I of the BC2 F. seed

~

--"';;'C"

Bul. Agron. (29) (2) 50 - 58 (2001)

lntrogression of Bcterial Leaf Blight ResistanceGenefrom
OryzaMinuta J:B. Presl. Ex C. B. Presl. into New Rice Type(Oryzasativa L.)
BuangAbdullahl),D. S. Brar2)and A. L. Carpena2)

~

..,...

ABSTRACT

F 1 Hybrids, backcrossprogenies,advancedintrogressionlines (2n=24) and monosomicalien addition lines
or MAALs (2n=25) were successfullyproduced following embryo rescue betweenan elite new plant type (NPT)
breeding line of Oryza sativa (2n=24, AA) and a wild species,O. minuta (2n=48, BBCC). F 1 hybrids performance
wereintermediatebetweentheparents. TheF 1 hybrids had 36 chromosomesindicating having 12 chromosomeA from
O. sativa and 12 Band 12 C from o. minuta. THE BACK CROSSprogenies had different chromosomenumber

indicating abnormal meiosisof the hybrids and back crossprogenies. Plant with 2n=24 and 25 chromosomeswere
obtained in BC4FI. The hybrids and backcrossprogenies were susceptibleto bacterial leaf blight (BB). However,
severalof the 2n=24 plants derivedresistantplant to bacterial leaf bligkt race 1 of the Philippines races. Thegene is
differentfrom introgressedgeneintio ricefrom O. longistaminata(Xa21)andfrom o. Minuta Acc. 101141.
.

I

Key words: Oryzaminuta, MAALS,Bacterial leaf blight

I

.
!

INTRODUCTION

;

I

c
1

I

Rice is important cerealfor one third of the world
population. During the last three decades,world rice
production has doubled from 257 million tons in 1966
to 563 million tons in 1998. More than 90 percentof
rice is produced and consumedin Asia. To meet the
growing need of an ever increasinghuman population,
50% more rice is neededby 2025, hence,rice varieties
with higher yield potentialare neededto meetthe global
need. To achieve this, IRRI is exploring a new plant
type (NPT) of rice which could increase the yield
potential by 20% (Khush, 1995). The NPT has the
characteristicsof few but all productive tillers, large
panicles, 200-250 grains per panicles, sturdier stems,
deeperroot system, thick and dark green leaves and

maturity of 115-120days. Although major increasesin

important reservoir of useful genes for rice
improvement(Sitch, 1990;Brar and Khush, 1997).
A numberof pathogensand insectsattack the rice
plants. One of the major rice diseasesis bacterialblight
(BB). Bacterial blight of rice causedby Xanthomonas
oryzae pv. Oryzae is one of the important rice
productionconstraints(Mackill, 1986). It reducesgrain
yield to varying levels dependingupon the stageof the
crop affected and degreeof susceptibility of cultivar.
Lossesdue to bacterial blight in the tropics are higher
than in temperateregions (Mizukami and Wakimoto,
1969) because of the prevalence of more virulent
populationsof the pathogen(Buddenhagenand Reddy,
1972). Xanthomonas oryzae pv. Oryzae (Xoo)
produces bacteriocin like substanceson solid media
(Mew, 1987). The proteinsfunction assignal molecules
whith affect host-specificplants responsessuch as cell

rice productionhave occurred,severalbiotic and abiotic
stresseslimit rice production. Moreover, diseasesand
insect pest are a continued threat, particularly due to
changesin insect biotypes and pathotypes. There is,
thus, an urgent need to broadenthe rice gene pool and
identify new genesfor resistanceto major diseasesand
insect pests from diverse sources. Wild speciesare

division, water soaking(i.e.,filling of the cellular spaces
in the leaf mesophyllwith water insteadof air) and the
hypersensitive response. The first evidence for
pathogenicspecializationwasreportedin 1979by VeraCrus and Mew. They recognizedfour bacterial groups
where interaction was confined to a specific cultivarisolatecombination. "Race" was adoptedto classify the

R

bacterialisolates. Eachracehasspecificvirulenceto

I

.*.

I) Balai Penelitian Bioteknologi Tanaman Pangan, JI. Tentara Pel ajar No. 3Bogor
2) International Rice Research Institute

.

!i!c

.

'i

!

50

.
.

'0,
.

,
.

--

Bul. Agron. (29) (2) 50 - 58 (2001)

varieties with different resistancegenes following a
gene-for gene conceptin the host pathogeninteraction.
Race 1 of BB was the most prevalentin the Philippines
in the late 1960' s and early 1970's (Khush et al.,
1988), Most of the IRRI varieties had Xa4 that
converedresistanceto BB race I. Therefore, races2
and 3 have becomemore prevalent in the Philippines.
.. .
.
In the PhilippInes,

10 race~ a~e currently recognized

,'*

~
-

AA, BB, BBCC, CC, CCDD, EE, FF, GG, and HHJJ
(Vaughan 1994; Aggarwal et ai" 1997). Of the two

as femaleparentwith O. minuta (Acc. 101089)as male,
F] plants from seedsobtained were produced through

.
cultivated

embryo

.

.
species,

o. glaberrima

O.sativa

,
IS grown

worldwide

,

is cultivated in limited area of West

,

.

.'

whrtebacked planthopper (WBPH),
bacterial blIght
.
(BB), blast and sheath blIght, However, several
crossability barriers such as high sterilitY, limited
homoeologouschromosomepairing and recombination
betweenthe genomesof cultivated and wild species
.

,

restrict

alien

mtrogresslon

'

and

I

to

produce

as th e

recurrent parent.

The standard embryo rescue procedure used in
Wide Hybridization Laboratoryof IRRI was followed in
this experiment. The panicles were sprayed with 75
ppm gibberilic acid (GA]) solution one day after
II ' t. S
.
t . d ti th

Sl.
po ma Ion, praYIngwas con mue or ree succes ve
days, Fourteendays after pollination, spikelets were
harvestedand surfacesterilized in sodium hypochlorite
solution (20 % of the commercialgrade) supplemented
with 2 drops of Tween-20. After washing them in
sterilized

water,

'

the

delicate

young

embryos

were

'

The fertile plants derived through embryo rescue
selfed to produce introgression lines. Selfed
dd .t .
I.
f
. I'
.

progenies 0 monosomic a ten a I Ion me (MAAL s)
and introgression lines (2n=24) in the genetic

transferred (Khush et ai" 1977; Khush et al., 1990;
Amante Bordeoset al., 1992; Jenadan Khush, 1990;

background of NPT were used for evaluation of the
transferof useful genesfrom O. minuta into rice.

Oamalcio et al., 1995, 1996; Brar and Khush , 1997).
f th .
..
(1)
Th
.fi b"
e speci IC 0 ~ectlves 0
e InvestIgatIon were
to
produce introgression lines (2n=24) from O. sativa x O.

.
. .
.
,
Morphological Characteristics of o. sativa x o. mmuta
H brids
Y

t

a

ti II
.
0 owIng

b

ac

k

.
crossIng

an

d

em

b

ryo

rescue

.
..
procedures,and (2) to evaluate mtrogresslon lInes
producedfor resistanceto bacterialleaf blight.

~
- j

used

,
,,
BPH, blast, and cytoplasmIc male stertllty have been

.

}~

then

rootsweretransplanted
to soil in pots,

mmu
,'"

were

rice and severalwild species(Jenaand Khush,1989;

breakdown

-

. ~

these

transferof useful genesinto cultivated plants (Brar and
Khush, 1986, 1997). One of approachesinvolves the
useof embryo rescuecan be usedto producewide-cross
derivatives.
.
.,
.
of . Interspecific hybrids, monosomIc
. A number
..
alien addition lInes (MAALs) and advanced
introgressionlines have beenproducedfrom crossesof

hybrid

Multani et al., 1994; Brar and Khush, 1997). Some
.
,were
useful genes for resIstanceto grassy stunt ViruS,BB,

..

~

..

and

It d .
t
.
d d
excIse an ISOa e usIng a s ereomlcrosc
ope under
aseptic conditions under sterile air-flow cabinet. The
isolatedembryoswere cultured in Y4MS medium and
incubatedin the dark (25 °C) until germination. The
seedlingswere then incubated in a lighted incubation
room up to the three-leafstage. Thesethree leaf stage
dl.
It d . Y h' d I' .d
I tl.
see mgs were cu ure m os I a Iqui so u on
(Yoshida et ai" 1976) in the IRRI phytotron for 10-15
days. Later, the healthy seedlingswith well-developed

and

~
;""

.

rescue

advanced b ack cross progenies
..
usIng NPT

whreas

Africa, One of the wild species,namely, o. minuta J,S.
Presl.Ex C.B, Presl. (2n = 48, BBCC) possesses
genes
for resistance to brown planthopper (BPH)

.

Production of Hybrids and BackcrossProgenies
Th
t .
t. t.
.
presen
Iga Ion was
out Wide
fr om
August e 1995
to mves
September
2000came
in dthe

Ogawa, 1987;Yamamotoand Ogawa, 1990).
Th
0
h' h I .
d . b I
e genus ryza to w IC cu tlvate rice e ongs
..
..
hasmore then 20 wIld specIes.TheseWIld specIeshave
2n = 24 or 48 chromosomesrepresentingnine genomes,

.

different to BB races of Japan(SakaguchI,1967;

j

Production of Advanced Backcross: Progeniesfrom
the CrossofO. sativax O. minuta

PI t B d.
G
t.
H b .d. t . L b t
y n lza Ion a ora ory, an ree lng, ene ICSand
fth I t
t. I R.
B. h .
(PBGB)D . . .
10Cemlstry
Ivlslon 0 enema lona Ice
R
h I t.
esearc ns Ie,
tut (IRRI) L os Banos, L aguna,
Ph.I..
Th. E
.
ti
d th
d t.
Ilppmes.
IS xpenment ocuse on e pro uc Ion
.,..
of a seriesof mtrogresslonlInes (2n=24) from the cross
of O. sativa x O. minuta. Crosseswere made between
an elite breedingline, NPT (IR65600-81-5-3-2)of rice

(Angeles,personalcommunication). Somegeneshave
.
.. .
same reaction to BB races of the PhilippInes but

.j
i

MATERIALS AND METHODS

Data

on

morphological
.

characteristics
.

of

the

parentsand hybrids were recorded. Five plants were
taken at random and data on plant height, number of
tillers leaf length and width Panicle length floret
"
,

i.

BuangAbdullah, D. S. Brar and A. L. Carpena

51

.

.

!

-'---,-'

I

i

Bul. Agron. (29) (2) 50 - 58 (2001)

I

! ,,;-.,

I

!

length, awn length, and number of floret per panicle
were recorded.Growth habit, grain shattering,and color
of leaf, apiculus,stigma,and awnswere also observed.
Pollen and Floret Fertility
P II fi rt.l.ty
dt
. d ti
t I
500
0 en e I I was e ermIne rom a east
II
.
fr
h f
t h b .d
d
po en grams
om
eac 0. paren s' y rI S, an
.
backcrossprogenies. The splkeletswere collectednear
th . d .
d. t I ta. d . h 2 01
.
an eslsa Imme la e y s me WIt
"/0 acetocarmme.
Sta' d d
d
II
.
d fi ' I
me an roun po en
gramswere counte as ertl e
h.1 th
ta. d I. htl t . d d h . I d
W lee
uns me ,. Ig Y same . an
s rive e ones
.
.
were countedas sterile. Pollen fertIlIty was determIned
. h fi II . fi
I
usIngt e 0 owIng ormu a :

'1,
';::
Ii!
""

Pollen fertility (%) =

Numberof fertilepollengrains

FI t fi rt.l.
d
. d fr
fi
. I
ore e I Ity was etermme om Ive panic es
of eachplant using similar formula as follows:
Number of filled florets

X 100
Totalnumberof florets

Meiotic Chromo.vome
Analysis
"

. ~hromosome analysIs was carried ou~ from
meIotIc pollen mo~hercells of paren~, F, hybrids ~nd
backcross progenies, Young panIcles representIng
different stageswere collected, between8:00 to 10:00
AM. Thesepanicleswere fixed at room temperaturein
a fresh solution of acetic-alcohol(I part glacial acetic
acid and 3 parts of 95 % ethanol) to which traces of
ferric chloride were added. After 24 hours of fixation,
panicleswere transferredto 70 % ethanoland storedat
4 °c until used. Squasheswere madefrom anthersin 2
% acetocarmine.Chromosomenumber of parents,FI,
BC1F., BC2F" BC3F., and BC4F), progenies at
d. k'
.
d
ta h
I
la mesls an me p ase .

P d

ifH b

.

rq uctlon 0

'd

y rl san

d B kc
P
,
ac ross rogemes

. f. hybrids.On the everage,56.1% seedsettingwas
obtained(906 out of 1616pollinated florets). However,
only 35.4%of the seedsfrom the crossbetweenNPT of
rice and O. minuta had embryos(Table I). After 10 15 days of pollination, embryos were excised and
cultured on MS medium. Out of the 321 embryos
cultured, only 37.7% germinatedinto F, seedlings. In
this study,seedsettingin hybrids betweenO. sativa and
O. minuta was much higher than in previous studies.
(Sitch et al., 1990)reported4% seedset while (Mariam
et ai" 1996)obtained14.8%seedset.
~. oroe:enies.The F. hybrids were backcrossed
to NPT using the latter as pollinator. On the average,
10.8%florets set seedand majority (94.6%) of the seeds
did not have embyo but were only watery. Of the 19
BC. .F} progenies from. original F. hybrids ~nd
c?lchlcme-treatedF) hyb~lds. They rep°.rt~d a lIttle
hIgher BC) F) productIon ~om colchlcme-treat~d
to

untreated

hybrids,

0.12

and

0.03 '10

Alien introgressionlines derived from 0, sativa x
0, minuta along with parents were evaluated for

(Amante-Bordeoset ai" 1992), and 1.2 and 0.63%
(Mariam et al., 1996),respectively. Amante-Bordeoset
al., (1992) obtained 6 BC.F, plants from 47,300
pollinatedflorets.
oroe:enies.BC,F. plantsof crossesbetween
NPT and O. minuta Acc. 10I 089 were pollinated with
tha recurrent panent to produce BC2FI. Of 16,682
florets pollinated, 14.5%set seed(Table I). Less than

introgression
of resistance
to bacterialleaf blight (BB).

I % of the BC2F. seedhad embryos. Only 8,plants

The parents, hybrids, and backcross progenies were
inoculatedwith six Philippine races(1,2,3,4, 5,6) of
Xanthomonas oryzae pv. Oryzae (Xoo) while introgressionlines were inoculatedwith threeraces(I 6 9)
Seven Philippine races of Xoo: PXO61 (r~c~ I);
PXO86 (race 2); PXO79 (race 3); PXO71 (race 4);
XOl12 (
5). PXO099 (
6) d XO
(
P
race,
race an P 339 race

could be produedfrom 16,862pollInated florets m BC2
F, The BC2 F) plant were produced from a BC) F)
plant, IS .14531. J~naand Kh~sh (1989),rep?rtedthat.
seed set m O. sativa x 0, mmuta officmahs crosses
rangedfrom08.8to 17-,3%,while Multani et al. (1996),
0.57 and 2.4'10,respectIvely.Tw~nty-threeBC2F. plant
were producedfrom 35,000 pollmatedflorets (AmanteBerdeoset ai" 1992).

~.

52

IntrogresiGenKetahanan
terhadap..,

.

..

~
i

.
ProductIon of Advanced Backcross ProgenIesfrom
Cross ofO. sativax O. minuta

compared

Evaluation of Introgression Lines for the Transfer of
Alin Genesfor Resistanceto Biotic Stresses

.

REUL TS AND DISCUSSIONS

X 100

Totalnumberof pollengrains.

Florey fertility (%) =

9) were used for inoculation. The bacteria were
transferred to slants of potato semisynthetic agar
medium and incubatedat 30 °c for 3 days. Inoculum
was preparedby suspendingthe bacterial mass with
sterilizedwater to a concentrationof about 109cells/ml,
Leaf bladeswere inoculatedby the clipping technique
.
..
(Kauffman
ai" 1973)
at themaxImumtlllerlng stage.
. et
.
EvaluatIon
for resIstance
was done at 14 days after
.
.
.
InoculatIonby lesIon length measurement. Plant were
..
.
classIfied as resIstant, moderately resIstant or
.
...
susceptIblebasedon lesIon length. resIstant,(R ) -- o6.0 cm. moderateI resIstant
.
(MR) -.
- 6 I - 8.,O. and
y
:bl (S) 8 O
susceptl e
>. cm.

.
.C
~

,
i~.

..
.

"

II
1\

Bul. Agron.(29)(2) 50- 58(2001)

"

1,;::1

ri
,
~

"c:- F"
-

"

1

c-

Tabel I.

Pro.ductionof hybrids and backcross.
progeniesfrom a crossbetweennew plant type (NPT) rice female, O.

sativa(IR65600-81-5-3-2)
andO.mmuta.
-

Femaleparent

Seed

Cross

Florest

combination

--. . .FI hybrid:
NPTx o. minuta
(101089)

Set

SeedWith

Produced

Pollinated

Gene- Chromo
ration
-some
no. (2n)

Seedling

(No)
(No.)

(%)

(No.)

Parent

24

1616

906

56.1

~1f:1::'
(NPT-x 101089)xNPT

F)

36

3297

356

10.8

19

~2f:I::'
- \AiHDISI453-1 x NPT
- WHDISI525-1 x NPT

BC)
BC)

35
40

9642
7220

1509
1096

15.6
15.2

16862 2605

Total
~f:I::'
- WHDISI668-1 x NPT
- WHDISI669-1 x NPT
- WHDISI963-1 x NPT

Embryo

Embryo

BC2
BC2
BC2

33
34
36

321 35.43

(No.)

(%)

121

37.7

5.34

6

31.6

24
0.0

1.59
0.00

8
0

. 33.3
0.0

14.5

24

0.92

8

33.3

528
1375
781

9.9
36.7
20.6

26
57
2

4.92
4.15
0.30

3
8
0

11.5
14.0
0.0

18945 2799

14.8

37

1.38

II

21.6

5304
3746'
3790

Total

(%)

BC.F) :
~-- WHDISI874-1
x NPT

BC]

26

257

17

30.0,

27

35.06

21

77.8

f:%:

- WHDIS1875-1
x NPT
- WHDIS1878-1
x NPT
- WHDIS1878-2
x NPT

BC]
BC]
BC]

31
24
26

134
316
438

0
186
222

0.0
58.9
50.7

0 0.00
2 1.08
15 51.80

0
0
54

0.0
0.0
47.0

~::
c

-- WHDIS1881-1
WHDIS1881-2xx NPT
NPT

BC]
BC]

29
-

469
411

15
59

3.2
14.4

0
0

0.00
0.00

0
0

0.0
0.0

2025

559

27.6

44

2579

75

52.1

:"""
~,

'~

.

~l-. r
i;

Total

Table2. Morphological characteristicsof parentsand hybrids of a crossbetweenNPT rice, O. sativa (IR65600-81-5-32) and O. minuta (Acc.IOI089)
Trait
Plantheight (cm)
No. of tiller
Leaf length (cm)
Leaf width (cm)
Paniclelength(cm)

.

.

O.sativa
100.94
9.80
62.38
2.12
25.16

Floretlength(mm)

6.00

Awn length
No. of floret/panicle
Leaf color
Apiculus color
Stigmacolor
Awn color
Grain shattering
Growth habit

awnless
264.60
dark green
purple
purple
awnless
not
hatteringerect

BuangAbdullah,D. S.BrarandA. L. Carpena

.

F) Hybrids
96.26
28.20
37.32
1.88
23.56

5.80
2.52
116.60
dark green
purple
purple
colorless
shattering
intermediate

O. Minuta
97.28
112.40
17.78
1.48
17.28
4.60
2.30
98.80
dark green
purple
purple
colorless
shattering
spreading

53

.

-

Bul.

Agron.

(29)

~-E!
cross

NPT

the

(1.4

%

were

florets,
of

seeds

obtained).

were

22.8

(2n=24)

plants,

BC2F

and

however,

I).

plants

could

I,

only

2n=31

Six
were

Of

the

be

0

to

4.4

meiosis

%

NPT.

2n=24

was

also

produce

any

1881-1)

with

seeds

not
plants

BC4

to

F)

x

O.
I 089)

In

O.

lack

viable

3.02

in

Figure

In

and

and

this

and

BC4

O.

F]

minuta

I.

minuta

Of

O.

sativa

x

O.

Hybrids
Th

b
y

.d
n

s

.
mterme

were

d

.

b

h

late

etween

t

.
elr

",

t .
h
. t.
I
t h . h
I
f
parens m manyc aractensICS,e. g., p an elg t, ea

1

I
th
d
. dth
. I
I
h
d
eng
an
WI
, panIc
e
engt,
an
grow
(T
b I 2)
Th
k
d "
.
h
..
a e
.
e
ey
Istmguls
mg
c aractenstlcs
.
.
I d d
d
II
I
mmuta
mc
u e
narrower
an
sma
er
eaves,
d
h tt
.
f
d .
see
s a enng,
presence
0
awn,
sprea
mg

r

habit,

th

h
a

h.

,
,

and
rt

d

reduced
th

t

h

floret
b

.d

repo
e
a
y
n
so.
.
.1
t
th
. Id
Slml
ar
0
e WI
paren,

Ch

b
romosome

num

size.
0

.

t

0

sativa
.
. mmuta.

d
er.

an

Mariam

f

x

.

f

b .
It
0
.

0
h
eavy.

backcross
analysis
2n=48

number

derivatives
showed
in

O.

of
was

2n=24

an

(1996)

O.

were

obtained

al,.

1992;

al.

(1992)
with

were

et

al.,

produced

25,

also

F)

Mariam

and

obtained

australiensis

15

two

others

from

the

(Multani

et

al.,

and

pollen
2%
On

so

did

plants

with

sterile.

2n=26

fertility)

to
to

1.64

40%

early

pollen

while

the

male

were

partly

between

O.

Multani

et

sativa

(0%

and

O.

(1994).

unstained

to

31.9%

fertility

was

them

are

progenies

had

(0%

sterile

floret

to

sterile.

. .

sterIlIty

al.

BC]F,

of

sterile

. .
Complete

in

The
0

cross

were

3)

fertility

3).

some

back

fertility
others

Pollen

pollen
but

hybrids
(Tabel

showed
high

and

sativa

fertility,

2.2%)

(Table

(12.7-97.5%),

hybrids

to

%).

4.06%

O.

pollen
The

(0.4

Relatively

All

88.5%

chromosomes

BC4F)

than

(0
1.42

by
fertility

average,

and

fertility

BC)F1s
from

determined
spikelet

respectively.

pollen

fertility.
in

as
and

fertility

93%

85.4%,

low
the

ranged

spikelet

showed
and,

BC2F1

fertility

acetocarmine

pollen
australiensis

High

pollen)

of

was
..
sterIlIty

pollen

of

.

fertIlity)

hybrids

hybrids

reported

by

(99.57

between

O.

to

and

O.

mmuta

was

also

reported

by

Manam

sativa
et

al.

(1996).
The

morphological

characters

of

all

BC4F)

were

.1 '
ertl

similar

to

rice.

Most

of

the

plants

plants
.
with

24

grains

of

parents,

F)

hybrids

and

Chromosome
in

O.

F I

hybrids

was

normal

most

BC4F)

plants

observed

were

fertile

sativa

and

had

to

sterile
were

diseases

(12.7-97.5%

selfed
for
and

and

fertility)
the

introgression

BC4F2
genes

(Table
seeds
for

3).

were

used

resistance

Thus
for
of

insects.

!

36

parentswith O. sativaand O. minutarespectively.In

in

they
screening

chromosomes;

the

two

~c
54

IntrogresiGen Ketahananterhadap...

.

'

Ity

t

meiosis

and

.

i.,

indicating

sativa

growt

al.

fi
oret

studied.

The

double

colchicine-treated

7

O.

monosomic

chromosomes
were NPT type in appearance.Pollen

chromosomes

minuta.

on

very

partially
Chromosome

and

minuta

99.37%
h

.
mmuta

djl
en

MAALs

sativa

89.6%

were

II
po

et
O

29.

O.

recorded.
O.

less

h
e

and

had

(DMAALs),
O.

from

had
51

was

chromosomes

chromosomes,

in

pollen

Characteristics

24

lines,

lines

derived

et

of

found
Morphological

2n=24

normal.

plants

Therefore,

and

between

Amante-Bordeos

27

showed

interspecific
x

cross

et

with

were

seed

in

the

with

stainability

(2n=25)

obtained

producing

the

that

not

23

univalents.

addition

40

BC]F)s

while

introgression

(Amante-Bordeos

1996).

was

bivalents,

two

II

indicating

these

(MAALs)

progenies

Data

not

(WHDIS
%

MAALs

(IR65600-81-5-3-2)

shown

did

embryos.

and

were
in

plant

only

(2n=24)

minuta

sativa
is

Another

plants

BC2F1

with

and

as
lines

respectively.

in

and

designated

-26;

of

and

36,

progenies

12

35

to

24

plants,
with

alien

33

IS had

backcross

bivalents

had

had

BC4F

BC4F)

12

BC)F)s

1,994).

1878-1)
sterile

and

monosomic

cross

backcrossing

75

addition

with

with

F)s

were

plants

2n=26

upon

highly

set

and

Procedure

hybrid

seed

F)

1874-

and

with

hybrids

BC4

1875-1)

(WHDIS

be

lines

sativa

progenies.

set

progeny.

abortive

75

(WHDIS

(WHDIS

chromosomes

introgression
O.

did

to

parent

possessing

plant

the

found

29

were

study,

of

plants

each
F)

24

recurrent

BC2F)s

chromosomes

alien

from

having

florets,

These

BC]

One

to

pollinated

1878-2)

chromosomes

with

backcrossed

2025

A

plants

six

eight
26,

in

they

BC2F1

seedset

progeny,

seventy-five

2n=25

(1992)

two

to

2n=24

used

al.

24

The

The

3 plants

from

BC3FI

obtained.

WHDIS

(10

of

et

seedset

I).

plants

florets.

out

had

minuta,

chromosomes.

,..

%

progenies.

(Tabel

of

plants

II

cross

of

embryos

only

back

chromosomes,

(Table

without

BC]F1,

the

Out

seeds

Amante-Bordeos

100

chromosomes

all

set

F)

pollinated
2

BC],

BC]

was

In

from
backcrossed

I plants.
~.1E!

36

%

from
In

reported

14.8
seeds

plants
were

produce

18,945

produced

F)

to

the

from

backcrossing.

BC2
101089

parent

produced

(2001)

minuta

majority
of

plants

58

The

O.

pollinated

However,

in

x

recurrent

18,945

-

50

progenies.

of

with

(2)

,

.
Bul. Agron. (29) (2) 50 - 58 (2001)

Table 3. Chromosomenumber and pollen fertility in the parents,hybrid, and backcrossprogeniesof a cross between
NPT rice, O. sativa (IR65600-18-5-3-2)and O. minuta (101089).
.
Lme

I

IR65600-18-5-3-2

rl~
'FI
.,

c"t'c..

,:'

Numberof Plant
Observed
I

Chromosome
Number(2n)

Fertility (%)1
Pollen (Range)
Floret (Range)

24

93.0

BCjF1

I
25
3

48
36
35-40

88.5
0.4-2.2
0.0 -1.6

85.4
0.0
0.0

BC2F1
BC]F1
BC4F,

4
II
75

33-36
24-29
24-26

0.0 -4.1
0.0 - 31.9
12.7-97.5

0.0
0.0
0.0-91.5

O. minuta (101089)

-

89.6

,"

k

Table 4. Reaction of BC4F2plants derived from a cross betweenNPT rice, O. savita (IR65600-81-5-3-2)and O.
minuta 101089)to race I of bacterialblight, 14 daysafter inoculation.
G
enet.
ra Ion

M atena
. I

i

i

.

i
I.

l
-

,

.

~~;'

,-

I

BB LESION
(14 DAI ) 1
Cm
Score

Morphological Traits

Parent.FI~I
IR65600=81-5-3-1
O. minuta (101089)

Parent
Parent

24
48

44.34
1,4

R
R

o. minuta (101141)

Parent

48

0.02

R

HWDIS1270-7

FI

36

13.6

S

HWDISI453-1
Introgression lines
HWDISI958-5-1
HWDISI958-5-8
HWDIS1958-9-1
HWDISI958-9-3
HWDISI958-9-10
HWDISI958-9-11
HWDIS1958-10-4
HWDISI958-10-5
HWDISI958-10-6
HWDISI958-10-9
HWDISI958-19-3
HWDISI958-19-6
HWDIS1958-19-7
HWDISI958-19-12

BC1

35

22..3

S

spreading,long and wide leaves

BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2

24
24
24
24
24
24
24
24
24
24
24
24
24
24

5.94
5.10
4.54
4.62
4.72
3.64
5.56
5.24
4.76
5.02
4.34
4.72
2.28
5.28

R;.
R
R
R
R
R
R
R
R
R
R'
R
R
R

NPTtype, mediumdensepanicle
NPTtype, late maturity
NPT type
NPT type
NPTtype party fertile
NPTtype, awned,sterile
NPT type, late maturity
NPTtype,latematurity
NPTtype, late maturity
NPT type, late maturity
NPTtype,latematurity
NPT type, late maturity
NPT type, mediumdensedpsnicle
NPT type

HWDIS 1958-22-2

BC4F2

24

4.70

R

NPT type, late maturity

HWDISI958-22-3
HWDISI 958-29-2
HWDIS1958-29-5
HWDIS1958-29-9
HWDISI958-34-4
HWDISI958-34-5
HWDISI958-4-3
HWDISI957-17-4
HWDIS1959-5-2
HWDIS1959-5-3

BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2
BC4F2

24
24
24
24
24
24
24
24
24
24

5.30
2.40
4.56
1.50
2.38
4.16
3.88
4.02
4.96
5.67

R
R
R
R
R
R
R
R
R
R

NPT type, short
NPT type, very late maturity
NPT type
NPT type, awned
NPTtype, very late maturity
NPTtype
NPT type,late,densepanicle
NPTtype, mediumdensedpanicle
NPT type, very late maturity
NPT type, mediumdensedpanicle

HWDlSI959-5-11

BC4F2

24

4.96

R

IRtype,latematurity

24

2.20

R

semidwarf variety

24

14.06

S

semi dwarf variety

-

IR 54 (R control)
:f$f"

Chromosome
No. (2n)

-

IR24 (S control)

')DAI = days after inoculation; 0

NPT
spreading,small tillers, narrow, short
leaves
spreading,small tillers, medium, leaves
medium

spreading,
intermediate
leaf

- 6 cm = R (resistant);6.1 - 8.0 cm = MR (moderatelyresistant);> 8.1 = S

(susceptible).

}(~~c:

BuangAbdullah,D. S.BrarandA. L. Carpena

55

~\
~

L

.

.

""~C7"""

i

Bul. Agron. (29) (2) 50 -

58 (2001)

(~:i~!f,:' "-:0

;,

Table 5. Reactionof BC4F3 lines derived from a crossbetweenNPT rice, O.savita(IR65600-8]-5-3-2) and O. minuta
G
Material

;

i;j

j

Chromoe~e-

ration

BB LESION:

some

t ~-~;i

(14 DAI).

No. (2n)
,
"

Cm

f

Morphological Traits
Score

'"

"",,-,
"ct;

]R65600-8]
-5-3-2
Parent.
F)- and
BC1-

Parent

24

28.15

S

NPT line, wide, long thick leaves

f

o. minuta (] 0] 089)

Parent

48

].2

R

spreading,small tillers, narrow, short

" """,,

o. minuta (]O] ]4])

Parent

48

0.01

R

leaves
spreading,
small tillers, medium,short

.."

HWDIS]270-7
HWDIS] 453-]
HWDIS]958-]9
Introgression lines

F1
BC.F1
BC4F]

36
35
24

12.4
20.1
]0.9

S
S
S

leaves
spreading,intermediateleaf
spreading,long and wide leaves
NPTtype,
NPT type,

HWDIS] 958-5-]

BC4F3

24

] 1.0

S

NPT type,

HWDIS1958-9-]
HWDIS]958-9-3
HWDIS]958-9-]0
HWDiS]958-10-5
HWDIS1958-]0-6
HWDIS]958-]0-9
HWDIS]958-]9-3
HWDIS]958-]9-6
HWDIS1958-]9-7
HWDIS1958-]9-12
HWDIS]958-22-2
HWDIS1958-22-3
HWD]S1958-22-22
HWDIS] 958-29-5
HWDIS] 958-29-8
HWDIS] 958-29-9
HWDIS]958-29-3]
HWDIS] 959-5-2
HWDIS]959-5-3
HWDIS]959-5-]]
HWDIS] 957-4-3
HWDIS]958-]7-4

BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3
BC4F3

24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24

11.9
]2.9
8.8
]6.7
10.5,
]3.0'
4.5
]5.0
4.9
5.9
] 1.2
]7.3
5.8
7.9
22.3
9.2
7.]
9.6
]3.5
13.1
5.7
8.0

S
S
S
S
S
S
R
S
R
R
S
S
R
MR
S
S
MR
S
S
S
R
MR

NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPTtype,
NPT type,
NPT type,
NPT type,
NPT type,
NPT type,
NPT type,
NPTtype,
NPT type,
NPTtype,
NPTtype,
NPT type,
NPTtype,

-

IR54(R control)

-

IR24 (S control)
I)

24

2.4

R

semidwarfvariety

24

26.0

S

semidwarf variety

*;:,.
,00
.,

i
r

;
-

OAI = daysafter inoculation;0 - 6 cm = R (resistant);6.] - 8.0 cm = MR (moderatelyresistant);
> 8.] = S (susceptible).
-

.

:.,
..

,
;~'fl
.,

56

;

IntrogresiGenKetahananterhadap...

'I

.

,

f

r--

.j"

-

!
!
,
I

,
;

it.

I

Bul. Agron. (29) (2) 50 - 58 (2001)

~ "I

I

been transferred from O. minuta (Acc.10 I 089) into

Alien Genesfor Resistanceto Bacterial Leaf Blight

O.sativa (NPT). However, this gene differs from the
gene transferred in the previous studies (AmanteBordeoset al., 1992;Mariam et al., 1996). Sinceall F1
hybrid and first backcrossprogenieswere susceptibleto
BB race I and the resistant plants were found in the
segregatingpopulation(BC4F2),the gene for resistance
to BB race I is, therefore, a recessive one. The
introgression lines were resistant to BB race I, but
susceptibleto race 6, further supportingthe conclusion
that the gene introgressedfor BB resistanceis different
from previousgenestransferredfrom wild species,such

The parents, O.sativa (NPT), IR 65600-81-5-3-2
and O. minuta (Acc. 101089) F. hybrids, and BC
progenieswere inoculated with six Philippine racesof
bacterial leaf blight (BB). The NPT was found to be
susceptibleto all the six races while O. minuta was
resistant. The hybrids were found to be susceptibleto
all six races. Similarly, all BC,F) to BC4F]plants of
were also susceptibleto 6 BB races. Forty-four lines of
BC4F2derived from BC4F) plants with 24 and 25

t'

.i

"

chromosomeswere trasplantedin the screenhouse.The
plants were inoculated with race I and race 9. The
NPT, O. minuta, and BC4plantswere resistantto race9.
The NPT parent was susceptibleto race I while O.
minuta was resistant. Twenty-six plants from II of the
44 lines tested showed resistance. These plants had
lesion length of 1.50 to 5.96 cm 2 weeks after
inoculation(Table 4).
The BC4F3progeniesderived from resistantBC4F2
plants were inoculated with races I and 6 BB in the
screenhouse. Although BC4F21ines
were not testedfor
resistanceto race 6, BC4F3lines derived from plants
reistantto BB race I were testedfor resistanceto race6.
All BC4F3lines were found susceptibleto race 6. Five
lines showedresistanceto race I (lesion length lessthan
6.0 cm), three, moderateresistance(7.1 and 8.0 cm );
and 20, susceptibility (11.2- 22.3 cm) (Table 5).
Amante-Bordeoset al. (1992) reportedthat all hybrids
of O.sativa x O. minuta Acc. 101141were resistantto
race I to 6 of BB of the Philippines. However, a BC2
plant with 24 chromosomeswas only resistantagainst
races2, 3, 5, and 6 of BB of the Philippines.The rice
parent, IR31917-45-3-2, was susceptible to race 6,
moderately susceptible to races 2, 3, and 4, and
resistantto races I and 5.
The rice parent had Xa4 gene for BB and,
therefore, was resistant to races I and 5. Similarly,
Mariam et al. (1996) found that all hybrids of O.sativax
O. minuta Acc.10 1141 were resistantto five Malaysian

c ,

-.~'i

Evaluation of Introgression Lines for the Transfer of

isolatesof BB.

.

However, a BC2 plant with 24

AggarwaL, R.K, D.S Brar,. and, G.S, Khush. 1997.
Two new genomes identified in the Oryza
complex based on molecular divergence using
total genomic DNA hybridization. Mol. Genet.
254:1-12.
Amante-Bordeos, A.D., L.A. Sitch, R. Nelson, R.D.
Damalsio, R.D. Oliva, H. Aswidinoor, and H.
Leung, 1992. Transfer of bacterial leaf and blast
resistancefrom the tetraploid wild rice Oryza
minuta to cultivated rice, O.sativa. Theor. Appl.
Genet.84:345-354.
Brar, D.S. and,G.S.Khush. 1997.Alien introgressionin
rice. Plant.Mol. BioI. 35:35- 47.
Buddenhagen,I.W. and A.P.K. Reddy. 1997.The host,
the invironment, Xanthomonas oryzae and
reseacher.In: Rice Breeding. International Rice

Research Institute, Los

Banos, Laguna,

...

Damalcio, R.D., D.S. Brar, T. Ishii, L. A. Sitch., S. S,
Virmani, and S. G. Khush. 1995. Identification

.

(Acc.10 I 089) were susceptibleto racesI to 6 of BB the
Philippines.
of 44 BC4F2
tested
for reaction
to BB
races
I and 6,Out
26 plants
of II lines
were
found resistant

and transfer of a new cytoplasmic male sterility
source
from
O.perennis
Euphytica
82:221
- 225.into indica rice (0. sativa).

to race I. No plants were resistantto race 6. in BC4F3
generation,of the 26 plantsresistantto race I, five lines
showedresistance,3 moderateresistance,3 moderate
susceptibility and the others susceptibility to race I.
Theseresultsshow that a genefor resistanceto BB has

BuangAbdullah,D. S.BrarandA. L. Carpena

.

Philippines,P.O.Box 933, Manila, p.289- 295.

Damalcio, R.D., D.S. Brar, S.S. Virmani, and G. S.
Khush. 1996. Male sterile line in rice (Oryza

savita)developed
with O.glumaepatula
cytoplasm.
IRRN 21 (I) : 21 - 23.

57

..

-

REFERENCE

chromosomeswas found resistant to isolates XO66,
XO99, XO257, and XO319 and moderatelyresistantto
XO100, in this study, all hybrids, BC.F" BC2F1,BC3F.
and BC4F)progenies of O.sativa (NPT) x O. minuta

i

I
I

as Xa21 from O.longistaminatawhich is resistant to
races I to 6 {Khush et al., 1990) as well as transferred
from O. minuta Acc.101141)which is resistantto race
2, 3, 5 and 6 (Amante-Bordeoset al., 1992). The
results suggest that the gene introgressed from O.
minutaAcc.10 I 089 into NPT is new.

,

Bul. Agron. (29) (2) 50 - 58 (2001)

',!
i:~

I

,~ij
r:;~j
ili
Jill

Jena, K.K. and G. S. Khush. 1989. Monosomic alien
addition lines of rice: production, morphology,
~y:~~~gy,and breedingbehavior. Genome32:449

Mew, T.W. 1987.Currentstatusand future prospectsof
researchon bacterial blight of rice. Ann. Rev.
Phytopathology25:359- 382.

.
Jena, K.K. and G. S. Khus~. 1.990.Introg,resslonof
genes from Oryza officmahs Well ex Watt to
cultivated rice, O. sativa L. Theor. Appl. Genet

Mizukami, T. and S. Wak~moto.19~9. Epid~miology
and control of bacterial leaf blight of rice. Ann
Rev.Phytopathology7: 51 - 82.

80:737 - 745.

I

Multani, D.S., Jena, K.K., Brar, D.S., De Los Reyes,

Kauffman,H.E., A. P. K. Reddy,S. P. Y. Hsien, and S.
D. Merca .1973. An improved technique for
evaluating resistance of rice varietas to
Xanthomonas oryzae. Plant. Dis. Red. 57:537 541.
Khush, G.S. 1995. Modem varieties - their real
contribution to food supply. Geo Journal 35
(3):275- 284.
Khush, G.S., E. Bacalangceand T. Ogawa. 1990. A
new gwnw for resistanceto bacterial leaf blight
from O. longistaminata. Rice Genetics Newsl.s
7:121- 122.

"i

t

T

Khush, G.S., K. C. Ling, R. C. Aquino, J. M and
Aguiero. 1977. Breeding for resistanceto grassy

stuntvirus in rice. Ini : PlantBreedingPapersI
(46). Proc. 3'd Int. CongoSabrao6, Canberra,

B.G., Angeles, E.R. and Khush, G.S. 1994.
Developmentof monosomic alien addition lines
and introgression of genes from Oryza
australiensisDomin. To cultivated rice O. sativa
L. Theor.Appl. Genet.88:102- 109.
Ogawa, T. 1987. Gene symbols for resistance to
bacterial leaf blight, 1987. Rice GeneticsNewsl.
4: 41-43.
Sakaguchi.S. 1967.Lingkage studieson the resistance
to bacterialleaf blight, Xanthomonasoryza (Uyeda
; et Ishiyama) Dowson in rice. Bull. Natl. Inst.
Agric. Sci. Japan16: 1 - 18.
Sitch, L.A. 1990. Incompatibility barriers operating in
crossesof Oryza sativa with related speciesand
genera. In: Gene Manipulation in Plant

ImprovementII. (Gustafson,J. P., ed.). Plenum
Press,
NewYork,p.77 - 96.

Australia,p.3 - 9.
Khush, G.S., D. J. Mackill and G. S. Sidhi. 1988.
Breedingrice for bacterialblight resistance.In: the
International Workshop on Bacterial Blight of
Rice. International Rice ResearchInstitute, Los
Banon,LagunaPhilippines,P.O. Box 933, Manila,
p. 1 - 18.
Mackill. D.L. 1986. Varietal improvementfor rainfed
lowland rice in South and SoutheastAsia: Result
of a survey. In Progressin rainfed lowland rice.
International Rice ResearchInstitute, Los Banon,
LagunaPhilippines,P.O. Box 933, Manila. p. 115
-144.

Vaughan, D.A., 1994. The wild relatives of rice. A
Genetic ResourcesHandbook. International Rice
Research Institute, Los Banon, Laguna
Philippines,P.O. Box 933, Manila, 137pp.
Vera-Cruz, N. and T. W. Mew. 1979. Variability of
Xanthomonasoryzae specity in infection of rice
differentials.Phytopathology69: 152- 155.
Yamamoto, T. and T. Ogawa. 1990. Inheritance of
resistancein rice cultivars, Toyonishiki, Milyang
23 and IR24 to Myanmar isolatesof bacterial leaf
blight pathogen.JARQ 24: 74 - 77.

,

Mariam, A.L.A. A. Zakkri, M. C. Mahanan and M. N.

Normah. 1996. Interspecific hybridization of
cultivated rice O.sativa L. with the wild rice,
O.minutaPresl.Theor. Appl. Genet.93:664- 671.

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