Beruflich Dokumente
Kultur Dokumente
233
2001 Kluwer Academic Publishers. Printed in the Netherlands.
Key words: Cotton (Gossypium hirsutum) fibre, Ovule culture, Plant growth regulators, Secondary cell wall
Abstract
The effect of plant growth regulators on the secondary wall thickening of cotton fibre was studied. The results
indicated that the GA S and iP + iPA levels in the fibre of field-grown cotton plants remained almost constant but
the IAA and ABA levels changed considerably during fibre development. Although the change in both IAA and
ABA levels seemed not to be closely related with the rate of cellulose accumulation, there was still a relationship
between the ratio of ABA to IAA and secondary wall thickening. In in vitro studies, ABA (50 molL 1) mark-
edly enhanced the accumulation of dry matter and cellulose in the fibre cell wall during secondary wall thick-
ening, but no similar effect was observed with NAA, GA 3 or kinetin treatments. The role of ABA in secondary
wall thickening of cotton fibre is discussed.
Abbreviations: ABA abscisic acid, DPA days post anthesis, DW dry weight, ELISA enzyme-linked im-
munosorbent assay, FW fresh weight, GA 3 gibberellic acid, GA S gibberellins, IAA indoleacetic acid,
iP(A) isopentenyladen(os)ine, NAA naphthalene acetic acid, SD standard deviation
ening and ABA had an adverse effect (Kosmidou- treatment were examined and ten seeds from every
Dimitropoulou 1986). However, some investigators flask were selected at random. The seeds were washed
reported that GA 3 and IAA had no effect on cellulose several times with distilled water, then the fibres were
synthesis, kinetin inhibited it slightly, but ABA was manually removed from the seeds. The fibres were
stimulatory (Francey et al. 1989; Jaquet et al. 1982; weighed after being oven-dried to a constant weight
Pillonel and Meier 1985). It is important to obtain at 70 C.The weight of fibres was expressed in terms
new information on the roles of plant growth regula- of dry weight per seed. Each data point represented
tors on cellulose synthesis and secondary wall thick- the mean SD.
ening. Hence the present study investigated the levels The cellulose content of the fibres was determined
of endogenous IAA, GA S, ABA and iP + iPA in the as described by Updegraff (1969).
developing fibres of field-grown cotton plants and The extraction, purification and determination of
also explored the effect of NAA, GA 3, kinetin and endogenous levels of IAA, GA S, ABA and iP + iPA
ABA on secondary wall thickening of the cultured fi- by an indirect ELISA technique were performed as
bre. described by He (1993). The samples were homoge-
nised in liquid nitrogen and extracted in cold 80%
(v/v) methanol with butylated hydroxytoluene (1
Materials and methods mmolL 1) overnight at 4 C. The extracts were col-
lected after centrifugation at 10000 g (4 C) for 20
Gossypium hirsutum L.cv Zhongmiansuo-12 plants min, the extracts were passed through a C 18 Sep-Pak
were grown in the field in Beijing, China. The cul- catridge (Waters, Milford, MA) and dried in N 2. The
tural practices, including irrigation and application of residues were dissolved in PBS (0.01 molL 1, pH
fertilisers and insecticides, were conducted on the 7.4) in order to determine the levels of IAA, GA S,
usual basis. On the day of anthesis, individual flow- ABA and iP + iPA. Microtitration plates (Nunc) were
ers were tagged, and healthy bolls were harvested for coated with synthetic IAA, GA S, iP + iPA or ABA-
the analysis of cellulose content, as well as the levels ovalbumin conjugates in NaHCO 3 buffer (50
of IAA, GA S, ABA and iP + iPA in the fibres. To min- mmolL 1,pH 9.6) and left overnight at 37 C. Oval-
imise the effect of environmental variations, data bumin solution (10 mg/ml) was added to each well in
were collected from flowers that had bloomed during order to block nonspecific binding. After incubation
as narrow a period as possible. for 30 min at 37 C, standard IAA, GA S, ABA,
Freshly harvested bolls were opened with a scapel. iP + iPA, samples and antibodies were added and in-
Intact ovules were taken at 2 DPA. At 9 DPA and cubated for a further 45 min at 37 C. The antibodies
subsequently, the fibres were separated manually against IAA, GA S, ABA and iP + iPA were obtained
from the ovule. Both freshly-separated fibres and in- as described by Weiler et al. (1981). Then horserad-
tact ovules were weighed and then frozen in liquid ish peroxidase-labelled goat antirabbit immunoglobu-
nitrogen and stored at 20 C for later analysis. lin was added to each well and incubated for 1 h at
For in vitro studies, fertilised ovules at 2 DPA were 37 C. Finally, the buffered enzyme substrate (ortho-
excised from bolls collected when the wilting corolla phenylenediamino) was added, and the enzyme reac-
became dried-up and the stigma began turning brown. tion was carried out in the dark at 37 C for 15 min,
A set of 20 ovules derived from the same boll were then terminated using 3 molL 1 H 2SO 4. The absor-
cultured in a modified medium of Beasley and Ting bance was recorded at 490 nm. Calculations of the
(1973), which contained the basal medium, but su- enzyme-immunoassay data were performed as de-
crose was used instead of fructose. It was also sup- scribed by Weiler et al. (1981). In this study the per-
plemented with 5 molL 1 NAA and 1 molL 1 centage recovery of each hormone was calculated by
GA 3. Ovules were cultured for 14 days and, at 16 adding known amounts of standard hormone to a split
DPA, were transferred to a subculture medium modi- extract. Percentage recoveries were all above 90%,
fied by adding 5 gL 1 activated charcoal. At 30 DPA, and all sample extract dilution curves paralleled the
ovules were transferred to further subculture media standard curves, indicating the absence of nonspecific
modified by adding one of a series of concentrations inhibitors in the extracts.
of NAA, GA 3, kinetin or ABA instead of 5 molL 1
NAA and 1 molL 1 GA 3. Each treatment consisted
of at least ten flasks. At 44 DPA, six flasks of each
235
Figure 1. Fibre length vs. Boll age. Each data point is the mean of Figure 2. Changes in cellulose content of fibres against boll age.
three replicates. Vertical bars representSD. The curve of the fi- Each data point is the mean of three replicates. Vertical bars
bres grown on cotton plants is defined by representSD. The curve of the fibres grown on cotton plants is
Y= 0.0001X 4 0.0107X 3 + 0.266X 2 0.5563X + 0.0535. The r 2 co- defined by Y=0.0005X 4+0.0351X 30.6045X 2+3.5161X+1.621.
efficient is 0.9991. The curve of the fibres cultured in vitro is de- The r 2 coefficient is 0.9885. The curve of the fibres cultured in vitro
fined by Y= 0.0002X 4 0.0133X 3 + 0.2815X 2 0.5612X 0.0027. is defined by
The r 2 coefficient is 0.9981. Y=0.0002X 4+0.0158X 30.1674X 2+0.8778X+1.1916. The r 2 co-
efficient is 0.9944.
Results
Discussion