Dihaploid plants of roses (Rosa x hybrida, cv Sonia) obtained by parthenogenesis induced using irradiated pollen and in vitro culture of immature seeds

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Dihaploid plants of roses (Rosa x hybrida, cv Sonia)

obtained by parthenogenesis induced using irradiated

pollen and in vitro culture of immature seeds

J. Meynet, R. Barrade, A. Duclos, R. Siadous

To cite this version:

J. Meynet, R. Barrade, A. Duclos, R. Siadous. Dihaploid plants of roses (Rosa x hybrida, cv Sonia)

obtained by parthenogenesis induced using irradiated pollen and in vitro culture of immature seeds.

Agronomie, EDP Sciences, 1994, 14 (3), pp.169-175. �hal-02703199�

Plant

_breeding

Dihaploid plants

of

roses

(Rosa

x

hybrida,

cv

_’Sonia’)

obtained

_by

_{parthenogenesis}

induced

_using

irradiated

_pollen

and

in

vitro

culture of immature seeds

J

_Meynet

R

Barrade

A Duclos

R

Siadous

1

INRA,

Station d’Amélioration des Plantes Florales, La Gaudine, F83370

_{Saint-Aygulf;}

2

Commissariat

à

_{l’Énergie Atomique,}

Service de

_{Radioagronomie,}

CEN de Cadarache, F13108 Saint-Paul-lez-Durance, Cedex, France

(Received

30

September

1993;

accepted

10

January 1994)

Summary -

’Sonia’ var rose ovules

_{produced plants}

_through

in vitro culture after

_being

_pollinated

with

irradiated

_{(gamma rays) pollen.}

A

_500-Gy

minimum dose was sufficient to inactivate

_pollen

and induce in situ

_{parthenogenesis;}

in vitro culture was _{necessary for}

_embryo

rescue. The

_{dihaploid plants}

_originated

from small

_embryos

which

_occupied

_only

a

_part

of the

_carpel

_{cavity; they}

were

_{clearly distinguished}

from

tetraploid plants by

miniaturization of all _organs. The

_{dihaploid plants}

were observed under

_glass-house

conditions until

_flowering

_presented

a normal

_{gynoecium. They}

_produced

a small amount of

_pollen

of reduced

but

_regular

_size;

_growth

and

_development

were faster than the

_tetraploid

controls in summer.

haploidy / Rosa

x

_{hybrida/ pollen}

irradiation

_{/ gamma}

_rays

Résumé - Obtention de

_{plantes dihaploïdes}

de rosier

_(Rosa

x

_hybrida,

cv « _Sonia

_{») après}

parthéno-genèse

induite par

l’emploi

de

_pollen

irradié et culture in vitro de

_graines

immatures. Des ovules de

_rosier,

variété « Sonia », cultivés in vitro

après pollinisation

avec du

_pollen

irradié aux _{rayons gamma} ont évolué en

_plantes

entières. Le

_pollen

inactivé efficace pour induire une

_{parthénogenèse}

in situ doit subir une dose minimale de 500

_Gy

et la culture in vitro

_paraît

_{indispensable}

_pour assurer la survie des

embryons.

Les

_plantes

_dihaploïdes

_proviennent

de

_{petits embryons}

_n’occupant

_{qu’une partie}

de la cavité

carpellaire,

elles se

_distinguent

nettement des

_{tétraploïdes}

_par une miniaturisation de tous les _organes.

Les

_{plantes dihaploïdes}

observées en serre

_jusqu’à

leur floraison

_présentent

un

_gynécée

normal,

elles ne

produisent

que très peu de

_pollen

d’un diamètre réduit mais

_régulier.

La croissance et le

_{développement}

de ces

_plantes

sont

_plus

_rapides

en été que ceux de

_plantes

_{tétraploïdes}

témoins.

haploïdie/Rosa

x

_{hybrida/irradiation}

de

_{pollen/rayons}

_gamma

INTRODUCTION

Modern

roses

with

constant

_{flowering belong}

to

Rosa

x

_{hybrida (2n}

= 4x =

28)

generally

considered

an

_{autotetraploid}

_species

(Berninger,

1992).

Although

this is

commer-cially

one

of the

_{major species}

that

_undergo

selection processes,

the

_genetic

structure

and the

_heredity

of its traits remain unclear.

On the other

hand,

new

_objectives

of

se-lection aim

at

_obtaining

an

insect-

and

dis-* Correspondence

and

_reprints.

t Deceased.

ease-resistant

or tolerant material.

_Hence,

botanic

_diploid

_germplasm

can

be used

as

the

_genetic

source.

_Finally,

the

_present

tetra-ploid

level,

the result of

200

_{years of}

uncon-trolled

_{interspecific}

_crossing

is

not

necessarily

the best

_ploidy

level for

_breeding

efficiency.

For all these

reasons,

it is

impor-tant to

obtain and

_analyse

the

_dihaploid

(2n = 2x= 14)

of cultivated

roses,

since this

work has never been undertaken before.

Tabaeizadeh and Khosh-Khui

₍₁₉₈₁₎

ob-tained

_{only haploid}

calluses

from in

vitro

culture of

R

damascena and

R

_hybrida

an-thers. The

_diploid

roses

_{produced by}

Reimann-Philipp (1981)

after

_backcrossing

of the

triploid

(R

multiflora x R

_hybrida)

with R

mul-tiflora

_{(2n = 14)}

were

both

_genetically

and

phenotypically

closer

to the

_diploid

_parent,

in

spite

of the

_genetic

_exchange

between

the

initial

_parents,

as

indicated

_by

the author.

The

above results

show

that

we need a new

method of

_{haploidization.}

In our

_work,

we

associated

_{parthenogenesis}

induced

_by

the

use

of irradiated

_pollen

and

_embryo

res-cue

_by

in vitro culture. This combined method

of

_{haploidization}

was

first

_developed

_by

Pan-dey

and

_Phung

₍₁₉₈₂₎

on

different

_species

of Nicotiana

and

_{by Raquin}

₍₁₉₈₅₎

on

Petunia. Sauton and Dumas

de

Vaulx

_(1987),

Doré

₍₁₉₈₉₎

and

_Zhang

et

al

₍₁₉₈₈₎

success-fully applied

this

method

to

musk

melon,

cab-bage

and

_{apple, respectively.}

MATERIALS

AND

METHODS

The ’Sonia’

_variety

was chosen as the female

parent

because of its

_ability

to flower in all

sea-sons, and its

_high

_productivity

of seeds. Pollen

was collected on a mixture of cultivated

_genotypes

since no

_{morphological}

markers could be used to

definitively

distinguish dihaploids

from

_hybrids.

The donors were cultivated on

_perlite

substratum under

_glass-houses

which were maintained at

least

14°C

and

_opened

at

22°C.

The anthers removed

_just

before anthesis were

_placed

in Petri dishes in a ventilated

_drying-room

at

27°C

for 24

h and

_{powdered pollen}

was then used

_immediately

for

_{experimentation.}

Irradiation and

_pollination

Collected

_pollen

was

_subjected

to _{gamma rays}

using

a Cobalt 60 source. Treatment was per-formed at the Centre de Cadarache on the

follow-ing

dates:

-

4 doses

_(0,

500, 1 000,

1 500 and 2 000

_Gy)

on

May

26, 1992;

- 3 doses

(0,

250, 500,

and 750

_Gy)

on

_July

2,

1992.

The flow rate _{of gamma rays} was 33.5

_Gy/min

for the 250

_Gy

dose and 150

_Gy/min

for all the other doses.

The

_half-open

flowers were emasculated and

isolated 1 d before

_pollen

irradiation. Pollination

was carried out in

_{saturating quantities during}

the

warm hours of the afternoon on the

_day

of

_pollen

irradiation and the

_{following day.}

After

_being

polli-nated,

the flowers remained covered for 10 d. For each

trial,

control flowers were

_{pollinated by}

non-irradiated

_pollen.

Pollen

_germination

was tested in vitro on a solidified medium as used

_by

Gudin et

al

_(1991).

Embryo

rescue and in

vitro culture

The

_protocol

of this work was based on a

prelimi-nary

experiment

conducted in 1991 on in vitro

culture of immature

_embryos

_(unpublished

-results).

In

1992,

the fruits were collected on

2

dates,

21 and 35 d after

_pollination.

The

_hips

collected after 21 d were soaked in 70% ethanol for 30 s and then 20 min in calcium

hypochloride

(15 g·

l

-1

),

followed

_by

2 rinses in sterile water. The fruit were then

_aseptically

opened

and

_only

_enlarged

ovules were removed. One half of the ovules was

_placed

_directly

in

culture,

whereas the other half was dissected under the binocular

_microscope

and

_placed

in culture without the

_tegument.

The fruits obtained 35 d after

_pollination

were

opened,

the collected seeds were disinfected in the same _way as the

_hips

and

_put

in culture without dissection. The

_pericarp

was

already

lignified

so that extraction of the

_embryos

was

very difficult.

The seeds were

_placed

on MS400 a

_Murashige

and

_Skoog’s

₍₁₉₆₂₎

medium modified to half

strength,

with 400

_mg·l

_-1

ammonium nitrate in-stead of 1 650

_mg·

_l

_-1

and

_supplemented

with 5%

sucrose.

_They

were

_randomly

distributed on 4 different hormone combinations:

In each 5.5 cm Petri

dish,

a maximum of 5 seeds were

_placed,

and then the dish was closed

with a

_plastic

film and left in the dark at 27°C.

After 3 months the

_embryos

extracted from seeds

were cultured on medium I with a 16

_h

photope-riod

₍₃₅

_μmol·m

_-2

_·S

_-1

_).

The

_plants

obtained were

multiplied

on a solid MS 400 medium

supple-mented with 3% sucrose and 2.22

_μM·l

_-1

BA.

In vitro

_rooting

took

_place

in Sorbarods

(cel-lulose

_{transplantation}

_plugs

from

_Baumgartner

basal

_liquid

medium

_{(6 ml/tube)}

enriched with 2.46

_μM·l

_-1

IBA. The rooted

_plants

were then

transferred to the

_glass

house on rockwool cubes.

In

situ matured fruit

and seeds

Some fruits were left in situ until

_complete

matu-rity,

and their seeds

_{germinated according}

to the

technique

devised

_by

Gudin et al

_(1990).

Observation of

_plants

obtained

The chromosome number was checked on root

tips, using

the same method as described

_by

Lespinasse

and Salesses

₍₁₉₇₃₎

for

_apples

and pears. The

specific

characteristics of the different

ploidy

levels were detected

_{by cytological}

and

morphological

measures.

RESULTS

The

influence of irradiation

on

the

_germination

of

_pollen

and

_development

of fruit and seeds

Pollen

_put

in culture on

the

_day

of irradiation

germinated

at 15-20%

except

for

the

one

ir-radiated

at 2000

_Gy,

which

reached

5% and

produced

shorter tube

_growth.

After

3 d

under

laboratory

conditions

all

_pollen

had

germi-nated

_except

that

irradiated

at 2000

_Gy.

One of the effects of

_pollen

irradiation

was

the

_yellowing

and

_drying

of

the

fruit,

which started

after the

third

week of

_pollination

(table I).

Ovule

_enlargement

was

observed between

the 21st

and the

28th

_day,

while

on

the 35th

day

all the ovules had

_enlarged

and the

peri-carp

was

_{considerably lignified.}

All the

fruit

examined contained

seeds,

in

_spite

of the

different irradiation doses

_{(table I).}

However

the number

of seeds

_(N)

varied with the dose

(D) following

a

linear model for the different

experimental

doses.

The number of seeds

was

correlated

directly

with fruit

_diameter,

r = 0.59

after

21

d

and

r = 0.84

after

35 d,

and

especially

with

fruit

_weight,

r = 0.81

after

21 d

and

r = 0.96

after 35 d.

The size of the seeds extracted from

ma-ture

fruit varied

_considerably

with the

various

pollen

treatments

_{(fig 1). Only}

the control and the

seeds

_resulting

from

_pollination

with

250-Gy-irradiated

pollen germinated

with

a success

of

43 and

_14%,

_respectively

(table II).

Immature

_embryo

rescue

in vitro

The nature

of the difficulties encountered

differed

with

the

_stage

of

_{sampling. The}

per-centage

of

_germination

with control ovules

was 9.2% on

_{average, and}

was 13.8%

for

in-tact

_ovules,

and

_only

4.4%

for

ovules

_stripped

of their

_teguments.

The

_percentage

of

success

observed

for control seeds 35 d after

pollination

was better

_(23%),

but was

fol-lowed

_by

_fungal

contamination

identified

as

Alternaria in

about

50%

of the material.

Embryos

were

observed

in

all

test

samples,

except

those treated with

₂

000-Gy-irradiated

_pollen.

The

_{embryos resulting}

from

pollen

irradiated

at 500

_Gy

and above

were

2-4-fold shorter than those

_resulting

from

control or

_{250-Gy-treated}

_pollen,

where

the

embryo occupied

all the

_carpel

_cavity.

Embryos germinated

on

all the media

tested;

the data showed no effect of the

different hormone combinations.

In

some cases 3

sub-cultures

on

medium

I were

_necessary

to obtain

_{green cotyledons}

followed

_by

the

appearance

of the

first leaf

and

_elongation

of

the seed root.

Thus,

we

obtained

_{(table III)}

59

_plants

from control

pol-len,

1

_plant

from

250

_Gy,

3

_plants

from

500

Gy,

1

_plant

from

750

_Gy

and

2

_plants

from

Description

of

_plants

obtained

The 250

_{Gy pollen produced}

3

_embryos

in vitro and 6 seeds in

situ

that matured and

germinated

and then

_{yielded only}

one

_plant

for

each

condition

_(in

situ

and in

_vitro).

These

plants

were

similar

to

the control but their

growth

rate was

_slower,

with

some

abnormali-ties

_(thick

leaf

_blade,

_irregular

leaflets,

and

absence

of

_stigma

in one

_case).

The

chro-mosome number

of the tested

_plant

was 28.

However,

the 6

_plants

_{produced through}

pollination

at 500

_Gy

and

_{higher pollen}

ir-radiations had a different in vitro

_morphology

from the control. When the first leaf

_appeared

(fig 2),

all organs,

leaflets,

petioles

and

stems had much smaller sizes.

_However,

their

multi-plication

rate was

_highly

variable.

_Only

one

plant

remained unable

to root

_up

to this

_point.

In

_comparison

with the

control,

the first

3

plants

flowered

at the same time and their summer

_growth

and

_flowering

were

_very

ac-tive,

but with smaller leaves and

_pollen,

stoma

_length

and

_chloroplast

number

in all

5

_plants

_(fig

_3,

table

_IV).

The chromosome counts were

often difficult. The

_dihaploid

state

_(2n

= 2x =

14)

was

demonstrated

on 2

plants

(figs

4 and

_5).

DISCUSSION

AND

CONCLUSIONS

All

the

_plants

obtained after

_pollination

with

pollen

irradiated

at 500

_Gy

and above

have

common

_{morphological}

traits,

which

are

clas-sically

used

as

_ploidy

indices;

2 have

the

di-ploid

number

of chromosomes.

_{Consequently,}

the

_500-Gy

dose of

_pollen

irradiation

is

suffi-cient

to induce

the

_{parthenogenetic}

Treatment

at 250

_{Gy produced high}

mor-tality

rates

_{and very slow}

_growth

of

_plants.

This established

_phenomenon

is

known as

the

_’Hertwig

effect’

_(Pandey

and

_{Phung, 1982).}

On the other

_hand,

all

_pollen

irradiated

above

500

_{Gy and up}

to 2 000

_Gy

_gave

ma-ture fruit

and

_enlarged

ovules unlike

_apples

where

the

flower fell after

_pollination

with 1

_{500-Gy-irradiated pollen}

_(Zhang

et

_al,

_1988).

Hence

the

_pollen

of Rosa

_hybrida

_appears

less

sensitive

to irradiation than

_{apple pollen.}

The

_embryos

and

seeds

_developed

and

germinated

in

vitro under

a constant

tempera-ture

of

27°C

with no cold treatment since

there

was no

_{dormancy phase}

in our

experi-mental conditions.

The

_percentage

of

_{diploid plants}

obtained in

the first trial

was 1.6

_plants

on

_average

for

100

_enlarged

ovules

in

culture,

and

6.7

plants

for

100

flowers

_{pollinated by}

inacti-vated

_pollen.

Further

_{optimization of the}

pro-cedure should be followed

_{giving priority}

to:

-

increasing

the

_number

of

seeds

_{per fruit}

through testing

of

_genotypic

and

_{physiological}

inductive factors

_occurring

before and

at

the

time

of

_pollination;

-

increasing

the

number

of

viable

_embryos

in

_enlarged

ovules

_by

suitable horticultural

growing

conditions,

especially just

after

polli-nation;

-

improving

in vitro

_embryo

rescue,

partic-ularly

by

a

better control of

_fungal

contami-nations.

The

_possibility

of

_using

the

_dihaploid

mate-rial in selection

_{programs depends}

on its

fer-tility.

Observation of the

first

flowers of

a

other

_plants

in

late

summer did not

_help

us

verify

this

_point.

The presence of

a

normal

_gynoecium

and

regular

but

rare

_pollen

is

rather

_encouraging.

ACKNOWLEDGMENTS

The authors thank J

_{Eychenne (student}

at

INP,

Toulouse)

for her

_{participation}

in the

_plant

material

observations,

P Venard

_(INRA,

Station de

Patho-logie, Antibes)

who was kind

_enough

to

_provide

the

_microscope

_photos

and L Kevorkian for

trans-lating

this article into

_English.

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Y,

Chevreau E

₍₁₉₈₈₎

Ob-tention de

_plantes

_haploïdes

de

_pommier

(malus

x domestica

_Borlch)

issues de

parthéno-genèse

induite in situ par du

pollen

irradié et

culture in vitro des

_pépins

immatures. CR Acad