Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation

Lolium temulentum L. (Darnel ryegrass) has been proposed to be used as a model species for functional genomics studies in forage and turf grasses, because it is a self-fertile, diploid species with a short life cycle and is closely related to other grasses. Embryogenic calluses were induced from mature embryos of a double haploid line developed through anther culture. The calluses were broken up into small pieces and used for Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors were used to infect embryogenic callus pieces. Hygromycin was used as a selection agent in stable transformation experiments. Hygromycin resistant calluses were obtained after 4–6 weeks of selection and transgenic plants were produced in 10–13 weeks after Agrobacterium-mediated transformation. Fertile plants were readily obtained after transferring the transgenics to the greenhouse. Transgenic nature of the regenerated plants was demonstrated by Polymerase chain reaction (PCR), Southern hybridization analysis, and GUS staining. Progeny analysis showed Mendelian inheritance of the transgenes. The transformation system provides a valuable tool for functionality tests of candidate genes in forage and turf grasses.     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Factors Affecting Plant Regeneration from Tissue Cultures of Chinese Leymus (Leymus chinensis)

Chinese leymus (Leymus chinensis Trin.) is a perennial grass of the Gramineae, which is widely distributed in China, Mongolia and in Russian-Siberian. In order to explore the potential of biotechnology for genetic improvement of this forage grass, an efficient tissue culture system was established and the factors affecting plant regeneration were evaluated. Immature inflorescence segments 3–5 mm in length from eight accessions were cultured on N6 medium supplemented with 2.26–22.60 µM 2,4-D. The callus induction frequency ranged from 72.11 to 82.19%. Shoots were differentiated from the calli on N6 medium containing 4.65 µM kinetin and 4.44 µM BA. Viable regenerants were developed on hormone-free medium. Normal plants were obtained after natural vernalization in the field. The plant regeneration frequency in Chinese leymus was associated with different genotypes and different combinations of growth regulators in medium. The concentration of 2,4-D in the callus induction medium had a strong effect on successive plant regeneration. Relatively higher concentrations of 2,4-D (i.e., 9.04 and 22.60 µM) were more favorable to the plant regeneration than lower ones (i.e., 2.26 and 4.52 µM). This is the first report on plant regeneration in vitro in L. chinensis.     

Establishment of a plant regeneration system for wheatgrasses (Thinopyrum,Agropyron and Pascopyrum)

Wheatgrasses (Thinopyrum, Agropyron and Pascopyrum spp.) are a ubiquitous group of cool-season grasses used throughout much of the semiarid temperate world. In order to explore the potential of biotechnology to accelerate conventional breeding efforts, we developed an efficient plant regeneration system for different wheatgrass species: tall wheatgrass [Thinopyrum ponticum (Podp.) Liu and Wang], intermediate and pubescent wheatgrass [Thinopyrum intermedium (Host) Barkw. and D.R. Dewey], crested wheatgrass [Agropyron cristatum (L.) Gaertner], and western wheatgrass [Pascopyrum smithii (Rydb.) Löve]. Embryogenic callus was induced from mature and immature embryos and immature inflorescence with an induction frequency in the range of 0.5–8.3% for the different wheatgrass species tested. Individual embryogenic calluses were then used to establish single genotype-derived suspension cultures. Efficient plant regeneration was achieved from the established em-bryogenic suspensions with regeneration frequencies in the range of 20–65% for tall wheatgrass, 21–40% for intermediate and pubescent wheatgrasses, 32–51% for crested wheatgrass, and 25–48% for western wheatgrass. The cell suspension-derived wheatgrass plants were fertile and phenotypically normal in the field. The efficient plant regeneration system provides a solid basis for genetic transformation of wheatgrasses.     

Floral inhibition in red fescue (Festuca rubra L.) through expression of a heterologous flowering repressor from Lolium

Extension of the vegetative growth phase through delay of flowering is an important goal in today's breeding programs of both forage and turf grasses. In forage grasses, the stem and inflorescence production comprise a significant reduction in the digestibility, nutritional value and productivity of the crop, and in turf grasses the stems that start to emerge during the growth season suppress the formation of new shoots and affect the quality, density and persistence of the sward. We have tested the potential of the strong floral repressor LpTFL1 from perennial ryegrass (Lolium perenne L.) to manipulate the transition to flowering in red fescue (Festuca rubra L.), a cool-season turf grass. Expression of LpTFL1 from the constitutive maize ubiquitin promoter represses flowering in red fescue, and the flowering repression phenotype correlates well with the level of LpTFL expression. Transgenic lines showing low to intermediate expression of LpTFL1 flowered approximately two weeks later than the controls, and transgenic lines showing very high LpTFL1 expression levels still remained non-flowering after exposure to natural vernalization conditions (Danish winter) in two successive years. There were no other phenotypic effects associated with the LpTFL transgene expression during vegetative growth. However, there was a tendency towards an LpTFL1-mediated reduction in stem length among the flowering lines. Expression of a truncated LpTFL, caused by transgene rearrangements during the transformation, lead to increased flowering and stem production and a decrease in panicle size. This is to our knowledge the first report on full inhibition of floral development in a commercially important grass species.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> 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. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Forage and Turf Grass Biotechnology

Referee: Dr. Ian Ray, Plant Breeding and Genetics, Department of Agronomy & Horticulture, New Mexico State University, MSC 3Q, P.O. Box 30003, Las Cruces, NM 88003-8003 Forage and turf grasses are the backbone of sustainable agriculture and contribute extensively to the world economy. They play a major role in providing high quality and economical meat, milk, and fiber products and are important in soil conservation, environmental protection, and outdoor recreation. Conventional breeding contributed substantially to the genetic improvement of forage and turf grasses in the last century. The relatively new developments in genetic manipulation of these species open up opportunities for incorporating cellular and molecular techniques into grass improvement programs. For some commonly used forage and turf species, significant advances have been achieved in the following areas: (1) establishment of a tissue culture basis for the efficient regeneration of fertile and genetically stable plants, (2) generation of transgenic plants by biolistic transformation and direct gene transfer to protoplasts, (3) recovery of intergeneric somatic grass plants by protoplast fusion, (4) development of molecular markers for marker assisted selection, and (5) sequencing of expressed sequenced tags and the development of DNA array technologies for gene discovery. Although difficulties still exist in genetic manipulation of these recalcitrant monocot species, impressive progress has been made toward the generation of value-added novel grass germplasm incorporating traits such as improved forage quality. The joint efforts of molecular biologists and plant breeders make the available biotechnological methods a useful tool for accelerating forage and turf grass improvement.     

Efficient<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation of soybeans using an embryonic tip regeneration system

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l^–1 N⁶-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l^–1 BAP and 0.2 mg l^–1 indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.Abbreviations GUS -Glucuronidase - T-DNA Transferred DNA - BAP N⁶-Benzylaminopurine - IBA Indolebutyric acid     

Orchardgrass (<Emphasis Type="Italic">Dactylis glomerata</Emphasis> L.) EST and SSR marker development,annotation, and transferability

Orchardgrass, or cocksfoot [Dactylis glomerata (L.)], has been naturalized on nearly every continent and is a commonly used species for forage and hay production. All major cultivated varieties of orchardgrass are autotetraploid, and few tools or information are available for functional and comparative genetic analyses and improvement of the species. To improve the genetic resources for orchardgrass, we have developed an EST library and SSR markers from salt, drought, and cold stressed tissues. The ESTs were bi-directionally sequenced from clones and combined into 17,373 unigenes. Unigenes were annotated based on putative orthology to genes from rice, Triticeae grasses, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Of 1,162 SSR markers developed, approximately 80% showed amplification products across a set of orchardgrass germplasm, and 40% across related Festuca and Lolium species. When orchardgrass subspecies were genotyped using 33 SSR markers their within-accession similarity values ranged from 0.44 to 0.71, with Mediterranean accessions having a higher similarity. The total number of genotyped bands was greater for tetraploid accessions compared to diploid accessions. Clustering analysis indicated grouping of Mediterranean subspecies and central Asian subspecies, while the D. glomerata ssp. aschersoniana was closest related to three cultivated varieties.     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Genetic Transformation of Maize Elite Inbred lines

An efficient transformation system was developed for maize (Zea mays L.) elite inbred lines using Agrobacterium-mediated gene transfer by identifying important factors that affected transformation efficiency. The hypervirulent Agrobacterium tumefaciens strain EHA105 proved to be better than octopine LBA4404 and nopaline GV3101. Improved transformation efficiencies were obtained when immature embryos were inocubated with Agrobacterium suspension cells (A₆₀₀ = 0.8) for 20 min in the presence of 0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivation was performed in the acidic medium (pH5.4) at 22 °C in the dark for 3 days. Using the optimized system, we obtained 42 morphologically normal, independent transgenic plants in four maize elite inbred lines representing different genetic backgrounds. Most of them (about 85%) are fertile. The transformation frequency (the number of independent, PCR-positive transgenic plants per 100 embryos infected) ranged from 2.35 to 5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to three copies of the transgene were integrated into the maize nuclear genome. About 70% of the transgenic plants received a single insertion of the transgenes based on Southern analysis of 10 transformed events. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This system should facilitate the introduction of agronomically important genes into commercial genotypes.     

Efficient and stable regeneration from protoplasts of <Emphasis Type="Italic">Cyclamen coum</Emphasis> Miller via somatic embryogenesis

Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 × 10⁵ protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.     

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless at low <Emphasis Type="Italic">Agrobacterium</Emphasis> density

Highly efficient genetic transformation protocols and the regeneration of transgenic plants of Sugraone and Crimson Seedless grapevines (Vitis vinifera L.) were achieved from embryogenic calli co-cultured with low Agrobacterium tumefaciens densities. The sensitivity of embryogenic cultures to kanamycin, as well as the effect of Agrobacterium strains, C58(pMP90) or EHA105, and the bacterial concentration (0.06 or 0.2 at Optical Density OD₆₀₀) on transformation efficiency were studied. Embryogenic cultures showed different kanamycin sensitivities and the total suppression of embryo differentiation at 20 and 50 mg/l kanamycin for Crimson Seedless and Sugraone, respectively. sgfp gene expression was evaluated in callus co-cultured with each bacterial strain. Although GFP transient expression was higher with A. tumefaciens EHA105 in both cultivars at the beginning of the culture, there were no significant differences 28 days post-inoculation. However, the concentration of Agrobacterium did affected transformation efficiency: 0.06 OD₆₀₀ being more effective for the transformation of Crimson Seedless and 0.2 OD₆₀₀ for Sugraone. By following the optimised procedure, 21 and 26 independent transgenic plants were generated from Sugraone and Crimson Seedless respectively, three to five months post-infection. PCR analyses were carried out to verify the integration of the sgfp and nptII genes into grapevine genome and the stable integration of the sgfp gene was confirmed by Southern blot.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

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.     

Recent advances in genetic transformation of forage and turf grasses

Summary Forage and turf grasses are critical to sustainable agriculture and contribute extensively to the world economy. Tremendous progress has been made in genetic transformation of forage and turf grasses in the past decade. The rapid advancement of cellular and molecular biology and transgenic technology provides novel methods to accelerate and complement conventional breeding efforts. This review summarizes the latest developments in genetic transformation methods and the applications of molecular techniques for the improvement of forage and turf grasses.     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of meadow fescue (<Emphasis Type="Italic">Festuca pratensis</Emphasis> Huds.)

Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved. The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines, the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated one or two copies of the uidA gene. C. Gao and J. Liu contributed equally to the work.     

The development of an efficient cultivar-independent plant regeneration system from callus derived from both apical and non-apical root segments of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.)

Summary Callus induction and later plant regeneration were studied in four widely grown garlic (Allium sativum L.) cultivars from Europe. Root segments from in vitro plantlets were used as starting material. In addition to cultivar effects, the effects of auxin and cytokinin levels and the position of the segments on the root were studied. There were no statistically significant differences among cultivars for the number of root segments that induced callus in the two series of experiments. The average induction frequency was 34.7% in the first series of experiments. Callus induction on apical root segments was significantly higher compared to callus induction on non-apical root segments in the second series of experiments. Two months after callus induction, callus lines were transferred to a regeneration medium consisting of Murashige and Skoog basal medium supplemented with 30gl⁻¹ sucrose and 1 mgl⁻¹ (4.6μM) kinetin. Calluses derived from different experiments were quite uniform with respect to their regeneration potential. Also it was found that our regeneration system was cultivar-independent. The average shoot regeneration frequency was 17.9% in the first series of experiments. Highly significant differences were found in the frequency of shoot regeneration among different callus induction treatments. When the cytokinin 6-(γ,γ-dimethylallylamino)purine (0.1mgl⁻¹∶0.5 μM) was present during callus induction, shoot regeneration ranged from 30.10 to 47.60%. Shoot regeneration from callus induced on non-apical segments was higher, although not significant, compared to callus induction from apical root segments in the second series of experiments. All in all, an efficient callus induction and plant regeneration system was developed from both apical and non-apical segments taken along the entire length of the roots. This system has potential to be used for garlic transformation.