitation treatment. These results suggest that MJ elicitation is beneficial for eleutheroside accumulation in the embryogenic cell suspension cultures.

ISOLATION AND CULTURE OF GRAPE PROTOPLASTS FROM EMBRYOGENIC SUSPENSION CULTURES AND LEAVES OF VITIS VINIFERA AND VITIS ROTUNDIFOLIA

ISHS International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species ISOLATION AND CULTURE OF GRAPE PROTOPLASTS FROM EMBRYOGENIC SUSPENSION CULTURES AND LEAVES OF VITIS VINIFERA AND VITIS ROTUNDIFOLIA

Triploid banana cell growth phases and the correlation of medium pH changes with somatic embryogenesis in embryogenic cell suspension culture

Embryogenic cell suspensions of Musa AAA and AAB genomic groups were cultured in a maintenance culture medium for 17 generations (lasting for 238 days). The cell growth phases and medium pH changes were also observed correspondingly. Three major growth phases of AAA genomic group have been focused, namely cell releasing, proliferation and globularization phases. During almost all the subculture generations the cell stocks of AAB ‘Raja’ were continuously characterized by proliferating cell aggregates while the globularization phase occurred only for short duration. The medium acidity levels of the cell stocks of AAA ‘Pei-Chiao’ and ‘Robusta’ were commonly scattered in a wider range of pH 3.3–5.3, while the AAB ‘Raja’ were deviated in a narrow range of pH 4.0–4.6. After subculture, culture medium showed biphasic pH changes, which were drastic pH falls followed by an auto-regulated steady-state level. The steady-state pH values in each of the three growth phases (i.e. cell releasing, proliferation and globularization phases) were of 3.3–4.0, 4.0–4.8 and 4.8–5.3 respectively. Morphological bipolarity and the efficiency in the formation of somatic embryos have been thoroughly discussed. Reported research indicates that the condition of pH below 4.6 may prevent the development of embryogenic cells towards polar growth.

Development of an embryogenic suspension culture of bitter melon (Momordica charantia L.)

Abstract We have optimized a system for the somatic embryogenesis via embryogenic suspension cultures in bitter melon (Momordica charantia L.). Friable calli could be induced in 30-day-old leaves on semi-solid MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.] medium supplemented with 1.0 mg/l 2,4-D. Large number of globular embryos (24.6%) were noticed when the calli was subcultured in liquid medium containing 1.5 mg/l 2,4-D. The complete removal of 2,4-D in the later stages of culture, stimulated their further development to heart and torpedo stages. Microscopic examination revealed the ontogeny of somatic cell development via the formation of cell clusters, which then enlarged to pro-embryos, and gave rise to heart and torpedo stages within a period of 2 weeks. Somatic embryos successfully germinated on agarified MS medium with no additional growth regulators. An effect of media, other components and stimulating factors such as carbohydrates, amino acids has also been evaluated for somatic embryogenesis. The full strength MS medium containing 50 mg/l PVP and 40 mg/l glutamine was effective to achieve a high frequency of somatic embryo induction, maturation and further development. An average of 6.2% young plants was achieved from friable callus and was phenotypically normal. To our knowledge, there is no published report on somatic embryogenesis of bitter melon (M. charantia L.) via embryogenic callus or cell suspension cultures. These results are likely to facilitate genetic transformation of bitter melon.

Transgenic Phalaenopsis plants with resistance to Erwinia carotovora produced by introducing wasabi defensin gene using Agrobacterium method

Transgenic plants over-expressing wasabi defensin gene were successfully produced in Phalaenopsis orchid by Agrobacterium-mediated transformation method. Embryogenic cell suspension culture of Phalaenopsis Wataboushi '#6.13' was infected with A. tumefaciens strain EHA101 carrying a plasmid containing wasabi defensin gene and selectable marker nptII, hpt genes. Plantlets were regenerated through somatic embryogenesis from the calli selected on hygromycin- containing medium. Transformation of plantlets with wasabi defensin gene was confirmed by PCR analysis. Southern blot analysis confirmed successful integration of 1-4 copies of the gene. Production of the 5 kDa wasabi defensin protein with varying levels was confirmed in the leaf extracts of different transgenic clones using Western blot analysis. Most of the transgenic plants showed strong resistance to Erwinia carotovora, which causes soft rot disease in the control plant. These results suggest the usefulness of this gene for conferring the resistance to various diseases of Phalaenopsis and possibly other orchids.

Establishment and plant regeneration of somatic embryogenic cell suspension cultures of the Zingiber officinale Rosc.

Somatic embryogenic cell suspension cultures of four ginger cultivars were established. Somatic embryogenic calli were induced from ginger shoot tips on MS agar medium supplemented with 1.0 mg l −1 2,4-D and 0.2 mg l −1 Kn, which contained only half concentration of NH 4NO 3. Rapid-growing and well-dispersed suspension cultures were established by subculturing this kind of callus in the same liquid MSN medium. The suspension cultures (about 1–2 mm in diameter) were placed on the MSN agar medium for callus proliferation. Thereafter embryogenic callus (1.5 cm 2) was transferred to solid media (MS + 0.2 mg l −1 2,4-D + 5.0 mg l −1 BA + 3% sucrose + 0.7% agar). Somatic embryos produced shoots and roots, and shoots developed into complete plantlets on solid MS medium supplemented with 3.0 mg l −1 BA and 0.1 mg l −1 NAA. The relationship between the DW of suspension cultures and pH changes in medium is also discussed. The suspension cultures still kept their vitalities after subculture for 8 months.

Establishment of embryogenic cell suspension cultures of garlic (Allium sativum L.), plant regeneration and biochemical analyses

Embryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1-4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1-0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared.

Establishment of embryogenic cell suspensions and plant regeneration of the dessert banana ‘Williams’ (Musa AAA group)

Summary‘Williams’ is one of the most important dessert banana cultivars in the World. Improvement through genetic engineering depends on the development of embryogenic cell suspension (ECS) cultures. Yellow, compact meristematic globules and yellow-whiteish, friable embryogenic calli were induced from scalps (i.e., the top layer of highly proliferating meristem cultures). About 10% of induced explants gave rise to embryogenic calli. Embryogenic calli were used subsequently to establish ECS cultures. After 3 months of selection and sub-culturing, homogeneous ECS cultures with a high regeneration capacity were obtained. More than 80% of the cell culture consisted of embryogenic cell aggregates. Regenerable somatic embryos emerged about 3 weeks after the ECS was plated onto regeneration medium. Their weight increased 4.8- to 13-fold after 8 weeks of culture. The number of somatic embryos regenerated per ml settled cell volume (SCV) of embryogenic cells (i.e., somatic embryo recovery frequency) ranged between 0.92 – 2.17 105, depending on incubation and regeneration conditions. The conversion frequency from somatic embryo to plant varied between 42.5 – 55.5%, and was independent of regeneration conditions.