Embryogenic Cell Suspension Research Articles

Propagation of pineapple (Ananas comosus L.) embryogenic cell suspension is regulated by LEAFY COTYLEDON1 gene AcoLEC1–1

Pineapple (Ananas comosus L.) is an important tropical fruit. Traditional breeding methods take about 15 years, but transgenic breeding using embryogenic cell suspension (ECS) can significantly shorten this cycle. Four different calli types were produced using the pineapple crown's middle stem part as an explant: water-like, solid, gemmiform, and crisp. The fastest multiplication was observed in suspension cultures obtained from the loose, crispy, granular, white, or yellow callus. The highest cell density in the liquid medium was achieved with MS+1.5 mg/L+4 mg/L6-BA+300 mg/L hormone combination. Inclusion of 11 mmol/L 2-morpholinoethanesulfonic acid (MES) stabilized the medium pH. The optimum sucrose concentration for pineapple propagation using ECS was 5 g/L. The expression of seven proteins in ECS in the medium containing 5 g/L sucrose was more than 5.0 times higher than in the medium containing 3 g/L. The expression of transcription factor NFYB/HAP3 (or LEAFY COTYLEDON1, LEC1) was the highest in the medium. The most prevalent AcoLEC1–1 transcripts were found in the embryogenic callus, and their patterns correlated well with ECS volume. AcoLEC1–1 played a key role in the process of sucrose-regulated ECS propagation.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

An efficient embryogenic cell suspension culture system through secondary somatic embryogenesis and regeneration of true-to-type plants in banana cv. Sabri (silk subgroup AAB)

Vinblastine synthesis under the influence of CaCl2 elicitation in embryogenic cell suspension culture of Catharanthus roseus

Vinblastine synthesis under the influence of CaCl2 elicitation in embryogenic cell suspension culture of Catharanthus roseus

Biotechnological and molecular genetic methods in grape breeding

The development of new grape varieties with improved quality and resistance against environmental factors using biotechnological methods is a pressing task in the context of import substitution policy implementation. Due to the high heterogeneity of grape varieties, techniques for regenerating plants from embryogenic callus by somatic embryogenesis have been developed only for a small number of genotypes. This work presents a versatile approach to the development of globular, heart, and torpedo embryos in various grape genotypes. Experiments were carried out using cultures of colchicine embryogenic cell suspensions and somatic globular embryos of the E-342 hybrid form in liquid media. The developed methodology allows the protocols of embryogenesis and polyploidy induction to be effectively improved. The versatility of the presented methodology for polyploidy induction and somatic embryogenesis in grape plants is ensured by a fourfold reduction in the concentration of inorganic nitrogen and vitamins in the NN and PG media and the addition of organic nitrogen and other biological active substances at the stages of embryogenic callus development, colchicination, and development of somatic embryoids in liquid media. The methods of indirect ploidy determination by the properties of the grape leaf epidermis were studied and implemented. Nine promising somaclones were preliminary selected based on a set of four features. It was established that full-fledged transgenic grape plants can only be obtained by the method of transformation, which involves the production of embryogenic callus from a leaf explant, its subsequent passage into a liquid culture to obtain a liquid embryo culture, and the transformation of this culture. In total, 300 leaf explants of the Libya variety and 1 100 globular embryos of the Podarok Magaracha variety were transformed. As a result, six callus kanamycin-resistant lines of the Libya variety and six lines of transgenic plants of the Podarok Magaracha variety were selected. Following genotyping based on nine nuclear microsatellite loci, seven unknown forms with a seedless phenotype were identified. Among the population of hybrid seedlings, hybrids having alleles associated with seedlessness were revealed using the VMC7f2 and p3_VvAGL11 MAS markers.

Cryo-derived plants through embryogenesis showed same levels of vinblastine and vincristine (anticancer) in Catharanthus roseus and had normal genome size

Cryopreservation of rare plant materials is an important approach for preserving germplasms and is a good added concept to tissue banking. The preservation of embryogenic cell suspensions is even more valuable as the tissues facilitate in producing millions of embryos, plantlets and generates transgenics en masse. Catharanthus roseus is a medicinally important plant that produces a variety of anticancerous phytocompounds and needs conservation of alkaloid producing cell lines. In this study, embryogenic tissue banking has been attempted in C. roseus by the two-step cryopreservation method combining cryoprotection and dehydration. Prior to plunging into liquid nitrogen (LN), the tissues were exposed to osmotic—and cryoprotective agents. Two osmotic agents (sugar and sorbitol) and three cryoprotective compounds, polyethylene glycol (PEG), dimethyl sulfoxide (DMSO) and glycerol were used at varying concentrations to protect cells from freezing damages. Both sucrose and sorbitol increased callus biomass post-cryopreservation; the influence of sucrose was however, more prominent. Embryogenic tissue treated in medium with 0.4 M sucrose for 2 days followed by 5% PEG for 2 h showed maximum viability before (83%) and after (55%) cryopreservation, high regrowth percentage (77%) and produced an average 9 cell colonies per Petri dish. Additionally, dehydration (1–5 h) was tested to reduce water content for improving viability and regrowth of cryopreserved embryogenic cells. Among the various tested cryoprotective conditions, the highest (72%) viability was observed following the combination of treatments with 0.4 M sucrose (2 days),10% PEG (2 h) and dehydration (2 h). Maximum regrowth percentage (88%) and 12 colonies/petri dish was noted in combination of 0.4 M sucrose + 5% PEG. The cryopreserved calli differentiated into somatic embryos (52.78–54.33 globular embryos/callus mass) in NAA (0.5 mg/l) and BAP (0.5–1.0 mg/l) added media. Plantlets were successfully regenerated from cryopreserved tissue and the 2C DNA was estimated through flow cytometry. The genome size of cryopreserved regenerant was 1.51 pg/2C, which is similar to field-grown Catharanthus plants. Vinblastine and vincristine levels were nearly the same in mother plant’s and frozen (cryopreserved) leaf tissue. The post cryopreservation embryogenesis protocol may be used for continuous production of plants for future applications.

Development of fusarium wilt resistant mutants of Musa spp. cv.Rasthali (AAB, Silk subgroup) and comparative proteomic analysis along with its wild type.

Induced mutagenesis using embryogenic cell suspension (ECS) explants with toxin based screening is an effective tool to create non-chimeral Fusarium wilt resistant mutants in banana. Global proteomics unravel the molecular mechanism behind resistance. Race 1 of Fusarium wilt is a serious threat to Musa spp. cv.Rasthali (AAB, Silk subgroup) which is a choice variety traditionally grown in most of the south East Asian countries. Resistant gene introgression into susceptible varieties through conventional breeding has several limitations and the predominant ones being sterility and long generation time. Under such circumstances, induced mutagenesis combined with toxin based in vitro screening remains as the viable alternative for the development of fusarium wilt resistant Rasthali. Therefore, induced mutagenesis was attempted by using ethylmethane sulfonate (EMS) in embryogenic cell suspension (ECS) of Rasthali followed by in vitro screening for fusarium wilt resistance using new generation toxins and pot screening through challenge inoculation with Foc race 1. This ultimately resulted in the identification of 15 resistant lines. Global proteomic analysis in one of the resistant mutant lines namely NRCBRM15 and its wild type revealed 37 proteins, of which 20 showed differential expression. Out of 20 proteins, nineteen were significantly abundant in NRCBRM15 and only one was abundant in wild Rasthali. A total of nine genes based on protein expression were further validated using quantitative real time polymerase chain reaction (qRT-PCR). Annotation results revealed that some of the genes namely Enolase, ATP synthase-alpha subunit, Actin 2, Actin 3,-glucanase, UTP-glucose-1-phosphate uridylyltransferase, Respiratory burst oxidase homolog, V type proton ATPase catalytic subunit A and DUF292 domain containing protein are involved in diverse functions such as carbohydrate metabolism, energy production, electron carrier, response to wounding, binding proteins, cytoskeleton organization, extracellular region, structural molecule and defense.

Up-regulation of a stress-responsive endochitinase VvChit-IV in grapevine cell cultures improves in vitro stress tolerance.

Chitinases are pathogenesis-related proteins, which play an important role in plant growth regulation, defense mechanism, and stress tolerance. Embryogenic cultures from Vitis vinifera cv. Tempranillo exposed to in vitro stress exhibited the expression of an extracellular class IV endochitinase VvChit-IV. Phylogenetic and conserved motif analyses provided insights into the evolutionary relationships of chitinases. A computation-based investigation showed conserved domains and illustrated a chitin-binding site for chitin cleavage with a catalytic domain of glycoside hydrolase. Interestingly, gene expression pattern showed a differential expression of VvChit-IV associated with embryonic stress response to in vitro conditions. In response to in vitro stress, transcript level of VvChit-IV increased in embryogenic calli and cell suspensions and peaked at 1.5 and 3 folds, respectively, when compared to an internal reference gene. Evidence of tissue culture stress-induced endochitinase was reported here for the first time indicating that in vitro stress could mitigate elicitor application to induce chitinase expression and can stimulate an immune response against abiotic constraints. Data showed that up-regulation of VvChit-IV was associated with a substantial increase of H2O2 and proline without significant change in malondialdehyde content suggesting that the H2O2 signaling network might trigger a priming effect to boost the defense response against environmental stress. Endochitinase activation in plant stress mitigation was thus highlighted to improve tolerance through attenuation of oxidative stress. This study revealed that the grapevine endochitinase is promising for enhancing coping-oriented adaptation and abiotic stress tolerance, which gives new insights into its feasibility for use in cross-tolerance and crop improvement.

Banana somatic embryogenesis and biotechnological application

As one of the most important economic crop for both staple food and fruit widely cultivated in tropics and subtropics, banana (<italic>Musa</italic> spp.) is susceptible to a plethora of abiotic and biotic stresses. Breeding cultivars resistant to abiotic and biotic stressors without adverse effects on yield and fruit quality are the objectives of banana improvement programs. However, conventional breeding approaches are time-consuming and severely hampered by inherent banana problems (polyploidy and sterility). Therefore, genetic transformation is becoming increasingly popular and can provide rapid solutions. Numerous efforts have been made to develop superior banana cultivars with better resistance to abiotic and biotic stresses and optimum yields using genetic modification strategies. Somatic embryogenesis (SE) through embryogenic cell suspension (ECS) cultures is an ideal recipient system for genetic transformation in banana. The purpose of this paper is to review the current status of banana somatic embryo research, clarify the way of banana somatic embryo induction and culture, and summarize the main influencing factors in the process of somatic embryogenesis. At the same time, their applications in breeding technologies such as cryopreservation, protoplast culture, genetic transformation and gene editing were also summarized, in order to provide reference for the research and practical application of banana somatic embryogenesis in the future.

Somatic Embryogenesis in Banana (Musa spp.).

Bananas (Musa ssp.) are among the world's most important crops. In terms of gross value of production, they are the fourth most important global food crop and have an important socioeconomic and ecological role. Somatic embryogenesis (SE) is a developmental process, in which somatic cells differentiate into embryos which eventually develop and regenerate into plants. SE is exploited to generate a large quantity of very high economic value, genetically identical and disease-free plantlets. In bananas, the use of shoot apexes of axillary buds to induce SE resulted an alternative for plant regeneration through embryogenic cell suspension (ECS). The protocol has been scaled up to commercial laboratories for tissue culture (biofactories) for production of planting materials. The genetic stability of regenerated plants and high yields obtained under field conditions demonstrate the feasibility of scaling up this biotechnological protocol and adapting it to commercial production of planting materials to mitigate a critical bottleneck in the value chain of this important crop.

RNAi Mediated Silencing of ftf1 Gene Enhances Resistance in Banana to Fusarium Wilt

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is a devastating disease of banana. Race 1 of Fusarium wilt is a serious threat to Musa spp. cv. Rasthali (AAB, Silk subgroup), which is a choice variety traditionally grown in most of the South East Asian countries. The investigation was an attempt to study whether RNAi-mediated expression of small interfering RNAs (siRNAs) targeted against ftf1 (fusarium transcription factor1) gene in transgenic banana could impart resistance against Foc race 1. Partial sequences comprising sense and antisense sequences of ftf1 gene were assembled as siRNA construct in a binary vector background and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium mediated genetic transformation. Presence of RNAi construct in transgenic banana plants were confirmed by polymerase chain reaction assay. Forty- six transformed lines derived from RNAi-FTF1 were found to be free of external symptoms of Foc after 3 months of challenge inoculation. Seventeen transgenic lines continued to resist the Foc race 1 even after six months of post inoculation. The present study confirmed that the host induced RNAi-mediated gene silencing of a fungal gene could confer resistance against Foc in banana.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Genetic transformation of apical meristematic shoots in the banana cultivar ‘Williams’

Bananas and plantains (Musa spp.) are among the most critical socioeconomic crops globally, being a staple food for millions of people in the tropics and an essential component for the export market, including the subtropics. Besides conventional breeding, genetic improvement of bananas and plantains could be performed through genetic engineering and new breeding techniques. Furthermore, plant tissue culture is essential for these technologies, including developing embryogenic cell suspensions and in vitro plants. The transient and stable genetic transformation could be performed from in vitro plants, shortening Musa transgenic lines development compared to genetic transformation while using embryogenic cell suspension. In this study, a genetic transformation protocol was established from banana apical meristems for the ‘Williams’ cultivar (genotype AAA). The protocol was based on the co-cultivation of the explants (whole in vitro plants or bisected meristematic tissues derived from in vitro plants) with Agrobacterium tumefaciens strain LBA4404 harboring two binary vectors denominated pLVCIBE1 (cassette: MabHIPP promoter::luc2::Tnos, P35S::hpt::Tnos) and pLVCIBE2 (cassette: P35S::luc2::Tnos, P35S::hpt::Tnos), independently. The stable genetic transformation was obtained by subculturing in vitro banana plants in selection medium (12.5µg/mL of hygromycin) for 8 weeks from bisected meristematic tissue transformation. Genetic transformation was confirmed in vivo with the use of the luciferase reporter gene system. Furthermore, PCR was performed on DNA extracted from leaves of regenerated transgenic in vitro plants after 8 weeks of selection, confirming stable genetic transformation. Therefore, genetic transformation was achieved in the apical meristematic tissue of in vitro banana plants with co-cultivation of Agrobacterium tumefaciens.

Genome-scale analyses of polyketide synthases in banana: Phylogenetics and expression profiling forecast their candidacy in specialized metabolism.

Plant type III polyketide synthases (PKSs) are associated with various functions in plant growth, development and defense by providing a multitude of polyketide scaffolds for diverse specialized metabolic pathways (SMPs). To decipher banana PKSs involved in specialized metabolism, genome-wide comparative analyses were conducted with A (Musa acuminata) and B (Musa balbisiana) genomes of banana. Both genomes retained eight chalcone synthases (CHSs), seven curcumin synthases (CURSs), three diketidyl-CoA synthases (DCSs) and one anther specific CHS (ASC). Segmental (42%) and tandem (37%) duplication events majorly flourished the banana PKS family. Six of 19 PKSs of A genome (designated as MaPKSs) showed relatively a higher expression in the root, corm, sheath, leaf and embryogenic cell suspension (ECS) of banana. To determine the defense response of MaPKSs and to highlight their candidacy in various SMPs, expression profiling was conducted by qPCR in ECSs treated with 100/200μM of jasmonic acid (JA) and salicylic acid (SA) at 24/48h. Maximum and subordinate expression induction of MaPKSs was apparent respectively against JA and SA treatments. Notably, most MaPKSs achieved their peak expression within 24h of JA and the total flavonoid content was reached maximum within 24h of JA/SA elicitations. Considering the homology, phylogeny, and expression levels in each analyzed sample (n=13), three CHSs, three DCSs along with three CURSs and one ASC were selected as most promising candidates respectively for flavonoids, phenylphenalenones and sporopollenin biosynthesis in banana. Our findings provide a first-line resource to disclose the functions of banana PKSs involved in distinct SMPs.

Retraction Note to: In vitro somatic embryogenesis from immature female flower of Musa AAB cv. Chenichampa and molecular analysis of transcript factors (TFs) during somatic embryogenesis

Somatic embryogenesis (SE) is a process where somatic embryos can form differentiated tissues and regenerate into new plants. Efficient SE and liquid culture system for large-scale production of banana cv. Chenichampa is presented. The embryo maturation media (M3) was modified with different organic additives and high-frequency somatic embryos were observed in M3 media supplemented with CH: Gln (100:150 mg/L) resulting in a tenfold increase in somatic embryo formation as compared to the control. The generated plantlets were hardened and polymerase chain reaction (PCR) with SSR primers were used to confirm the genetic fidelity of the plantlets. To understand the molecular mechanism of SE, expression patterns of transcript factors (TFs) in respect of embryogenic calli (EC) and embryogenic cell suspension (ECS) was also studied. Transcript factor (TF) such as BBM has been reported to play a significant part in converting explants to EC. The higher expression of TFs MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the EC and MaBBM2 and MaWUS2 in ECS, suggested that these genes could play a crucial role in SE. The protocol developed in this commercially important banana cv. Chenichampa could be highly useful for large-scale micropropagation and genetic manipulation studies which can be adapted to other cultivars lacking in male flowers. An efficient micropropagation system using SE for banana cultivar Chenichampa (Musa AAB) is presented. Differential expression profiles of TFs during various stages of SE studied. Organic additives enhanced the SE and this optimized protocol will serve as a platform for developing efficient propagation system for banana cultivars that lack male flowers.

Molecular analysis of ERF subfamily genes during coffee somatic embryogenesis

With ample cultivation and economic potential, coffee is one of the main commodities in the world. Despite its vast cultivation, there remain problems related to its productivity as a consequence of pests and diseases, questions regarding its physiology, and difficulty with its propagation. Due to such challenges, the application of biotechnological tools, such as tissue culture and molecular markers, has become essential in the search for improvements to coffee cultivation. Among tissue culture techniques, somatic embryogenesis is considered the best form of micropropagation in vitro since it is possible to produce plants that are identical to the parent plant. It is therefore essential to identify the genes involved in the process of somatic embryogenesis, as well as to understand their functions. Different studies have shown the expression of ethylene response factor (ERF) genes in different tissues and under different conditions, mainly in response to biotic and abiotic stresses. However, little is known about their role during somatic embryogenesis. Thus, this study is aimed to identify and analyze the expression patterns of ERF subfamily genes during Coffea arabica L. somatic embryogenesis. The EST-contig6, 9, and 27 were confirmed to be the ethylene response factor 8, 13, and 12 genes from Coffea arabica L., respectively, and their relative expression analysis suggests that they may be involved in coffee somatic embryogenesis. The expression patterns of ethylene response factor 8 and 13 in embryogenic calluses and embryogenic cell suspensions showed that these genes can potentially act as somatic embryogenic markers.

Induction of Ced9 mediated anti-apoptosis in commercial banana cultivar Rasthali for stable resistance against Fusarium wilt.

Anti-apoptotic gene Ced-9 enhanced resistance against Fusarium oxysporum f. sp. cubense (Foc) in the susceptible banana cultivar Rasthali by arresting tissue necrosis. The embryogenic cell suspension of banana cultivar Rasthali was stably transformed with Ced-9 gene and transformed lines were regenerated independently. The putative transgenic lines were analyzed with PCR using gene primers and further subjected to Southern blot to estimate copy number. The root-challenge bioassay with Foc showed 17-51% Vascular Discoloration Index in independent transformants compared to untransformed banana cv Rasthali (98% VDI). Four transgenic events showed a higher level of resistance over a period of 6months. Overcoming tissue necrosis is the most ideal method to avoid Fusarium multiplication and spread in banana. Oxidative stress-induced cell necrosis is prevented by the activation of antiapoptotic pathways by Ced-9 and is proving to be an effective method to control this dreaded disease. This is the first report from India on the generation of transgenic banana cultivar Rasthali expressing antiapoptotic Ced-9 gene for resistance to Fusarium wilt.

Phytochemical Analysis and Establishment of Embryogenic Cell Suspension and Agrobacterium-mediated Transformation for Farmer Preferred Cultivars of West African Plantain (Musa spp.).

Banana and plantain are among the foremost staple food crops providing food and livelihood to over 500 million people in tropical countries. Despite the importance, their production is hampered due to several biotic and abiotic stresses. Plant tissue culture techniques such as somatic embryogenesis and genetic transformation offer a valuable tool for genetic improvement. Identification and quantification of phytochemicals found in banana and plantain are essential in optimizing in vitro activities for crop improvement. Total antioxidants, phenolics, flavonoids, and tannins were quantified in various explants obtained from the field, as well as in vitro plants of banana and plantain cultivars. The result showed genotypic variation in the phytochemicals of selected cultivars. The embryogenic cell suspensions were developed for three farmer-preferred plantain cultivars, Agbagba, Obino l’Ewai, and Orishele, using different MS and B5-based culture media. Both culture media supported the development of friable embryogenic calli (FEC), while MS culture media supported the proliferation of fine cell suspension in liquid culture media. The percentage of FEC generated for Agbagba, Obino l’Ewai, and Orishele were 22 ± 24%, 13 ± 28%, and 9 ± 16%, respectively. Cell suspensions produced from FECs were successfully transformed by Agrobacterium-mediated transformation with reporter gene constructs and regenerated into whole plants.

Highly Efficient Biolistic Transformation of Embryogenic Cell Suspensions of Banana Via a Liquid Medium Selection System

An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.

Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.)

BackgroundSwitchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration.ResultsRegenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines.ConclusionThe new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.