Z.Y. Wang

Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells

Abstract Transgenic forage-type perennial ryegrass (Lolium perenne L.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using a chimeric hygromycin phosphotransferase (hph) gene construct driven by rice Actl 5′ regulatory sequences. Parameters for the bombardment of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric β-glucuronidase (gusA) gene driven by the CaMV 35S promoter. For the recovery of stably transformed clones, hygromycin selection using liquid and solidified media was tested. Initial selection in liquid culture medium allowed for a higher, compared with continuous plate selection using solid medium, recovery efficiency of transformed hygromycin resistant clones. Plants were regenerated from 23% of the hygromycin resistant calli obtained. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the transgene in transformed adult perennial ryegrass plants was confirmed by Northern analysis and a hygromycin phosphotransferase enzyme assay.

Viability and longevity of pollen from transgenic and nontransgenic tall fescue (Festuca arundinacea) (Poaceae) plants

Pollen is an important vector of gene flow in plants, particularly for outcrossing species like tall fescue. Several aspects of pollination biology were investigated using pollen from transgenic and nontransgenic plants of tall fescue (Festuca arundinacea Schreb.), the most important forage species worldwide of the Festuca genus. To effectively assess in vitro pollen viability in tall fescue, an optimized germination medium (0.8 mol/L sucrose, 1.28 mmol/L boric acid and 1.27 mmol/L calcium nitrate) was developed. Treatment with relatively high temperatures (36° and 40°C) and high doses of UV-B irradiation (900-1500 μW/cm(2)) reduced pollen viability, while relative humidity did not significantly influence pollen viability. Viability of pollen from transgenic progenies (T1 and T2) was similar to that from seed-derived control plants. Pollen from primary transgenics (T0) and primary regenerants (R0) had various levels of viability. Hand pollination using the primary regenerants and transgenics revealed that no seed set could be obtained when pollen viability was lower than 5%. Pollen from transgenic progenies and nontransgenic control plants could survive up to 22 h under controlled conditions in growth chamber. However, under sunny atmospheric conditions, viability of transgenic and nontransgenic pollen reduced to 5% in 30 min, with a complete loss of viability in 90 min. Under cloudy atmospheric conditions, pollen remained viable up to 240 min, with about 5% viability after 150 min. This report is the first on pollen viability and longevity in transgenic forage grasses and could be useful for risk assessment of transgenic plants.

Protoplast culture and generation of transgenic plants in red fescue (Festuca rubra L.)

Abstract Embryogenic callus cultures, derived from mature embryos, were initiated for three cultivars of red fescue ( Festuca rubra L.). Morphogenic suspension cultures were established for different genotypes of red fescue cvs. ‘Roland’ and ‘Gondolin’, 5 months after their initiation. Suspension cultures of red fescue cv. ‘Roland’ showed the capacity to regenerate green plants efficiently over a period of 14 months. Similar behavior, concerning growth and plant regeneration, was observed for embryogenic cell suspensions when thawed and re-established after cryopreservation for their long-term storage. Protoplasts isolated from these highly morphogenic suspensions and cultured in agarose beads using nurse cells formed microcalli with 2 × 10 −3 overall plating efficiency. More than 85% of the protoplast derived microcalli grew further and allowed for regeneration of green plantlets in vitro. Forty representative plants from protoplasts were established in soil and grown under greenhouse conditions. To develop transgenic red fescue plants, experiments using direct gene transfer to protoplasts, polyethylene glycol treatment and a chimeric phosphinotricin acetyltransferase gene driven by rice actin 1 5′ sequence were performed. Upon selection with 50 mg/l phosphinotricin, resistant clones were obtained with 10 −6 overall transformation frequency and several transgenic plants were recovered and grown in soil. Stable integration of the transgene in the genome of plants regenerated from resistant callus clones was shown by Southern hybridization analysis. Expression of the transgene in mature plants was demonstrated by phosphinotricin-herbicide spraying.

Plants from cell suspension-derived protoplasts in Lolium species

Abstract An efficient system for green plant regeneration from protoplasts in different Lolium species: L. multiflorum var. westerwoldicum, L. multiflorum var. italicum, L. perenne L. and L. x boucheanum is described. The protocol is based on established single-genotype derived embryogenic cell suspensions, crypreservation for long-term storage of suspension cultures and a protoplast bead-type culture system including nurse cells. After screening over 200 genotypes each for different cultivars in the different ryegrasses, single-genotype derived embryogenic callus cultures were obtained. These cultures allowed to establish embryogenic suspensions for at least every second cultivar evaluated in the different ryegrasses considered. Conditions required for efficient recovery of embryogenic suspension cultures upon cryopreservation have been investigated. Plant regeneration and protoplast performance in culture from frozen-thawed suspension cultures was similar as for non-cryopreserved primary cultures. For the different ryegrasses studied, similar behavior and cultivar-dependence was apparent when comparing plant regeneration directly from embryogenic suspension cultures with regeneration from corresponding protoplasts. Regeneration of over 150 green plants from protoplasts out of two experiments each in L. multiflorum, L. perenne and L. x boucheanum was achieved. Thirty protoplast-derived plants from Westerwolds ryegrass were evaluated concerning their fertility: they are both male and female fertile, and set seeds. A RAPD analysis provided evidences for limited newly-induced genetic variation among independent protoplast-derived ryegrass plants.

Cryopreservation of embryogenic cell suspensions in Festuca and Lolium species

A reproducible method is described for the cryopreservation of single-genotype derived embryogenic cell suspensions established from different forage grass species belonging to the genera Festuca and Lolium: F. arundinacea, F. pratensis, F. rubra, L. multiflorum, L. perenne and L. × boucheanum. The protocol allowed for a long-term availability of highly regenerable embryogenic suspension cultures to be used directly for biolistic transformation approaches and as a source of totipotent protoplasts. Evaluation of different parameters, such as cryoprotectant composition, pre-freezing osmotic adaptation of suspension cultures, cooling regimes, and post-thaw washing of cryopreserved embryogenic cultured cells, revealed species-specific differences on post-thaw growth, and led to improved procedures for the storage in liquid nitrogen of the embryogenic suspension cells. These procedures allowed for 40–70% of the cryopreserved cells to re-initiate post-thaw growth, to maintain their embryogenic character and to regenerate into mature plants. Embryogenic cell suspensions were re-established within 2 months from frozen thawed cultures. Tall fescue plants were regenerated from cryopreserved and re-established embryogenic cell suspensions and their protoplasts at frequencies comparable with those of the original non-frozen cultures. The genetic stability of representative cryopreserved cultures and corresponding regenerated plants was assessed for F. arundinacea.

Transgenic ryegrasses (Lolium spp.) with down-regulation of main pollen allergens

Ryegrass pollen (Lolium species) is a widespread source of air-borne allergens and is a major cause of hayfever and seasonal allergic asthma, which affect approximately 25% of the population in cool temperate climates. The main allergens of ryegrass pollen are the proteins Lol p 1 and Lol p 2. These proteins belong to two major classes of grass pollen allergens to which over 90% of pollen-allergic patients are sensitive. The functional role in planta of these pollen allergen proteins remains largely unknown. Here we describe the generation and analysis of transgenic plants with reduced levels of the main ryegrass pollen allergens, Lol p 1 and Lol p 2 in the most important worldwide cultivated ryegrass species, L. perenne and L. multiflorum. These transgenic plants will allow the study of the functional role in planta of these pollen proteins and the determination of potential for development of hypo-allergenic ryegrass cultivars.

Culture of Vegetative and Floral Meristems in Ryegrasses: Potential Targets for Microballistic Transformation

Summary An in vitro culture system allowing the regeneration of fertile plants from isolated vegetative meristerns has been established for Lolium multiflorum (Italian ryegrass) and L. perenne (perennial ryegrass). In addition, early immature inflorescences before initiation of stamen primordia have been isolated and in vitro cultured in L. multiflorum . Development of flower meristems from cultured early immature inflorescences to normal in vitro matured spikelets led to the production of viable pollen. Both protocols have been worked out as a basis for microtargeting-mediated transformation of cells in vegetative and floral meristems for these important forage grass species. A description based on scanning electron microscopy of different developmental stages of vegetative, transition and floral apices of L. multiflorum allowed the identification of potential target meristems appropriate for particle bombardment. Plant regeneration from 79 % and 99 % of the aseptically cultured target vegetative meristems, isolated with 1–4 leaf primordia from greenhouse-grown plants, was achieved in Italian and perennial ryegrass, respectively. A stepwise culture system for in vitro development of early immature inflorescences bearing target floral meristems until maturation of floral organs in 80 % of the cases and production of 44 % viable pollen has been optimized for Italian ryegrass. Approximately 10 % of the crosses made with pollen of anthers obtained from in vitro cultured early immature inflorescences led to seed setting. Ballistic microtargeting to vegetative meristems and to floral meristems of early-staged immature inflorescences allowed for delivery of particles to cells in first and second cell layers, and for transient β-glucuronidase expression of a rice act1-uidA chimeric gene construct in the microtargeted cells.

Intergeneric symmetric and asymmetric somatic hybridization in Festuca and Lolium

Intergeneric symmetric and asymmetric somatic hybrids have been obtained by fusion of metabolically inactivated protoplasts from embryogenic suspension cultures of tall fescue (Festuca arundinacea Schreb.) and unirradiated or 10-500 Gy-irradiated protoplasts from non-morphogenic cell suspensions of Italian ryegrass (Lolium multiflorum Lam.). Genotypically and phenotypically different somatic hybrid Festulolium mature flowering plants were regenerated.

Genetic Transformation in Festuca arundinacea Schreb. (Tall Fescue) and Festuca pratensis Huds. (Meadow Fescue)

Tall fescue (Festuca arundinacea Schreb.) is a wind-pollinated, highly self-infertile polyploid perennial cool-season forage, turf, and conservation grass. It is indigenous to Europe, also naturally occuring on the Baltic coasts throughout the Caucasus, in western Siberia, and extending into China. Introductions have been made into North and South America, Australia, New Zealand, Japan, and South and East Asia (Barnes 1990).

Field Evaluation and Risk Assessment of Transgenic Tall Fescue (Festuca arundinacea) Plants

Tall fescue (Festuca arundinacea Schreb.) is an outcrossing hexaploid grass species widely grown for forage and turf purposes. Transgenic tall fescue plants were generated by biolistic transformation of embryogenic cell suspension cultures of the commonly used cultivar Kentucky-31. T1 and T2 progenies were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analysis of the progenies revealed stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue. Agronomic performance of the primary transgenics and primary regenerants under field conditions were generally inferior to seed-derived plants, with primary transgenics having fewer tillers and lower seed yield. However, no major differences between the progenies of transgenics and the progenies of seed-derived plants were found for the agronomic traits evaluated. The addition of a selectable marker gene in the plant genome seems to have little effect on the agronomic performance of the regenerated plants. No indication of weediness was observed for the transgenic tall fescue plants. An experiment on pollen dispersal has also been carried out using transgenic tall fescue in a central plot, surrounded by untransformed recipient plants in a wagon wheel design. The highest transgene frequencies, 0.88% at 50 m and 0.59% at 100 m, were observed north of the central plot, the prevailing wind direction. Issues regarding experimental design for gene flow studies and future directions on risk assessment of forage and turf grasses are discussed.