An efficient method for the regeneration of wheat (Triticum aestivum) from anther cultures.

Bull. Rech. Agron. Gembloux U987122 (4)' 301-314

An

efficient method

for

(Triticum

aestivum)

the regeneration

of

wheat

from

anther

cultures

by

L.J. ZnnNc(*)(**), C. ANcEnu(***), P. LreotvRr(*),

P.

Serlleun(**)

and J. Sanaar-(*)

Abstract

An efficient procedure to obtain high yield of green wheat plantlets from anther culture is presented. The technique was applied to 4 spring genotypes and 2 winter genotypes, using a modified potato-2 medium [CHueNc et al., l9'78), supplemented with glutamine, with sucrose being pretreated with activated charcoal, and agarose replacing agar.

The total average percentage of anthers yielding at least one callus increased from 9.1 9o (with the original potato-2 medium) to 21 .6 7o vith our modified medium, while the total average percentage of regenerated green plants raised from 2.5 Yo (50 green plants from 2,040 anthers) to 6.9 Vo (369 green plants from 5,350 anthers).

Sucrose pretreatment of excised anthers greatly increased the rate of callus induc-tion for 2 winter genotypes (Fl hybrids).

Introduction

In some plant species, haploids are easily produced from anther cultures. However, for most gen€ra, including grasses, the rate of successful haploid production is low [Prcar.D et a\.,1978;Mrao et

a\.,1978;Bl.rrreu,et

ql,,

l98l;

Bulr-ocx et a\.,1982; FoRoucsr-WEHn et a\.,1982; GeNovESr and CoLLTNS, 1982; RrNes, 19831.

(*) Laboratoire de Pathologie Végétale. Faculté des Sciences Agronomiques de I'Etat. Passage

des Déportés, 2. 8-5800 Crvgloux (Belgique).

(**) Chaire de Cénétique et d'Amélioration des Plantes. Faculté des Sciences Agronomiques de

I'Etat. Passage des Déportés, 2. 8-5800 Grrrsloux (Belgique).

(+*+) Centre de Lutte Intégrée en Phytopathologie. Faculté des Sciences Agronomiques de I'Etat. .Avenue Maréchal Juin, 13. 8-5800 GrNrsr-oux (Belgique).

302 ZHANG L.J., ANCEAU C., LEPOIVRE P., SEILLEUR P.. SEIV{AL J.

In

wheat, breeding of doubled-haploids _{is currently limited by the low} frequency of plantlet production per anther, specially in eiire breeding rines

[Burrocx

et a|.,1982; PrcaRo and De BuysER, 1977], thus making

it

dif-ficult to be used effectively by plant breeders

_[Gnrrrrxc,

1975].

Potato medium rvas much more effective in pollen callus induction than MS or Maiji

I

medium _[Research Group 301, 1976], and has been widery used

in many laboratories for anther cultures

ol

wheat, barley and rye

_[\\rxzel

et a|.,1977; De Buvsen and HexRy, 1979,1980; Scri-TEFFER el at.,1979;

SuNorRi-eND et al., 1979; Bur_locx et al., 19821.

Cnu.c.Nc et

al.

119781 designed a new porato medium named potaro-2 medium. The yield of pollen calli r'irh potaro-2 medium increased tivofold as

compared to medium _{N6 [CHu,} 19;-8] or Nlaiji

l.

porato-2 medium has been

gradually adopted by many _{laborarories [OuveNc.} 1986].

Our paper deals

*'ith

a modified medium, shotl.ing greater efficiencf in regenerating green plantlets than the original potaro-2 medium

for

6 wheat genotypes.

2. Material and methods

2.1. N{ATERIAL

Six genotypes ol field-groçn wheat _{Triticam aesûvum) u'ere used as anther donors, i.e. spring genotypes Sabine, Line7, Atys and Echo, and *'inter geno-types

Fl

_{hybrid [(Zemon} x V.P.

Ml)

Gema] x _[(Zemon

x

Mer.50-B2t) Gema]

HIl972, from the < Station d'Amélioration des Plantes, Gembloux - Belgium >

and N.R.P.B. 824997.H.1986.Fl

ol

NrcxeRsoN

(\\'t

and W2 respectively).

2.2. METHODS

Donor plants were sown in mid-Nor-ember for *inter genotypes and

late-April for

spring genotypes. No chemical treatmenr rvas applied

in

the test plots during the grorving period of donor plants.

Preliminary tests were carried out ro shorv pos-iible variations betrveen

pollen callus formation capacities of main spike or branched spikes

of

the same plant. In further experiments only the main spikes u-ere used as donors. we used anthers containing the mid- or late- uninucleate microspores, as described by

Hr

and _{ouv.q.xc tl981l. Anther} stage \ïas defined according to that of the majority of the pollen. Young spikes were disinfected with 0.1q0 mercuric chloride for 8 min followed b-v 4 *'ashings in sterile distilled water [cHUANc et

al.,

1978]. After disinfection, intact anrhers were immediately removed from the spikes and placed in 9 cm diameter Petri dishes (about 120 anthers per dish) containing 30 ml

ol

solidified meCium.

1t

's

n

I

RECENERATION OF WHEAT FROM ANTHER CULTURES 303

In

some experiments, excised disinfected anthers were dipped

for

1-2

hours prior to inoculation in a 0.8

M

sucrose solution prepared as follows:

sucrose (273 e) rvas dissolved in

I

I distilled water in which 30 g activated char-coal was added; the preparation was then autoclaved at 120'C for 20 min, and filtered;the filtered solution was autoclaved again at 110'C for 20 min, and used after cooling.

The potato extract and the original potato-2 medium were prepared as described by Cnuaxc et ql. _ll978l. The detailed composition of the potato-2 medium is as folloii's: 1090 aqueous potato extract, 10aM Fe.EDTA,

I

mgll

thiamine

HCl,

L5

mg/|2,+D,0.5

mg/l kinetin, 1000 mgll KNO3, 100 mgll (NH4)2SO4, 100

mg/l

Ca (NO:):.4H2O, 125

mgll

MgSOl7}{2O, 200 mg/l KH2PO4, 35 mg/l

KCl,

9-1090 sucrose, 0.55-0.7V0 agar.

2.2.1. Preparation of

A

medium

The modified potato-2 medium (A medium) contained the components

of

the

original medium, except

for

being supplemented

with

200 mg/l

glutamine, and semi-solidified with agarose (type

VII,

5 g/l) instead of agar. Also sucrose, prepared as a20Vo aqueous solution, was autoclaved at 120"C

for 20 min in the presence of 2Vo activated charcoal;

it

was then filtered to remove the charcoal, and added to make 990 in the final

A

medium.

The components, to make

I

litre of medium (except for the potato

ex-tract), were dissolved in 600 ml distilled water and sterilized by ultrafiltration (0.2 pm filter).

Potato extract rvas diluted into 400 ml and autoclaved with agarose at

110'C for 20 min; its temperature was lowered to about 50"C before mixing rvith the above preparation to make

I

litre of

A

medium.

2.2.2. Culture conditions

Callus induction was carried out in the dark, or in dim light, at 27oC +

loC.

Plant regeneration was obtained

at

26"C under 1500

lux

and

a

photoperiod

of

12 hours.

2.2.3. Culture temperature and _{pollen formation}

In

order

to

establish the optimal temperature

for

callus induction and green plant regeneration, anthers were placed comparatively at 25" _, 26" ,27o ,

304 ZHANG L.J., _{ANCEAU C., LEPOIVRE P., SEILLEUR P., SEMAL J.}

2.2.4. Regenerqtion of plantlets

calli,

about 1 mm, were _{transferred onto a regeneration medium}

con-sisting

of

MS

medium

_[MunasHrcE

and

Sxôoc,

1962]

wirh

rhe macroelements

_{at half}

_{concentration, naphtaleneacetic acid}

lNea)

at

0.5

mgll,

kinetin at 0.5

mg/l

and agar (Difco Bacto agar) at7.5 g/1.

when reaching 3 cm long, regenerated _{rooted àr} rootless plantlets were removed from _{the regeneration medium, and transferred into test tubes} con-taining a _{root-inducing liquid medium [scuaenngn et}

_at.,

_{rgg4l aerated by} permanent shaking at 80-90 _{rpm in a} gyrotary shaker, with a photoperiod

oî

16 hours light at 1500 lux. planrlets _{were maintained in this meàium}

until they developed _{sufficient roots for potting.}

cytological analysis _of microspore embryogenesis _{during in vitro culture} was based on the method described by HeNnv et at. [19g4], modified as

follows. After 0, 6, 8, 10, 12, 16 days of culture, g rand-omlychosen viable anthers were removed from each of 3 petri dishes per treatment, and fixed in acetic acid-alcohol

(l

:3) for observation.

For analysis of the percentage of anthers containing viable microspores, about 50 anthers were used

for

each treatment.

In

order

to

determine the developmental stage and the percentage _{of viable microspores, a sampling}

_of

200 microspores taken from each anther (or from a mixture of 4 anthers) were observed. _{Microspores were considered}

_to

_{be alive rvhen their nuclei and} cytoplasm stained well in acetic-carmin.

cytological analysis

of

regenerated green plants was performed accor-ding to the technique of HucoRNs _[19g7].

3. Results and discussion

3.1. EFFECT OF CULTURE MEDIUM

comparison

of

A

medium and potato-2 _{medium is shown}

_in

_{Table I.} Pollen callus induction

on

A

medium was 2-3

fold

higher than

that

on potato-2 medium, with all genotypes used.

The enhanced frequency of green shoots differentiation was mainly due

to the increase in responsive anthers frequencies on A medium,

The superiority

of

medium

A

over _{the original potato-2 medium may} result from sucrose pretreatment _{with activated charcàal.}

charcoal might have absorbed _{5'-hydroxymethylfurfural, a growth}

in-hibitor

produced

by

sucrose degradation during autoclave sierilization

[wnarHenug{D et

al.,

1979). Also, as suggested by Bour-av _[lg7g], char-coal might bring some _{components favourable to growth and development}

_of

the explants, such as monophenylamines.

However, agarose may have played a favourable role as well, since agar is not the most suitable agent for _{anther culture [JoneNssoN, tôao].}

i

*

==

Lid,H ÈE

gùÈË-Ë

koÉ-5 sOo C d À \ooo\o\ Nt.lÊ NC{ro rO\r\O O\ \O NO rÔ Ooe !q*

5Ss

d oôo€ O\€ô\<i O'N AN

o!5.u-b

I<

9

U = bs d s z2d N _o-æ€ h€æN fia'ldô I \oo @s Oa hu= t -o* N À -ôiOr r*6h hæao c)Ô æo o:o

494

Z 6'ii N À

\qqn

æ I N d À O-rôôrTh çhær O\d h6 r€ æ u >ic -ç â: o l: a)Ê6=Y t i:4 a = J:i=u!r

z.â

N æ6Sæ ô-rQ 6d\oo ridÔ NNdÊ

{

O a o:o =?d ZY d ohrô rô !ec

z9y.

N 0. ôd.i...l Ndr6 drrr r:fN€ o o o o 9r - o .= o >!

;5fû

dd a.l EoÉ àgE

z

Yà -2

2@ ? É ôd g.q Ê_: o=

Àt

a * é :

RECENERATION OF WHEAT FROM ANTHER CULTURES 305

NÔ l-5 N r

=

c6 ..q! o Në n6 id o.;

zg

.Y 9" à =coi ÈN 6 =--Vr, 'r: r-OLqO rqY!

;So

e ç i è

^2.-I

H>:

x d! rtsl l À 9>c-:Fæ9 ^OL+Y .. ! Ér- O il ç::â:

^

_^,

ori9 i

É

_=À

Ë

*.9..-

'

FArA= =o.9 .;ge*e É 3

br;

<9 V Èflilo llL-do

<Ê.>ta

F N o ô <d E É o a .o À O I o .o F ) l

306 ZHANG L.J., _{ANCEAU C., LEPOIVRE P,, SEILLEUR P.. SEMAL J.}

3.2. EFFECT OF SUCROSE PRETREATMENT

wANc et

al.

u9811 treated excised wheat anthers with 0.g

M

sucrose

prior to culture. Their results showed that this treatment significantly increa-sed the induction frequency of pollen calli. In our experiments, pretreatment of excised anthers with 0.8 M sucrose (pretreated _{with activated charcoal) had} no effect with four homozygotic spring genotypes.

_A

_{favourable effect rvas} obtained, however, with hybrid winter genotypes, _{rvhich showed an increase} of the rate of callus induction raising from

l6.l

go _{(rvithout sucrose treatment)} to 28.990 (with sucrose treatment) for genotype

wl,

_{and from 3.g vo to 6.3v0} for genotype w2, while the average percentage of regenerated green plantlets raised from 3.9v0 to 6.8 9o for w1 and from I .9vo r.o 3.g go for w2 (Table II).

c1'tological study

of

genotype

_wl

(Table

III),

showed

that

sucrose pretreatment _{of excised anthers cultured on A medium increased the} percen-tage

of

anthers containing living microspores. No modifications

of

the sur-vival rate of microspores, and of the average number of nuclei per surviving microspore, rvere observed.

PaN e/ _{ol. ll983l} shor.ved that early developmenr _{of pollen} was related to the viability of anther wall tissue; growth conditions and genotype of anthers donor plant may play a role in microspore callus induction, by affecting (or controlling) the activities of anther wall tissue _[OuvaNc, 19g6] .

In

our

tests,

the

increased frequency

of

anthers containing viable microspores, when pretreated with 0.8

M

sucrose, _{may be linked either to} heterozygotic or winter genotypes.

3.3. EFFECT OF TEMPERATURE

For all 6 genotypes we have studied, culture temperatures

of

26oc to 28oc were favourable both for callus induction and green plantlets regenera-tion capacity. Typical data for

w1

and sabine are presented _{in figure}

l.

In-creasing temperature

for

callus induction above 2goC

did not

modify significantly the frequencies of callus formation, but suppressed the capacity

for green _{plant differentiation.}

our

results are in general agreement with those of Research Group 301 119771, showing that, although pollen callus yield increased with temperature, yet the capacity of calli to differentiate into green plantlets _decreased

accor-dingly. The

studies

of

_{ouveNc et}

a/.

[19g3] also indicated

that

the temperatures suitable for wheat anther culture ranged _{lrom 26oc to 30oc,} ac-cording to the genotype of anther donor plants.

C

REGENERATION OF WHEAT FRON'I ANTHER CULTURES o o o

i.

$ .3 a

{

o o = d o È c ij] I _o F

e3

^= a É o!

o*

r ææ r N r É@ d oq â æ æ

O-<o.

a z . b-=! o .q=2, O _{€ ô\} a æ_{c æ} æ O 3 N IN a.l €N N € d r a_N €ô r ô € d ô. OO

i-ç:.

c-Lo ôq)E J= 6 n r N rN ôt N ôiN .+N N .l $ I q ôi e É, r N_{r €} s _e

ç

Uo N or N O€ O N_s O€ 9z u É@ 2 i=â c o È o _É Ê o (, -ô cd 6 r J o FT d 'É il o o ,o d o a

308 ZHANG L.J., ANCEAU C,, LEPOIVRE P., SEILLEUR P,, SEMAL J.

Table III. _- Pollen evolution in vitro with or without pretreatmeni with 0.8 M sucrose

(Genotype Wl medium A)(1).

3.4. EFFECT OF DONOR

SPIKE

i

In our hands, frequencies of callus formation in anthers from the

main

l

spikes were always higher than those of the branch spike for all 5 genotypes used (Figure 2).

3.5. EFFECT OF INOSITOL

Preliminary experiments with genotype Sabine (Table IV) indicated that

additionofinoSitol(l00mg/l)inourcallusinductionmediumincreasedboth

the number of calli per responsive anther and the number of green

plants.

.

,,€

;

3.6. NUCLEAR STATE OF REGENERANTS

Cytological studies of 409 green plants regenerated from anthers showed

about809ohaploids,thedihap1oidizationofwhichiscurrentlyunderin. vestigation (Table V). Observation Pretreatment with 0.8 M sucrose

Age of culture (days)

ol6lsllol12116

9o of anthers containing living microspore with 100 99 72 68 49 38 without 100 95 5l 37 t( l9 9o of living microspore in living anthers with 98.0 54.2 18.0 9.1 3.8 l.l without 98.0 32.0 1 1.3 4.5 al 0.9

Average number of nuclei per

living microspore

with I 1.8 2.6 4 8

>8

without I 1.6 3.0 4 8

>8

REcENERATIoN oF wHEAT FRoM AIsrrrgRCULTURES 309

s---{

oF---'-o

cO N (.o l/\

H

H

FÔ ZA 2t*

ziE

t!vF lôz Ll=< A,L J. 8 M sucrose )m the main 5 genotypes dicated that reased both een plants. ters showed y under in-z

5t

-=

=d L.À J-)6 1u iu ,-a -d

yb

'-,

É É,a >Ê l! .i z'É o o ç € c€ À o o d o À O o 6 o O -ô o & I I -; o il l!

@

i":@

o

'29 ôF- _ ttz*; ) 12116 49 38 25 t9 J.ô l.t 2.1 0.9 8 >8 8 >8 a ?t t: !z

^ :'.

>

tô

.Ï oo

310 ZHANG L.J., ANCEAU C., LEPOIVRE P., SEILLEUR P., SEMAL J :

{s

o. -ô t o 'â d -: LU dq

a-'=û

(> :e Oo q= LA È6 oÔ. .:d o 6 i-I I ôi @ Ll q t-z trl (, a F )Z v:l Jv)

*-22

7<

.id.

zc0

r-t! z

J

EI z U)

Nt:]

t'iic

^ô

-q2 z--iiz

REGENERATION OF WHEAT FROM ANTHER CULTURES 311

Table IV.

- Effect of inositol on the regeneration of green plants from Sabine anthers

(l) See table L

Table V.

- Cltological analysis of regenerated green plants

{':e

(1)

See table I c.

--o o. ôo

;=

56_d> ,OU = ?-!E oô. o o 5 I .i 9 a il

:

Medium Cultured anthers Respon-sive anthers Calli or embryos observed Calli/ responsivt anther Green plants Albino plants Green plant/ I 00 cultured anthers Medium A(1) t20 36 6l 1.1 7 29 5.8 Medium A + 100 g inositol/l 120 3l 96 3.1 12 15 10.0 Genotl'pe Total number of regenerated green plants

Haploids Diploids Aneuploids

Haploids/ 100 regenerated green plants Sabine 9'7 t4 9 '16.3 wl (1) 99 93 3 J s3.9 Line ,7 '79 _{64 9 6} 8l .0 At)'s 63 45 15 J 71 .4 Echo 50 +s 1 6 86.0 w2 (1) 21 19 0 2 90.5 Total 409 338 29 82.6

312 ZHANG L.J., ANCEAU C., LEPOIVRE P,, SEILLEUR P., SEMAL J.

Acknowledgments

We thank M. MrulrvaNs for his help, and J. Denercr for efficient technical assistance. This research was sponsored by the <Institut pour I'Encouragement de la Recherche Scientifique dans I'Industrie et l'Agriculture>, (I.R.S.I.A.), Brussels, Belgium.

Résumé

Une méthode efficace de régénération du froment de printemps (Triticum aestivum) à partir de cultures d'anthères

La technique de culture d'anthères décrite, appliquée à deux génotypes de froment d'hiver et à quatre génotypes de froment de printemps, a permis d'obtenir un taux important de plantes chlorophylliennes.

Le milieu utilisé est dérivé du milieu potato-2 de Cnuaxc et ol. 119781 par

addi-tion de glutamine, utilisation de saccharose traité au charbon de bois actir'é et rempla-cement de I'agar par de I'agarose.

Le pourcentage total moyen d'anthères produisant au moins un cal est passé de

9,1% (avec le milieu potalo-z original) à21,6V0 avec le milieu modifié, tandis que le pourcentage total moyen des plantes chlorophylliennes régénérées passait de 2,5 9o (50 plantes vertes pour 2040 anthères) à 6,9V0 (369 plantes vertes pour 5350 anthères).

Le prétraitement au saccharose des anthères excisées a augmenté considérable-ment le taux de callogenèse pour les génotypes de froment d'hiver (hybrides Fl).

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dt'i"i;;;;;''gou'

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