by by Enkhchimeg Vanjildorj Supervisor Prof. Lee, Hyo-Yeon College of Applied Life Sciences, Cheju National University, Jeju , Korea College of Applied Life Sciences, Cheju National University, Jeju , Korea Overexpression of Arabidopsis ABF3 Gene Enhances Tolerance to Drought & Cold in Transgenic Lettuce (Lactuca sativa L.) & Bentgrass (Agrostis mongolicum)

"flavonoid 3', 5'- hydroxylase" GMO-Transgenic Blue Roses 'Fausse Beauté' 'Détresse Bleu' 'Incrédulité' "blue gene" + Herbicide resistant bar gene

GMO-Transgenic Golden Rice Normal rice Golden rice 2 Golden rice 1 You can not build PEACE on the empty stomach….

Drought/dehydrationCold/freezing heat heat Water excess Biological invaders High-salinity Plants must ADAPT or DIE ! ! ! ! ! ABA hormone

Inhibit germination by high salt Inhibit germination by high salt Inhibit the development of young seedling by high sugar Inhibit the development of young seedling by high sugar Exhibit drought tolerance than wild-type plants Exhibit drought tolerance than wild-type plants Low/high temperature tolerance than wild-type plants Low/high temperature tolerance than wild-type plants Oxidative stress tolerance than wild-type plants Oxidative stress tolerance than wild-type plants Hypersensitive to ABA Hypersensitive to ABA Slightly delayed germination Slightly delayed germination Mild growth retardation than that of wild type Mild growth retardation than that of wild type ABF3 gene regulations in Arabidopsis: Kang et al., (2002) The Plant Cell, Vol. 14,

Objectives: Objectives: 1. To establish an efficient in vitro regeneration system in lettuce and bentgrass 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 2. To transform ABF3 gene to the lettuce and bentgrass via Agrobacterium tumefaciens 3. To determine the transgene transmission and expression in T1 progeny 3. To determine the transgene transmission and expression in T1 progeny 4. To enhance tolerance to drought and cold in transgenic lettuce and bentgrass 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 in vitro regeneration system Establishment of gene transformation system Establishment of gene transformation system In vivo test (drought & cold) In vivo test (drought & cold)

Family: Compositae Genus: Lactuca S pecies : Lactuca sativa L. Common name: Lettuce Genotype name: Chongchima Type: Leaf type lettuce Usage: Food and Home garden What is the Chongchima lettuce?

ABA-regulated genes Promoter ABRE (CACGTGGC) DNA ABF3 (Gene product) Interacts with ABRE (transcription factor) Transactivates ABRE- containing reporter gene Multiple stresses ABA hormone Schema of ABF3 (ABA-responsive elements (ABRE) Binding Factor) gene expression Choi et al., (2000) The Journal of Biological Chemistry. Vol. 275:

H P C 7-day-old explants: C: Cotyledon, P: Petiole H: Hypocotyl Lactuca sativa L. cv. Chongchima

A1B1C1 A2B2C2 rooting shooting Cotyledon Petiole Hypocotyl Regeneration from different explants of Chongchima lettuce

Shoot regeneration on cotyledon explants of lettuce Chongchima *Values indicate the mean of three replications±SE

RBRB NOS TER Gfp::gus Ubi-PRO ABF3 Arbc S TER 35SPRO HPH 35S TER LBLB SmaI EcoRI XhoI HindIII BamHI SacI ABF3 probe (2 kb) HindIII A part of the T-DNA region of vector pCUMB

Selection1 Rooting Selection2 Transformation stages of Lactuca sativa L. genotype Chongchima via Agrobacterium tumefaciens

Co- cultivated explants (A) Produced Hyg R calli Produced Hyg R plants Produced Hyg R and GUS+plants (B) T.E. (%) (B/A) Transformation efficiency (T.E.) of transgene

21 days later Morphology and fertility of T 0 transgenic plant

Transgene segregation to T 1 progenies WT ubi::ABF3 WT ubi::ABF3

A Cotyledon Flowers Seedlings Segments of stem Seeds 2-week old 4-week old Root of adult plant pappus ovary GUS expression on T 1 putative transgenic plants

WT WT kb 25 µg gDNA was digested by HinDIII, and hybridized with 32 P labeled ABF3 probe Gene integration is confirmed by Southern blotting

Drought assayCold assay WT ubi::ABF Wild-type Transgenic Drought and Cold tolerance assays

T 2 plants at -5±1 ℃, 50±5% relative humidity with strong wind for 48 h After 2 weeks since moving into the glasshouse Wild-type T2 plants Cold test at the open field in the winter

Conclusions To choice of genotype and explants type are a critical factors to consider in lettuce tissue culture. 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. 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. 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 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. 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 in vitro regeneration system Establishment of gene transformation system Establishment of gene transformation system

What is Agrostis mongolicum & its usage? Family: Poaceae Genus: Agrostis Species : Agrostis mongolicum Common name: Mongolian bentgrass ROADSIDE GRASSING & GREENING SPORTS FIELD GOLF COURSE LIVESTOCK PASTURE & FORAGE LAWNS

Friable Regenerable, green-spotted Watery Brown-spoted Compact Callus types derived from mature seeds

TDZ and NAA combination effect on shoot regeneration of Agrostis mongolicum TDZ (mg/L) * Values indicate the mean ± SE of three replicates Callus Type TDZ (mg/L) NAA (mg/L) Number of shoots/callus

Transformation stages of Agrostis mongolicum via Agrobacterium tumefaciens Shoot regeneration & selection Callus induction Pre-cultivation Infection Infection Co-cultivation Co-cultivation Bacteria Elimination Bacteria Elimination Putative transgenic plant growing in the glasshouse Root induction & selection R: resistant, S: susceptible R S S S

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) M WT M WT bp M WT M WT A B M: Ladder marker (Takara) M: 1 kb Plus DNA Ladder WT: wild-type and lanes 1-5: putative transgenic lines 500 bp

2 kb WT Gene integration is confirmed by Southern blotting 25 µg gDNA was digested by EcoRV, and hybridized with 32 P labeled ABF3 probe

Five type of callus was determined, callus type 2 displayed significantly higher shoot regeneration capacity 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 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 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 The morphology of transgenic plants were normal in terms of shape of whole plants Conclusions

Future study Introducing draft inducible geneIntroducing draft inducible gene Introducing herbicide-resistant geneIntroducing herbicide-resistant gene

Thank you for your attention! Acknowledgments