Plant Science 160 2001 247 – 257
Hormonal status of maize initial explants and of the embryogenic and non-embryogenic callus cultures derived from them as related
to morphogenesis in vitro
Vı´ctor M. Jime´nez
1
, Fritz Bangerth
Institut fur Obst-, Gemu¨se- und Weinbau
370
, Uni6ersita¨t Hohenheim, D-
70593
Stuttgart, Germany Received 24 February 2000; received in revised form 26 June 2000; accepted 5 September 2000
Abstract
Endogenous hormone levels indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins
1, 3
and
20
[GAs], zeatinzeatin riboside [ZZR] and N
6
[D
2
-isopentenyl] adenineN
6
[D
2
-isopentenyl] adenosine; [iPiPA] were analysed in immature maize zygotic embryos of two maize Zea mays L. genotypes, known for their distinct ability to generate embryogenic E callus. No differences were
found among genotypes in the hormone contents of the embryos. These embryos were also used as initial explants to establish callus cultures. E and non-embryogenic NE calli were obtained from the competent genotype A188, while only NE callus was
produced by the incompetent one B73. The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. When analysing the endogenous hormone levels in the various callus types generated in each
genotype, it was found that only differences in the IAA levels accounted for variations in the morphogenic properties of the calli. Higher levels of endogenous IAA were typical of embryogenic callus cultures. It was also observed, that a loss in the embryogenic
competence of the calli, due to a prolonged time of culture, occurred concomitantly with a reduction in the IAA levels, practically to the levels found in the non-embryogenic calli. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Phytohormones; Radio-immunoassay; Somatic embryogenesis; In vitro culture; Zea mays L. www.elsevier.comlocateplantsci
1. Introduction
Green and Phillips [1] were the first to report the successful regeneration of complete plants from
maize tissue cultures. In all previous attempts only non-competent callus was obtained. Since then,
techniques and culture media adequate for stimu- lating the embryogenic response in this plant have
been improved, as well as the comprehension of the mode of action of several factors that influence
the establishment, growth and differentiation of embryogenic callus cultures reviewed by Bajaj
[2]. Although regenerable maize callus cultures can be initiated from many meristematic tissues of
the plant, immature embryos are the best source of callus with regeneration competence [2].
Different callus types can be induced in maize, i.e. Type I, II and NE callus [3]. Type-I callus is
generally white and compact and seems to be a further advanced differentiation step of Type-II
callus. This latter type is soft, white or pale yellow, friable, can usually retain totipotency after long
periods of time in culture, and is similar to the embryogenic cultures of model plant species such
as Daucus and Nicotiana spp. [4 – 6]. NE callus is translucent, does not show any sign of organisa-
tion and produces only roots when the 2,4-D concentration in the culture medium is reduced [7];
this is the typical callus obtained in non-embryo- genic lines [6].
Corresponding author. Tel.: + 49-711-4592350; fax: + 49-711- 4592351.
E-mail address
:
fkbanguni-hohenheim.de F. Bangerth.
1
Present address: CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica.
0168-945201 - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 8 2 - 4
As in tissue culture of most plant species, in maize, the genotype plays a very important role in
the establishment, growth and subsequent differ- entiation of callus cultures. There are several re-
ports in which differences among genotypes in callus formation rates have been found in this
species [4,8 – 11].
Some attempts have been carried out in certain species to associate the endogenous hormone lev-
els of explants and cultures derived from them with their regeneration competence [12 – 14]. To
the best of our knowledge, the only report in maize is that of Carnes and Wright [15], in which
they investigated endogenous hormones, such as indole-3-acetic acid IAA, abscisic acid ABA,
zeatin Z, zeatin riboside ZR, N
6
[D
2
-isopentenyl] adenine iP and N
6
[D
2
-isopentenyl] adenosine iPA, in immature kernels of genotypes reported
to have different levels of competence. To understand the hormonal regulation during
somatic embryogenesis in maize might permit the application of this developmental process to a
broader spectrum of genotypes, which may be very useful for clonal propagation, multiplication of F1
hybrids and especially genetic transformation in maize [7]. Efficient procedures for obtaining sus-
pension cultures and protoplasts from embryo- genic
callus, which
are capable
of plant
regeneration, enables cell technologies to be ap- plied, along with conventional methods, for speed-
ing up the selection process and making it easier [16].
The aims in this study were to establish embryo- genic and non-embryogenic callus lines from com-
petent and incompetent maize genotypes and to measure and relate endogenous hormone contents
in these lines to their embryogenic competence. Furthermore, the endogenous hormone levels of
the immature zygotic embryos and the endosperm of competent and incompetent genotypes were
compared.
2. Material and methods
