[12] and Delbreil et al. [13] described the produc- tion of long-term habituated embryogenic lines H
lines growing through repeated secondary em- bryogenesis on hormone-free medium with a very
high potential for plant multiplication which can be used for genetic transformation of asparagus
[14]. Plants regenerated from H lines exhibited an increased embryogenic capacity compared to the
control plants. For three of these lines, the high embryogenic capacity was transmitted to the
progeny, following a Mendelian pattern, providing evidence for a dominant monogenic mutation that
improved somatic embryogenesis [15,16].
An essential aspect of in vitro plant recovery is the conformity of regenerated plants. In the case
of somatic embryogenesis in asparagus, little at- tention was paid to somaclonal variations with
only a few exceptions concerning variations in ploidy levels [17 – 20]. As synthetic auxins have
often been considered as largely responsible for polyploidisation [21], H lines maintained on hor-
mone-free medium could give a material less sub- ject to ploidy level changes than embryogenic lines
maintained in the presence of auxin, as usually used for asparagus [8 – 10,17 – 20]. The indication
of the mutational origin of H lines cited above was another example of somaclonal variation and
questioned about the function of corresponding genes. Genes implicated in the embryogenic capac-
ity have been identified in alfalfa [22], in maize [23,24] and in Dactylis glomerata [25], but their
function has not been defined. A relation between embryogenic capacity and hormonal metabolism
has been found in alfalfa [26] and wheat [27] where the embryogenic capacity could be regulated by
the ratio of abscisic acidIAA.
The aim of this study was to characterize several H lines including two previously demonstrated
mutant lines for future plant production. Growth modalities of the lines were compared by weight
measurements, sieving of the cultures for embryo size and cytological analysis of growing tissues.
Second, the intensity of secondary embryogenesis of various lines was tentatively related to their
hormonal content and was compared to the hor- monal composition in vegetative tissue and culti-
vated apices of the embryogenic mutant and wild type plants. Third, the ploidy level and its stability
in embryogenic calli and regenerated plants were examined by flow cytometry to define long term
usable H lines.
2. Materials and methods
2
.
1
. Plant material The habituated embryogenic lines H lines used
were derived from eleven genotypes: two female clones CO1 and CO3 and four male clones 8,
186, DDNO5 and JMal provided by J. Marionnet GFA Soings en Sologne, France; two female
clones A1 and A2, two male clones A3 and A4 provided by Asparagus bv Horst, Holland and
81A a F1 hybrid provided by INRA Versailles, France. The H lines derived from these genotypes
are indicated in the text and named as following: for instance A2L2 was the H line n° 2 obtained
from the genotype A2. The H lines 8L1 [15], A3L3 and A4L1 [16] carry a dominant ‘high embryo-
genic’ mutation that enables vegetative tissue to develop somatic embryos when cultivated on hor-
mone free medium.
2
.
2
. Cultures of habituated embryogenic lines Isolation of H lines was described previously
[13,16]. Briefly, shoot apices dissected from adult plants were cultured 1 month on MSN basal
medium containing
naphthaleneacetic acid
NAA 10 mg l
− 1
then were subcultured on basal medium for development of H lines. Basal
medium consisted of Murashige and Skoog macronutrients [28], Nitsch micronutrients [29],
Nitsch and Nitsch vitamins [30], 2 sucrose and 0.7 agar Biomar. The pH was adjusted to 5.7
before autoclaving. Most of the H lines were maintained 2 years by subculturing every month
on basal medium. For subcultures, embryogenic callus 0.1 g containing mainly elongated and
mature embryos coming from a precedent culture were plated on 3MM paper Whatman and cul-
tured on 20 ml basal medium in a Petri dish. For recovery of H lines without an auxin treatment,
shoot apices or nodes of diploid regenerated plants A4L1 and 81AL2 were cultured on basal medium.
All cultures were put in a growth chamber with 16 h per day fluorescent light providing 40 – 70 mmol
m
− 2
s
− 1
at 25°C and 70 relative humidity. To determine the distribution of embryo devel-
opment stages, the embryogenic calli were dissoci- ated in distilled water then sieved on various
meshes 0.2, 0.4, 0.8 and 1.6 mm. The fresh weight of each fraction was measured. The 0.2 – 0.4
mm fraction contained globular embryos, the 0.4 – 0.8 fraction globular and bipolar embryos, the
0.8 – 1.6 mm fraction bipolar elongated embryos and the 1.6 mm and over fraction mature embryos
with a chlorophyllous cotyledon. Four repetitions corresponding to four Petri dishes were made.
2
.
3
. Embryo con6ersion For plant recovery, 0.1 g callus from H lines
was plated in Petri dishes on basal medium con- taining 36 g l
− 1
maltose and solidified with 10 g l
− 1
Phytagel [8]. After 1 month of culture, mature embryos were transferred to glass pots containing
30 ml of germination medium: MSN solidified with 2 g l
− 1
Phytagel. Later 1 month, plantlets were transferred to test tubes containing 20 ml of
the same medium where they developed during 2 months. Plantlets were then transferred to the
greenhouse.
2
.
4
. Histological analysis Embryos were fixed overnight in a 0.2 glu-
taraldehyde, 0.4 formaldehyde solution, rinsed three times in water and dehydrated in successive
ethanol solutions from 10 to 100. They were embedded in Technovit 7100 and cut at 3 – 4 mm.
Sections were stained with toluidine blue.
Scanning electron microscopy was carried out using a Phillips 625M microscope. Samples were
fixed by cryodesiccation in a Cryostans System CT 1500 or dehydrated by critical point method after
glutaraldehyde fixation and ethanol dehydration.
2
.
5
. Hormone analysis
2
.
5
.
1
. Plant material All plants and calli used for hormone analyses
were diploid. The hormonal content of 8 H lines 1 year old was analysed in callus samples collected 2
weeks after subculture. Plants regenerated from the mutant H line A4L1 and wild type plants from
the clone A4 were compared for their hormonal content in different tissues. Cladophylls of mutant
and wild type plants were collected on stems at the end of growth. One to seven mutant and wild type
plants were analysed. Buds of young spears 15 – 20 cm high collected on mutant or wild type
plants 1 year old, grown in greenhouses, were dissected and cultured on basal medium for 0, 7,
14 or 25 days. Samples
were frozen
in liquid
nitrogen, lyophilised and stored 1 month at room tempera-
ture in a desiccator. Before extraction each sample was ground with a ball mill.
2
.
5
.
2
. Extraction, purification and fractionation Extractions were performed at 4°C in darkness
for 60 h from about 40 mg of tissue powder in 5 ml of 80 aqueous methanol supplemented with
BHT butylhydroxytoluen 40 mg l
− 1
as antioxi- dant.
3
H-ABA and
3
H-IAA were added to the extracts to measure extraction efficiency. A prefil-
ter 0.2 mm connected to a Sep-Pak cartridge was equilibrated with 10 ml of 80 aqueous methanol
before sample loading. Eluates were reduced by rotary evaporation and taken up with 0.2 formic
acid up to 500 ml and injected into a C18 Macheray-Nagel liquid chromatography HPLC
column. Elution was performed at 0.8 ml min
− 1
with a HPLC System gold, Beckman with a 0.2 formic acidmethanol gradient. Retention time of
ABA abscisic acid, ABA-GE abscisic acid glu- cose ester, IAA indoleacetic acid, iP isopen-
tenyladenine,
iMP isopentenyladenosine
monophosphate, iPA isopentenyladenosine, Z zeatin and ZR zeatin-9-riboside were deter-
mined by separate injection of pure compounds Sigma as standards. A total of 40 fractions of 0.8
ml were collected. They were evaporated to dry- ness in a speed-vac concentrator, methylated with
250 ml of diazomethane in ether, evaporated again to dryness and finally taken up with 1.5 ml dis-
tilled water with 0.2 g l
− 1
NaN
3
as preservative. Aliquots 50 ml of fractions were submitted to
scintillation counting in order to determine ABA and IAA recovery, or to enzyme-linked immuno-
sorbent assay ELISA.
2
.
5
.
3
. ELISA procedure The whole procedure was described by Julliard
et al. [31]. ABA and ABA-GE were measured using anti-ABA monoclonal antibodies LPDP
229, IAA using anti-IAA polyclonal antibodies LPDP 47, iP, iPA and iMP using anti-iPA poly-
clonal antibodies LPDP 5, and ZR and Z using anti-ZR polyclonal antibodies LPDP 17. Microt-
itration plates were coated with ABA, IAA, iPA or ZR conjugated to ovalbumin. After washing
the plates five times, 50 ml of the fractions or 50 ml of solutions containing different concentrations of
methylated ABA, IAA, iPA, or ZR standard were
added followed by 50 ml of anti-hormone antibody solution. Plates were incubated 2 h at 4°C in
darkness. After washing, anti-hormone antibodies bound to the plates were quantified by means of
an anti-mouse antibody for ABA Sigma and anti-rabbit antibodies for IAA, iPA and ZR linked
to a peroxidase system Sigma. The peroxidase substrate ABTS: 2,2-azino-bis 3-ethylbenzthaz-
oline-6-sulfonic acid, diluted in a perborate buffer, was added and optical density was mea-
sured at 405 nm. The measures were repeated 5 times for each sample.
2
.
5
.
4
. Statistical analysis Hormone levels in H lines and in explants from
embryogenic and wild type plants were analysed using a Fisher test at P = 0.05 or a Student test at
P = 0.05.
2
.
6
. Ploidy le6el analysis through flow cytometry Ploidy level analyses were conducted with H
lines calli and regenerated plants. Callus samples were generally taken from H lines 1 month after
subculture, corresponding to the end of the growth phase. For asparagus plants, aerial parts including
stem fragments and cladophylls were used. About 0.1 g of fresh matter was chopped with a razor
blade in 600 ml Galbraith buffer [32] containing 0.5
Triton X-100
and 0.01
M sodium
metabisulfite, sieved through a 30 mm filter. RNase was added to 10 mg ml
− 1
and BET to 50 mg ml
− 1
. Plants from the eight genotypes were used as
controls, and tomato plants Lycopersicon esculen- tum as a reference. For each sample, 5000 nuclei
were analysed on a cytometer EPICS V from Coultronics France with an Argon laser 400 mW,
514 nm [33]. Samples were considered as diploid when a peak of 2C nuclei was observed and as
tetraploid when the peak corresponding to 2C nuclei was absent and a peak of 4C nuclei was
observed.
3. Results
