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RESE RCH ART ICLE

High efficiency transforn1ation

of

n1aize (Zea mays

L.)

n1ediated

by

Agrobacterium tumefaciens

Yuji Ishida

,

Hideaki Saito, Shozo Ohta, Yukoh Hiei, Toshihiko Komari, and Takashi Kumashirot

Plant Breeding and

Genetics

Research

Laboratory, Japan Tobacco Inc., 700 Higashibara,

Iwata, Shizuoka

438, Japan.

tPresent

address:

Agribusiness Division,

Japan

Tobacco Inc.,

2-1 Toranomon 2-chome, Minato-ku,

Tokyo 105, Japan.

*Corresponding author e-mail: [email protected]).

Received 29 December 1995; accepted

28

February 1996.

Transformants

of maize

inbred

A 188 were

efficiently

produced from immature embryos cocultivated

wi th

grobacterium

tumefaciens that carried super-binary vectors.

Frequencies

of

transformation

(independent t ransgenic plants/embryos)

were

between

5 and 30

. Almost

all transformants

were

nor

mal

in m

orphology

, and

more than

70%

were

fertile. ~ t b l e

integration, expression

,

and inheritance

of

the

t r

ans

genes

were

confirmed

by molecular and genetic

analysis.

Between one and three copies

of

the

transgenes w ere integrated

with

little rearrangement, and

the

boundaries

of

T-DNA were similar

to

those

in transgenic dicotyledons and rice. F1 hybrids between A188 and five

other

inbreds were transformed

at

low frequencies.

Keywords: transformation, maize, Agrobacterium tumefaciens

Application

of

Agrobacterium-mediated method

of

gene transfer

has until now been limited to dicotyledonous plants, although this

method of gene delivery to higher plants has advantages, such as the

transfer

of

relatively large segments

of

DNA with little rearrange

ment, and integration

of

low numbers

of

gene copies into plant

chromosomes. Although monocotyledons are not the natural hosts

of

Agrobacterium tumefaciens , infection

of

maize and other cereals

with A tumefaciens has been attempted in various laboratories.

Competence of A. tumefaciens in infection

of

maize was first

indicated in the studies of agro-infection by Grimsley et al. ' , in

which eDNA of maize streak virus was delivered to maize plants by

A. tumefaciens and the plants became systemically infected. Gould

et al. ' inoculated shoot apices of maize with A. tumefaciens and

obtained a few transgenic plants, and Shen et al.• observed expres

sion

of

a 13-glucuronidase (GUS) gene delivered to maize shoots by

A. tumefaciens. The studies

of

the Agrobacterium methods in other

cereals also provided indications

of

successful transformation'-' .

For example, Mooney et

al.

' produced transformed cells from

wheat embryos cocultivated with A. tumefaciens, and Chan et al.

obtained a few transgenic rice plants by inoculating immature

embryos with

A. tumefaciens

.

However, the transformation fre

quency in these methods was rather

low,

and some

of

the studies

did not provide sufficient molecular and genetic evidence

of

pro

duction

of

transgenic plants. Consequently, these methods have

not

been widely adapted.

Recently, Hiei et al. ' reported a method to efficiently produce

transgenic plants from rice calli cocultivated with A tumefaciens.

They claimed the choice

of

starting materials, tissue culture condi

tions, bacterial strains, and vectors were essential in efficient gene

transfer. Here

we

describe a method to efficiently transform maize

by cocultivation

of

immature embryos with A tumefaciens. We pro

duced a large number

of

transformants

of A188

and demonstrated

stable integration, expression, and inheritance

of

transgenes.

Results

Infection

and

selection. Immature embryos

of

maize inbred line

NATURE BIOTECHNOLOGY VOLUME 14 JUNE 1996

A188 were cocultivated with

LOX

10 ' cfu/ml

of A

tumefaciens

LBA4404(pSBl31) (Fig.

l

in

LS-AS

medium. The embryos were

between 1.0 and 1.2

mm

in length, and between 80o/o and lOOo/o

of the immature embryos expressed GUS after cocultivation

(Fig. 2A). The immature embryos were transferred to a selection

medium, LSDl.5, containing phosphinothricin (PPT). PPT

resistant calli emerged from between 38o/o and

90o/o

of the imma

ture embryos (Table 1, Fig. 2B) and expressed GUS uniformly

(Fig. 2C). These calli were typical type I calli, which are compact

clusters of relatively organized cells' . A large number of shoots

were regenerated from the PPT-resistant calli that were transferred

to a regeneration medium containing PPT (Fig. 2D), and showed

strong expression of GUS in the leaves (Fig. 2E). PPT-resistant,

GUS-positive plants were obtained from >11.8% of the immature

embryos initially cocultivated (Table

l .

Efficiency of transforma

tion was remarkably consistent from experiment to experiment,

and

as

many

as

44 independent transgenic plants were obtained in

a single trial (experiment no.

6)

in the best case. In the following

experiments, all of the parameters were identical to those above

unless otherwise indicated.

Oth

er tissue types. Type I

ca

lli' produced from immature

embryos

of

A188 were infected with LBA4404(pSB 131). Although

. most

of

the calli expressed GUS after cocultivation, few PPT

resistant cells were obtained during selection. Shoot tips

of

A188

and

cells in a suspension culture from cultivar 'Black Mexican

Sweet'

BMS)

were also infected with LBA4404(pSB13l), but

GUS

expression was only observed in a few tissue pieces. Therefore,

infection

of

these tissues

was

not studied further.

Stage of immature e

mb ry

os. Immature embryos

of

A188

between

1.5

and 2.0

mm

and between 2.0 and

2.5

mm in length

were also tested.

GUS

expression was detected in most

of

the

immature embryos after cocultivation, and the PPT-resistant calli

derived from such immature embryos showed uniform expression

of

GUS. Stable transformants, however, were obtained at a very low

frequency (Table

1).

Concentrationof inoculum

. Infection with >5.0X 10 ' cfu/ml

745



ARTIC

LE

E

B H B EH

I

ARi l

I

55

55

11

GUS

I

I BR

TNOS lntr

on

TNOS

1Kb

1.

T-DNA

of

pSB131. Abbreviations:

BR

,

right

border; BL, left

r; GUS,

3

-glucuronidase; BAR, phosphinothricin acetyltrans

; 355, 35S p

romoter;

TNOS, 3 signal of nopaline synthase;

BamHI; E, EcoRI; H, Hindlll

LBA4404(pSB 131) resulted in large clusters

of

GUS-expressing

but no trans

l.O X

10

' cfu/ml, PPT-resistant cells were obtained

<10

of

the immature embryos. PPT-resistant cells were not

was

<

LOX 10

' cfu/ml.

Media based on

th

e N6

medium

. Many media for maize tissue

ed from the N6 medium

1

. Immature embryos

of

were cocultivated with LBA4404(pSB131) in N6-AS Jpedium

transferred to a selection medium, N6Dl.5 , containing PPT.

of

the

not

. Therefore, media contain ing

LS

salts are superior

to

all

further experiments.

Hygr

om

ycin

re

sis

tan

ce. Another selective-marker, a hygro

sistance gene, was tested. When immature embryos

of

' , most

of

. The

re then cultured on a hygromycin-containing medium,

1. Effic

iency of

maize transformation.

and resistant calli were obtained 8 weeks after cocultivation. Plants

were regenerated from the calli

on

hygromycin-containing

medium

and showed strong expression

of GUS

in the leaves. The

frequencies

of

transformation by hygromycin selection were con

sistent,

and

transformants were obtained from 5% to 10%

of

the

immature embryos (Table l.).

ther

s

tra

ins of

A

tum f ciens Immature_embryos

of

A188 ·

were infected with LBA4404(piG 121Hm)'

and

other strains

of

A

tumefaciens Expression

of

GUS after the cocultivation was

found at high frequencies, but the level

of

expression was consid

erably lower

than

in those

immature embr

yos infected with

LBA4404(pSB131)

or

LBA4404(pTOK233). Thus, use

of other

strains

was

not studied further.

the

r

genotyp

es

of ma

ize. Five inbred lines (W117, W59E,

A554, W153R, and H99) and

five

F1 hybrids (W117 x

A188,

W59E

x A188, A554 x A188, W153R x A188, and H99 x

A188

) were exam

ined. Immature embryos of these genotypes were cocultivated with

LBA4404(pSB131). GUS expression was detected in most

of

the

immat

ure embryos after cocultivatio'n, and transformed plants

were obtained from all

F1

hybrids. The frequencies

of

transforma

tion

of

F1 hybrids varied from 0.4% to 5.3% (Table 1), whereas no

transformants were obtained from the inbred lines.

Characterizati

on of th

e

plant

s

in

the

RO

gen

era

tion. A total

of

120

independent, PPT-resistant, GUS-positive plants

of A188

from

immature embryos infected with

LBA4404

(

pSB131

) were grown in

a greenhouse. Almost all

of

the plants were normal in morphology

(Figs.

2F,

2G, and 2H) and the majority (

about

70 %)

of them

produced

as

many seeds

as

seed-derived control plants by sel -

pollination (Fig. 21) .

Thirty-three

of

the transformed plants were analyzed by

Southern hybridization. Isolated DNA

was digested with BamHI or

EcoRI

and

allowed

to

hybridize with bar

and

gus probes. Both

genes were detected in all

of

the

RO

plants analyzed, whereas no

hybridization signal was detected in the nontransformed plants

Number

of immature embryos

Produced

Produced Produced

antibiotic-

antibiotic- antibiotic-

resistant,

Size of

immature

em

bryos

(mm)

Experiment

no.

Inoculated

resistant resistant GUS+ Frequency

xA1

88

X A188

R x A188

Vector

pSB131

pSB131

pTOK233

1.0--1 .2

1.5-2.0

2.0--2.5

1.0--1

.2

1.0-1 .2

1

2

3

4

5

6

7

8

9

10

1

1

1

2

1

1

2

3

4

(A)

callus

44 28

52

33

51

46

70

56

76 30

369 200

121 46

27

15

36 26

77

38

57

11

156

33

112

36

114

26

104 44

247

46

284 69

21 9 18

7-2

15

22

5

22 1

19

2

plants plants (B)

(B

/A

, )

9 6

13.6

10 7 13.5

13 7 13.7

26

14

20.0

12 9

11

.8

71 44

11

.9

33 20

16.5

8 5

18.5

18

11

30.6

32

16

20.8

0 0 0.0

3 2 1 .3

8

4

3.6

10 6 5.3

1 1

1.0

7

5

2.0

2 1

0

.4

4

3

1.4

8 7 9.7

2 2 9.1

1

4.5

2 5.3

NATURE BIOTECHNOLOGY VOIIIMF 14 .IIINF 1QQR

. .



Figure 2. Transgenic cells and plants derived from

the

immature

embryos

of

maize inbred A188 infected with

A tumef i ns

LBA4404(pSB131). (

A)

Expression

of

GUS

after

infection. The

i

mmature embryos

were

stained wi

th

5 bromo

-4-

chloro 3

indolyl 3-D-glucuroni

de

(X-Giuc) after 3 days

of

cocultivat ion.

B)

PPT-resistant calli derived

from the

immature embryos. The

immature embryos were plated on a selective medium after

infection. The ph

otograph was

taken after 3

weeks

of selection.

C)

Expression

of

GUS in PPT-resist

ant

calli . Proliferated calli

were stained

with

X-Giuc after 7 weeks of selection. (D) Plant

regeneration

from

PPT-resistant calli. The photograph

was

taken

3 weeks

after the

selected calli had been plated on PPT-contain

ing regeneration

med

ium. (E) Expression o f GUS in

the

eaf

of

a

transformant. Excised leaf of a transformed plant (upper) and a

nontransformed plant (lower) were

st

ained

with

X-Giuc. (F) A

transformed

plant

at

fl

owering

st

age. (G) A tassel

of

a trans-

formed

plant.

H)

Silk exposure

from

an

ear of

a transformed

plant.

I)

Harvested ears

of

a

trans

formed plant (upper) and a

nontransformed plant (lowe

r)

. Both ears were obtained by self

pollination. (J) Test

of

the progeny

for

resistance

to

Basta® Seed

derived young plants

of trans

formed (left) and nontransformed

(right) plant were sprayed with a 0.2% Basta®s

olut

ion. The pho

tograph

was

taken 2 weeks

after the

application

of the

herbici

de

.

(Figs. 3A, 3B, 3C , and 4).

As

expected from the T-DNA map of

pSB131 (Fig.

1),

digestion

of

the DNA with BamHI yielded various

band

sizes longer than 1.9 kb that hybridized to the

bar

probe,

and various

band

sizes longer than 2.3 kb that hybridized to the

gus

probe (Figs. 3A and

3B

) . The

number

of hybridizing bands

reflected the copy number of the trans genes in the plant genome,

which varied from one to three (Figs . 3A, 3B, and Table 2).

Nineteen

of

the 33 plants contained a single copy

of

the

bar

gene,

and

23

plants contained a single copy

of

the

gus

gene. Because the

EcoRI sites are located very close to either border

of

the T-DNA

of

pSB131, detection

of

a 5.4-kb EcoRI fragment in this analysis

strongly indicated integration

of

an intact copy

of

the T-DNA.

(F

ig. 3C). Thirty-one

of

the 33 plants contained a 5.4-kb EcoRI

fragment hybridizing to the

bar

probe (Table

2)

. Therefore, it

is

likely that approxi-mately 40%

of

the

tran

sformants carried a

singly copy

of

the intact

T-

DNA.

Analysis of

th

e T-DNA

boundaries

The junction regions

of

introduced DNA and plant genome were cloned from several

plants that contained a single copy

of

the 5.4-kb EcoRI fragment

by the inverse PCR method

•

. Sequence analysis revealed that the

junctions were located in

or

near the 25-bp repeats (

Fig

. 5) . This

observation

is

similar to the results from the analysis

of

the

junctions in dicotyledons · and rice'. However, only four

of

the

10

right junctions sequenced were at the site found in tobacco trans

formants, and it

is

not clear why the right boundaries in maize

appeared

less

precise than those in dicotyledons and rice.

Inheritance

of

marker

genes. Selfed progeny

of 40 of

the

120

transformants

of

A188 grown in the greenhouse were examined for

PPT resistance and GUS expression (Table

3)

. Resistant and sensi

tive seedlings were distinguishable 6 days after the application

of

Basta® The sensitive plants died within 2 weeks after the treatment,

while the resistant plants were

as

healthy

as

non reated plants (Fig.

2J) . PPT resistance and

GUS

expression were strongly linked and a

segregation ratio

of

3: 1 for both traits (resistant:sensitive and posi

tive:negative) was observed for 28

of

the

40

lines. A

few

lines

showed strange

seg

r

eg

ation ratios of

1:1

and 1:3 (Table 3).

DNA was extracted

fro

m the R1 progeny

of

transf6rmants 131,

NATURE BIOTECHNOLO

GY

VOLUME 14 JUNE 1996

RESE RCH

A RTIC LE

238, 248, and 249, shown in Tables 2 and 3, and analyzed by

Southern hybridization. The

bar

gene

and

the

GUS

gene were

present in the PPT-resistant, GUS-positive progeny

and

absent

from the sensitive, negative progeny (Fig . 5. The data for

bar

are

not

presented.). The bands that were identical in size to

the bands detected in the R1 plants were also present in their

respective parents.

Discussion

The method

of

maize transformation reported here

is

efficient and

reproducible. Only 10 weeks were needed to obtain transformed

plants from infected immature embryos, and several lines

of

evidence show that the transgenes are stably incorporated int o

maize genome. Clear Mendelian transmission of the T-DNA to the

progeny was demonstrated by genetic analysis. Drug-resistan ce

and

GUS

expression were tightly linked,

and

segregation

of th

e

T-DNA was confirmed by Southern hybridization. Sequence

analysis

of

the junctions between T-DNA and plant DNA in the

maize transformants revealed

that

T-DNA boundaries in maize

were similar to those in dicotyledons and rice .

In

Agrobacterium mediated

gene transfer, expression

of

D A

segments in

Agrobacterium

attached to inoculated tissues

or

in

other contaminating microorganisms needs to be carefully distin

guished from expression

of

integrated foreign DNA. Here we take

advantage

of

a

GUS

gene

that

contains in the coding region

an

intron

that is not

expressed in bacterial cells . Thu

s, th

e strong

expression

of

GUS

we

observed in the immature embryos after the

cocultivation with

A. tumefaciens

and in PPT- or hygromycin

resistant maize plants was not due to bacterial contam inati

on

.

A large

number of

transformants were analyzed by So uth

ern

hybridization, and the size

of

BamHI fragments hybridi

ze

d to the

probes differed from plant to plant, indicating random insertion of

the transgenes into maize chromosomes.

Various transformation techniques have often been

as

sociated

with aberrations in morphology, fertilit y, and other agronomically

important

characteristics '•- . In this stud

y,

almost

all

of

the 120

independent transgenic maize plants characterized

in

detail were

747



RESEARCH ART ICLE

A

kb

3

9.6

6.6-

4.

3

2.

3

2.

B

c

<::.<

-

2.0-

U.b-

1 2 3 4 6 7 8 9 1 1 1 c

Figure 3. Southern blot analysis of transformed plants RO generation). DNA extracted from PPT-resistant and GUS-positive plants was digested

with BamHI

A

B) or EcoRI C), and allowed to hybridize to the

b r A, C)

or

gus

B) probe. Lane C, non-transformed control plant; lanes 1 14,

transformed plants

No.

176, 185, 187, 191, 194, 197, 198, 238, 239,

241

, 244, 245, 248, and 249 shown

in

Table

2)

regenerated from PPT-resistant

calli, which were derived from independent immature embryos infected with LBA4404 pSB131).

Table 3.

Genet

ic analysis

of

independent

transfor

mants

pro

duced

by LBA4404 pSB131).

PPT resistance

GUS expressi

on

Number of

Number of

plants in R1 plants in R1

Transformant generati

on

Ratio

generation Ra t io

RO) R s

R:S

x'

+

c

+

:

x'

Table 2.

Copy number of

transgenes

in maize

transformants pro-

37

9

3:1 0.72 23 23

duced by LBA4404 pSB131).

5

39

13

3:1 0.00

37

15 3:1

0.41

6 46 5

15:1 1.10 42 9

3:1

1.47

Copy number

24 36 15

3:1 0.53 33 18 3:1

2.88

53

15 41

14 42

Transformant

b r

GUS

5.4kb EcoRI*

55 39

17 3:1 0.86 40

16 3:1 0.38

68 38

16

3:1 0.62 38 16 3:1 0.62

2

2

+

75 45 10

3:1 1:36 3 33 19

3

2

2

+

76 34

21 28 6

21

7 2 1

+

77 48 7 48 7

23

2 2

+

79

34 22

34 22

25 3

1

+

80

44 12

3:1 0.38 44 12 3:1 0.38

33

2 1

+

83

42 14

3:1 0.00 41 15 3:1

0.10

42

1 1

+

91 38 15

3:1 0.31 38 15 3:1

0.31

47

1 1

+

93

46 18 3:1

0.33 46 18 3:1 0.33

131

2 2

+

102 19 5

3:1

0.22 9

5

3:1 0.22

176

1 2

+

105 39

15 3:1 0.22 36 16 3:1

0.92

185

1 1

+

115

43 13 3:1

0.10 43 13 3:1 0.10

187

1 1

+

123

42 12 3:1

0.22 42 12 3:1 0.22

191

1 1

+

124

35

14 3:1 0.33 35

14 3:1 0.33

194

1 1

+

125 37 9

3:1

0.72 37 9

3:1 0.72

197

1 2

+

126 37

12 3:1 0.01 37 12 3:1

0.01

198

1 2

+

131 49

1 15:1 1.54 49 1

15:1 1.54

238

1 1

+

133

41 13 3:1

0.02 41 13 3:1 0.02

239

2

1

+

134 29 9

3:1 0.04 29 9 3:1 0.04

241 3 3

+

136

31

19 9 22 19

244

1 1

+

139

25 15 3:1

3.33 25 15 3:1 3.33

245

1

2

+

140 38 12

3:1

0.03

38

12 3:1

0.03

248

2

1

+

144

39

17

3:1 0.86 39 17 3:1 0.86

249

2 2

+

145

51

1

15:1 1.66 49 2 15:1 0.47

252

1 1

154 37 14

3:1 0.16 37 14 3:1 0.16

253

1 1

156 28 8

3:1 0.15 27 8 3:1 0.09

258

1 1

+

191

14 8 3:1

1.52 14

8

3:1 1.52

263

2 1

+

197 17 8

3:1 0.65 17 8 3:1 0.65

282

1

1

+

238 20

10 3:1

1.11

20

10 3:1 1.11

289

1

1

+

241 22 5

3:1

0.60 22 5 3 : 0.60

293

1 1

+

244 24 6

3:1 0.40 24 6 3:1 0.40

295

1

1

+

245 26 6

3:1 0.67 26 6 3:1

0.67

291

2 2

+

248 24 5

3:1 0.93 24 5 3:1

0.93

294

2

+

249

27

3

15:1 0.72 27 3

15

:1

0.7

•oetection

of

5.4-kb Eco RI fragments which contained most

ofthe

T-D

NA R resistant; S sensitive;

C

chimeric expression. Ratios that

gi

ve the smallest

region in the Southern

blot

analysis.

x values are shown together with the x values .

748

N4TIIRI= RlnTI=r.I INnl nr V l in li 1<1 .II IN =

1<NR



l

eft bo

rder

re pe

at

DNA

Ri &ht border repeat

oT

T3

GCTGGTGGCAGGA

TAT ATTGTGGTGT

AAACMATT

----

TTCAGTTT

AAACTATCAGTGTTTGACAGGATAT ATTGGCGGGTAAACCT

AA

G

. 2

--acsagcacaaaaccATATTCTGGTGT

WCWTT

----TTCAGTTT C

TAT

CAGTGTTTGA

a g a g a a t g c c a ~ : a c c t t a t a g c

47 -- t tga tat st

tt

catat gaGGTGT C TT---- TTCAGTTT CTATCAGTG

TTTGA ;aacatcact

cc gactaugt

ca tt

- -

185

--tat

ggcc acGGA T TATG TGGTGT C TT----TTCAGffi AAACTATCAT

acu&cgggcggggttacaggctgccc

ccaccg --

194 --aq;ctgcacaagagacgc&a&aUUAAACAA.ATT

----TTCAGffi

AAACTATCA T t a a a c a a g c c a g a c . g a g c ~ : a l l : t t t tattttat--

23

8 --gt atattctctagtgtcaaccacca tct t

AAATT

----

TT

C

AGm

ct cctatctatactcccge&cttatcaatgtccctagat&t caa--

244 -- cttaatcatctcATATA TTGTGGTGTAAACAAATT-- --TTCAGffiW attaataaggcatgtt

tt

aac tgcgaaacatcauacgaca--

258

----TTCAGTTTAAACTATCAGTGffiGA tacaagaaaccattsauaaacattcc--

282 --gtgcgtaggtgtAT T

ATTGTGGTGTWCWTT ----TTCAGagcai&catggtg&tgatgguacaattattattccttt&t&cct :g

--

2

89

--

acaacttctgctgccgtTTG

T

GGTGT

A

C

AAATT

----

TTCA

GffiAAAC

T

ATCAG

TG

fi

GAaagggtcacatactactac

tactcctc

--

293 -- t

gt

cgatcacGGAT T TTGTGGTGT C TT

---

-TTCAGffiAAccc tgaagaacctgtgt aaA:ctgt caatgaagt t ctcgacgc--

Figure 4. Sequence

analysis

of T-DNA/plant DNA

junctions

.

Se q

uences

of

the

junctions found in

selected

maize

transformants

are shown below the

sequences

of the T-DNA borders of pTiT37.

Sequences presumably originated from maize genomic DNA are

s hown

in

lowercase letters. As the Southern blot analysis indicated

these plants each contained a single copy of the

T-DNA;

the left and

right border sequences in a plant possibly

corresponded

to the ends

of a singleT-DNA segment and are

presented

here in such a way. The

left junction in transformant 258 was not determined.

nor

mal in

morphology

and

70o/o

were

fully fertile. Furthermore

Southern hybridization and sequence analysis ofT-DNA

bound

ar ies revealed that a majority (around

70o/o) of

the maize transfor

m ants contained a

single

copy

of

the T-DNA with no notable

rearrangements,-and

no

transformants contained more

than

th ree

cop ies of the T-DNA.

Fac

to r

s

that

affect

the

efficiency

of

transformation include the

ty

pes

and s

tage

s of

ma i

ze

ti s

s

ues

infected , the concentration of

A.

tumefaciens,

composition

of

the media for tissue culture selec

tion marker genes, kinds

of

vectors, and the

maize

genotype. This

m

ultiplicity of factors

is probably the reason that transfo rmation

metho ds mediated by

A. tumefaciens have

not

been

readily

deve

loped

. It was relatively easy to find conditions for good GUS

expression after the coculti

va

tion

,

but

drug

- res

istant

cells

were

e

lecte

d in only a few insta

nces

. Therefore GUS expression da ta

di d not necessarily help adjust infection parameters. It is possible

Tabl e 4.

Media for

culture

of

maize tissues.

edium

LS

-inf

-AS

0 1.5

z

ti2

LSF

inf

AS

01 .5

Composition

LS major salts and LS

min

or salts, 0.5 mg /L nicotinic acid,

0.5 mg /L pyridoxine HCI, 1.0 mg /L thiamine HC I, 100 mg /L

myo -inositol, 1.0 g/L casamino acid, 1.5 mg /L 2,4-o,

68.5 g/L sucrose, 36.0 g/L glucose, pH 5.2

LS major salts,

LS

minor salts, 0.5

mg

/L nicotinic acid ,

0.5

mg

/ L pyridoxine

HCI

, 1.0 mg /L thiamine HCI , 100 mg /L

myo-inositol, 700 mg /L L-proline, 1.5 mg /L 2,4-o, 20 g/L

sucrose, 10 g/L gl ucose, 500

mg

/L

MES

, 100 iJM acetosy

ringone, 8 g/L agar, pH 5.8 -

LS-AS

medium without glucose and acetosyringone, plus

250 mg /L cefotaxime, pH 5.8

LS01

.5 medium without 2,4-o, plus 5.0 mg /Lzeatin, pH 5.8

Half-strength

LS

major salts of LS01 .5 medium without

2,4-o and

L

pro

line

, pH 5.8

N6 major salts, N6

mi

nor

sa

lts and N6 vitamins, 1.0 g/L

casamino acid, 1.5

mg

/L 2,4-D, 68.5 g/L sucrose, 36.0 g/L

glucose,

pH

5

.2

N6

major salts,

N6

minor salts, N6 vitamins, 700 mg /L

Lproline, 1.5 mg /L 2,4-o, 20 g/L sucrose, 10 g/L glucose,

500 mg /L MES, 100 iJM acetosyringone, 8 g/L agar, pH 5.8

N6-AS medi

um

without glucose and acetosyringone, plus

250 mg /L cefotaxime, pH 5.8

JU

RE

BIOTECHNOLOGY

VOLUME 14 JUNE 1996

k

23

-

9.6-

6.6-

RESE RCH ARTI

CLE

0.5- ..._ ._ ...

Figure 5. Southern blot analysis of the R1 progeny of transformed

plants 131 , 238, 248,

and

249

shown

in Table 2

and

3. DNA extracted

from

RO

plants (lane 1); PPT-resistant, GUS-positive R1 progeny (lanes

2-6 for transformant 1

31

, lanes 2-4 for transformants 238, 248, and

249); PPT-sensit ive, GUS -negative R1 progeny (lane 7 for transformant

131 , lane 5 for transformants 238, 248, and 249); and nontransformed

control plant (lane C) was

digested

with BamHI, fractioned by electro

phoresis, transferred to a nylon

membrane

, and allowed to hybridize

to t

he us

probe.

th at the main hurdle

in

transformation was not in delivery

of DNA

fragments

into plant cells,

but

in recovery

of

cells that

acquired

the

T-DNA in

their chromosomes.

pTOK23

3 and pSB131 belong to a class of vectors called super

binary vectors. These vectors carry

the

virB, virC,

and

virG of

A281, a strain hig

hly

efficient in transformation of higher plants" .

It is evident that super-binary vectors are very useful in

ma

ize

transformation.

Although

pSB131 was

apparently higher

than

pTOK233 i.n the efficiency

of

transformation this may be

because

various parameters were first optimized for PPT selection .

The

present study

and

the

previous study

of

rice transforma

tion

by Agrobacterium have

provided strong

support for

the

hy

pothesi

s that

T-DNA

is

transferred from

Agrobacterium to

dicot

yle

do n

s

and monocot

y

ledons by

an

identical molecu

l

ar

mechanism. Therefore

being

monocotyledonous is

no longer

a

reason to restrict the application of Agrobacterium-mediated gene

transfer techniques to

other important

cereal crops.

Experimental

protocol

Plant materials.

Mai

ze inbred lines A188, W117, W59E, A554, W153R, H99,

and cultiver BMS were supplied from the National Institute of Agribiological

Resources of japan. F1 hybrids were obtained by c ross-pollinat ion in a

greenhou

se.

Immature

em

bryos of l.0-1.2 mm in length were aseptically

excised from kernels of plants grown in a greenhouse. Such immature

embryos were generall y obtained between 9 and 14

days

after pollination

(DAP

),

depending on env ironmental fac tors. For the study of optimal stages,

immature embr

yos

of 1.5-2.0 mm (11-16

DAP

) and of2.Q-2 .5 mm (13-18

DAP )

wer

e prepared. Ty

pe

I calli of A188 and a susp ension culture of BMS

cells were

prepared according to the procedure previously described" .

Bacterial strains and plasmids.

A tumefaciens

strain

LBA4404(pTOK233 ) has been previously described' . The T

-D NA

of

pTOK233

contained a hygromycin-resistance gene hpt), a kanamycin-resis

tance gene, and a gene for

GUS,

which has an intron in the

N-

terminal

region of the coding sequence and is connected

to

the 35S promoter of cauli

flower mosaic virus" . This intron-gus gene exp resses GUS activity in plant

cells but not

in

the ce

ll

s of A. tumefac iens

1

. pSB131 was co nstructed

as

fol

lows:A PPT-resistance gene

(bar)

connected to the 35S promoter was

excised

as

a 2.2-kb Hindlll-EcoRI fragment from pDE110 (r

ef.

25) and inserted

between the Hind II and EcoRI sites

of

pTOK246 (manuscript in prepara

tion), which consisted of the origin of replication of pBR322, a sp ectino

mycin-resistance gene, and the border fragments ofT-DNA, to generate

pSB25 . The in tron-gus

was

transferred

as

a 3.1-kb Hindlll fragment from

pGL 2

-IG

'

to

pSB25

to

give pSB3l.

pSB31

was then introduced

to A. tumefa

cien

s strain

LBA4404

(

pSB1

) (manuscript

in

preparation) by bacterial mat-

749



RESE RCH ARTICLE

ings" . Bacteria carrying the cointegrate from pSB1 and pSB31 are designated

pSB131. pSB1 is a wide host range plasmid that contained a region

of

homol

ogy to pSB31 and a 15.2-kb Kpnl fragment from the virulence region of

pTiBo542. Thus, pSB 131 contained the

bar

and intron gus in the T-DNA.

Infection

. LBA4404(pSB131) and LBA4404(pTOK233) were grown for

3 days on

YP

medium (5 giL yeast extract, 10 giL peptone, 5 giL NaCl, 15 giL

agar,

pH

6.8) supplemented with 50 mg/L spectinomycin (for pSBl31 )

or

50 mg/L hygromycin (for pTOK233). The bacteria were collected with a

platinum loop and suspended at a density

of

l.O X

10

' cfu/ml in LS-inf

medium or N6-inf medium. When necessary, bacterial suspensions of differ

ent

densities were prepared. The immature embryos were washed once with

LS-inf

or

N6-inf media. The immature embryos were immersed in the bacter

ial suspension, stirred for 30 sec with a vortex mixer (Vortex Genie

2,

Scientific

Industries) for thorough immersion, and allowed to stand for 5 min. No

apparent disruption

or

wounding

of

the immature embryos was observed.

The immature embryos were cultured on LS-AS medium

or

N6-AS medium

in the dark

at

25°C for 3 days (Table 4). During the incubation, the embryo

axes

were in contact with the medium and the scutella were exposed to air.

Selection and regeneration of

transformants.

After the cocultivation, the

immature

embryos were transferred to LSD1.5

or N6Dl.5

media supple

mented

with 5 mg/L PPT (for infection with pSB131) or 10 mg/L hygro

mycin (for infection with pTOK233) for selection

of

transformed cells. After

2 weeks

of

incubation at 25°C in the

dark

, the

immature

embryos were

subcultured on LSD1.5

or N6Dl.5

media supplemented with

10

mgiL PPT

or

30 mg

/1

hygromycin at 25°C in the dark for 3 weeks. Clusters

of

cells that

proliferated from the

immature

embryos

and

showed the characteristics

of

the type I calli' were excised with scalpel and cul tured o n media

of

the same

composition at 25°C in the

dark

for 3 weeks. Calli proliferated from the

culture were excised again and cultured on

LSZ

medium supplemented with

5 mgiL PPT

or

30 mgiL hygromycin at 25°C under continuous illumination

(a

bout

50 ~ J m o l m·' sec'). All media for selection and regeneration con

tained 250 mg/L cefotaxime for elimination

of A. tumefaciens. Regenerated

plants were transferred to 1/2LSF

medium,

and incubated

under

the same

condition for 2 weeks (Table

4)

. The plants were transferred to soil in pots

and grown in a greenhouse.

Assay for GUS activity. Expression of GUS in maize calli and plants was

examined by a calorimetric assay using substrate

5-bromo-4-chloro-3-

indolyl[3-D-glucuronide X -Gluc) described by Hiei et al.'.

Test of

the

progeny for resistance

to

PPT. The selfed progeny R1 genera

tion) of

transformed

plants were grown for 8 days in a greenhouse, and

0.2% Basta®(Hoechst, Frankfurt, Germany) solution was applied to the

leaves with a writing brush

or

sprayed to the leaves. Basta® s a commercial

formulation

of

glufosinate, which is the

ammonium

salt

of

PPT. Resistance

was scored 6 days after the treatment.

Isolation of DNA

and Southern

hybridization. DNA was extracted from

leaf tissu

es

of

RO

and

R1

plants by the procedure described by Komari et

al.

" .

Ten mg

of

DNA were digested with BamHI

or

EcoRI

and

fractionated on

a 0.8% agarose gel by electrophoresis at 1.5 V/cm for 15 h. Southern

hybridization was carried

out as

described by Sambrook et al. . The bar

probe was prepared by PCR from pSB25 using primers

5'

-ATGGACC

CA

GAACGACGCCCG-3' and

5'-

TCAGATCTCGGTGACGGGCAG-3'. The gus

probe was prepared by PCR from pBI221 (ref. 29) using primers

5'

-ATGT

TACGTCCTGTAGAAAC-3' and 5' -ATGGTGCGCCAGGAGAGTTG-3'. The

reaction mixture (50 ~ J - 1 for PCR consisted

of

1 ng

of

template DNA, 50

mM

KCl, 10 mM Tris-HCl (pH 8.3),

1.5

mM MgCI,, 0.2 mM each of dGTP,

dATP,

dTTP

and

dCTP, 1

unit

of Taq DNA polymerase

and 10 pmol

each

of

primers. Thermal cycling for 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C

was

performed for

35

cycles.

Sequencing of border regions of

the inserted

T-DNA. Junction regions of

the introduced T-DNA

and

maize genomic DNA were analyzed using an

inverse PCR

method

". Genomic DNA was digested with either

one of

BamHI, Sal , Xbal, and Sac , circularized by self-ligation, relinearized

by digestion with EcoRI

and Hindiii, and

used

as

a template. PCR was

performed as described above except for the use of 250 ng of template

DNA. Primers for analysis of the right boundari es were I-1: 5' -CGTTGCG

GTTCTGTCAGTTCCA-3', GUS: 5' -TCACGGGTTGGGGTTTCTA

C-3'

,

nos : 5·• ATCATCGCAAGACCGGCAAC-3', and primers for ana lysis of

the left boundaries were LS1: 5' -TCAGTACATTAAAAACGTCCGCA-3', bar:

5' -CAGCTGGACTTCAGCCTGCC-3', nos-F: 5' -GGTGTCATCTATGTTAC

TAG-3'. The amplified fragments were subcloned into pCRII (Invitrogen,

San Diego,

CA)

and sequenced by Applied Biosystems (Foster City, CA) 373A

DNA sequencer.

750

cknowledgments

The

authors thank Jan

Lee

mans for providing us with pDEll

0

Tomoaki Kubo

and Sumio Iwai

for

helpful discussions and

advice

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