Annals of Botany 84 : 337–342, 1999
Article No. anbo.1999.0922, available online at http:\\www.idealibrary.com on
The Micronutrient Boron Causes the Development of Adventitious Roots in
Sunflower Cuttings
P. J O S T E N and U. K U T S C H E R A*
FB 19 Pflanzenphysiologie, UniŠersitaW t Kassel, Heinrich-Plett-Str. 40, 34109 Kassel, Germany
Received : 6 November 1998
Returned for revision : 15 February 1999
Accepted : 17 May 1999
Three-day-old light-grown sunflower seedlings were de-rooted and incubated in nutrient solutions that either
contained or lacked boric acid (B). In the absence of B, in the majority of the seedlings, no adventitious roots were
formed. The micronutrient B caused the development of numerous roots in the lower part of the hypocotyl. The effect
of B occurred without the supply of any phytohormones. A dose-response curve of B-induced rooting yielded an
optimum concentration of 0n1 m boric acid. Histological studies revealed that cell divisions occurred in the control
but no root primordia developed. In cuttings that were incubated in B (0n1 m) root primordia were observed that
rapidly developed into well-differentiated adventitious roots. Sunflower cuttings that were planted with their cut end in
vermiculite that was moistened with nutrient solutions without B degenerated after several weeks. In the presence of
B the cuttings formed numerous adventitious roots that entirely replaced the tap root system of intact seedlings. The
rooted cuttings developed into sturdy adult sunflower plants. Our results are discussed with respect to the possible
role of B in the evolution of vascular from prevascular plants.
# 1999 Annals of Botany Company
Key words : Adventitious roots, boron, cuttings, organogenesis, sunflower seedlings.
INTRODUCTION
Seventy-five years ago, Warington (1923) reported that a
continual supply of boron (B) is necessary in very low
concentrations to grow healthy broad bean plants. It is now
well established that B is an essential micronutrient for the
normal growth of all higher plants and nitrogen-fixing
cyanobacteria. However, in spite of decades of intensive
research, the primary function of B in the metabolism of
higher plants is still not known (Marschner, 1995). Boron is
the least well understood of all eight essential mineral
micronutrients, despite the fact that B deficiency symptoms
have been described in detail. B deficiency causes many
anatomical, physiological and biochemical changes ; however, it is difficult to distinguish between primary and
secondary effects (Shorrocks, 1997). In the absence of B,
root growth of intact plants is rapidly inhibited. This
suggests that the micronutrient may be required for the
maintenance of cell division, cell enlargement or both of
these processes. Recent experiments with squash plants and
cultured tobacco cells that were grown in the absence or
presence of boric acid led to the hypothesis that B may play
a role as a structural component of the growing (primary)
cell walls in developing plant tissues (Hu, Brown and
Labavitch, 1996).
Non-woody stem cuttings are suitable systems to study
organogenetic processes such as the formation of adventitious roots. Numerous reports have shown that the
plant hormone auxin has a central role in the initiation and
growth of these organs. In several well-investigated plant
* For correspondence. Fax j49 561804-4009, e-mail Kut!hrz.unikassel.de
0305-7364\99\090337j06 $30.00\0
species, such as Phaseolus Šulgaris or P. aureus, a supply of
B is essential for root development in stem cuttings of lightgrown seedlings (Hemberg, 1951 ; Ali and Jarvis, 1988).
Based on these findings, Jarvis (1986) proposed a model of
adventitious root formation in which B may have a role in
the control of the level of endogenous auxin. This detailed
hypothetical scheme of events that may lead to the formation
of lateral roots is largely based on results obtained with
mung bean cuttings. However, numerous studies with lightgrown de-rooted sunflower seedlings revealed no role of
exogenous B in the initiation of adventitious roots (Fabijan
et al., 1981 a ; Fabijan, Taylor and Reid, 1981 b ; Liu and
Reid, 1992). Since the growth and differentiation of
sunflower seedlings is under continuous investigation in our
laboratory (Kutschera, 1990 ; Jucknischke and Kutschera,
1998) we were interested in whether or not B is required in
the formation of adventitious roots in this important crop
species. In this report we describe a system to study the
exogenous factors that control the formation of adventitious
roots in light-grown seedlings.
MATERIALS AND METHODS
Achenes of sunflower (Helianthus annuus L. ‘ Giganteus ’)
were soaked for 1 h in distilled water prior to sowing in fine
granular vermiculite that was moistened with distilled water
(Kutschera, 1990). The seedlings were raised at 25 mC under
continuous light supplied by warm white fluorescent tubes
at an irradiance of approx. 100 µmol m−# s−" (PAR). Growth
took place in closed plastic trays at 99 % relative humidity.
Cuttings were made from 3- or 4-d-old seedlings of average
size by severing the entire root at the base of the hypocotyl.
# 1999 Annals of Botany Company
Josten and Kutschera—AdŠentitious Roots in Sunflower Cuttings
RESULTS
Effect of boron on the deŠelopment of adŠentitious roots
The experiments described in this section were carried out
with 3-d-old light-grown sunflower seedlings that were
raised in vermiculite moistened with distilled water. Under
these growth conditions hypocotyls were 18–25 mm in
length and elongated at an average rate of approx.
0n7 mm h−" (Kutschera, 1990). After removal of the roots at
the base of the straight hypocotyls, stem cuttings were
placed in Petri dishes that either contained distilled water or
nutrient solution (pB). After a period of 4 d in white light
the cuttings were analysed (Fig. 1). Under all conditions the
hypocotyls were curved ; the stems were significantly longer
than at 3 d after sowing. In the absence of nutrient solution
(H O) the cotyledons were only slightly larger than at the
#
start of the treatment and the hypocotyls had developed
severe necrosis (brown, soft tissues). In complete nutrient
solution (jB) the cotyledons and primary leaves were much
larger than in the water control. Numerous adventitious
roots were present in the basal 5 mm-region of the green,
sturdy hypocotyl. In cuttings that were floated on nutrient
solution that contained no boron (kB), the epicotyl and
primary leaves were less developed than in the presence of
B. The hypocotyls were green and sturdy but no or very few
adventitious roots were detected.
Quantitative data on the effect of B on the initiation of
adventitious roots in cuttings of sunflower seedlings are
F. 1. Representative cuttings from 3-d-old light-grown sunflower
seedlings that were incubated for 4 d in distilled water (A), nutrient
solutionjboric acid (B) and nutrient solution kboric acid (C). Note
that in the presence of boric acid (0n1 m) numerous adventitious roots
are present (B).
12
25
Number
Fm
10
20
8
15
6
10
4
Fresh mass (mg)
The cuttings were either placed into Petri dishes which
contained 50 ml medium (distilled water or nutrient solution) or planted upright with the cut end of the hypocotyl
5 mm deep in moist vermiculite. Cuttings were kept for
1–7 d in continuous white light as described above (25 mC,
99 % relative humidity). The complete nutrient solution
(jB) was composed of : 5 m Ca(NO ), 5 m KNO , 2 m
$
$
MgSO , 1 m KH PO , 22n8 µ FeNaEDTA (12–14 %),
%
# %
18 µ MnCl ;4 H O, 1n6 µ ZnCl , 0n5 µ CuCl ;2 H O,
#
#
#
#
#
0n2 µ Na MoO ;2 H O and 0n1 m H BO . In some
#
%
#
$ $
experiments the concentration of boric acid was variable
(0n001–10 m), in other experiments no H BO was added
$ $
(nutrient solutionkB). The phytohormones indolebutyric
acid (IBA), gibberellic acid (GA ) and 6-furfuryl-amino$
purine (kinetin) were prepared from stock solutions (0n01 )
that contained dimethylsulfoxide (DMSO). The final
solutions contained the corresponding growth hormones
(10 µ), nutrient solution and 0n1 % DMSO (pB). After
1–7 d of incubation, cuttings were removed from the Petri
dishes or vermiculite, washed with distilled water and
blotted dry. Thereafter, the number of adventitious roots
was counted. The fresh mass of entire cuttings and the
severed adventitious roots was measured. For light microscopy, cross sections through the basal 5 mm-region of
the cuttings were fixed, dehydrated and embedded as
described by Kutschera (1990). The sections were stained
with Ruthenium red (100 m).
All experiments were repeated at least six times with new
batches of sunflower seedlings. In Figs 1 and 3–8 representative samples are depicted.
Number of adventitious roots per cutting
338
5
2
0
0
0
0·001 0·01 0·1
1
Boric acid (mM)
10
F. 2. Effect of boric acid on the number ($) and fresh mass of the
adventitious roots (#) of 3-d-old sunflower cuttings that were incubated
for 4 d in nutrient solutions (pboric acid). Data are means of six
independent experiments, with ten cuttings each. Bars represent s.e.m.
shown in Fig. 2. At a concentration of 0n001 m boric acid
a significant enhancement in the number of roots was
observed compared to the control (kB). 0n1 m H BO
$ $
proved an optimal concentration for the initiation of
organogenesis ; at higher concentrations (1k10 m) boric
acid inhibited adventitious root development.
Effect of boron on root initiation
In a previous study it was shown that adventitious roots
in sunflower cuttings originate from primordia that develop
in the region between the vascular tissues of the hypocotyl
(Fabijan et al., 1981 a). However, the role of boron was not
analysed. Specifically, it is not clear whether or not
primordia develop in the absence of B. Therefore, we
analysed cytological changes during the period prior to the
rapid growth of adventitious roots. Figures 3 and 5 show
Josten and Kutschera—AdŠentitious Roots in Sunflower Cuttings
that 2 and 3 d after excision cell divisions were detected in
the interfascicular parenchyma cells of the hypocotyls. In
the absence of B no or very few root primordia were
detected. In cuttings that were incubated for 2 d in the
presence of boric acid (0n1 m), well-developed root
primordia could be observed in the cortex of the hypocotyl
(Fig. 4). By the third day, the juvenile adventitious roots
had protruded through the cortex and the epidermis (Figs 6
and 7). The structure of a representative adventitious root
that had pierced the epidermis is shown in Fig. 7. The root
cap and apical meristem were fully differentiated at this
early stage of organ development. In summary, the results
shown in Fig. 3–7 demonstrate that the development of root
primordia is dependent on the supply of boron.
Effects of phytohormones on root deŠelopment
Numerous studies have shown that the naturally occurring
phytohormone auxin plays a central role in the initiation
and development of adventitious roots in stem cuttings
(Jarvis, 1986). The experiments shown in Figs 1 and 2 were
carried out with nutrient solutions, i.e. in complete absence
of exogenous auxin. Since it is well known that non-woody
stem cuttings are highly responsive to exogenously supplied
auxins and other plant growth substances, the effects of
three phytohormones were analysed. Our objective was to
determine whether these plant hormones, applied at a
concentration that causes an efficient growth response, can
replace the effect of B. The auxin indolebutyric acid was
added to the medium at a concentration of 10 µ. Four days
after the start of the treatment the cuttings were analysed. A
significant thickening of the hypocotyls was observed, but
no stimulation of cell enlargement occurred (data not
shown). These effects were independent of B. In the absence
of B no adventitious roots were observed. In the presence of
B, root development was inhibited by the exogenously
supplied auxin (Table 1). Gibberellic acid (10 µ) caused a
significant enlargement of the hypocotyls and an inhibition
of the B-induced root development. In cuttings that were
incubated for 4 d in the presence of kinetin (10 µ)
cotyledons showed a large increase in size (data not shown).
The growth of adventitious roots was entirely suppressed by
this phytohormone. Taken together, data in Table 1
demonstrate that the micronutrient B can not be replaced
by one of these three phytohormones.
AdŠentitious root deŠelopment and the surŠiŠal of the
cuttings
The experiments described above (Figs 1–7 ; Table 1) were
carried out with 3-d-old sunflower cuttings that were
incubated in Petri dishes. The effect of B on adventitious
root development was analysed under these unnatural
conditions. In order to elucidate the possible ecological role
of the soil micronutrient boron the following experiments
were carried out. Four-d-old light-grown seedlings, raised in
wet vermiculite (kB), were de-rooted. The cuttings were
placed in an upright position 5 mm deep in vermiculite that
was moistened with nutrient solutionpB. Representative
results are shown in Fig. 8. In the control (kB) few or no
339
adventitious roots were observed. The cuttings ceased to
grow and died within 1–2 weeks of the start of the
treatment. In the presence of B numerous adventitious roots
occurred. These rooted cuttings developed into juvenile
plants. To investigate whether these seedlings without a
main root are capable of developing a sturdy stem with
leaves, and finally flowering, individuals such as those
depicted in Figs 1 B and 8 B were planted into vermiculite
that was moistened with a complete nutrient solution (jB).
Under a 12 h white light\12 h dark regime, a relative
humidity of 70 % and a daily photon flux density of
120–150 µmol m−# s−", the cuttings developed into sturdy
adult plants that flowered 3–4 months after transfer into the
new substrate (results not shown). This finding demonstrates
that sunflower cuttings survive in the absence of a main
root ; adventitious roots can replace all the functions of the
tap-root system of intact sunflower plants.
DISCUSSION
Under natural conditions, boron deficiency is a widespread
nutritional disorder, because, as a consequence of heavy
rainfall, boric acid is readily leached from acid soils.
Moreover, availability of B decreases under drought
conditions and as a consequence of a rising alkalization of
the soil (Marschner, 1995). Boron toxicity, on the other
hand, has also been observed, notably in plants growing in
arid and semi-arid regions of the world (Marschner, 1995).
If the availability of B is insufficient, a rapid cessation or
complete inhibition of root growth occurs (Jarvis, 1986). At
B concentrations that are too high, a number of toxicity
symptoms have been described that are not yet well
understood. Hence, a bioassay for a quantitative investigation of B-deficiency and -toxicity would be valuable
(Marschner, 1995).
In the present study light-grown sunflower seedlings were
used. As pointed out by Shorrocks (1997), sunflower is one
of the most responsive and sensitive plant species to B
application. Our results demonstrate that cuttings from deetiolated seedlings raised in the absence of B (i.e. in
vermiculite moistened with distilled water) are a suitable
system for the analysis of this problem. In the presence of all
macro- and micronutrients (pB) the induction and suppression of adventitious roots, which develop exclusively at
the base of the hypocotyl, can be studied experimentally
(Fig. 1). Our dose-response curve (Fig. 2) demonstrates that
at boric acid concentrations in the range 0n01–0n1 m
organ formation reached a maximum ; at 10 m H BO the
$ $
rooting response was entirely inhibited. These effects
occurred in the absence of exogenous phytohormones such
as auxins, i.e. endogenous levels of phytohormones were
obviously sufficient. To investigate the B-dependent processes that lead to the formation of adventitious roots in derooted sunflower seedlings, we analysed histological changes
during the early phase of organogenesis. In the control
(kB), cell divisions were detected in the tissues between the
vascular bundles of the hypocotyl (Figs 3 and 5), but no or
very few root primordia were observed. The organization of
primordia and the subsequent growth and differentiation of
these organs was only detected in the presence of B. We
340
Josten and Kutschera—AdŠentitious Roots in Sunflower Cuttings
3
4
5
6
F. 3–6. For legend see facing page.
Josten and Kutschera—AdŠentitious Roots in Sunflower Cuttings
341
T     1. Effects of the phytohormones indolebutyric acid (IBA), gibberellic acid (GA ) and 6-furfurylamino-purine (kinetin)
$
on the initiation and growth of adŠentitious roots in stem cuttings of 3-d-old sunflower seedlings.
Mean number of
roots per cutting
Treatments
Control
IBA
GA
$
Kinetin
Fresh mass of the
roots per cutting (mg)
kB
jB
kB
jB
0n9p0n2
0
0n8p0n3
0
9n4p0n3
3n7p0n2
6n3p0n6
0
0n4p0n1
0
0n15p0n1
0
22n9p1n4
3n0p0n2
6n8p0n5
0
Data are the meansps.e of six replications with six cuttings each.
Cuttings were incubated for 4 d in nutrient solution (pboron, 0n1 m) that contained 0n1 % DMSO (control) or one of the three phytohormones
at concentrations of 10 µ (and 0n1 % DMSO) each.
F. 8. Effect of boric acid (0n1 m) on the development of adventitious
roots in sunflower cuttings. Four-d-old light-grown seedlings were derooted and placed with the cut end in vermiculite that was moistened
with nutrient solution without (A) or with (B) boric acid (0n1 m). The
photograph was taken 7 d later.
F. 7. Light micrograph of a cross section through the basal part of the
hypocotyl of a sunflower cutting that was incubated for 3 d in nutrient
solution (j0n1 m boric acid). At this time, juvenile adventitious roots
had protruded through the cortex (C) and epidermis (E). The roots
originated between the vascular tissues (V) ; meristem (M) and root cap
(R) were well differentiated (Ruthenium red, i533).
suggest that cell divisions were induced under the influence
of endogenous auxins that were provided by the cotyledons
of the cuttings. The micronutrient boron stimulated the
meristematic activity of the cells and caused elongation and
differentiation of the juvenile roots.
F. 3–6. Light micrographs of cross sections through the region between two vascular bundles in the basal part of the hypocotyls of sunflower
cuttings (see Fig. 1). The cuttings were treated as follows : 2 and 3 d in nutrient solution – boric acid (Figs 3 and 5) ; 2 and 3 d in nutrient
solutionjboric acid (0n1 m) (Figs 4 and 6). In the absence of boric acid cell divisions were observed between the vascular bundles (V) (arrows
in Figs 3 and 5). In the presence of boric acid primordia (P) and adventitious roots (A) developed which protrude through the cortex (C) of the
hypocotyl (Figs 4 and 6). (Ruthenium redi1350).
342
Josten and Kutschera—AdŠentitious Roots in Sunflower Cuttings
Our data are incompatible with the results of Fabijan et
al. (1981 a, b) on adventitious rooting in sunflower cuttings.
These authors used 6-d-old sunflower seedlings that were
grown in a 23 mC day\18 mC night-cycle with a 16 h photoperiod. The achenes were sown in moist vermiculite or
a ground baked clay potting medium. After excising the
roots under water the cuttings were placed into various test
solutions and rapidly developed root primordia. This effect
occurred without the addition of boric acid. We suggest that
the vermiculite or the clay potting medium was moistened
with tap water which may have contained a sufficient
amount of boric acid. It is also possible that the test
solutions were contaminated with traces of B, although we
consider this unlikely. Our experiments clearly demonstrate
that, in the absence of boron, cuttings from 3- and 4-d-old
sunflower seedlings grown in continuous white light do not
develop adventitious roots. In addition, it should be pointed
out that in our control cuttings (kB) the formation of root
primordia was largely suppressed. Under the growth
conditions used in this study these physiological processes
are strictly dependent on the availability of the micronutrient
B, as postulated by Jarvis (1986).
Figure 8 demonstrates that de-rooted sunflower seedlings
placed with the cut surface of the hypocotyl in vermiculite
moistened with nutrient solution (jB) rapidly developed
numerous adventitious roots. In the control (kB) no or
very few roots were formed. The rooted sunflower seedlings
(jB) survived, whereas the rootless cuttings (kB) degenerated after several weeks. This finding indicates that the
micronutrient B, which is present in most soils in sufficient
concentrations (Marschner, 1995), induces new roots at the
cut surface of the stem. Hence, this soil micronutrient is
capable of inducing new organs. This result corroborates
and extends a hypothesis on the role of B in the origin of
vascular plants proposed by Lewis (1980). He postulated
that the presence of B was a pre-requisite for the evolution
of vascular from pre-vascular plants because one of the
primary roles of the micronutrient concerns the biosynthesis
of lignin, the role of the phytohormone auxin, and the
development of xylem. In addition, Lewis (1980) pointed
out that the first roots to develop in the evolving vascular
plants would today be classified as adventitious. Since the
development of adventitious roots was found to be enhanced
by borate it was suggested that the soil micronutrient B may
have contributed to the exploitation of the sub-aeral
environment. This hypothesis is supported by our finding
that the growth of adventitious roots in light-grown
sunflower cuttings is caused by B and that newly rooted
seedlings survive and develop into adult plants. In accordance with Lewis (1980) we suggest that in the course of
evolution of the tracheophytes B was one of the environmental factors that contributed to their success on land : the
ability to exploit the soil for ancorage, water and minerals
may have been made possible by the presence of borate.
A C K N O W L E D G E M E N TS
We thank Mrs. I. Diebel and C. Frohmuth for technical
assistance. This work was supported by a grant from the
Fonds der Chemischen Industrie (Frankfurt\M.).
L I T E R A T U R E C I T ED
Ali AHN, Jarvis BC. 1988. Effects of auxin and boron on nucleic acid
metabolism and cell division during adventitious root regeneration.
New Phytologist 108 : 383–391.
Fabijan D, Taylor JS, Reid DM. 1981 b. Adventitious rooting in
hypocotyls of sunflower (Helianthus annuus) seedlings. II. Action
of gibberellins, cytokinins, auxins and ethylene. Physiologia
Plantarum 53 : 589–597.
Fabijan D, Yeung E, Mukherjee I, Reid DM. 1981 a. Adventitious
rooting in hypocotyls of sunflower (Helianthus annuus) seedlings.
I. Correlative influences and developmental sequence. Physiologia
Plantarum 53 : 578–588.
Hemberg T. 1951. Rooting experiments with hypocotyls of Phaseolus
Šulgaris L. Physiologia Plantarum 4 : 358–369.
Hu H, Brown PH, Labavitch JM. 1996. Species variability in boron
requirement is correlated with cell wall pectin. Journal of
Experimental Botany 295 : 227–232.
Jarvis BC. 1986. Endogenous control of adventitious rooting in non
woody cuttings. In : Jackson MB, eds. New root formation in plants
and cuttings. Dordrecht : Martinus Nijhoff Publishers, 191–222.
Jucknischke A, Kutschera U. 1998. The role of cotyledons and primary
leaves during seedling establishment in sunflower. Journal of Plant
Physiology 153 : 700–705.
Kutschera U. 1990. Cell-wall synthesis and elongation growth in
hypocotyls of Helianthus annuus L. Planta 181 : 316–323.
Lewis DH. 1980. Boron, lignification and the origin of vascular
plants—a unified hypothesis. New Phytologist 84 : 209–229.
Liu JH, Reid DM. 1992. Auxin and ethylene-stimulated adventitious
rooting in relation to tissue sensitivity to auxin and ethylene
production in sunflower hypocotyls. Journal of Experimental
Botany 43 : 1191–1198.
Marschner H. 1995. Mineral nutrition of higher plants. 2nd edn. San
Diego : Academic Press.
Shorrocks VM. 1997. The occurrence and correction of boron
deficiency. In : Dell B, Brown PH, Bell W, eds. Boron in soils and
plants : ReŠiews. Dordrecht : Martinus Nijhoff Publishers, 121–148.
Warington K. 1923. The effect of boric acid and borax on the broad
bean and certain other plants. Annals of Botany 37 : 629–672.