Plant Science 154 2000 143 – 151
Differences in the contents of total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from
Medicago arborea L.
Ana Belen Martin
a
, Yolanda Cuadrado
a
, Hilario Guerra
b
, Piedad Gallego
a
, Oscar Hita
a
, Luisa Martin
d
, Ana Dorado
c
, Nieves Villalobos
a,
a
Departamento de Fisiologı´a Vegetal, Facultad de Biologı´a, Uni6ersidad de Salamanca, A6da del Campo Charro sn
Edificio de la F. de Farmacia , E-
37007
Salamanca, Spain
b
Departamento de Fisiologı´a Vegetal, Facultad de Farmacia, Uni6ersidad de Salamanca, E-
37007
Salamanca, Spain
c
Departamento de Estadistica, Facultad de Economia y Empresa, Uni6ersidad de Salamanca, E-
37007
Salamanca, Spain
d
Laboratorio de Fisiologı´a Vegetal, Facultad de Biologı´a, Uni6ersidad Complutensede Madrid, E-
28040
Madrid, Spain Received 21 May 1999; received in revised form 17 November 1999; accepted 22 November 1999
Abstract
The total sugars, reducing sugars, starch and sucrose in embryogenic and non-embryogenic calli from explants cotyledons, petioles, hypocotyls and leaves obtained from Medicago arborea L. seedlings were evaluated. Total sugars were the major
components in the calli and no significant differences between embryogenic and non-embryogenic calli were observed. In contrast, important differences between the embryogenic and non-embryogenic calli were observed for reducing sugars, the highest levels
being observed in embryogenic calli. The highest starch levels were found in non-embryogenic calli developed in MS medium. During the development of somatic embryogenesis very low starch levels in the callus were found. During the first months of
culture, no significant differences in the sucrose content were found between calli that produced embryos and those that did not. The most important differences in sucrose were seen between calli transferred to medium F0, which had the greatest embryogenic
capacity, and those transferred to medium F6, which inhibited embryogenesis. In the latter case, an increase in sucrose was observed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Medicago arborea; Total sugars; Reducing sugars; Starch; Sucrose; Embryogenic calli; Non-embryogenic calli www.elsevier.comlocateplantsci
1. Introduction
Medicago arborea L. subsp. arborea is an ever- green shrub that may be considered a forage plant
in dry areas. One of the possible expressions of the totipotent
character of plant cells and tissues is their ability to regenerate into plants via embryogenesis and
for organogenesis. In vitro plant tissue culture normally requires an external source of carbon.
This is usually supplied in the medium as sucrose [1,2]. A part of the sucrose in the medium must
first be degraded extracellularly and the resulting hexoses are then taken up by the cells [3]. Sucrose
is an important hexose storage source and is nor- mally the main carbon source for the synthesis of
polysaccharides, after their hydrolysis by sucrose synthase [4] andor invertase [5]. Sucrose hydroly-
sis, in the cytosol, is carried out by both acid and alkaline soluble invertases [5] to provide, as they
become necessary, the hexoses used as major sub- strates for the synthesis of structural and storage
polysaccharides [6,7].
Abbre6iations
:
2,4-D, 2,4-dichlorophenoxyacetic acid; DW, dry weight; F0, kinetin-free MS medium with 2,4-D 0.5 mg l
− 1
; F6, MS medium supplemented with 2,4-D 0.5 mg l
− 1
and KIN 0.5 mg l
− 1
; H8, MS medium containing 2,4-D 2 mg l
− 1
and KIN 2 mg l
− 1
; KIN, kinetin; MS, medium of Murashige and Skoog 1962. Corresponding author. Tel.: + 34-23-29-4471; fax: + 34-23-29-
4515. E-mail address
:
crtagugu.usal.es N. Villalobos 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 2 5 1 - 4
Different authors have reported the presence of starch in plant cells at the beginning of the in vitro
developmental process [1,8,9]. The possible role of this polysaccharide in these processes remains un-
clear, although it has been suggested that it could act as an energy source or as an essential osmotic
agent for development [10].
Here, we report the variations occurring in con- tent of total sugars, reducing sugars, starch and
sucrose in undifferentiated and embryogenic calli. The calli studied were obtained using four types of
explants from M. arborea L. subsp. arborea seedlings and four different culture media which
induced several responses 1 basal MS medium; 2 F6 medium both induced non-embryogenic
calli; 3 H8 medium and 4 F0 medium both induced embryogenic calli, except when leaves are
used as explants.
In the course of the study of somatic embryoge- nesis, the analysis of metabolic differences between
embryogenic and non-embryogenic calli could be of relevance. We initiate these studies by determin-
ing the total sugars, reducing sugars, starch and sucrose content in calli, with the aim to determine
a possible relationship between sugar contents and somatic embryogenesis.
2. Materials and methods
