Embryogenic Cell Aggregates Research Articles

Efficient regeneration and transformation of Spathiphyllum cannifolium

An efficient and stable transformation system was developed based on embryogenic cell suspension culture by Agrobacterium tumefaciens-mediated method in Spathiphyllum cannifolium. Embryogenic calli induced from leaves on MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L thidiazuron were incubated in liquid medium with identical growth regulators for suspension cultivation. The plantlet regeneration rate of embryogenic suspension cell aggregates (ESCAs) incubated on MS medium achieved 100 %. The ESCAs were transformed using A. tumefaciens strain EHA105 harboring a vector pCAMBIA1301 containing the β-glucuronidase (gusA) reporter gene and the hygromycin phosphotransferase II (hpt II) selection marker gene. The optimized transformation conditions were as follows: infiltration of ESCAs with Agrobacterium for 10 min under 60 kPa vacuum pressure, followed by co-cultivation on medium containing 200 μM acetosyringone for 3 days. In total, 858 plants were regenerated from 1200 infected ESCAs. Analyses of randomly selected regenerated plants by GUS assays, PCR and dot blot showed that 91.30 % were transgenic. Southern blot analyses revealed that the copy number of T-DNAs integrated into the genome ranged from one to three. This newly developed transformation system provides a basis for the future development of genetic engineering techniques to improve flower color and enhance the disease resistance of peace lily.

Highly efficient in vitro tetraploid plant production via colchicine treatment using embryogenic suspension cultures in grapevine (Vitis vinifera cv. Mencía)

In vitro chromosome doubling techniques have been used to produce polyploids in several species, including table grapes, but limited research in this field has been performed using winegrapes. An efficient procedure was established to induce tetraploid grapevine plants by colchicine treatment of embryogenic cell aggregates (ECAs) grown in winegrape cultivar (cv. Mencia) suspension cultures. Colchicine treatment caused a significant decrease in the survival and embryogenic potential of the ECAs compared with the control. However, plantlets were regenerated and assessed for ploidy level by flow cytometry. Almost all plants not treated with colchicine maintained their ploidy level, whereas colchicine treatment resulted in a high variation in ploidy level. Colchicine at 0.2 % was the most effective concentration for obtaining tetraploid plantlets (25 % tetraploids). No chimeric or mixoploid plantlets were detected in this study. Based on the unique cell origin of grapevine somatic embryos, our system prevented obtaining chimeric plants. This protocol allows an increase in grapevine genetic diversity and could be a valuable tool for improving this crop. To our knowledge, this is the first report on in vitro chromosome doubling by colchicine treatment of embryogenic cell aggregates growing in grapevine suspension cultures.

Secondary multiplication of somatic embryos in banana (Musa spp. AAA) in semisolid medium: effect of the culture vessel type and sterilization method

The objective of this work was to evaluate the influence of the type of culture vessel, and sterilization method on secondary multiplication of somatic embryos of the banana cultivar Grande naine (AAA) in semisolid culture medium. From 200 µl of embryogenic cell aggregates, somatic embryos in the globular stage were formed, which were used in the experiments to compare the types of culture vessel (glass and plastic) and the form of sterilization – humid heat (autoclave) and chemical (Vitrofural®). The evaluations of the number of somatic embryos that were multiplied were done at 15 and 20 days of culture. The results obtained with respect to the multiplication phase were highly promising, 40.25 and 80.15 ± 5.0 somatic embryos at 15 and 20 days of culture in glass vessel with plastic cover and using the autoclave as the sterilization method. The results presented in this work open the door for the use of this phase in the mass propagation protocol of this crop via somatic embryogenesis.

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine

Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7% ± 1.3% for anthers and 4.8% ± 2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9% ± 4.8% to 83.9% ± 8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7% ± 9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.

Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells.

Ploidy instability in long-term in vitro cultures of Coffea arabica L. monitored by flow cytometry

Somatic embryogenesis of Coffea arabica L. has been mainly carried out in liquid medium for clonal and mass propagation of elite lines. This in vitro system involves suspension cultures of embryogenic aggregates, with high multiplication rate and unorganized growth. These characteristics are linked to the occurrence of somaclonal variation (SV), especially considering that cell aggregates are usually maintained for long periods in media supplemented with the synthetic auxin 2,4-dichlorophenoxyacetic acid. Because SV detection has been considered essential in in vitro tissue cultures, flow cytometry (FCM) was applied to verify ploidy instability in embryogenic cell aggregates of C. arabica, throughout successive subcultures. FCM allowed us to detect the occurrence of non-true-to-type aggregates in all samples collected after approximately 4 months in liquid medium. These aggregates showed octaploid and/or aneuploid cells, with DNA ploidy level being corroborated by chromosome counting. Considering this result, we recommend a limit of <4 months for true-to-type mass propagation of C. arabica cell aggregate suspensions. Besides, FCM was an important tool to detect SV at an early stage of tissue culture in this species, proving to be very useful for quality control in clonal propagation and in the introduction of somaclones to breeding programs.

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma2 and PCM, significantly affected the number of microcalli produced, which was 90 × 103 per Petri dish on Ma2 with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 103 per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma2 and the frequency of embryogenic cell aggregates was higher among microcalli on Ma2-medium. However, no cell division was observed in protoplasts cultured on Ma2 in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.

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.

A protocol for consistent, large-scale production of fertile transgenic rice plants

A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates, when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure, transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0 plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies.

Somatic embryogenesis in liquid cultures of a tetraploidAlstroemeria

A simple and efficient procedure was developed for regeneration of a tetraploid cultivar ofAlstroemeria (A. pelegrina x A. psittacina) via somatic embryogenesis in liquid cultures. Embryogenic callus induced from mature zygotic embryos, cultured on MS medium supplemented with 40 µM NAA and 20 µM kinetin, was used as inoculum for liquid cultures. Pre-culture of the callus on MS medium supplemented with 80 µM NAA for two days was essential for cell proliferation in the liquid medium. Embryogenic cell aggregates, obtained by sieving through a 750 µm nylon mesh, continued to proliferate in media containing 10 or 20 µM NAA and 10 or 20 µM kinetin. When transferred to a semi-solid half strength MS medium supplemented with casein hydrolysate, cell aggregates successfully differentiated into plantlets which later grew to maturity under greenhouse conditions.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens

Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Advanced propagation systems for biomass species: a model system based on sweet potato

A method for somatic embryo production of sweet potato ( Ipomoea batatas (L.) Lam.) has been developed. The first step of somatic embryogenesis was to obtain embryogenic callus from 0.2 mm apical domes with 1–2 leaf primordia on medium containing 10 M 2,4-dichlorophenoxyacetic acid (2,4D). Selective proliferation of embryogenic callus has been obtained on solid media containing 10 μM 2,4-D and 1 μM benzylaminopurine (BAP) and in liquid media containing 5 μM 2,4-D. The fraction of suspension cultures larger than 710 μM was commonly used to produce embryos. Cultures were recultured every 2 weeks in liquid media and every 6 weeks on agar media. The formation of embryos was triggered by transferring embryogenic calli or cell aggregates from nutrient media containing 2,4-D to fresh media without 2,4-D. Late torpedo and cotyledonary stage embryos had the highest potential for plant formation. The automated production of synthetic seed in combination with fluid drilling technology could render economically feasible the production of sweet potato for biomass.

Improved Production Procedures for Somatic Embryos of Sweetpotato for a Synthetic Seed System

Embryogenic callus of sweetpotato [Ipomoea batatas (L.) Lam.] disassociates in liquid medium to form a heterogeneous population of embryogenic and nonembryogenic cell aggregates of varying sizes. To improve embryo production, such cell aggregate populations were obtained by manually fragmenting calli 5 to 10 mm in size into liquid medium. The resulting suspensions were analyzed and the embryogenic fraction identified. The percentage of embryogenic aggregates and the percentage of aggregates forming embryos decreased with decreasing aggregate size. Thus, 76% of the 710- to 1000-μm-diameter aggregates but only 14% of the 180- to 250-μm aggregates had embryogenic potential. However, only 20% of the 710- to 1000-μm aggregates and only 2% of the 180- to 250-μm aggregates actually formed embryos. Conversely, embryogenic callus and embryo production per milligram of cultured embryogenic callus increased quadratically with decreasing aggregate size. Individual torpedo-stage embryos were produced from cell aggregates 180 to 250 μm in size.

Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63μm mesh openings and the composition of each fraction was determined. Cellular units larger than 355 μm were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 μm. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 μm fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 μM 2,4-D. The 63–125 μm fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.

Plantlet differentiation from callus protoplasts induced from Citrus embryo

Embryos from young seeds of three Citrus species, ‘Washington’ navel orange ( Citrus sinensis (L.) Osbeck), yuko ( C. yuko Hort. ex. Tanaka) and ponkan ( C. reticulata Blanco), were cultured on a Murashige and Skoog medium with 0.16 M sucrose, 0.5 g l −1 malt extract and 50 μM kinetin. One to three months after inoculation, whitish friable callus was produced from the hypocotyl region of the embryos. After sub-culture for about 2 years, those callus lines maintained an embryogenic capacity. Protoplasts isolated from the callus produced embryogenic cell aggregates in all media tested. Globular and torpedo-shaped embryoids were also produced in the media with 1 μM zeatin or without any growth regulators. Most of them regenerated to entire plantlets after transplanting onto a Murashige and Skoog agar medium containing 1 μM GA 3 and 0.06 M sucrose.

Ultrastructural study of different types of callus from leaf expiants ofAesculus hippocastanum L.

The cell ultrastructure in three types of callus obtained from leaf explants ofAesculus hippocastanum L. has been studied. Remarkable differences have been shown between the cells of the forerunner E1 callus and those of the callus arising from it, according to the culture conditions. The peculiar characteristics of E1 are the scarcity of intercellular spaces and the occurrence of autophagic vacuoles in the cells. An embryogenic friable callus (E2) is formed in time when E1 is maintained on solid culture medium. The E2 cells show cytological features typical of a higher metabolic level and contain starch. Diffused middle lamella digestion leads to the detachment of small embryogenic cell aggregates consisting of vacuolated parenchymatous-like cells and small meristematic cells which may be regarded as embryoids initials. Shaking E1 in the same liquid medium and subsequent culture on solid medium lead to the differentiation of a non-embryogenic callus (NE), whose cells are very large and highly vacuolated, devoid of starch and with organelle-rich cytoplasm. The NE callus shows a high degree of growth, but does not attain embryogenic competence in time.

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.