Overexpression of Arabidopsis ABF3 Gene Enhances Tolerance to Drought & Cold in Transgenic Lettuce (Lactuca sativa L.) & Bentgrass (Agrostis mongolicum) by Enkhchimeg Vanjildorj Supervisor Prof. Lee, Hyo-Yeon College of Applied Life Sciences, Cheju National University, Jeju 690-756, Korea
GMO-Transgenic Blue Roses 'Fausse Beauté' 'Incrédulité' 'Détresse Bleu' "blue gene" + Herbicide resistant bar gene "flavonoid 3', 5'- hydroxylase"
GMO-Transgenic Golden Rice Golden rice 1 Golden rice 2 Normal rice You can not build PEACE on the empty stomach….
Drought/dehydration Cold/freezing High-salinity heat Biological invaders Water excess Plants must ADAPT or DIE ! ! ! ! !........ ABA hormone
ABF3 gene regulations in Arabidopsis: • Inhibit germination by high salt • Inhibit the development of young seedling by high sugar • Exhibit drought tolerance than wild-type plants • Low/high temperature tolerance than wild-type plants • Oxidative stress tolerance than wild-type plants • Hypersensitive to ABA • Slightly delayed germination • Mild growth retardation than that of wild type Kang et al., (2002) The Plant Cell, Vol. 14, 343-357
Objectives: • 1. To establish an efficient in vitro regeneration system in lettuce and bentgrass • 2. To transform ABF3 gene to the lettuce and bentgrass via Agrobacterium tumefaciens • 3. To determine the transgene transmission and expression in T1 progeny • 4. To enhance tolerance to drought and cold in transgenic lettuce and bentgrass
Section I.LETTUCE (LACTUCA SATIVA L.) • Establishment of in vitro regeneration system • Establishment of gene transformation system • In vivo test (drought & cold)
What is the Chongchima lettuce? Family: Compositae Genus: Lactuca Species : Lactuca sativa L. Common name: Lettuce Genotype name: Chongchima Type: Leaf type lettuce Usage:Foodand Home garden
Multiple stresses Interacts with ABRE (transcription factor) ABF3 Promoter ABRE (CACGTGGC) ABA-regulated genes DNA (Gene product) Transactivates ABRE-containing reporter gene ABA hormone Schema ofABF3 (ABA-responsive elements (ABRE)BindingFactor) gene expression Choi et al., (2000) The Journal of Biological Chemistry. Vol. 275: 1723-1730
Lactuca sativa L. cv. Chongchima 7-day-old explants: C: Cotyledon, P: Petiole H: Hypocotyl C P H
shooting A1 B1 C1 rooting A2 B2 C2 Regeneration from different explants of Chongchima lettuce Cotyledon Petiole Hypocotyl
Shoot regeneration on cotyledon explants of lettuce Chongchima *Values indicate the mean of three replications±SE
ABF3 probe (2 kb) L B R B 35SPRO HPH Gfp::gus 35SPRO Ubi-PRO 35S TER NOS TER ABF3 Arbc S TER EcoRI SacI HindIII BamHI SmaI HindIII XhoI XhoI A part of the T-DNA region of vector pCUMB
Transformation stages of Lactuca sativa L. genotype Chongchima via Agrobacterium tumefaciens Selection1 Selection2 Rooting
Co-cultivated explants (A) Produced HygR calli Produced HygR plants Produced HygR and GUS+plants (B) T.E. (%)(B/A) 83 53 9 9 10.8 Transformation efficiency (T.E.) of transgene
21 days later Morphology and fertility of T0 transgenic plant
WT ubi::ABF3 Transgene segregation to T1 progenies
Cotyledon Seedlings A 2-week old 4-week old Seeds Segments of stem Flowers Root of adult plant GUS expression on T1 putative transgenic plants pappus ovary
WT 101 104 107 108 2 kb Gene integration is confirmed by Southern blotting 25 µg gDNA was digested by HinDIII, and hybridized with 32P labeled ABF3 probe
WT ubi::ABF3 WT ubi::ABF3 Drought assay Cold assay 68.5 51.2 31.7 10 Wild-type Transgenic Drought and Cold tolerance assays
Cold test at the open field in the winter T2 plants at -5±1 ℃, 50±5% relative humidity with strong wind for 48 h Wild-type T2 plants After 2 weeks since moving into the glasshouse
Conclusions • To choice of genotype and explants type are a critical factors to consider in lettuce tissue culture. • Incorporation of both exogenous kinetin and NAA are essential, and cytokinin to auxin ratio 5:1 and 10:1 is the most effective on shoot regeneration of lettuce cotyledon explants. • All transgenic plants and their progenies were fertile and morphologically normal, and gene transformation efficiency was relatively higher. • The transgene inherited in a normal Mendelian pattern in one out of the nine T1 progenies. • The transgenic plants displayed more tolerant to drought and cold than that of wild-type plants, suggesting that overexpression of Arabidopsis ABF3 is functional in lettuce.
Section II.MONGOLIAN BENTGRASS (AGROSTIS MONGOLICUM) • Establishment of in vitro regeneration system • Establishment of gene transformation system
What is Agrostis mongolicum & its usage? Family:Poaceae Genus:Agrostis Species :Agrostis mongolicum Common name:Mongolian bentgrass LAWNS ROADSIDE GRASSING & GREENING LIVESTOCK PASTURE & FORAGE GOLF COURSE SPORTS FIELD
Callus types derived from mature seeds 1 3 5 Compact Watery 2 4 Brown-spoted Regenerable, green-spotted Friable
0 0.05 0.5 1 2 TDZ (mg/L) 0 30 25 20 0.05 15 NAA Number of shoots/callus (mg/L) 10 0 0.05 5 0.1 0.2 0.1 0 0 0.05 0.5 1 2 TDZ (mg/L) 0.2 TDZ and NAA combination effect on shoot regeneration of Agrostis mongolicum * Values indicate the mean ± SE of three replicates Callus Type 2
Transformation stages of Agrostis mongolicum via Agrobacterium tumefaciens • Infection • Co-cultivation • Bacteria Elimination Callus induction Pre-cultivation R S S S Shoot regeneration & selection Putative transgenic plant growing in the glasshouse Root induction & selection R: resistant, S: susceptible
Morphology of ubi::ABF3 transgenic Agrostis mongolicum 3-month-old plants growing in the grasshouse adjusted at 25℃, photoperiod of 18/6 h and at 70±10% of humidity
PCR products using hygromycin (A) and GUS gene primers (B) B A M WT 1 2 3 4 5 M WT 1 2 3 4 5 900 bp 500 bp M: Ladder marker (Takara) M: 1 kb Plus DNA Ladder WT: wild-type and lanes 1-5: putative transgenic lines
Gene integration is confirmed by Southern blotting WT 1 2 3 4 5 2 kb 25 µg gDNA was digested by EcoRV, and hybridized with 32P labeled ABF3 probe
Conclusions • Five type of callus was determined, callus type 2 displayed significantly higher shoot regeneration capacity • TDZ+NAA combination was effective, however the highest number of shoots per callus was observed when medium containing 0.05 mg/L TDZ • Transgenes were stably integrated in the genomes of putative transgenic plants • The morphology of transgenic plants were normal in terms of shape of whole plants
Future study • Introducing draft inducible gene • Introducing herbicide-resistant gene
Acknowledgments Thank you for your attention!