Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.)

Key message

With a novel elite genotype, SA37, and an improved transformation protocol, it is now possible to routinely and efficiently engineer switchgrass using biolistic transformation.

Abstract

Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 μm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.     

A simplified protocol for genetic transformation of switchgrass (Panicum virgatum L.)

The increasing interest in renewable energy has attracted more research attention on biofuels. In order to generate sustainable amount of biomass feedstock from dedicated biofuel crops such as switchgrass they need to be genetically improved. Genetic transformation is one of the techniques to achieve this goal. The aim of our study was to devise a simplified protocol for switchgrass genetic transformation. We have used NB(0) as the basal medium and mature seeds of the cultivar Alamo as the starting material. The nutrient medium used and scutellum-derived callus are fashioned after rice genetic transformation protocols. We obtained friable calluses, which were similar to the type II calluses in other monocotyledonous species. Calluses were amenable for Agrobacterium-mediated genetic transformation with at least 6?% transformation efficiency. The concentration of hygromycin was optimized for successful selection of transgenic calluses. The Green Fluorescent Protein gene was used to monitor and demonstrate successful genetic transformation. Compared to the previously published methods for genetic transformation of switchgrass, our protocol is simpler and equally efficient. KEY MESSAGE: An efficient, simplified switchgrass genetic transformation method with NB(0) basal medium and mature seeds as inoculum was developed. The appropriate concentrations of hormones and selection agent are described.     

Somatic embryogenesis and Agrobacterium-mediated transformation of turmeric (Curcuma longa)

Turmeric (Curcuma longa L.) is a rhizomatous species belonging to the Zingiberaceae and known both for its culinary and medicinal uses. Based on an efficient tissue culture and somatic embryogenesis system that we established, we have developed a reliable Agrobacterium-mediated transformation protocol for this species. Calli derived from turmeric inflorescences were used as source tissues for transformation. Factors affecting transformation and regeneration efficiency were evaluated, including callus induction and culture conditions, Agrobacterium strains, co-cultivation conditions, selection agent sensitivity and bacterial elimination, and transformant selection. Optimized transformation conditions were identified, including: use of Agrobacterium strain EHA105 with plasmid pBISN1 for infection; a modified B5 medium system for callus induction, subculture, co-culture and selection; and MS media for transformant regeneration. Transgenic plants and their vegetative (clonal) progeny stably expressed the transgene as indicated by GUS assay, PCR and Southern blot analysis. In addition, a transient gene expression system was developed that involves Agrobacterium infiltration of young turmeric leaves followed by in vitro regeneration of plantlets. This approach established that a MADS-box-GFP fusion protein was localized to the nucleus of turmeric cells. The stable transformation and transient expression systems described herein offer opportunities for assaying gene function in turmeric and for improving turmeric properties.     

Disruption and overexpression of Arabidopsis phytosulfokine receptor gene affects cellular longevity and potential for growth

Phytosulfokine (PSK), a 5-amino acid sulfated peptide that has been identified in conditioned medium of plant cell cultures, promotes cellular growth in vitro via binding to the membrane-localized PSK receptor. Here, we report that loss-of-function and gain-of-function mutations of the Arabidopsis (Arabidopsis thaliana) PSK receptor gene (AtPSKR1) alter cellular longevity and potential for growth without interfering with basic morphogenesis of plants. Although mutant pskr1-1 plants exhibit morphologically normal growth until 3 weeks after germination, individual pskr1-1 cells gradually lose their potential to form calluses as tissues mature. Shortly after a pskr1-1 callus forms, it loses potential for growth, resulting in formation of a smaller callus than the wild type. Leaves of pskr1-1 plants exhibit premature senescence after bolting. Leaves of AtPSKR1ox plants exhibit greater longevity and significantly greater potential for callus formation than leaves of wild-type plants, irrespective of their age. Calluses derived from AtPSKR1ox plants maintain their potential for growth longer than wild-type calluses. Combined with our finding that PSK precursor genes are more strongly expressed in mature plant parts than in immature plant parts, the available evidence indicates that PSK signaling affects cellular longevity and potential for growth and thereby exerts a pleiotropic effect on cultured tissue in response to environmental hormonal conditions.     

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10⁶ protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.     

Establishment and characterization of long-term embryogenic maize callus and cell suspension cultures

Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 × B73, and hybrid B73 × A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 × A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5–7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 × A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.     

Clonal propagation of someFreesia cultivars through tissue culture

A propagation method for someFreesia cultivars usingin vitro cultures is described. Bud and root formation was evoked in callus tissue culture on a modified Murashige and Skoog’s medium. Reconstituted plants were transferred into soil and cultivated in the glasshouse.     

Optimization of biological and physical parameters for biolistic genetic transformation of common wheat (Triticum aestivum L.) using a particle inflow gun

The parameters for delivery of expression cassettes to cells of wheat morphogenic callus induced from immature embryos were optimized. Three systems (gradation, delayed, and regeneration) for in vitro selection of transgenic wheat tissue using the bar gene, providing resistance to the herbicide phosphinothricin (PPT), were compared. The efficiency of gene delivery to the cells competent for plant regeneration was assessed by comparing the number of spots transiently expressing uidA gene (encoding β-glucuronidase) per unit surface of the morphogenic calluses treated under various conditions. The selection systems in question were evaluated by comparing the transformation efficiency frequencies. The optimal parameters for wheat biolistic transformation using a particle inflow gun were determined, namely, the distance between the particle source and the target tissue (12 cm) and helium pressure during the shot (6 atm). The optimal time of callus tissue development on the medium inducing callus formation was determined (10–14 days). Comparison of the three selection variants demonstrated that the regeneration system was the most efficient for producing true transgenic plants of common wheat.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes. II. Dexamethasone enhanced longevity and maintenance of morphology.

Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1 micronM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethason supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture.     

Micropropagation of <Emphasis Type="Italic">Arnebia hispidissima</Emphasis> (Lehm). DC. and production of alkannin from callus and cell suspension culture

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.     

Screening Wheat Genotypes for High Callus Induction and Regeneration Capability from Immature Embryo Cultures

Selecting the explant genotypes is crucial step in in vitro culture and Agrobacterium-mediated transformation system due to its host range specificity. Immature embryos of five winter and three spring wheat (Triticum aestivum L) cultivars were evaluated for tissue culture response in three callus initiation media. MS medium containing 2,4-0 (2 mg ml^-1) plus B5 vitamins (MSB5), MS medium containing 2,4-0 (1 mg ml^-1) with no vitamins (MS1GC) or MS medium containing picloram (2.2 mg ml^-1) and 2,4-0 (0.5 mg ml^-1) plus MS vitamins (CM4C) were used for callus initiation. Percentage of callus induction varied widely with the genotype and initiation medium used, with values ranging from 5.7% to 100%. Embryogenic capacity of genotypes was evaluated by number of somatic embryos formed from cultured immature embryos. Bob White (spring) and NE92458 (winter) were equal and most embryogenic; Pronghorn and 2137 (both winter) were the poorest. CM4C medium was found to be the best medium for initiating embryogenic callus among three culture media tested. A standard regeneration procedure was used. The genotypes with the highest regeneration efficiencies were Bob White, Fielder and NE92458, (1.8, 1.4 and 1.6 plantslexplant, respectively).     

In Vitro Long-Term Storage of Date Palm

A reliable method for the long-term conservation of date palm tissue cultures is described. In vitro shoot bud and callus culture were successfully stored for 12 months at 5°C in the dark. At this conditions high percent of cultures remained viable without serious signs of senescence. However, the growth rate decreased as storage period increased. The role of sorbitol as osmotic agent in storage was examined. Health shoot bud cultures were obtained after 6 months of storage on medium containing 40 g dm^–3 sorbitol. This period extended for 9 months in case of callus cultures.     

Type II callus production and plant regeneration in tropical maize genotypes

A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally, four media combinations of 15 or 30 μm dicamba with or without 88 μm AgNO₃ were used to study the effect of dicamba and AgNO₃ on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 μm and when AgNO₃ was added to the medium. A synergistic effect between 88 μm AgNO₃ and 30 μm dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best tissue culture performance and plant regeneration capacity. Received: 5 October 1996 / Revision received: 21 April 1997 / Accepted: 9 May 1997     

Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection

Perennial ryegrass is one of the most widely cultivated grasses in temperate regions. However, it is recalcitrant for in vitro manipulation. In this study, various parameters affecting Agrobacterium tumefaciens-mediated infection were tested to optimize transformation efficiency in perennial ryegrass. The effects of heat shock and maltose concentration during Agrobacterium infection were evaluated along with variations in callus induction medium, bacterial infection media and callus age. Our results suggest that Agrobacterium infection at 42 °C for 3 min and co-cultivation of Agrobacterium-infected callus on a high maltose medium (6 %) significantly enhances the transformation efficiency in perennial ryegrass. The most optimal conditions proved to be use of four-month-old embryogenic callus induced on a modified N6 medium, infected with Agrobacterium grown on a modified Murashige and Skoog (MSM) medium, and a 42 °C heat shock treatment followed by the co-cultivation of the Agrobacterium and the callus on medium containing 6 % maltose (instead of 3 %). Using this optimized protocol, we were able to increase the transformation efficiencies for regenerated plants from approximately 1 % to over 20 %. Significant improvement in rice stable transformation efficiency was also observed when the optimized conditions were applied to this important cereal, indicating the method described here may apply to other monocots as well.     

Influence of form of activated charcoal on embryogenic callus formation in coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis>)

Development of micropropagation protocols for Cocos nucifera has progressed slowly. Activated charcoal is included in the culture medium of each protocol, mainly to prevent tissue browning. Charcoal production procedures can affect the properties of different brands. In this study, eight types of activated charcoal were evaluated for their effects on free 2,4-dichlorophenoxyacetic acid level, pH, conductivity, and osmolarity of the culture medium and on the frequency of embryogenic callus induction. Moreover, the effect of particle size of the optimum charcoal type on embryogenic callus development was also studied. Charcoal type had a significant effect on (Y3) culture medium properties. Free 2,4-D was highest in Reactivos y Productos Químicos Finos-containing medium and pH was lowest in MERCK-containing medium. Charcoal type also influenced embryogenic callus induction, with acid washed for plant cell and tissue culture-, DARCO- and United States Pharmacopeia-containing media promoting ~60% embryogenic callus, but with different optimal 2,4-D concentrations. Particle size profiles varied among all charcoal types, although small particle fraction (<38 μm) was abundant in all. Use of small particle fractions produced higher frequencies of embryogenic callus (70%) than either large particle or whole charcoal fractions.     

Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase

Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 × 10⁵ spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 × 10⁷ spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 × 10⁷ spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl₂, medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o-methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis.     

Somatic embryogenesis and morphogenesis in callus derived from the epiblast of immature embryos of wheat (Triticum aestivum)

Factors affecting the formation of embryogenic callus from the epiblast of immature embryos of wheat (Triticum aestivum L.) are described. Embryos were incubated on a modifed Murashige and Skoog medium with the scutellum in contact with medium. Callus formation from the epiblast was affected by the type of cultivar used, the stage of embryo development, and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium. The number of embryos forming embryogenic callus ranged from 10% to 72%, depending on the cultivar. Embryo development was classified into five distinct morphological stages and higher yields of callus were produced using embryos excised at stages II and III. Concentrations of 2,4-D higher than 1 μM were required for the formation of embryogenic callus. Epiblast callus gave rise to embryoids which differentiated into multiple plants at a high frequency when placed on hormone-free medium.     

Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

An improved cytokinin bioassay using cultured soybean hypocotyl sections

This paper describes a modified soybean (Glycine max) tissue culture bioassay for cytokinins. Soybean hypocotyls were grown under sterile conditions and sliced into 1-mm sections. Sections were cultured for 5,9,13, or 22 days on a callus medium with zeatin or other cytokinins. The fresh weight of sections increased with the cytokinin concentration from 0.0005 to 1 mum zeatin; 2-fold concentration differences were readily distinguishable at 9 days. The assay should prove to have several advantages over the conventional soybean callus bioassay including convenience, lower variability between tissue samples, and improved resolution. Its specificity is comparable to that of the soybean callus bioassay.     

BAGY2 Retrotransposon Analyses in Barley Calli Cultures and Regenerated Plantlets

The stability of aging barley calli and regenerated plantlets from those calli was investigated by the BAGY2 retrotransposon-specific IRAP technique. Mature embryos of barley (Hordeum vulgare cv. Golden Promise) were cultured in Murashige and Skoog medium supplemented with 4 mg/L dicamba and maintained on the same medium for 45 and 90 days. Two IRAP-based primers were used, and the levels of variation of DNA isolated from 45- and 90-day-old calli and regenerated plantlets were found to be increased 0–21%, depending on the mature embryo material and the age of the callus. It has been observed that culture conditions cause genetic variations and evident BAGY2 retrotransposon alterations. Internal domains of BAGY2 were also analyzed by qPCR, and copy numbers were found to be increased. These findings are expected to contribute to understanding of how retrotransposons affect features like tissue culture (especially callus tissue) formation and genetic engineering studies.