J. Nagel

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.

Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells

Summary Transgenic forage- and turf-type tall fescue ( Festuca arundinacea Schreb.) and red fescue ( F. rubra L.) plants have been obtained by microprojectile bombardment of embryogenic suspension cells using chimeric hygromycin phosphotransferase ( hph ) gene constructs driven by CaMV 35S promoter or rice actin 1 5 regulatory sequences. Bombardment parameters of embryogenic suspension cultures with the particle inflow gun were partially optimized using transient expression assays of a chimeric β -glucuronidase ( gusA ) gene construct. 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 twofold — compared with continuous plate selection using solid medium — recovery efficiency of transformed hygromycin resistant clones. Plants were regenerated from 35 % and 85 % of the hygromycin resistant calli obtained in tall and red fescue, respectively. The transgenic nature of the regenerated plants was demonstrated by Southern hybridization analysis. Expression of the trans gene in transformed mature tall and red fescue plants was confirmed by northern analysis and hygromycin phosphotransferase enzyme assay.

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.

Expression of a sulphur-rich sunflower albumin gene in transgenic tall fescue (Festuca arundinacea Schreb.) plants

Abstractu2002Transgenic tall fescue (Festuca arundinacea Schreb.) plants have been generated that express foreign genes encoding a rumen-stable protein rich in sulphur-containing amino acids. The aim was to improve the protein quality of a forage grass for ruminant nutrition. Chimeric sulphur-rich sunflower albumin (SFA8) genes, including an endoplasmic reticulum retention signal (KDEL), were constructed under the control of constitutive (CaMV 35S) and light-regulated (wheat Cab) promoters. These constructs were introduced into the tall fescue genome by microprojectile bombardment of embryogenic suspension cells. The sunflower albumin transgenes stably integrated into the plant genome as demonstrated by Southern hybridization analysis. The transgenic tall fescue plants produced the expected transcript, and the corresponding sulphur-rich SFA8 protein accumulated up to 0.2% of the total soluble protein in individual transgenic 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.

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.