IlIA Advantages and Disadvantages of the Use of

in Vitro/in Situ Produced DH Maize Plants

M. BECKERT)

1 Introduction

The selection of lines is an ancient practice in maize breeding. New hybrids have
been derived through crossing of lines, which are better adapted to agronomic
conditions. The current method of deriving new lines is to self-pollinate plants
during a period spanning eight to ten generations ofinbreeding in order to obtain
a high homozygotic level. Due to strict conditions for registration, the release of
new lines in Europe requires a very high level of homozygosity.
If doubled haploid (DH) line production could be mastered in maize, it
would accelerate the process ofvarietal release by up to 2 to 3 years. This breeding
strategy could also produce more efficient schemes of selection because of the
experimental precision of using nonsegregating material and because of the
ability to analyse the genetic components ofcharacteristics. This would lead to a
better understanding of the genetic potential of possible new lines (Gallais 1988).
Before using such a technology for the production and release of new lines,
certain technical and genetical requirements must be fulfilled:
1. A cost efficient great number ofDH plants must be produced.
2. There must be no genotypic incompatibilities.
3. Diploid plants with high fertility levels are necessary.
4. There must be molecular, cytological, and phenotypical stability in DH plants.
5. The method should not select undesirable traits.
6. The process should save time or produce better "genetic progress" per unit of
time.
Current investigations are aimed at evaluating and comparing different
systems in situ (androgenesis and gynogenesis) and in vitro (androgenesis) for
the production of haploid maize plants. This chapter outlines and makes
comparisons between both systems for breeding purposes.

IINRA, Domaine de Crouelle, 63039 Clermont-Ferrand, France

Y. P. S. Bajaj (ed.), Maize

© Springer-Verlag Berlin Heidelberg 1994
202 M. Beckert

2 Androgenesis in Vitro

Chinese scientists were the first to obtain androgenetic maize plants via anther
culture (Anonymous 1975). Since this pioneering work, numerous teams in
China, the U.S.A. and Europe (Miao et al. 1981; Genovesi and Collins 1982; Dieu
and Beckert 1986; Kuo et al. 1986; Petolino and Jones 1986) have been interested
in defining the genetic and environmental factors leading to a greater
androgenetic response. Until recently, the increase in response has been some-
what slow. Nevertheless, it was clear from early publications that a large part of
the success in obtaining androgenetically regenerated maize plants was con-
nected to some specific genetic background capability.

2.1 Genetic Components of Androgenetic Aptitude

Screening of a very large number of different ancestries made clear that genetic
variation existed for characteristics such as the percentage of androgenetic
anthers produced, the percentage of androgenetic embryos produced and the
ability of these embryos to regenerate as fertile plants (Beckert 1986). In the early
part of this work, haploid plants were generated and progenies were derived after
selfing double chromosomed spontaneous plants. The androgenetic response of
the progenies of these DH lines were directly evaluated at line level or through
comparison with elite agronomic lines.
From a qualitative point of view, it was clear that the androgenetic process
produces lines with a very high androgenetic capability, and there was a high level
ofexpression in hybrid structures including those ofelite lines such as F244 which
showed little androgenetic aptitude (see Fig. 1). It was also possible to obtain
hybrid structures revealing a very high heterosis effect. The androgenetic anther
percentage response of the DH5 x DH7 hybrid, for example, was often as high
as 75. Other lines derived through androgenesis did not display such a great
androgenetic aptitude. It is possible that some sporophytic determination js
involved in androgenetic induction (Barloy et al. 1989).
Using a more quantitative methodology, an evaluation of heritability of the
percentage ofandrogenetic anthers was carried out. This was done with factorial
crosses using five original DH lines with varying response levels and five other
lines derived by classical selection. Heritability was found to be relatively strong
(0.76) in a trial where the general mean was about 10%. An evaluation of the
same parameters in different lines (Afele and Kannenberg 1990) gave similar
results. Using classical models it was then possible to draw some conclusions
about various genetic components. It appeared that additive genetic variance
was greater than nonadditive genetic variance (Barloy 1990). This was recently
confirmed (Afele and Kannenberg 1990) for characteristics such as the percent-
age ofandrogenetic anthers produced or the capability ofandrogenetic embryos
to generate plants. Normal or reciprocal cross-results do not vary considerably
for these traits.
This first step in genetic analysis was continued with a more precise study
including a characterization of recombinant DH lines derived through
androgenesis from the DH5 x DH7 hybrid.
The Use of in Vitro/in Situ Produced DH Maize Plants 203

Androgenetic embryo yield

per plated anther

• DH5 x DH7

·DH5

• DH7

2 * F244 x DH7

* F244 x DH5

• DH7 x DH9

-(( MBS 847 x DH 5

-(( MBS 847 x DH7

-((MBS 847
.*F244
H9
o 10 20 30 40 50 60
Percentage
of responsive
anther

Fig. 1. Relationship between lines and hybrid combinations for androgenetic aptitude

The results for the percentage of androgenetic anthers produced by these

recombinant lines are given in Fig. 2. It is clear that androgenesis itself does not
select for high response levels. It was possible to obtain both, lines with the high
heterotic value of the hybrids, and also lines with very little potential. This
suggests there are complementary genetic systems in the parental lines which act
together.
The regeneration potential of androgenetic embryos was evaluated for these
lines (Fig. 3). These experiments did not reveal any strong genetic correlation
between the two characters, and it was easy to recombine a high level of
androgenetic anther response with a high capability of plant regeneration from
androgenetic embryos.
204 M. Beckert

Percentage
of responsive
anthers

80

•
•
o• DH5 x DH7
60
•
•
•
••
40
•:*
.• r
DH5

DH7
••

,.., .. #
•
20

..""
., ••
••••
o •
Ranking lines following increase of yield

Fig.2. Analysis of percentage of embryogenic anthers in DH lines derived from DH5 x DH7

Because ofthe results ofthe 2nd cycle ofDH androgenetic lines it seems likely
that in vitro androgenesis selects for higher levels of regeneration capability in
comparison with parental lines.
This sample of recombinant lines indicates that a small number of genes or
a cluster ofgenes differentiate both parental lines from androgenetic traits. Using
Snape's formula (Snape et al. 1984), it appears that only four or five groups of
genes may be different from both parents for induction and regeneration from
androgenetic embryos.
Before extending such an estimation to the genetic variability of maize in
general, it must be searched for different genetic backgrounds. As a first step,
back-cross experiments were made using DH5 and F244 as parental +/- for
androgenetic lines (Hardy et aI, 1991). Evaluation of the hybrids obtained by
crossing regenerated DH plants, derived from the one-way cross with the non-
responding F244 line, reveals the same general observation. Calculation gives
also a low number of genes (two or three).
The Use of in Vitrolin Situ Produced DH Maize Plants 205

Regeneration potential
of androgenetic embryos

60
• •• •
• • •
• ••
50

•
40 •• •
••
• • •• • ••• • •
30
•• ••
••••• •
• •• •
tl'DH7

20
•• •
• • • •••• • *
10 • •• o DH5 DH5 x DH7

•
o •
o 20 40 60 80 Percentage of
responsi ve anthers
Fig. 3. Analysis of the relationship between percentage of responsive anthers and regeneration
potential of androgenetic embryos in DH lines derived from DH5 x DH7

Recently, a genetic approach, using genetic maps with RFLP markers closely
spaced and covering the genome, has been used on material derived from H99,
Pa91, and Fr16, a dent maize background responding to androgenesis compared
to the nonresponding line B73. Two major and two minor quantitative trait loci
(QTL) account for 57% of the genetic variability in androgenetic embryo-like
structure production (Cowen et al. 1992). Using same technology on the res-
ponding DH5, DH7lines, compared to the nonandrogenetic responding AI88
and Mo 17 lines, M urigneux et al. (I 993b) pointed out other chromosomal
regions for the same kind of characters. These works indicate that, in fact,
androgenesis is certainly a complex genetic process, from the microspore to the in
vitro regenerated plantlets. Numerous genes are expressed during these morpho-
genetic steps.
A more precise identification or localization of the genes involved would
simplify back-cross experiments to material with greater agronomic potential.
206 M. Beckert

2.2 The Molecular Approach to Induction of Androgenesis

A set ofcompetent noncompetent pure lines, derived from a narrow genetic basis
for androgenesis, is a choice material for the characterization at a molecular level
of the critical steps of the androgenetic pathway. In a study using DH5 x DH7
hybrids and other lines, Vergne et al. (1990) reported that a specific protein (32
kDa molecular weight) is expressed only in anthers of highly androgenetic
genotypes. Its synthesis is largely increased after cold treatment during the first
steps of anther culture. There is also a correlation between the level of andro-
genetic response and the level of protein expression (Vergne et al. 1993).
This specific gene product seems to be related to the androgenetic process in
maize. It has not been detected during normal flowering or development of the
anthers on the tassel. The demonstration of the accumulation of the 32-kDa
protein in the anther during the cold pretreatment raises the question as to which
cells of the anther accumulate it. Comparison of protein extracted from isolated
microspores and from whole anther indicated that the accumulation takes place
in the locular space or in the sporophytic tissues of the anther, but not in the
microspore cells (Vergne et al. 1993).
This specific gene product seems to be related to the androgenetic process in
maize. It has not been detected during normal flowering or development of the
anthers on the tassle. This protein represents a putative marker ofanther culture
responsiveness in maize, and evaluation of back-crossed isogenic lines may give
also more precise information.

2.3 Media Improvements for Increased Yields of Regenerated Plants

During the androgenetic process it is normal to have macroscopic embryo

development before transferring embryos onto a medium containing 2,4-0,
which induces secondary callogenesis and regeneration (Petolino 1990). An early
trans-fer ofanthers, before macroscopic embryo formation, has been reported as
improving embryo regeneration within other species (Chambonnet 1985). This

Table I. General protocol and medium for maize anther culture

Step I Grow donor plants as well as possible

Step 2 Pick tassels just prior emergence from the whorl
Step 3 Control pollen developmental stage and choose right parts (uni- to early binucleate)
(Alexander scanning procedure)
Step 4 Cool treat at 7 DC for 7 days, flower wrapped in aluminum foil (pay special attention to
humidity)
Step 5 Plate anthers on G medium (25 anthers per 55 mm <p Petri dishes)
Step 6 Cool treat at 14 DC for 7 days (dark): avoid condensation in Petri dishes
Step 7 Then 28 DC for 3 weeks at low light intensity 13 to 22jlE/m 2/s (cool white spectrum)
Step 8 Transfer anthers on F medium for regeneration, 28 DC, 13 to 22jlE/m 2/s, 16 h 0/8 h N
Step 9 Transfer plantlets on P medium for leaf and root development 24/18 DC 16 h1day/night 8 h
175jlE/m 2/s (cool wl)ite spectrum)
Step IO Readapt regenerated plantlets with high relative humidity (90%) to soil and transfer to
greenhouse
The Use of in Vitro/in Situ Produced DH Maize Plants 207

Number of
Percentage of plant lets for
androgenetic
100 anthers (0)
anthers (*)

80 200

o
60 150

o
40
100

o
20 50

T A B

Fig. 4. Increase of regeneration efficiency with early transfer on low sugar-containing medium

Fig. 5. Androgenetic embryos with DH5 x DH7

208 M. Beckert

Table 2. Composition of culture media

MEDIA

G E P

Major elements (mg/I)

KNO J 2500 2500 2500
NH.NO J 165 165 165
KHlPO. 510 510 510
MgSO.,7HP 370 370 370
CaCI 2.2HP 176 176 176
Minor elements (mg/I)
NalEDTA 37.3 37.3 18.6
Fe SO•• 7Hp 27.8 27.8 13.9
MnSO•• 1HP 22.3 3.5 3.5
Zn SO•• 7Hp 8.6 1.5 1.5
HJBO J 6.2 1.6 1.6
KI 0.83 0.8 0.8
Na MoO•• 2Hp 0.25
Cu SO•• 5HP 0.0025
CoCl l .6HP 0.0025
Organic components (mg/I)
Glycine 7.70
Nicotinic acid 1.30 1.30 1.30
Thiamine HCI 0.25 0.25 0.25
Pyridoxine HCI 0.25
D-Pantothenic acid 0.25
Casein hydrolysate 500 500 500
Succinic acid 25 25
Inositol 100 100
Hormones (mg/I)
T.I.B.A. 0.1
Kinetin I
pH (before autoclaving) 5.8 5.8 5.8
Gelrite (mg/I) 2500 2500 2500
Sucrose (mg/I) 120000 25000 20000
Activated charcoal (mg/I) 5000

technique was tested with the maize androgenetic anthers for plantlet formation
Fig. 4 shows the comparative results of a classical treatment (T), with a second
treatment (A) where the anthers were transferred 3 weeks after plating and a third
treatment (B) where the anthers were transferred 4 weeks after plating. The
anthers were transferred from medium G to medium E (Tables I and 2). The DH5
x 0 H7 hybrid showed a slight decrease in the percentage ofandrogenetic anthers
produced and a dramatic increase in the number of regenerated plantlets per 100
anthers plated (Barloy and Beckert 1993). Similar results were obtained using
other genotypes (see Figs. 5 and 6).
Using this procedure, we can currently accelerate plantlet regeneration
without lowering the percentage of spontaneous chromosome doubling or
damaging plantlet development. By not trying to induce callus formation and
secondary embryogenesis, it is possible to avoid somaclonal variation, thus
 The Use of in Vitrolin Situ Produced DH Maize Plants 209

Fig. 6. Increase of regeneration using early transfer

Nurrber of DH lines.
9

CHARACTERS
8
- Number olleav8S,
7 -Height 01 plants,
-Height 01 ears,
-Male t1owering,
6 -Number of branches per tassle.

I I
-
F2 F546 A.F.D. on intra-
variance (first axe).

Fig. 7. Homogeneity of DH progenies

210 M. Beckert

saving time and giving a better picture of the genetic potential of a cross
(Murigneux et al. 1993).

2.4 Homogeneity of Progeny ofDH Androgenetic Plants

A high level of homogeneity of OH progeny is required for lines to be released

after breeding evaluation. Homogeneity was assessed after two generations of
selfing in order to suppress effects connected with seed quality. The study involved
the characterization of intra-variances between different OH lines for certain
agromorphological traits such as: plant height, the number ofleaves, the number
of tassel branches, etc.
Figure 7 is a diagramatic representation of all the lines analyzed. A factorial
discriminant analysis was carried out to testify the intra-variances between the
OH lines and the test lines (F 2 and F 546). The test lines were respectively 20 and
10 selfing generations after line release (Murigneux etal. 1993a).
This study clearly demonstrates that the level of homogeneity of OH
progeny is similar to that of lines derived by classical selection.

3 Comparison of the 'in Situ' and 'in Vitro' Systems

of Haploid Production

Maternal haploidy was developed by Coe (1957), as he discovered the Stock 6

strain which was used as a male pollinator. In contrast, the W 23 line carrying the
ig (indeterminate gametophyte) mutation was used as a female parent, which
developed androgenetic embryos. These embryos arose from the failure of
fertilization and the development of a reproductive male sperm cell (Kermicle
1969).
The detection of haploid plantlets was based upon dominant markers
leading to an accumulation of anthocyan pigments in inducing lines at the
embryo level, as well as at the aleurone level of the hybrid kernels. Using this kind
of markers, spontaneous haploids were selected (Chase 1974) and OH lines were
obtained and included in hybrids; but during various attempts at using these
markers on a larger scale, it became clear that their expression was too much
influenced by the genetic context of the complementary parent or by the general
seed maturation process. This could lead to an incorrect evaluation of plantlet
ploidy and a large number of presumptive haploid plants must be greenhouse-
grown.
In recent years, considerable work has been carried out by our group using a
specific detection method in order to evaluate genetic and environmental factors
which lead to a higher frequency of haploid induction. It has been clearly
demonstrated that the frequency of gynogenetic haploid induction is genetically
controlled and is heritable (Lashermes and Beckert 1988). A new line WS 14 was
derived through selection for its high frequency of gynogenetic induction when
compared with the Stock 6 line (Fig. 8).
The Use of in Vitro/in Situ Produced DH Maize Plants 211

6.--------------------,

25
IConfidence interval at 5 % level

1 I
I
c
1
0
co.
~:§4
_ 0-
jJ
l 1
00
0-.<=
c -
~ E3 •
1
U QI
:;>-
0
-0
T

I
c E
'Io
~Q.l
2 I
Fig. 8. Comparison of

I
0=
-0 - _
0
haploidy-induction potential of o c
stock 6 (0) and WS 14 (e) :z: ~ 1
used as male parent in different
QI
0-
{ ~
crosses 0

.....
....<>: ""
.
5
.
~
""
V")
CD
L;: '-'
<>: CD
~ ~
CD ....
<>:
5....
""
~
a::>
L;:
CD
~ ~
u-
Female parent genotype

Some agromorphological characters of the gynogenetic haploid lines were

analyzed and showed a high level of homogeneity and stability. For all of the
characters analyzed the levels of homogeneity were comparable with normal
selection-derived lines (Lashermes 1987).
Another comparison was drawn between SSD, F6, and DH lines to evaluate
any selection against certain characters due to the method itself. Using molecular
(isozymes) and morphological markers, DH lines and SSD F6 appeared very
similar in mean dispersion and marker frequencies (Lashermes et al. 1988).

4 Conclusion

It is difficult to compare the in vitro and in situ androgenesis systems, as they are
very different. Table 3 gives some general comparisons for in vitro versus in situ

Table 3. General comparison of in situ and in vitro systems of production of DH lines in maize

Parameters In situ gynogenesis In vitro androgenesis

Genotypic specificity Low Strong

Screening procedure Necessary Not necessary
Average production Low Low
Highest production 12 Haploids per cross I Haploid per plated anther
Spontaneous chromosome doubling Very low: 1% 20 to 30%
Seed set Bad Bad to good
DH homogeneity Good Good
DH stability Good Good
SSDIDH similarity Good Good
DH production cost Low Intermediate
212 M. Beckert

DH systems. The in situ system has advantages because of the nongenetic

specificity of the material from which we expect to generate haploid plants. We
think that genetic transformation of an inducing line with a specific marker
having strong expression at early development plantlet or embryo stage may
solve the problem of screening procedure in an elegant and reliable manner. In
situ androgenesis remains the choice method for cytoplasm transfer.
Nevertheless, the very high spontaneous chromosome doubling frequency
and the high seed set level in regenerated plants are important aspects of the in
vitro system. We may be able to localize or identify genes in the near future which
are involved in this pathway and transfer them into elite material, or, in a more
prospective and hypothetical manner, to induce nonresponding material with
products of these genes. With this in vitro technique, breeders may find some
interesting tools and the answers to many technical and genetical questions.

Acknowledgments. This research work was supported in part by LimagrainiBiocem and Rhone-
Poulenc Agrochimie. This study would not have been possible without the expert technical assistance
of J. Tucci. Dr. D. Barloy, Dr. P. Dieu, Dr. Ph. Lashermes and Dr. P. Vergne provided invaluable
contributions to this research.

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