T h e o r e t i c a l and A p p li e d G e n e t i c s 47, 109-114 (1976) 9 S p r i n g e r - V e r l a g 1976

Anther Cultures of Nicotiana tabacum L. Mutants J. Vagera, F.J.

Nov~k and B. Vyskot

C z e c h o s l o v a k A c a d e m y of S c i e n c e s , I n s t i t u t e of E x p e r i m e n t a l Bo t an y , P r a h a , E x p e r i m e n t a I Station O l o m o u c (Czechoslovakia) Summary. The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana t~ac~ L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther. The anthers from M I plants of a diploidized Nicotiana tabac~ haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M I sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concent ration.

Introduction A haploid system

in higher plants would be important

for studying spontaneous

and induced mutagenesis

well as for hybrid analysis. haploids

derived from

ations have been observed tion r a t i o s

During

the analysis

anther culture of hybrids from

the expected

as of devi-

segrega-

( M e l c h e r s and Labib 1970; M e l c h e r s 1972;

Opatrn~ 1973), m i s r e p r e s e n t i n g the r e s u l t s of a r e a l g a m e t i c s e g r e g a t i o n of the f a c t o r s . To v e r i f y the s u i t a b i l i t y of a n t h e r d e r i v e d h a p l o i d s f o r g e n e t i c a n a l y s i s we c o m p a r e d the t h e o r e t i c a l and o b s e r v e d p h e n o t y p i c r a t i o s in p o p u l a t i o n s of h a p l o i d s d e r i v e d f r o m h e t e r o z y g o u s c h l o r o p h y l l m u t a n t s of Nicotiana tabacum L. The c o n c l u s i o n s of t h e s e m o d e l t r i a l s h a v e a d e c i s i v e inf l u e n c e on the i n t e r p r e t a t i o n of s t u d i e s i n v o l v i n g s e g r e g a t i o n in h ap l o id p r o g e n i e s of p l a n t s with unknown genotypic constitution. In f u r t h e r t r i a l s , which w e r e r e l a t e d to o u r p r e v i ous work (Vyskot et a l . ,

1974), we s t u d i e d the d e v e l -

o p m e n t s of h a p l o i d s f r o m a n t h e r s of M 1 p l a n t s o b t a i n ed a f t e r the s e e d s had been t r e a t e d with i n c r e a s i n g c o n c e n t r a t i o n s of th e m u t a g e n s . In haploid p r o g e n i e s of M 1 p l a n t s we e s t i m a t e d not only the f r e q u e n c i e s of mutant p h e n o t y p e s but a l s o s o m e q u a n t i t a t i v e c h a r a c t e r i s t i c s of a n d r o g e n e s i s p r e v i o u s l y o m i t t e d .

Materials

and Methods

The chlorophyll mutants (ys) and White seedling kindly provided by prof. material for analyses of

Sulfur (Su), Yellow seedling (ws) of Niaotiana tabac~ L. , L.G. Burk, served as initial haploids. The mutation Sulfur

is i n c o m p l e t e l y dominant with a m o n o g e n i c c o n s t i t u tion and is l e t h a l at the h o m o z y g o u s dominant s t a g e . Heterozygous (Su/su) plants were utilized for anther c u l t u r e . The m o n o g e n i c m u t a t i o n Yellow s e e d l i n g is lethal at the h o m o z y g o u s l y r e c e s s i v e s t a g e . The p h e notype White s e e d l i n g , p r o d u c e d by two g e n e s with a d u p l i c a t e n o n - c u m u l a t i v e f u n c t i o n , is n o n - v i a b l e u n d e r n o r m a l c o n d i t i o n s in the h o m o z y g o u s r e c e s s i v e c o n d i t i o n . We u s e d p l a n t s which w e r e h o m o z y g o u s l y r e c e s s i v e in one of both the loci so that the m u t a t i o n White s e e d l i n g r e s p o n d e d as a s i n g l e r e c e s s i v e during s e g r e g a t i o n . A c c o r d i n g to the s e g r e g a t i o n of ys and ws m u t a n t s in a n t h e r c u l t u r e s , the t r i a l i n v o l v e d only don o r plants with h e t e r o z y g o u s g e n o t y p i c c o n s t i t u t i o n . P h e n o t y p i c f r e q u e n c i e s in h ap l o i d p r o g e n i e s w e r e s t u d ied using f i v e plants f r o m e a c h of the t h r e e t ype s of c h l o r o p h y l l m u t a n t . The c u l t u r e c o n t a i n e d 60 a n t h e r s from each plant. In f u r t h e r t r i a l s the i n i t i a l d o n o r m a t e r i a l was a d i p l o i d i z e d h a p l o i d of Nicotiana tabacwn L. c v . ' S a m sun' obtained through r e g e n e r a t i o n from tissue cult u r e of an a n d r o g e n i c h a p l o i d p e t i o l e . The s e e d s w e r e t r e a t e d with s o l u t i o n s of n - b u t y l m e t h a n e s u l p h o n a t e (BMS 5 m M , 1 0 m M , 2 0 m M , 4 0 m M ) and N - n i t r o so-N-methylurea (MNU- 0.1raM, 0.2raM, 0.4raM, -

0.8mM) for 24 hrs at 23 - 2 5 ~ C in the dark. T h e c o n trol w a s soaked in distilled water. The seeds w e r e then rinsed in running water and s o w n on moist garden soil. R a n d o m i z e d sets of 20 plants in each variant ( M I generation) w e r e planted out in the experimental field and the anthers w e r e taken from individual plants at the stage of uninuclear microspores. W e have cultivated 40 anthers from each M z plant, i.e. 800 anthers per variant, with the exception of 4 0 m M B M S and 0.8 mM MNU (240 anthers and 600 anthers, respectively). The experiment w a s carried out at r o o m temperature and under continuous illumination. The haploid plants w e r e evaluated during the course of the trial (detection of chlorophyll mutations) and also in one lot after 13 weeks' cultivation (evaluation of quantitative characteristics of androgenesis). The evaluation involved 3,255 haploid plants. The chlorophyll mutations w e r e classified according to the system proposed by L a m precht (1960). In all the trials the anthers w e r e cultivated on the m e d i u m of Nitsch (1969) with an addition of 0.1 m g / e [~ - indoleacetic acid (IAA).

110

J. Vagera,

F.J.

NovSk

and B. Vyskot:

Anther

Cultures

of Nicotiana

tabae~m

L. Mutants

Table I. Evaluation of segregation ratios in populations of haploid plantlets in the in vitro anther culture derived from heterozygous chlorophyll mutants of N. tabacwn L. Donor plant No.

Total of regenerants obtained 1 2 3 4 5

Theor. segreg, ratio

159 121 171 144 112

Total

79.5 60.5 85.5 72 56

707 1 2 3 4 5

Total 1 2 3 4 5

: : : : :

353.5

96 74 82 90 72

48 37 41 45 36

414

207

91 121 102 96 108

Total

Observed segreg. ratio 79.5 60.5 85.5 72 56

: 353.5 : : : : :

259

403

48 37 41 45 36

54 42 48 53 41

: 207

237

45.5 : 60.5 : 51 : 48 : 54 :

518

89 : 70 : 97 : 82 : 65 :

45.5 60.5 51 48 54

: 259

Results

2

P

70 51 74 62 47

2.27 2.98 3.09 2.78

: 304

13.86

: : : : :

2.89

0.30 O. 10 0.10 O. 10 O. 10

-

0.10 0.05 0.05 0.O5 0.05

0.001

42 32 34 37 31

1.50 1.35 2.39 2.84 1.39

0.30 0.30 0.30 O. 10 0.30

-

: 177

8.70

0.01

- 0.001

35 47 41 38 45

4.85 6.02 3.92 4.17 3.00

0.05 0.02 0.05 0.05 O. 10

-

: 206

21.69

56 : 74 : 61 : 58 : 63 : 312

X

0.10 0.10 0.10 0.05 0.10

0.02 0.01 0.02 0.02 0.05

0.001

strated by taking the average n u m b e r of haploid plants obtained per cultivated anther (Fig. 1). The concentra-

In anther

cultures

derived

from

cz~ plants of the mutations White

seedling

lower

than of green

biometric from

individual

an expected

plants.

mother

Although

plants,

ys and ws decreased

from

the level of pollen grains) resp.

mutant 0.36

Haploid during

haploids

mutation,

the mutagens mation

the values 0.75

influenced

in all the studied parameters.

the method

the total

their variances decreased with increasing concentraMNU

had no

0.43

(at

number of haploid

haploid plants per embryogenic anther decreased in all

genie anthers per M 1 plant was also dependent on m u tagen concentration. tion of 5 mM

and

resp.

for-

The efficiencyef

pared

BMS

haploids

MNU

of both the mu-

the variance. of embryogenic

anthers increasedthree-

and by 50 ~ in the variants with 5 mM and 20 mM

the number

BMS,

respectively,

The concentration of anthers

which

BMS,

when

com-

0.1 mM

MNU

gave

rise to

number

of true

by 75 %.

After leaves

relationship was recordedwith

with the control.

increased

at a concentra-

with increasedconcen-

All the applied concentrations

increased

10 mM

of

but decreased

(Fig.2).

fold, twofold

of true

stimulated

A similar

The number

of

of in vitro anther culture can be well demon-

BMS

It was

trations.

tagens

number

The average n u m b e r of

the treatments (Fig. 2). The average n u m b e r of e m b r y o -

and

0.25

average n u m b e r of haploids per cultivated anther with higher concentrations of M N U .

considered

14. The concentrations

the frequency

age n u m b e r s of haploid plants per cultivated anther and

its variability. There was a continual decrease in the

and continued

55, the highest

plant was

per cultivated anther and increased variability. Aver-

effect on the frequency of androgenesis but decreased

M l plants emerged

culture

stimulated the n u m b e r of haploid plants

with

of selection of

The highest

BMS

a significant

plants in the in

of 0.25,

tion 5 m M

tions of B M S . The application of 0.1 m M

alleles Su,

and 0.64,

from

of anther

was

the

the initial value of 0.50

until the 13 TM week. per anther

were

of mutant

to that of 0.43,

plantlets derived

per haploid

cases derived

results of segrega-

The coefficients

fitness 0.75,

the third week

to appear leaves

cultures).

was

in agreement

(at the level of haploid

alleles reached resp.,

in most

types when

by summarizing

(Tab. 1). The gene frequencies

vitro anther

seedling and

ratio 1 : 1, there was

tion in 5 plants of the same

0.40,

was

N. taba-

phenotypes

progenies,

in all three mutant

obtained

Yellow

of mutant

of haploid

gametic

disagreement values,

Sulfur,

the frequency

evaluation

heterozygous

13 weeks'

culture the mean

on haploid plants was

significantly

higher

in all

J. Vagera,

F.J.

Nov&k

and B.

Vyskot:

Anther

Cultures

of Nicotiana tabacum L. Mutants

111

300 250 200 "S 150 g~

!

- -

BMS MNU

- -

250

BMS MNU

-

~

o 200 -a g 150 2

100

100'

50

\

5O 5

I0

20

L

i

i

0.1

0.2

mM BMS 40 I

0.4. Concentrations

D

mMMNU 0.8

I

i

[

5

10

20

i

i

J

0.1

Fig. I. Average number of androgenic haploids per cultivated anther in the control and treated variants ({ = P < 0.05)

i

mM BMS 40 I

0.4 mM MNU 0.8 Concentrations A v e r a g e n u m b e r of e m b r y o g e n i c a n t h e r s p e r

Fig.2.

0.2

M z plant (full line) and that of haploid plants per embryogenic anther (dashed line) in the control and treated variants

80 j O

/ / /

/o

9 Contro[ D BMS [] MNU

60

/

i

I

o/

0

I/

t~ 4o

~4 2O

O

O

/

:.: :i: BMS I II= MNU

o/

{/

///l/z~.

~ 0~0

~0

IJ // ...o "

"

~_~--o /

/

~2

0

0510 20

40 0.10.204 mM0.8 C0ncenfrati0ns Fig. 3. Average number of true leaves on haploidplantlets after 13 weeks' cultivation in the control and treated variants (9 = P