Full text of DOI:10.1007/s003440000020

J Plant Growth Regul (2000) 19:423–428
DOI: 10.1007/s003440000020
© 2000 Springer-Verlag
Starch Accumulation Is Associated
with Adventitious Root Formation
in Hypocotyl Cuttings of
Pinus radiata
Mingshan Li and David W. M. Leung*
Department of Plant & Microbial Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand
ABSTRACT
This study of the internal physiology of adventitious
root formation in Pinus radiata was performed with-
out the potential complications from microbial con-
tamination and nutritional stress. Hypocotyl cut-
tings of radiata pine were cultured in half strength
MS nutrient medium supplemented with IBA (in-
dole-3-butyric acid), IBA + kinetin, kinetin, or with-
out phytohormones (control). Averages of 8.35 and
0.08 roots per cutting were formed in IBA and in
growth regulator–free treatments, respectively. No
roots were formed in IBA + kinetin, kinetin, or su-
crose-free treatments at day 30 after excision of hy-
pocotyls. Changes in fresh weight, sugar, and starch
content were measured at established developmen-
tal stages associated with adventitious root forma-
tion. Sucrose-supplemented medium was required
for higher levels of sugar, starch, and root formation
in rooting region of IBA-treated hypocotyls. Starch
accumulation, in particular, seems to have potential
as a biochemical marker before root primordium
emergence in light of the following observations in
the IBA treatment. Starch began to build up prefer-
entially in cells involved in or in close proximity to
potential sites of new root primordium formation
(that is, the cells on the inside of the cortex and the
pith) before any visible organized root primordia
and then began to disappear during root primor-
dium formation. The substantial starch accumula-
tion associated with IBA treatment was not observed
in the IBA + kinetin, kinetin alone, growth regula-
tor–free, sucrose-free treatments (nonrooting treat-
ments) or in the nonrooting region of hypocotyls
treated with IBA.
Key words: Adventitious root formation; IBA (in-
dole-3-butyric acid); Starch; Sucrose; Sugar; Pinus
radiata
importance in several southern-hemisphere coun-
tries (Smith and Thorpe 1975a), can be induced
with varying plant tissues, including hypocotyls
(Smith and Thorpe 1975b), mature shoots (Thulin
and Faulds 1968), micropropagated shoots from em-
bryos (Aitken-Christie and others 1985; Horgan and
Aitken 1981; Reilly and Washer 1977), seedling
shoot tips (Horgan and Aitken 1981), and mature
INTRODUCTION
Adventitious root formation in radiata pine (Pinus
radiata D. Don), a species of considerable economic
Received 21 December 1999; accepted 21 April 2000; online publication 15
September 2000
*Corresponding author; e-mail: d.leung@botn.canterbury.ac.nz
423

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Li and Leung
shoots (Horgan and Holland 1989). In New Zealand,
commercial clonal production of radiata pine plant-
lets relies on the capacity of seedling shoot-derived
clones to form adventitious roots under tissue cul-
ture conditions (Gleed and others 1995). However,
many clones are difficult to root, thus limiting the
potential development of otherwise desirable clones.
A better understanding of the internal physiology of
the rooting process and knowledge of potential bio-
chemical markers of this process in radiata pine
would be advantageous for further development of
clonal forestry based on radiata pine shoot cuttings.
Research on other species suggests that biochemical
changes, such as protein, enzyme, and carbohydrate
changes, are involved in the rooting process (Bhat-
tacharya 1988; De Klerk 1996; Gonza´lez and others
1991; Haissig 1986; Hand 1994; Jarvis 1986). To
date reports on radiata pine in this area, however,
are lacking.
Development of the present experimental system
for biochemical investigations into adventitious root
initiation in radiata pine was based on two factors.
First, many past studies were performed under non-
aseptic conditions, making it impossible to rule out
interference from microbial metabolism. Second, to
avoid studying root formation in starving or senes-
cencing tissue, a basal nutrient medium was consid-
ered appropriate. The choice of plant material was
based on a previous study showing that hypocotyls
of derooted radiata pine seedlings formed adventi-
tious roots in response to IBA (Smith and Thorpe
1975b). This is a simple and convenient method for
providing large amounts of experimental materials
compared with rooting from in vitro clonal shoot cul-
ture. This study examined changes in sugar and
starch levels of derooted hypocotyls of radiata pine
after treatment with IBA (indole-3-butyric acid, an
inducer of adventitious root formation), or kinetin
(counteracts IBA action).
maintained in a warm dark room (26°C) until seed-
lings emerged before transfer to a plant growth
room at 22°C with continuous lighting at 80 µE и
m-2 и s-1.
Uniform seedlings with hypocotyls about 1 mm in
diameter and 2.5–3 cm long were selected to prepare
cuttings. The original roots and part of the hypoco-
tyls of the seedlings were aseptically removed at 2.5
cm below the cotyledonary nodes and were dis-
carded. Then seven derooted seedling cuttings were
transferred to one tissue culture jar (250 mL, clear
polycarbonate plastic container from Labserv, Bio-
lab, New Zealand) and were cultured upright with
cut-ends of the hypocotyls inserted into 0.5–1 cm of
a basal medium comprised of half strength mineral
salts and vitamins as described by Murashige and
Skoog (1962) and 2 % (w/v) sucrose. This was
supplemented with 44.3 µM IBA, 46.5 µM kinetin, a
combination of these two growth regulators, or no
phytohormone. All media were set at pH 5.8, solidi-
fied with 0.8% (w/v) agar, and then autoclaved.
The lowermost 0.5 cm of hypocotyl tissue cut-
tings were harvested for all analyses because adven-
titious roots formed in this region. Fresh weight de-
termination involved at least 100 segments from
each treatment.
Determination of Carbohydrates
The methods of McCready and others (1950) and
Jermyn (1975) were used for sugar and starch ex-
traction and determination.
Starch Localization
Free hand-sections were stained for starch with an
I-KI solution (Gates and Simpson 1968).
RESULTS AND DISCUSSION
Rooting Response
MATERIALS AND METHODS
First, we examined different basal media, including
MS, 1/2 MS, 1/5 MS, GD (Gresshoff and Doy 1972)
modified by Reilly and Washer (1977), RIM (Ran-
cillac and others 1982), different IBA concentrations
and treatment times, and different sucrose concen-
trations to establish a reliable in vitro adventitious
rooting system. The best combination for root for-
mation was 1/2 MS nutrient medium with 0.8%
agar, 2% sucrose, supplemented with 44.3 µM IBA
for 10 days. Average number of roots per cutting
(mean root number of the total rooted and non-
rooted cuttings) and percentage of rooted cuttings
from this treatment compared with the same me-
dium without IBA were 8.35 vs. 0.08 and 95% vs.
Rooting Treatments under in vitro Condition
Seeds used in all experiments were collected in 1995
from the same population of open-pollinated Pinus
radiata D. Don trees grown in Canterbury, New
Zealand. Seeds were surface-sterilized in 70% (v/v)
ethanol for 30 s, rinsed briefly in sterile water, then
soaked in 50% (v/v) of a commercial bleach (con-
taining 31.5 g/L active sodium hypochlorite) for 30
min before being rinsed thoroughly with sterile wa-
ter. The sterilized seeds were sown in autoclaved
vermiculite in tissue culture jars and stratified in a
cold room (4°C) for 1 week. The jars were then

Starch and Adventitious Root Formation
425
Figure 1. Fresh weight changes in the rooting region of
Pinus radiata hypocotyls during the rooting process. Each
data point is the mean weight of at least 100 segments (the
lowermost 0.5 cm of the cuttings) from each treatment.
All treatments had media supplemented with 2% (w/v)
sucrose.
Figure 2. Changes in content in the rooting region of
Pinus radiata hypocotyls during the rooting process. Treat-
ments are IBA and/or kinetin (kin) and growth regulator–
free control (CT) with (+) or without (−) sucrose (suc).
Vertical bars represent mean ± SE value for three deter-
minations from three individual extracts. Where no bars
are shown, SE values are smaller than the legend symbols.
8% at day 30 after excision of the hypocotyls, re-
spectively. Root formation was not observed in the
medium supplemented with IBA + kinetin (callus
was formed instead), kinetin alone, or IBA minus
sucrose.
Light microscopic observation of developmental
changes (data not shown) indicated that the first cell
division, appearance of root primordium initials (a
cluster of meristematic cells), well-organized root
primordia (dome-shaped with organized root caps),
and root emergence occurred at days 4, 7, 10, and
13, respectively during in vitro culture of cuttings (Li
and Leung, unpublished observation). Results were
similar to those from experiments performed under
nonaseptic conditions (Smith and Thorpe 1975a).
Samples were harvested with reference to the his-
tologic observations.
tious root formation because a similar increase was
also observed in the nonroot-inducing treatment
(IBA + kinetin).
Free sugar levels dramatically increased during
the first day in all treatments, except in those with
sucrose-free medium, reaching different levels but
remaining relatively high throughout the experi-
ment (Figure 2). This initial increase and mainte-
nance of higher levels of free sugar in different treat-
ments after culturing is likely dependent on sucrose
rather than the type of plant growth regulator in the
media (Figure 2). However, it appears that sugar
content was higher in IBA treatment than in other
treatments throughout the process, suggesting that
free sugar content might be associated with root for-
mation. This is consistent with the findings of many
researchers (see review by Haissig 1974).
Fresh Weight and Soluble Sugar Changes
Changes in Starch
The fresh weight of the basal hypocotyl region in-
creased very little in kinetin and control treatments
throughout the duration of the experiment. In IBA
and IBA + kinetin treatments, fresh weight in-
creased slightly during the first 7 days of culture and
then increased sharply (Figure 1). This increase was
probably IBA-induced but not related to adventi-
Interestingly, starch content was very low at day 0
but increased sharply during the first day. After that
starch content remained almost constant in all treat-
ments except in IBA + sucrose (Figure 3). Under this
root-inducing condition, the increase in starch con-
tent continued until day 4, then starch remained

426
Li and Leung
Figure 4. Changes in starch content in the nonrooting
region of Pinus radiata hypocotyls during the rooting pro-
cess. Vertical bars represent mean ± SE value for three
determinations from three individual extracts. Where no
bars are shown, SE values are smaller than the legend
symbols.
Figure 3. Changes in starch content in the rooting re-
gion of Pinus radiata hypocotyls during the rooting pro-
cess. Treatments are IBA and/or kinetin (kin) with (+) or
without (−) sucrose (suc), and controls (CT). Vertical bars
represent mean ± SE value for three determinations from
three individual extracts. Where no bars are shown, SE
values are smaller than the legend symbols. The bars with
different letters are significantly different at p < 0.05. Mul-
tiple comparison test was performed using Tukey’s
method following ANOVA. Statistical analysis was per-
formed using the SPSS for Windows statistical software
package (SPSS Inc., Version 8.0, 1998).
note is that more starch did seem to accumulate in
IBA-treated rooting regions of hypocotyls than in
growth regulator–free treatments, particularly from
day 4 to day 7, even when sucrose was omitted from
the media (Figure 3). This much-reduced starch ac-
cumulation in the IBA minus sucrose treatment was
apparently associated with root primordium forma-
tion (Li and Leung, unpublished histologic observa-
tions) but was not sufficient for root emergence.
These data support the contention that nutrient de-
ficiency, in general, and carbohydrate status, in par-
ticular, should be important considerations in stud-
ies of adventitious root formation.
When cuttings were made, most cells were de-
void of starch, except for some endodermis cells
(Figure 5A). Starch grains were completely absent
from cortical cells. Initially, slight starch accumula-
tion occurred in endodermis and pith cells in hypo-
cotyl cuttings treated with IBA at day 1 (Figure 5B,
arrows). At day 4, starch deposition was more pro-
nounced in hypocotyl cuttings treated with IBA
(Figure 5C) than in nonrooting controls (growth
regulator-free) (Figure 5D), particularly in the pith
and the area around differentiating resin ducts or
so-called inner cortex (Smith and Thorpe 1975a). In
contrast, fewer starch grains were detected in the
cortex (Figures 5C,D).
constant until day 7 before subsequently declining.
This preferential accumulation, at critical times in
the rooting process, of substantial levels of starch in
the rooting region of IBA-treated (no kinetin) hy-
pocotyls and not in the nonroot-inducing treat-
ments suggests that starch accumulation before root
primordium organization could have potential as a
biochemical marker for this differentiation event in
radiata pine. This postulate is consistent with the
conspicuous absence of a predominance of starch
accumulation in the nonroot-forming region of hy-
pocotyls in the IBA treatment (Figure 4). Overall,
starch levels in all treatments were lower in the non-
root-forming than in the root-forming region of ra-
diata pine hypocotyls. It might be that the rooting
region was closer to the externally supplied sucrose.
This and the wider question of the potential sugar
uptake gradient along the hypocotyl require further
investigation.
Higher levels of starch accumulated in the hypo-
cotyls when cultured in sucrose-containing rather
than sucrose-free media (Figure 3). Another point to

Starch and Adventitious Root Formation
427
sig 1986; Ja´sik and De Klerk 1997). Very few starch
grains were observed in the section from the hypo-
cotyl cuttings of nonrooting controls (Figure 5H).
FURTHER DISCUSSION AND CONCLUSION
The close agreement of the temporal and spatial evi-
dence in this study suggests that starch accumula-
tion may be a biochemical marker for early critical
stages of root formation (that is, root initiation and
primordium formation) in hypocotyl cuttings of ra-
diata pine. Further research is required to evaluate
more fully this possibility.
Interestingly, starch accumulation has frequently
been correlated with de novo induction of shoot pri-
mordia (Mangat and others 1990; Thorpe and Mu-
rashige 1968). Furthermore, similar to our study,
the phytohormone-containing shoot induction me-
dia required the addition of sucrose (Burritt and
Leung 1996; Ramage and Leung 1996). It is there-
fore possible that organ initiation and development
might be fundamentally the same with respect to
energy requirements.
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