by by Enkhchimeg VanjildorjEnkhchimeg Vanjildorj

Supervisor Prof. Lee, Hyo-YeonSupervisor Prof. Lee, Hyo-Yeon

College of Applied Life Sciences, Cheju National University, Jeju College of Applied Life Sciences, Cheju National University, Jeju 690-756, Korea 690-756, Korea

Overexpression of Arabidopsis Overexpression of Arabidopsis ABF3ABF3 Gene Enhances Tolerance to Drought & Gene Enhances Tolerance to Drought &

Cold in Transgenic Lettuce (Cold in Transgenic Lettuce (Lactuca Lactuca sativasativa L.) & Bentgrass ( L.) & Bentgrass (Agrostis Agrostis

mongolicummongolicum))

Overexpression of Arabidopsis Overexpression of Arabidopsis ABF3ABF3 Gene Enhances Tolerance to Drought & Gene Enhances Tolerance to Drought &

Cold in Transgenic Lettuce (Cold in Transgenic Lettuce (Lactuca Lactuca sativasativa L.) & Bentgrass ( L.) & Bentgrass (Agrostis Agrostis

mongolicummongolicum))

"flavonoid 3', 5'- hydroxylase" "flavonoid 3', 5'- hydroxylase"

GMO-Transgenic Blue RosesGMO-Transgenic Blue Roses

''Fausse Fausse BeautéBeauté' '

'Détresse Bleu' 'Détresse Bleu' 'Incrédulité' 'Incrédulité'

"blue gene" + Herbicide resistant bar gene

GMO-Transgenic Golden RiceGMO-Transgenic Golden Rice

                                                 

Normal Normal ricerice

Golden rice Golden rice 22

Golden rice Golden rice 11

You can not build PEACE on the empty You can not build PEACE on the empty stomach….stomach….

Drought/Drought/dehydrationdehydration

Cold/Cold/freezingfreezing

heatheat

Water excessWater excess Biological Biological invadersinvaders

High-High-salinitysalinity

Plants must ADAPT or DIE ! ! ! ! !........Plants must ADAPT or DIE ! ! ! ! !........

ABA hormoneABA hormone

• Inhibit germination by high salt Inhibit germination by high salt • Inhibit the development of young seedling by Inhibit the development of young seedling by

high sugar high sugar

• Exhibit drought tolerance than wild-type plantsExhibit drought tolerance than wild-type plants

• Low/high temperature tolerance than wild-type Low/high temperature tolerance than wild-type plantsplants

• Oxidative stress tolerance than wild-type plantsOxidative stress tolerance than wild-type plants

• Hypersensitive to ABAHypersensitive to ABA• Slightly delayed germinationSlightly delayed germination• Mild growth retardation than that of wild typeMild growth retardation than that of wild type

ABF3ABF3 gene regulations in gene regulations in ArabidopsisArabidopsis::

Kang et al., (2002) The Plant Cell, Vol. 14, 343-357

Objectives:Objectives:

• 1. To establish an efficient 1. To establish an efficient in vitro in vitro regeneration regeneration systemsystem in lettuce and bentgrass in lettuce and bentgrass

• 2. To 2. To transform transform ABF3ABF3 gene to the lettuce and gene to the lettuce and bentgrass via bentgrass via Agrobacterium tumefaciensAgrobacterium tumefaciens

• 3. To determine the 3. To determine the transgene transmissiontransgene transmission and and expression in T1 progenyexpression in T1 progeny

• 4. To enhance 4. To enhance tolerance to drought and coldtolerance to drought and cold in in transgenic lettuce and bentgrasstransgenic lettuce and bentgrass

Section I.Section I. LETTUCE (LETTUCE (LACTUCA SATIVALACTUCA SATIVA L.) L.)

• Establishment of Establishment of in vitroin vitro regeneration systemregeneration system

• Establishment of gene Establishment of gene transformation systemtransformation system

• In vivoIn vivo test (drought & cold) test (drought & cold)

Family: Compositae Family: Compositae

Genus: Genus: LactucaLactuca

SSpecies : pecies : Lactuca Lactuca sativasativa L. L.Common name: LettuceCommon name: Lettuce

Genotype name: Genotype name: ChongchimaChongchima

Type: Leaf type lettuceType: Leaf type lettuce

UUsage:sage: FoodFood and and Home Home garden garden

What is the Chongchima What is the Chongchima lettuce?lettuce?

ABA-regulated genesABA-regulated genesPromoter ABRE Promoter ABRE (CACGTGGC)(CACGTGGC)DNADNA

ABF3ABF3

(Gene product)(Gene product)

Interacts with ABRE (transcription

factor)

Transactivates ABRE-containing reporter

gene

Multiple stresses

ABA hormoneABA hormoneABA hormoneABA hormone

Schema ofSchema of ABFABF33 ( (AABA-responsive elements BA-responsive elements (ABRE)(ABRE) BBindinginding FFactor) gene expressionactor) gene expression

Choi et al., (2000) The Journal of Biological Chemistry. Vol. 275: Choi et al., (2000) The Journal of Biological Chemistry. Vol. 275: 1723-17301723-1730

HH

PP

CC 7-day-old explants:7-day-old explants:

C: Cotyledon, C: Cotyledon,

P: PetioleP: Petiole

H: HypocotylH: Hypocotyl

Lactuca sativaLactuca sativa L. cv. Chongchima L. cv. Chongchima

A1 B1 C1

A2 B2 C2

rootingrooting

shootingshooting

Cotyledon Petiole Hypocotyl

Regeneration from different explants Regeneration from different explants of Chongchima lettuceof Chongchima lettuce

Shoot regeneration on Shoot regeneration on cotyledoncotyledon explants of lettuceexplants of lettuce

0

2

4

6

8

10

12

0 0.05 0.1 0.2 0.4 1.6

00.250.512

N

o.

of

shoots

/coty

ledon

Kinetin(mg/L)

NAA (mg/L)

Chongchima

*Values indicate the mean of three replications±SE

RB

NOSTER Gfp::gus Ubi-PRO ABF3

Arbc STER

35SPRO35SPRO HPH35S TER

LB

SmaSmaII

EcoEcoRIRI

XhoXhoII XhoXhoII

HinHindIIIdIIIBamBamHIHI

SacSacII

ABF3 probe (2 kb)ABF3 probe (2 kb)

HinHindIIIdIII

A part of the T-DNA region of vector A part of the T-DNA region of vector pCUMBpCUMB

Selection1Selection1

RootinRootingg

Selection2Selection2

Transformation stages of Transformation stages of Lactuca sativa Lactuca sativa L. L. genotype Chongchima via genotype Chongchima via Agrobacterium Agrobacterium

tumefacienstumefaciens

Co-Co-cultivated cultivated explantsexplants

(A)(A)

Produced Produced HygHygRR calli calli

Produced Produced HygHygRR plantsplants

Produced Produced HygHygRR and and

GUS+plants GUS+plants (B)(B)

T.E. T.E. (%)(%)

(B/A)(B/A)

8383 5353 99 99 10.810.8

Transformation efficiency (T.E.) of transgeneTransformation efficiency (T.E.) of transgene

21 days later21 days later

Morphology and fertility of TMorphology and fertility of T00 transgenic planttransgenic plant

Transgene segregation to TTransgene segregation to T11 progeniesprogenies

WTWT ubi::ABF3 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 TGUS expression on T11 putative putative transgenic plantstransgenic plants

WT 101 104 107 108 WT 101 104 107 108

2 kb2 kb

25 µg gDNA was digested by 25 µg gDNA was digested by HinHinDIII, and hybridized with DIII, and hybridized with 3232P labeled P labeled ABF3ABF3 probe probe

Gene integration is confirmed by Southern Gene integration is confirmed by Southern blottingblotting

Drought assay Cold assay

WT ubi::ABF3 WT ubi::ABF3

31.7

68.5

51.2

10

Wild-type Transgenic

Drought and Cold tolerance assaysDrought and Cold tolerance assays

TT22 plants at -5±1 ℃, 50±5% relative humidity with strong wind plants at -5±1 ℃, 50±5% relative humidity with strong wind for 48 hfor 48 h

After 2 weeks since moving into the glasshouseAfter 2 weeks since moving into the glasshouse

Wild-type T2 plantsWild-type T2 plants

Cold test at the open field in the Cold test at the open field in the winterwinter

Conclusions Conclusions • To choice of To choice of genotypegenotype and and explants typeexplants type are a critical factors are a critical factors

to consider in lettuce tissue culture. to consider in lettuce tissue culture.

• Incorporation of both exogenous Incorporation of both exogenous kinetinkinetin and and NAANAA are are essential, and cytokinin to auxin ratio essential, and cytokinin to auxin ratio 5:15:1 and and 10:110:1 is the is the most effective on shoot regeneration of lettuce cotyledon most effective on shoot regeneration of lettuce cotyledon explants. explants.

• All transgenic plants and their All transgenic plants and their progeniesprogenies were were fertilefertile and and morphologically normalmorphologically normal, and gene transformation efficiency , and gene transformation efficiency was relatively higher. was relatively higher.

• The transgene inherited in a The transgene inherited in a normal Mendelian patternnormal Mendelian pattern in in one out of the nine T1 progenies. one out of the nine T1 progenies.

• The transgenic plants displayed The transgenic plants displayed more tolerant to drought more tolerant to drought and coldand cold than that of wild-type plants, suggesting that than that of wild-type plants, suggesting that overexpression of Arabidopsis overexpression of Arabidopsis ABF3ABF3 is functional in lettuce. is functional in lettuce.

Section II.Section II. MONGOLIAN MONGOLIAN BENTGRASS BENTGRASS

((AGROSTIS MONGOLICUMAGROSTIS MONGOLICUM))• Establishment of Establishment of in vitroin vitro regeneration systemregeneration system

• Establishment of gene Establishment of gene transformation systemtransformation system

What is What is Agrostis mongolicumAgrostis mongolicum & its & its usage?usage?Family:Family: Poaceae Poaceae

Genus:Genus: AgrostisAgrostis

Species :Species : Agrostis mongolicumAgrostis mongolicum

Common name:Common name: Mongolian Mongolian bentgrass bentgrass

ROADSIDE GRASSING ROADSIDE GRASSING & GREENING& GREENING

SPORTS FIELDSPORTS FIELDGOLF COURSEGOLF COURSELIVESTOCK PASTURE & FORAGE LIVESTOCK PASTURE & FORAGE

LAWNLAWNSS

FriableFriableRegenerable, green-Regenerable, green-spottedspotted

WateryWatery

Brown-Brown-spotedspoted

CompacCompactt

11

55

44

33

22

Callus types derived from mature Callus types derived from mature seedsseeds

TDZ and NAA combination effect on TDZ and NAA combination effect on shoot regeneration of shoot regeneration of Agrostis Agrostis

mongolicummongolicum

0 0 0.050.05 0.5 1 0.5 1 22

0.050.05

00

0.10.1

0.20.2

TDZ TDZ (mg/L)(mg/L)

* Values indicate the mean ± SE of three replicatesCallus Type 2

0

5

10

15

20

25

30

0 0.05 0.5 1 2

00.050.10.2

TDZ (mg/L)

NAA (mg/L)

Nu

mb

er o

f sh

oots

/cal

lus

Transformation stages of Transformation stages of Agrostis Agrostis mongolicummongolicum via via Agrobacterium Agrobacterium

tumefacienstumefaciens

Shoot regeneration Shoot regeneration & selection& selection

Callus inductionCallus induction Pre-cultivationPre-cultivation

• InfectionInfection

• Co-cultivationCo-cultivation

• Bacteria Bacteria EliminationElimination

Putative transgenic Putative transgenic plant growing in the plant growing in the

glasshouseglasshouseRoot induction & Root induction &

selection selection

R: resistant, S: R: resistant, S: susceptiblesusceptible

R

SS

S

Morphology of Morphology of ubi::ABF3 ubi::ABF3 transgenic transgenic Agrostis mongolicumAgrostis 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 PCR products using hygromycin (A) and GUS gene primers (B)gene primers (B)

M WT 1 2 3 4 M WT 1 2 3 4 5 5

900 900 bpbp

M WT 1 2 3 4 M WT 1 2 3 4 55

AA BB

M: Ladder marker M: Ladder marker (Takara)(Takara)

M: 1 kb Plus DNA LadderM: 1 kb Plus DNA Ladder

WT: WT: wild-type and wild-type and lanes lanes 1-5: putative transgenic 1-5: putative transgenic lines lines

500 bp

2 kb

WT 1 2 3 4 WT 1 2 3 4 55

Gene integration is confirmed by Southern Gene integration is confirmed by Southern blottingblotting

25 µg gDNA was digested by 25 µg gDNA was digested by EcoEcoRV, and hybridized with RV, and hybridized with 3232P labeled P labeled ABF3ABF3 probe probe

• Five type of callus was determined, Five type of callus was determined, callus type 2callus type 2 displayed significantly higher shoot regeneration capacitydisplayed significantly higher shoot regeneration capacity

• TDZ+NAA combination was effective, however the highest TDZ+NAA combination was effective, however the highest number of shoots per callus was observed when medium number of shoots per callus was observed when medium containing containing 0.05 mg/L TDZ0.05 mg/L TDZ

• Transgenes were Transgenes were stably integratedstably integrated in the genomes of in the genomes of putative transgenic plantsputative transgenic plants

• The The morphologymorphology of transgenic plants were of transgenic plants were normalnormal in in terms of shape of whole plantsterms of shape of whole plants

Conclusions Conclusions

Future studyFuture study

•Introducing draft inducible Introducing draft inducible genegene

•Introducing herbicide-Introducing herbicide-resistant generesistant gene

Thank you for your attention!Thank you for your attention!

AcknowledgmentsAcknowledgments