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Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73401
* Corresponding author (zywang{at}noble.org).
ABSTRACT
Lolium temulentum L. (Darnel ryegrass) is closely related to major forage and turf species, is self-pollinated, diploid, and has a short life cycle. These traits make it suitable to serve as a model species for testing gene functions in grasses. A new L. temulentum line, NFLT12 (Reg. No. GS-7, PI 655941), was developed using anther culture on an F2 population derived from a cross of two parental L. temulentum lines. Haploid and doubled haploid plants were obtained and their tissue culture responsiveness and amiability to genetic transformation were tested. NFLT12 is highly responsive to tissue culture, is readily transformable and is expected to provide a resource for those using L. temulentum as a model species for functional genomics studies.
Abbreviations: MS, Murashige and Skoog
Lolium temulentum L. (Darnel ryegrass) has been proposed as a model grass species for functional genomics studies (Ge et al., 2007) because it is self-pollinated, is easy to grow, flowers in the greenhouse without vernalization, has a short life cycle, and is closely related to important forage and turf grasses in the Lolium–Festuca complex (Kirigwi et al., 2008; Mian et al., 2005). With vast and still rapidly expanding expressed sequence tag (EST) and genome sequencing information, it has been possible to clone large numbers of genes in a short time (Dixon et al., 2007). The roles of such genes need to be tested or confirmed by either overexpression or downregulation of the genes in transgenic plants. In grasses, functional characterization of genes has become a bottleneck because the generation of transgenic grass plants is still of low efficiency and time consuming and requires experience (Wang and Ge, 2006). In addition, many forage and turf species are outcrossing and polyploids and require vernalization to flower, which further complicates detailed analyses of gene functions, particularly in the progenies. The unique characteristics of L. temulentum make it a suitable species for analyzing gene functions in grasses (Ge et al., 2007; Wang et al., 2005).
Genotype plays an important role in plant tissue culture response (Henry et al., 1994; Spangenberg et al., 1998). It is often found that within the same species, some genotypes respond well to tissue culture, while others may not. The development of a tissue culture responsive and readily transformable line will facilitate the identification of genes of agronomical value. NFLT12 (Reg. No. GS-7, PI 655941) is a new L. temulentum line developed by anther culture. It has significantly improved tissue culture responsiveness, and a genetic transformation procedure has been established using this line.
Methods
To improve tissue culture response of L. temulentum, two relatively responsive accessions, PI165903 and PI219594, were crossed and hybrid seeds harvested. The seeds were germinated and grown into F1 plants and subsequent F2 plants in the greenhouse (16 h light, 390 µE m–2 s–1). Anther culture was performed using the F2 plants when pollen development was in mid- to late unicellular stage. When inflorescences just came out (
1 cm) of the flag leaf in the F2 plants, tillers were collected, cold treated for 3 to 5 d at 4°C, and surface sterilized with 70% ethanol. Anthers were isolated from the two lowest florets of each spikelet and plated on modified LS-3 medium (Linsmaier and Skoog, 1965; Opsahl-Ferstad et al., 1994). Anther-derived calluses were obtained after 3 to 5 wk of culture. Green shoots were obtained after transferring the calluses onto a regeneration medium—Murashige and Skoog (MS) basal medium (Murashige and Skoog, 1962) supplemented with 0.45 µM 2,4-D, 0.45 µM kinetin, and 6% (w/v) maltose and solidified with 0.3% gelrite. Haploid plantlets were established after transferring the green shoots to culture vessels containing hormone-free half-strength MS medium. Roots of the haploid plantlets were submerged in 0.34% colchicine solution for 1.5 h and then rinsed with tap water (Wang et al., 2005). The treated materials were transferred to the greenhouse and seeds were collected from the doubled haploid plants. One of the lines was selected and named NFLT12. Seed of NFLT12 was subsequently increased in the greenhouse.
Calluses were induced from mature embryos of the doubled haploid lines and the parental accessions, and frequencies of embryogenic callus formation were compared. The isolated embryos were placed on MS basal medium (Murashige and Skoog, 1962) supplemented with 22.6 µM 2,4-D, 3% (w/v) sucrose and 0.8% (w/v) agar. The experiment comprised three replications with at least 100 mature embryos plated for each replication. Analysis of variance was performed using GLM procedures of the SAS program (SAS Institute, Cary, NC). Differences were declared significant when P < 0.05. To further test the usefulness of the line for transformation, embryogenic calluses were infected with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA1301 and pCAMBIA1305.2 vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker gene and hygromycin as the selection agent. Hygromycin-resistant calluses were obtained after 4 to 6 wk of selection and transgenic plants regenerated in 10 to 13 wk after Agrobacterium-mediated transformation (Ge et al., 2007). Fertile plants and transgenic seeds were readily obtained after transferring the regenerants to the greenhouse.
Seeds of NFLT12 were planted in the greenhouse at Ardmore, OK, in 11.5- by 10-cm pots using a commercial potting mix (SB 100 bedding mix; SunGro Horticulture, Bellevue, WA) on 21 Jan. and 13 Feb., 2008. Supplemental light, at 900 µmol m–2 s–1, from high pressure sodium lamps was provided for a total photoperiod of 18 h. Temperature averaged approximately 20.8°C. A soluble fertilizer (Peters Fertilizer, The Scotts Co., Marysville, OH) was used frequently to maintain vigorous growth. Morphological data, including plant height, spike length, awn length and flag leaf length, width, and height, were gathered on 50 random plants per planting date (100 plants total) on or shortly after plants had headed, corresponding to the R3 stage (Moore et al., 1991). Seed was harvested in bulk from all plants on 13 May and 27 May 2008. Data provided are means ± SE.
Characteristics
Embryogenic calluses can be easily induced from mature embryos or seeds of the L. temulentum line NFLT12. The use of mature embryos or seeds as explants offers a significant advantage over the use of immature embryos required in many other monocot crops (Ge et al., 2007). The frequency of embryogenic callus formation in NFLT12 was 55.7 ± 1.5%, double the frequencies of parental lines. Embryogenic calluses could be infected by A. tumefaciens. After infection and antibiotic selection, hygromycin-resistant calluses were obtained. Transformed shoots and plants were obtained after transferring the resistant calluses onto regeneration medium. Transformation efficiency (the number of independent transgenic plants divided by the number of original calluses used) was 4.8%. Soil-grown transgenic L. temulentum plants were established in the greenhouse about 4 mo after transformation.
Seed of the original nontransgenic NFLT12 line was easily propagated in the greenhouse. Plant height averages 45.1 ± 1.5 cm, while flag leaf height, length, and width average 28.1 ± 1.0 cm, 14.6 ± 0.7 cm, and 5.2 ± 0.2 mm, respectively. Heading date occurs on average 45 ± 0.4 d after planting. Spike length averages 13 ± 0.4 cm, and seed weight is 100 seeds g–1. Generation time, measured from planting to seed harvest, ranges from 105 to 114 d.
Availability
Limited quantities of seed will be available for research purposes on request to the corresponding author for five years after registration with CSSA, after which NFLT12 will be available from the USDA National Plant Germplasm System (NPGS). The user would be expected to acknowledge the source when this material contributes to research publications or to the development of a germplasm or genetic stock.
Footnotes
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.
Received for publication January 21, 2009.
References
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