Plant Science 160 2000 15 – 26
Analysis of habituated embryogenic lines in Asparagus officinalis L.: growth characteristics, hormone content and ploidy level of
calli and regenerated plants
Anne Limanton-Grevet
a,b,c
, Bruno Sotta
d
, Spencer Brown
e
, Marc Jullien
a,
a
UMR INRAINA P-G Biologie des Semences, INRA Versailles, Route de Saint-Cyr,
78026
Versailles Cedex, France
b
Laboratoire
‘
in 6itro
’
, J. Marionnet GFA, Route de Courmemin,
41230
Soings-en-Sologne, France
c
Asparagus b6, Postbus
6219
,
5960
AE Horst, France
d
Laboratoire de Physiologie du De´6eloppement des Plantes, UMR de Physiologie Cellulaire et Mole´culaire des Plantes, Uni6ersite´ Pierre et Marie Curie
P VI , Tour
53
,
4
place Jussieu,
75252
Paris Cedex
05
, France
e
Cytome´trie, Institut des Sciences Ve´ge´tales, CNRS,
91198
Gif-sur-Y6ette, France Received 23 March 2000; received in revised form 26 July 2000; accepted 31 July 2000
Abstract
Habituated asparagus embryogenic lines derived from eleven genotypes were maintained on hormone-free medium and grew actively through secondary embryogenesis. Secondary embryos were of single cell origin and emerged from the transversal division
of some epidermal or subepidermal cotyledonary cells of primary embryos. The intensity of secondary embryogenesis was found to be variable between embryogenic lines. Plants regenerated from three of these lines have been previously demonstrated to carry
a mutation whose phenotype was the direct appearance of somatic embryos on apices or nodes cultured on hormone-free medium. Habituated lines of embryogenic calli and various tissues of embryogenic mutant and wild type plants were analysed for their
hormonal content in ABA, IAA, iP, Z and their metabolites ABA-GE, iPA, iMP, ZR. No significant difference was found between different embryogenic lines, except the level of iPA, or between cladophyll or apex cultures of mutant and wild type
plants. Flow cytometry analyses indicated only 34 of the embryogenic lines were diploid, most of the others being tetraploid, but 62 of regenerated plants from these lines were diploid. This indicated the process of maturation and conversion selected
diploid embryos in the embryogenic lines. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Asparagus officinalis L.; Somatic embryogenesis; Habituation; Mutation; Hormone; Flow cytometry; Ploidy level www.elsevier.comlocateplantsci
1. Introduction
Asparagus has a low multiplication rate using conventional methods [1,2]. In vitro clonal propa-
gation using shoot multiplication from cultured meristems and shoot tips leads to higher efficiency
but is very labour intensive [3]. Somatic embryos could be the most efficient method for clonal
micropropagation of plants [4] and thus could also be useful for asexual multiplication of asparagus.
Different procedures for somatic embryogenesis have been described in Asparagus officinalis from
different types of explants such as hypocotyls [5], terminal buds [6 – 8], stems [9,10], crowns of
seedlings [10], cladophylls [11] and mesophyll cell culture [12]. In most cases, embryogenic calli were
induced and maintained on a medium containing growth regulators, especially auxins and cy-
tokinins. Then, they had to be transferred on a medium with reduced hormone concentration to
express their embryogenic potential, allowing em- bryo development and plant conversion. Jullien
Corresponding author.
Tel.: +
33-1-3083-3074 + 33-1-4408- 1831; fax: + 33-1-3083-3099.
E-mail address
:
jullienversailles.inra.fr M. Jullien. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 5 6 - 3
[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
