Annals of Botany 85: 687±701, 2000
doi:10.1006/anbo.2000.1132, available online at http://www.idealibrary.com on
Relation between Nitrogen Status, Carbohydrate Distribution and Subsequent Rooting of
Chrysanthemum Cuttings as A€ected by Pre-harvest Nitrogen Supply and Cold-storage
U . D R U E G E *{, S. Z E R C H E {, R . K A D N E R{ and M . E R N S T {
{Institute for Vegetable and Ornamental Crops Grossbeeren/Erfurt e.V., Kuehnhaeuser Strasse 101, D-99189
Erfurt-Kuehnhausen, Germany and {University of Hohenheim, Emil-Wol€-Straûe 25, D-70599 Stuttgart, Germany
Received: 18 October 1999 Returned for revision: 23 December 1999
Accepted: 30 January 2000
This study investigated the relationship between internal nitrogen and carbohydrate distribution in chrysanthemum
cuttings of two cultivars (`Puma', `Cassa') when a€ected by nitrogen supply to stock plants (0.6, 1.5, or 4.0 g N m ÿ2
week ÿ1) and di€erent periods (2, 3, or 4 weeks) of dark cold-storage (0.5 or 58C), and adventitious rooting.
Concentrations of total nitrogen (Nt) and nitrate in cuttings and the levels of sugars, starch and fructan in di€erent
cutting parts (leaves, upper stem, and basal stem) were studied in relation to subsequent adventitious rooting at
natural radiation in a greenhouse. Increasing nitrogen supply resulted in substantially lower starch levels and higher
sucrose concentrations in leaves when cuttings were excised. Fructan concentrations were low and decreased with
increasing nitrogen levels. Starch completely disappeared from leaves and to a large extent from stems within the
shortest storage period. A less pronounced decrease in sugar concentration was observed, particularly in low-nitrogen
cuttings and the cuttings of `Puma'. The number and length of adventitious roots subsequently formed by unstored
and stored cuttings was positively correlated with initial Nt , and to a lesser extent with initial nitrate concentrations in
cuttings. Whereas rooting was not limited by pre-rooting concentrations of carbohydrates in the di€erent cutting
parts, the generally higher rooting capability of nitrogen-rich cuttings, a stronger nitrogen response of `Cassa', and
increased rooting at a particular harvest date, were associated with higher sucrose : starch ratios in leaves at harvest.
This re¯ected an increased assimilate export. By using this characteristic in a linear regression model, total variability
of root numbers, ranging from three±35 per cutting, could be predicted to 57% for the unstored and to 40% for all
cuttings. Increased basipetal transport of carbohydrates, of nitrogen compounds, and of auxins may be causally
# 2000 Annals of Botany Company
involved in these associations.
Key words: Adventitious rooting, nitrogen, sugars, carbohydrates, source-sink, partitioning, quality, storage,
cuttings, stock plants, chrysanthemum, Dendranthema grandi¯orum.
I N T RO D U C T I O N
Adventitious root formation of cuttings is substantially
a€ected by the initial nitrogen and carbohydrate status
(Haissig, 1986; Blazich, 1988; Veierskov, 1988). High
nitrogen supply to stock plants, meeting or even surpassing
the level necessary for maximum growth, has often been
observed to decrease subsequent rooting of cuttings
(Roeber and Reuther, 1982; Haissig, 1986; Henry et al.,
1992). Such e€ects have been discussed repeatedly in
relation to decreased carbohydrate levels and an altered
C : N ratio, which was ®rst suggested by Kraus and Kraybill
(1918) to be important for root formation. However, the
hypothesis of a predominating limitation of adventitious
rooting by low carbohydrate reserves and by a low C : N
ratio has received little experimental support, owing to the
large amount of con¯icting data (Hansen et al., 1978;
Leakey, 1983; Haissig, 1986; Veierskov, 1988; Leakey and
Storeton-West, 1992). In a recent study, rooting of chrysanthemum cuttings under natural radiation in a greenhouse
during spring and summer was found to be positively
correlated with initial nitrogen concentrations, and not
* For correspondence. Fax ‡49(0)36201 785222, e-mail Druege@
Erfurt.igzev.de
0305-7364/00/050687+15 $35.00/00
impeded by simultaneously lower sugar concentrations in
cuttings (Druege et al., 1998).
Nitrogen is needed for synthesis of diverse nitrogenous
compounds, but the promotive in¯uence of nitrogen in
root formation may also be manifested by the manner in
which it relates to carbohydrate content and metabolism
(Blazich, 1988). Adventitious rooting should be related to
individual carbohydrate pools in certain tissues rather than
to total content in cuttings (Haissig, 1986; Veierskov, 1988).
The ability to utilize carbohydrates eciently has repeatedly
been shown to be a crucial factor (Okoro and Grace, 1976;
Haissig, 1984; Tschaplinski and Blake, 1989) and has
already been included in a mechanistic model of adventitious root development in leafy cuttings (Dick and Dewar,
1992). Friend et al. (1994) emphasized that carbon allocation should be a crucial process in¯uencing root initiation
and development, and that knowledge of source±sink
relations of intact plants may add to the understanding of
these processes. Nitrogen supply strongly in¯uences carbon
allocation and partitioning in plants (Rufty et al., 1988;
Huber and Kaiser, 1996; Kaiser, 1997) and speci®c nitrogenous compounds like nitrate can act as strong signals
a€ecting these processes (Scheible et al., 1997). Considering
that ratios among di€erent carbohydrates indicate carbon
partitioning (Galtier et al., 1993), the distribution of speci®c
# 2000 Annals of Botany Company
688
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
carbohydrates within particular cutting parts at the time of
excision should re¯ect the carbon ¯uxes as a€ected by
nitrogen supply. Since cold-storage, which is a standard
procedure in the propagation of chrysanthemums, a€ects
the carbohydrate status of cuttings (Behrens, 1988), strong
interactions can be expected between pre-harvest nitrogen
supply and this post-harvest factor.
With these relationships in mind, and given the results
obtained in a previous study (Druege et al., 1998), the
present investigation was carried out to provide a more
detailed insight into the interactions between nitrogen
status and the distribution of non-structural carbohydrates
and their role in subsequent rooting. In addition to
cold-storage of cuttings, two cultivars were included to
investigate whether their di€erent rooting responses were
associated with particular nitrogen or carbohydrate
responses.
M AT E R I A L S A N D M E T H O D S
Plant material
Chrysanthemum (Dendranthema x grandi¯orum) (Ramat.)
Kitamura `Puma' and `Cassa' were planted in a greenhouse
in commercial peat (Einheitserde Typ P-Sinta, Patzer
Company, Germany) at a density of 100 plants m ÿ2. Plants
were pinched back to four leaves after 1 week and then
managed as stock plants over a 5-month period. Cuttings
were harvested every week, leaving the ®rst two leaves of the
axillary shoot on the stock plant. Photosynthetic photon
¯ux density (PPFD) measured outside the greenhouse was,
on average, 313 mmol m ÿ2 s ÿ1 (400±700 nm) per 16 h
daylength. Plants were shaded when natural radiation
exceeded 630 mmol m ÿ2 s ÿ1, and until 15 May a 16 h
daylength was maintained using additional lighting during
the midnight period (PPFD: 2 mmol m ÿ2 s ÿ1 at plant
height). Heating/ventilation setpoints for temperature
were 19/208C (day) and 17/188C (night), providing an
average air temperature of 20.28C. Average relative
humidity was 60.3% and water and nutrients were supplied
manually. Fertilization was carried out at weekly intervals
with the application of 10 l of nutrient solution per square
metre. Apart from nitrogen (see below), all plants received
the same adequate nutrient supply with the application of
0.6±0.9 g l ÿ1 Flory Basis 2 (Eu¯or-Company, Germany).
Experimental design
A factorial design was used for this experiment. Two
cultivars (`Puma', `Cassa') were combined with three nitrogen treatments as pre-harvest factors using three replicates.
Three hundred and seventy-eight stock plants were planted
per each replication plot (18 plots). The nitrogen dosages
(N-low, N-medium, N-high: 0.6, 1.5, 4.0 g N m ÿ2 week ÿ1,
respectively) were adjusted by combining a constant dose
of calcium nitrate (0.24 g l ÿ1, 14.5% nitrate-N ‡ 1%
ammonium-N ‡ 19% calcium) with di€erent doses of
ammonium nitrate (9% nitrate-N ‡ 9% ammonium-N).
Cuttings were harvested on three occasions from all of the
individual plots (21 Apr. 1997, 2 Jun. 1997, 14 Jul. 1997)
and either chemically analysed and rooted immediately as
described below, or ®rst stored in non-perforated polyethylene bags in the dark at two temperatures (0.5, 58C) for
three di€erent periods (2, 3, 4 weeks).
Determination of adventitious rooting
Adventitious rooting of 20 cuttings per treatment and
replicate (n ˆ 3) were studied in a greenhouse using perlite
as a rooting substrate. No fertilizers or plant hormones
were applied. In addition to using a fog-system, adequate
water conditions of cuttings were provided by intermittent
misting as described by Zerche et al. (1999). Average PPFD
during the rooting periods, measured outside the greenhouse, was 364 + 65 mmol m ÿ2 s ÿ1 (400±700 nm) per 16 h
daylength. Cuttings were shaded when natural radiation
exceeded 540 mmol m ÿ2 s ÿ1. Until 15 May, additional light
was supplied as described above for the stock plants. The
average, maximum and minimum air temperatures for the
entire experiment were 22.8, 33.7 and 18.18C, respectively.
After 12 d the number of roots per cutting and mean root
length were determined.
Chemical analysis
At harvest, 50 cuttings per treatment and replicate (n ˆ 3)
were sampled for the determination of total nitrogen
concentration in the dry matter (Nt ) of cuttings. Samples
were dried as described by Druege et al. (1998) and Nt
analysed with a CHN-Rapid analyser by using the Dumas
method (Ehrenberger, 1991). As total nitrogen in cuttings
during 4 weeks of cold-storage remained unchanged
(Druege et al., 1998), this data was used for all storage
combinations when calculating the regressions between
nitrogen content, carbohydrate characteristics and adventitious rooting. At harvest, and after the di€erent storage
intervals, 15 cuttings per treatment and replicate (n ˆ 3)
were shock frozen and stored at ÿ188C for determination of
nitrate concentrations in cuttings. Ten grams of homogenized samples were extracted in 100 ml of boiling distilled
water for 10 min and then ®ltered (Schleicher & Schuell;
Ref. No. 311652). Filtrates were made up to 250 ml by
adding distilled water, and after mixing 50 ml aliquots with
5 ml of an ionic strength adjustment liquid (0.9 mol
Al2(SO4)3.18H2O), nitrate concentrations were quanti®ed.
This was done by using an ion-selective electrode for nitrate
(NO 500/AT, reference: R 502, electrolytes ELY/IN/502,
0.02 mol (NH4)2SO4 , WTW-Weilheim, Germany) as
described by Kolbe and Mueller (1984) and modi®ed after
Cammann and Galster (1996). Matrix interferences were
excluded by preliminary dilution trials.
At harvest, and after the di€erent storage intervals, six
cuttings per treatment and replicate (n ˆ 3) were sampled to
determine carbohydrate concentrations in the di€erent
cutting parts. Samples were always taken between 1000
and 1200 h, when the collected or stored cuttings were
rooted, and the parallel samples of cuttings were separated
into leaves, upper stems and basal stems (1 cm). Plant
material was cut into small pieces (4 25 mm3), immediately
transferred to sealed polypropylene tubes containing cold
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
aqueous ethanol (80%, ÿ208C) and then stored in a freezer
below ÿ208C until analysis. Percentage of dry matter of
additional samples was determined gravimetrically after
drying for 24 h at 1058C. Sugars were extracted in 80%
ethanol as previously described (Druege et al., 1998), and
glucose, fructose and sucrose concentrations determined in
a microplate assay by an enzyme-coupled colorimetric
reaction (Hendrix, 1993) using glucose-6-phosphate
dehydrogenase (EC 1.1.1.49), hexokinase (EC 2.7.1.1),
phosphoglucose isomerase (EC 5.3.1.9) and invertase
(EC 3.2.1.26) (Sigma Chemical Company). For the
determination of starch and fructans, extraction residues,
from which sugars had been removed, were ground together
with a small amount of quartz sand. After 15 ml doubledistilled water per gram fresh weight had been added,
suspensions were incubated in a water bath at 1008C for 3 h
to extract the fructans (Meier and Reid, 1982) and to
gelatinize the starch (McRae, 1971). When the polypropylene tubes had been cooled to room temperature, 15 ml of
an amyloglucosidase suspension ( from Aspergillus niger,
EC 3.2.1.3, 1.5 U ml ÿ1 in 0.2 M acetate bu€er, pH 4.8) was
added per gram fresh weight. Closed tubes were incubated
at 608C for 40 h in the dark to allow complete starch
digestion. After ®ltration, the starch concentration was
determined via the glucose released. Fructan concentrations
were estimated colorimetrically (470 nm) by assaying for
fructosyl-residues according to Heisterrueber et al. (1994).
Representative ethanolic (80%) extracts from leaves
(n ˆ 54) and stems (n ˆ 54) of stored and unstored cuttings
of both cultivars were tested for potential interferences
between the sucrose measurements of the enzymatic assay
and the low-degree of polymerization fructans previously
discussed by Druege et al. (1998). This was done by
comparison with HPLC analyses as described by Chatterton et al. (1989) and Ernst et al. (1996). Fructans were
detected almost exclusively in stem extracts and did not
interfere with the enzymatic method, indicated by a high
correlation (r ˆ 0.87) between sucrose analysed by HPLC
and that estimated enzymatically.
According to Nt , the nitrate and carbohydrate concentrations were related to dry matter. In addition,
nitrate concentrations were calculated on a nitrogen basis
(nitrate-N), to allow for comparison with total nitrogen.
Statistical analysis
Data were analysed within the ANOVA/MANOVA and
Multiple Regression modules of the Statistica software
program (Statsoft, 1995). E€ects on nitrogen and carbohydrate characteristics were tested by analyses of variance
using harvest date as a potential interacting factor. Because
results were not substantially a€ected by storage temperature, the two temperatures were not di€erentiated. If signi®cant e€ects, independent of harvest dates, were found for
speci®ed storage durations, comparison of mean values was
carried out by using the Newman-Keuls test with a signi®cance level of at least P 4 0.05. Linear regressions and
correlations were calculated between concentrations of
nitrogen, nitrate and carbohydrate characteristics for
the di€erent cutting parts and number and length of
689
subsequently-formed adventitious roots. For this purpose,
the replicates were analysed individually, to cover the whole
variation of data.
R E S ULT S
Fresh weight, dry matter and nitrogen concentration of
cuttings
Fresh weight and dry matter concentrations of cutting
samples were similar for the di€erent nitrogen treatments
and cultivars, with medium nitrogen supply resulting in
only slightly higher fresh weight and lower dry matter
concentrations (Table 1). Average fresh leaf weight of
`Puma' (0.92 g) was higher (P 4 0.01) than from `Cassa'
(0.71 g), but not statistically signi®cant if the nitrogen
treatments were regarded individually (Table 1). As
expected, increasing nitrogen supply resulted in higher Nt
in the cuttings, which were on the same level for both
cultivars. Furthermore, nitrate concentrations in cuttings
were similar for both cultivars and strongly increased with
an increasing nitrogen supply. The relative increase in
nitrate was much higher than for total nitrogen, but the
relative proportion of nitrate within the total nitrogen pool
was small, constituting 10±12% at low and 21±22% at the
highest nitrogen supply (Table 1). Irrespective of the
cultivar and nitrogen supply, decreasing nitrogen concentrations during stock plant cultivation resulted in signi®cantly higher average Nt for the ®rst harvest date (5.5%)
compared with the last date (4.2%). In contrast, the highest
nitrate concentrations in cuttings (1.15% nitrate-N in dry
matter) were measured at the last harvest date.
Carbohydrate distribution as in¯uenced by nitrogen supply,
cold-storage and cultivar
With regard to the carbohydrate composition of the
di€erent cutting parts, strong interactions were found
between the nitrogen supply of stock plants and the
duration of cold-storage, as well as between cultivar and
storage duration. The response of leaf carbohydrates to
nitrogen supply and cold-storage is presented in Fig. 1.
Levels of glucose determined at harvest were not signi®cantly in¯uenced by nitrogen (Fig. 1A). Signi®cantly higher
fructose and sucrose levels were measured when cuttings
were collected from high-nitrogen-supplied stock plants,
even though fructose levels strongly varied between harvest
dates (Fig. 1B) and the di€erences in sucrose were rather
small (Fig. 1C). In contrast to fructose and sucrose levels,
increasing the nitrogen supply strongly decreased starch
concentrations in leaves, which also varied greatly between
harvest dates (Fig. 1D). Also, concentrations of fructans at
harvest, which were more than 20-fold lower than starch
concentrations, decreased when more nitrogen was supplied
(Table 2). While there was no clear in¯uence of cold-storage
on fructans, regardless of the cutting part studied, starch
completely disappeared from leaves within 2 weeks of
storage regardless of the nitrogen treatment (Fig. 1D). Coldstorage also resulted in a decrease in sugar concentrations
in leaves; this e€ect was more pronounced in the cases of
690
`Puma'
Nitrogen
treatment
N-low
N-medium
N-high
Fresh weightn.s.
(g)
Dry mattern.s.
concentration (%)
Leaves
US
BS
Whole cutting
0.86
0.15
0.10
1.11
13.5
10.5
9.4
12.8
Leaves
US
BS
Whole cutting
0.99
0.17
0.11
1.28
12.7
10.2
8.4
12.0
Leaves
US
BS
Whole cutting
0.92
0.19
0.11
1.22
14.4
11.5
9.8
13.5
Organ of cutting
`Cassa'
Nt
(% DM)
4.03a
4.91b
5.64c
Fresh weightn.s.
(g)
Dry mattern.s.
concentration (%)
0.39a
0.65
0.16
0.10
0.90
14.2
10.0
9.2
12.9
3.77a
0.45a
0.79b
0.78
0.18
0.12
1.08
12.6
9.8
8.3
11.7
4.83b
0.63ab
1.18c
0.71
0.18
0.10
0.99
13.4
10.1
8.9
12.3
5.62c
1.25c
Nitrate-N
(% DM)
Nt
(% DM)
Nitrate-N
(% DM)
n.s., Mean values not signi®cantly di€erent between nitrogen levels and cultivars;
Di€erent superscripts indicate signi®cant di€erences (P 4 0.05) between di€erent nitrogen levels for the same cultivar and between di€erent cultivars for the same nitrogen level; DM, dry
matter; US, upper stems; BS, basal stems. Mean values of three harvest dates.
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
T A B L E 1. Fresh weight, dry matter and nitrogen characteristics of parts (leaf, upper stem, basal stem) of chrysanthemum cuttings at harvest as in¯uenced by cultivar
and nitrogen supply to stock plants
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
20
A Glucose (NxSxH P_
< 0.01)
15
10
5
0
6
B Fructose (NxSxH P_
< 0.05)
Leaf carbohydrate concentration (mg g −1 dry matter)
5
4
3
2
1
0
12
C Sucrose (NxS P_
< 0.001)
10
8
6
4
2
0
70
D Starch (NxSxH P_
< 0.001)
60
50
40
30
N-low
N-medium
N-high
20
10
0
0
1
2
3
4
Duration of storage (weeks)
F I G . 1. E€ects of nitrogen supply (N) and cold-storage (S) on glucose
(A), fructose (B), sucrose (C) and starch (D) concentrations in leaves
of chrysanthemum cuttings. Average of two cultivars, three harvest
dates and two storage temperatures. Vertical bars represent LSD
(P 4 0.05) between nitrogen treatments at speci®ed storage durations.
In the case of signi®cant interaction between nitrogen, harvest date
(H) and storage duration, small symbols represent mean values per
harvest date.
691
glucose and fructose compared to sucrose (Fig. 1A±C).
For all sugars, the decrease was lowest in leaves of the
low-nitrogen treatment and highest in the high-nitrogen
cuttings. As a consequence, the ranking of sugar concentrations in leaves amongst the nitrogen treatments found at
harvest was reversed by storage, with low-nitrogen cuttings
having the highest glucose and fructose levels after 2 weeks
and the highest sucrose levels after 3 weeks of storage
(Fig. 1).
At harvest, the basal stems of cuttings contained more
sugars, but less starch, than the leaves (Fig. 2). With regard
to the reducing sugars in these cutting parts, no signi®cant
nitrogen e€ect could be observed at harvest (Fig. 2A,B).
Sucrose concentrations in the basal stems were highest with
the low nitrogen supply regardless of storage time, but the
di€erences were not statistically signi®cant if the unstored
cuttings were analysed separately (Fig. 2C). As was
observed for the leaves, the highest nitrogen supply resulted
in the lowest starch concentrations in the basal stems,
although the e€ect was rather small when compared to the
variation between harvest dates (Fig. 2D). In addition, the
fructan concentrations in these parts decreased if more
nitrogen was supplied to the stock plants (Table 2). As was
observed with the leaves, starch levels in basal stems were
strongly reduced by 2 weeks of cold-storage. However, a
slower decrease of sugar concentrations was observed
(Fig. 2A±C), and was less pronounced when compared
with the leaves (Fig. 1A±C). Glucose concentrations in
basal stems of low-nitrogen cuttings remained constant
during storage, while fructose levels increased. As a result, a
strong nitrogen e€ect, similar to that in the leaves, became
apparent after the cuttings had been stored, at which point
the low-nitrogen cuttings had the highest sugar concentrations (Fig. 2A±C). The di€erences were even larger in
basal stems when compared to the leaves (Figs 1, 2).
In addition to the interactions presented above, the two
cultivars showed di€erent responses to cold-storage. At
harvest, sugar concentrations in leaves and basal stems were
higher for `Cassa' than for `Puma' (Fig. 3). By contrast,
`Cassa' had lower starch levels (Fig. 3D) and, although not
statistically signi®cant, they were consistently lower for all
harvest dates. Cold-storage, which caused a dramatic loss of
starch in cuttings of both cultivars (Fig. 3D), resulted in a
much slower decrease of sugar levels in tissues of `Puma'
than in `Cassa' (Fig. 3A±C). Sugar concentrations in basal
stems of `Puma' remained either constant or increased
during storage, whereas the same concentrations in `Cassa'
rapidly decreased. As a result, after 4 weeks of storage,
`Puma' and `Cassa' had similar concentrations of reducing
sugars in their basal stems (Fig. 3A,B), `Puma' also had
higher sucrose concentrations (Fig. 3C). Similar results were
observed for the leaves (Fig. 3A±C). These interactions were
generally independent of nitrogen supply, except that the
di€erent storage response of fructose level in leaves of both
cultivars was less pronounced in the case of low-nitrogen
supply compared with the other nitrogen treatments (data
not provided). With regard to interactions between nitrogen
supply and cold-storage, as well as between cultivar and
cold-storage, the responses of reducing sugars and of starch
in the upper stems were similar to that noted in the basal
692
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
50
60
A Glucose (NxS P_
< 0.05)
40
50
30
40
30
20
20
10
10
0
0
10
B Fructose (NxS P_
< 0.01)
8
6
4
2
0
25
C Sucrose (N P_
< 0.001, S P_
< 0.001)
20
B Fructose (CxS P_
< 0.001)
8
15
10
Carbohydrate concentration (mg g -1 dry matter)
Carbohydrate concentration in basal stem (mg g -1 dry matter)
10
6
4
2
0
C Sucrose (CxS P_
< 0.001)
20
15
10
5
5
0
0
18
50
16
D Starch (NxSxH P_
< 0.01)
14
D Starch (leaves CxS P_
< 0.01,
basal stem CxSxH P_
< 0.001)
40
12
30
10
8
N-low
N-medium
N-high
6
4
2
0
A Glucose (CxS leaves P_
< 0.001,
basal stem P_
< 0.01)
0
1
2
3
4
Duration of storage (weeks)
F I G . 2. E€ects of nitrogen supply (N) and cold-storage (S) on glucose
(A), fructose (B), sucrose (C) and starch (D) concentrations in basal
stems (1 cm) of chrysanthemum cuttings. Average of two cultivars,
three harvest dates and two storage temperatures. Vertical bars
represent LSD (P 4 0.05) between nitrogen treatments at speci®ed
storage durations. In the case of signi®cant interaction between
nitrogen, harvest date (H) and storage duration, small symbols
represent mean values per harvest date.
‘Puma’, leaves
‘Cassa’, leaves
‘Puma’, basal stem
‘Cassa’, basal stem
20
10
0
0
1
2
3
4
Duration of storage (weeks)
F I G . 3. Interactions between cold-storage (S) and cultivar (C)
regarding glucose (A), fructose (B), sucrose (C) and starch (D)
concentrations in leaves and basal stems of chrysanthemum cuttings.
Average of three nitrogen treatments, three harvest dates and two
storage temperatures. Asterisks indicate signi®cant di€erences
between the two cultivars at speci®ed storage durations at the (*)
0.05, (**) 0.01 and (***) 0.001 levels. In the case of signi®cant interaction with harvest date (H), small symbols represent mean values per
harvest date.
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
stems. The response of sucrose levels in these cutting parts
was similar to that found in the leaves (data not provided).
As a result of the di€erent responses of sugars and starch
in leaves of harvested cuttings, their respective ratios were
also in¯uenced by nitrogen supply and cultivar. Increasing
nitrogen supply raised the sucrose : starch ratio in leaves at
harvest (Table 2). While in the case of the low nitrogen
treatment, sucrose : starch ratios in leaves were similar for
both cultivars, increasing the nitrogen supply led to
increasingly higher ratios for `Cassa' compared to `Puma'
(Table 2). A similar interaction was found for the ratios
between the reducing sugars and starch, as well as between
total sugars and starch in the leaves.
Relationship between internal nitrogen status and
carbohydrate distribution
Correlations, calculated with the internal nitrogen
characteristics as independent variables and carbohydrate
characteristics of the di€erent cutting parts as dependent
variables, generally highlighted the e€ects noted above
(Table 3). Sugar concentrations for the unstored cuttings, in
most cases, were either negatively correlated with Nt or did
not show any signi®cant relation to nitrogen concentration.
In contrast, fructose concentrations in leaves at harvest
were positively correlated with nitrogen level (Table 3).
However, this e€ect was not statistically signi®cant when
`Cassa' was considered separately (r ˆ ‡0.30). Irrespective
of data pools, sucrose levels in leaves of unstored cuttings
were positively correlated with total nitrogen (Table 3).
Similar, though relatively weak, regressions were calculated
for both cultivars, with `Cassa' having slightly higher
sucrose concentrations than `Puma' at the same nitrogen
level (Fig. 4A). Strong negative correlations were calculated
between Nt and both starch and fructan concentrations in
leaves of unstored cuttings (Table 3). In the case of starch,
similar, but lower, correlations were determined for the
upper stems (Table 3). Regressions for starch in the leaves
extended over a broad range of Nt and were similar for
both cultivars, with `Cassa' having slightly lower levels than
`Puma' at the same nitrogen concentration (Fig. 4B).
Because of the very high proportion of starch in leaves of
693
unstored cuttings (Fig. 1), total non-structural carbohydrate
concentrations in the same cutting parts were strongly
negatively correlated with Nt (Table 3). Strong positive
correlations were calculated between nitrogen concentration
and the sucrose : starch ratio in leaves of unstored cuttings
(Table 3). Corresponding to the interactions presented in
Table 2, linear regression lines document a stronger nitrogen
response of this characteristic for `Cassa' (slope: 0.15) than
for `Puma' (slope: 0.09), and in the case of `Cassa' the data
even indicate a steeper non-linear relationship (Fig. 4C).
Similar positive correlations were found for the ratios
between reducing sugars and starch and between total sugar
and starch in leaves at harvest. However, the correlations
were always less strong, irrespective of data pools (Table 3).
Correlations between nitrogen levels and carbohydrate
concentrations in the di€erent parts of stored cuttings were
generally negative or else absent (Table 3). The positive
correlations between both fructose and sucrose, and
nitrogen levels of harvested cuttings disappeared after
storage, and in the case of fructose, were even reversed
into a negative relationship (Table 3). Consistently negative
correlations were found between nitrogen concentration and
all individual and total carbohydrate concentrations in the
basal stems of stored cuttings.
In most cases, no signi®cant correlations were found
between nitrate concentrations and carbohydrate characteristics of unstored cuttings. Otherwise, correlations were
similar, but generally less pronounced, than for total
nitrogen. This was also the case for the stored cuttings
(data not provided). In contrast to Nt , no signi®cant
correlations were determined between nitrate and starch
concentrations and between nitrate and sucrose : starch
ratios in leaves of unstored cuttings of `Puma'. In the case
of `Cassa', these relationships were also less pronounced
(r ˆ ÿ0.57**, r ˆ ‡0.55**) compared with those calculated for total nitrogen (Table 3).
Subsequent rooting of cuttings as related to nitrogen and
carbohydrate status
Within the 12 d rooting period, only slight shoot
growth was observed. For both cultivars, and irrespective
T A B L E 2. Concentrations of fructans in di€erent cutting parts and ratios among selected sugars and starch in leaves at harvest
as in¯uenced by cultivar and nitrogen nutrition
`Puma'
`Cassa'
Carbohydrate status
Organ
N-low
N-medium
N-high
N-low
N-medium
N-high
Fructans (mg g ÿ1 DM)
Leaves
US
BS
1.70a
2.01a
5.66a
1.54ab
1.48a
1.62a
1.26ab
2.18a
1.17a
1.75a
1.77a
1.90a
1.05b
1.49a
1.61a
0.97b
1.39a
1.37a
Sucrose : starch ratio
Reducing sugars : starch ratio
Total sugars : starch ratio
Leaves
Leaves
Leaves
0.15a
0.23a
0.38a
0.18a
0.27a
0.45a
0.31b
0.41b
0.71b
0.19ab
0.41ab
0.60ab
0.30b
0.59b
0.89b
0.53c
1.09c
1.62c
Di€erent superscripts indicate signi®cant di€erences (P 4 0.05) between di€erent nitrogen levels for the same cultivar and between di€erent
cultivars for the same nitrogen level; reducing sugars ˆ glucose ‡ fructose; total sugars ˆ glucose ‡ fructose ‡ sucrose; US, upper stem; BS,
basal stem. Mean values of three harvest dates.
694
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
T A B L E 3. Correlation coecients, calculated from di€erent data pools, between total nitrogen concentrations (Nt) in
cuttings as independent variables and carbohydrate concentrations or ratios in dry matter of di€erent cutting parts as
dependent variables
Carbohydrate
concentration
or ratio
`Puma'
`Cassa'
After storage
(n ˆ 162)
At harvest
(n ˆ 27)
`Puma' ‡ `Cassa'
Organ
At harvest
(n ˆ 27)
Glucose
Leaves
US
BS
n.s.
ÿ0.40*
ÿ0.46*
ÿ0.70***
ÿ0.60***
ÿ0.59***
n.s.
n.s.
n.s.
ÿ0.71***
ÿ0.70***
ÿ0.70***
n.s.
n.s.
ÿ0.33*
ÿ0.71***
ÿ0.65***
ÿ0.64***
Fructose
Leaves
US
BS
‡0.49*
n.s.
n.s.
ÿ0.41***
ÿ0.24**
ÿ0.46***
n.s.
n.s.
n.s.
ÿ0.62***
ÿ0.34***
ÿ0.46***
‡0.28*
n.s.
n.s.
ÿ0.51***
ÿ0.29***
ÿ0.46***
Sucrose
Leaves
US
BS
‡0.59**
n.s.
ÿ0.55**
n.s.
ÿ0.24**
ÿ0.43***
‡0.54**
n.s.
n.s.
ÿ0.28***
ÿ0.23**
ÿ0.40***
‡0.53***
n.s.
ÿ0.40**
ÿ0.15**
ÿ0.22***
ÿ0.39***
Total sugars (TS)
Leaves
US
BS
n.s.
n.s.
ÿ0.62***
ÿ0.58***
ÿ0.54***
ÿ0.61***
n.s.
n.s.
n.s.
ÿ0.63***
ÿ0.63***
ÿ0.66***
n.s.
n.s.
ÿ0.37**
ÿ0.59***
ÿ0.59***
ÿ0.63***
Starch
Leaves
US
BS
ÿ0.75***
ÿ0.49**
n.s.
ÿ0.35***
ÿ0.38***
ÿ0.49***
ÿ0.82***
ÿ0.57**
n.s.
ÿ0.44***
ÿ0.38***
ÿ0.49***
ÿ0.76***
n.s.
n.s.
ÿ0.38***
ÿ0.37***
ÿ0.46***
Fructans
Leaves
US
BS
ÿ0.77***
n.s.
ÿ0.59**
n.s.
n.s.
ÿ0.30***
ÿ0.61***
n.s.
ÿ0.57**
n.s.
n.s.
ÿ0.21**
ÿ0.68***
n.s.
ÿ0.47***
n.s.
n.s.
ÿ0.25***
Total non-structural
carbohydrates (TNC)
Leaves
US
BS
ÿ0.66***
ÿ0.41*
ÿ0.62***
ÿ0.54***
ÿ0.53***
ÿ0.62***
ÿ0.72***
n.s.
n.s.
ÿ0.61***
ÿ0.62***
ÿ0.67***
ÿ0.68***
ÿ0.32*
ÿ0.44***
ÿ0.57***
ÿ0.57***
ÿ0.64***
Sucrose : starch ratio
RS : starch ratio
TS : starch ratio
Leaves
Leaves
Leaves
‡0.85***
‡0.59**
‡0.78***
‡0.75***
‡0.52**
‡0.64***
After storage
(n ˆ 162)
At harvest
(n ˆ 54)
After storage
(n ˆ 324)
‡0.69***
‡0.40**
‡0.52***
TS ˆ glucose ‡ fructose ‡ sucrose; TNC ˆ total sugars ‡ starch ‡ fructans; RS ˆ glucose ‡ fructose; US, upper stem; BS, basal stem;
n.s., non-signi®cant; ***P 5 0.001; **P 5 0.01; *P 5 0.05.
of cold-storage, the number and length of subsequentlyformed adventitious roots was positively correlated with
pre-rooting nitrogen concentrations in cuttings and, to a
lesser extent, with nitrate concentrations (Tables 4, 5).
However, regression slopes over the same broad range of
Nt were di€erent for the two cultivars and depended on
whether or not cuttings experienced cold-storage. The level,
and particularly the steepness, of the regression line was
higher for `Cassa' than for `Puma' with regards to the
number of roots formed by the unstored cuttings (slopes of
5.3 and 2.7, respectively). This indicates a higher rooting
capability and a stronger nitrogen response for `Cassa'
(Fig. 5A). On the other hand, similar regressions were
calculated for the root length of unstored cuttings of both
cultivars (slopes: 0.23, Fig. 5C). Whereas the regression
between Nt and the number of roots formed by `Puma' was
not signi®cantly a€ected by cold-storage (slope: 2.6,
Fig. 5B), the steepness of the respective regression of
`Cassa' was substantially decreased (slope: 3.1, Fig. 5B).
Thus, the stronger nitrogen response of rooting of `Cassa'
was reduced by storage, even though it still remained
slightly higher than `Puma'. In addition, the regression
between nitrogen level and root length of `Puma' became
substantially steeper after storage (slope: 0.36), indicating a
promotive in¯uence of cold-storage on root development,
particularly at high nitrogen levels. This e€ect was only
small for `Cassa' (slope: 0.29, Fig. 5D).
The correlations between pre-rooting carbohydrate
concentrations in the di€erent cutting parts of unstored
cuttings of the individual cultivars and the number of
adventitious roots were, in most cases, either insigni®cant
or else negative (Table 4). In contrast, signi®cant positive
correlations were calculated between fructose in the leaves
of `Puma' and sucrose in the leaves of both cultivars, and
root numbers. However, a causal relationship was not
evident from these correlations because the same sugar
concentrations also revealed positive correlations with
nitrogen level (Table 3), which showed a strong positive
correlation with root number (Table 4). The positive
relationship between fructose, as well as sucrose, concentrations and root number mainly re¯ects intercorrelations
with nitrogen, and becomes evident from the correlations
determined for the stored cuttings. For each cultivar, the
positive correlations between sucrose, as well as fructose,
and root number were reduced by storage and, in the latter
case, reversed into a negative relationship (Table 4). The
fact that root numbers of stored cuttings revealed strong
positive correlations to Nt , and at the same time were either
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
16
A Sucrose
14
12
Carbohydrate concentration (mg g-1 dry matter)
10
8
6
4
both cultivars: r = 0.53
‘Puma’: r = 0.59 ‘Cassa’: r = 0.54
2
0
120
B Starch
100
both cultivars: r = -0.76
‘Puma’: r = -0.75
‘Cassa’: r = -0.82
80
60
40
20
0
1.0
C Sucrose:starch
Ratio
0.8
both cultivars: r = 0.69
‘Puma’: r = 0.85
‘Cassa’: r = 0.75
0.6
0.4
0.2
0
2
3
4
5
6
7
Nitrogen concentration (% of dry matter)
F I G . 4. Linear regressions between total nitrogen concentration (Nt) in
cuttings and sucrose concentrations (A), starch concentrations (B),
sucrose : starch ratios (C) in leaves of cuttings at harvest. All
combinations of two cultivars, three nitrogen treatments, three harvest
dates and three replications (n ˆ 27 per cultivar).
negatively correlated or did not show any correlation to
carbohydrates (Table 4), demonstrates that decreased prerooting carbohydrate concentrations in the parts of
nitrogen-rich cuttings did not substantially impede their
improved rooting. Also, the negative correlations between
carbohydrate concentrations and subsequent rooting of
stored cuttings (Table 4), particularly with regards to the
basal stems, were always related to similar intercorrelations
between nitrogen concentration and sugars (Table 3). This
indicates that these relationships were not causal. The
average length of adventitious roots was similarly correlated for the individual cultivars as for root number, and
was similar to the pre-rooting carbohydrate concentrations
in the di€erent parts (Table 5).
Whereas pre-rooting carbohydrate concentrations in the
di€erent cutting parts did not a€ect rooting, which was
695
increased by higher nitrogen concentrations, the more roots
formed by nitrogen-rich cuttings, as well as the stronger
nitrogen response of `Cassa' (Fig. 5) were associated with
increased sucrose : starch ratios in leaves at harvest
(Fig. 4C). Furthermore, a higher rooting capability determined for the ®rst harvest date when compared to the
others corresponded to a higher sucrose : starch ratio
(Table 6). Consequently, strong correlations were calculated
between the sucrose : starch ratio in leaves at harvest and
root numbers irrespective of data pools (Table 4). It can be
seen from Fig. 6A that, even after combining the data
obtained from unstored cuttings of both cultivars, a highly
signi®cant regression covered both the principal positive
nitrogen e€ect as well as the stronger nitrogen response of
`Cassa'. While 23% of the variability in root numbers
formed by the unstored cuttings of both cultivars could be
predicted by Nt , (Fig. 5A), 57% of the same variability
could be explained by using the sucrose : starch ratio
(Fig. 6A). In addition, the rooting response of stored
cuttings increased with increasing sucrose : starch ratio at
harvest (Fig. 6B). As a result, 40% of the total variability in
root numbers, a€ected by the two cultivars, three nitrogen
treatments, three harvest dates, seven storage combinations
and three replicates, ranging from three±35 roots per cutting, could be explained using this characteristic (r ˆ 0.63),
whereas only 25% could be predicted from Nt (Table 4).
The less steep regression line of stored cuttings (Fig. 6B)
was mainly attributed to the higher storage sensitivity of
nitrogen-rich cuttings from `Cassa' (slopes: 25.2 vs. 10.9 for
unstored and stored cuttings, respectively), revealing the
highest sucrose : starch ratios in leaves at harvest (Fig. 4C).
Regarding root number, similar correlations to those
calculated for the sucrose : starch ratio were also determined
for the reducing sugar : starch ratio as well as for the total
sugar : starch ratio in leaves at harvest. However, these
correlations were generally lower if they were calculated
individually for the two cultivars (Table 4). In addition, the
root length of unstored cuttings was positively correlated
with the sucrose : starch ratio in leaves (Fig. 6C), even
though these relations were weaker when compared to the
regressions between nitrogen and root length (Fig. 5C).
DISCUSSION
With regard to carbohydrate levels, the most pronounced
e€ect of increasing nitrogen supply, and correspondingly of
increasing Nt , was a decrease in starch concentrations
in leaves at harvest. Sucrose concentrations in the same
organs increased slightly with an increase in the nitrogen
supply. In a previous hydroponic study, high nitrogen
supply decreased sugar concentrations in whole cuttings of
`Puma' at harvest, even though the e€ect was stronger after
additional cold-storage (Druege et al., 1998). The more
pronounced negative e€ect of nitrogen on sugars in the
previous study may be related to other cultivation factors.
These could include speci®c root zone stress conditions
(Druege, 1997), higher nitrogen assimilation capacity
observed in the hydroponically-grown stock plants (Zerche
et al., 1999) and unshaded cultivation. The response of
sugars to nitrogen level has often been found to be less
696
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
T A B L E 4. Correlation coecients, calculated from di€erent data pools, between pre-rooting concentrations or ratios of
carbohydrates, total nitrogen (Nt) and nitrate in the dry matter of di€erent cutting parts as independent variables and number
of subsequently-formed adventitious roots as dependent variables
`Puma'
`Cassa'
At harvest
(n ˆ 27)
`Puma' ‡ `Cassa'
Concentration or ratio
Organ
At harvest
(n ˆ 27)
After storage
(n ˆ 162)
Glucose
Leaves
US
BS
n.s.
ÿ0.41*
ÿ0.56**
ÿ0.51***
ÿ0.41***
ÿ0.47***
n.s.
n.s.
n.s.
ÿ0.54***
ÿ0.56***
ÿ0.55***
‡0.35**
‡0.45***
n.s.
ÿ0.40***
ÿ0.31***
ÿ0.42***
Fructose
Leaves
US
BS
‡0.46*
n.s.
n.s.
ÿ0.27***
n.s.
ÿ0.31***
n.s.
n.s.
n.s.
ÿ0.48***
ÿ0.40***
ÿ0.41***
‡0.56***
‡0.42**
‡0.52***
ÿ0.15**
ÿ0.20***
ÿ0.24***
Sucrose
Leaves
US
BS
‡0.44*
n.s.
n.s.
n.s.
ÿ0.21**
ÿ0.39***
n.s.
n.s.
ÿ0.23**
‡0.53***
‡0.50***
n.s.
ÿ0.32***
ÿ0.24***
ÿ0.36***
Total sugars (TS)
Leaves
US
BS
n.s.
n.s.
ÿ0.59**
ÿ0.42***
ÿ0.38***
ÿ0.49***
ÿ0.45***
ÿ0.50***
ÿ0.51***
‡0.49***
‡0.50***
n.s.
ÿ0.41***
ÿ0.32***
ÿ0.43***
Starch
Leaves
US
BS
ÿ0.63***
n.s.
n.s.
ÿ0.28***
ÿ0.19*
ÿ0.32***
ÿ0.82***
ÿ0.61***
n.s.
ÿ0.24**
ÿ0.19*
ÿ0.33***
ÿ0.59***
ÿ0.74***
ÿ0.38**
ÿ0.24***
ÿ0.21***
ÿ0.36***
Fructans
Leaves
US
BS
ÿ0.61***
n.s.
n.s.
n.s.
n.s.
ÿ0.22**
ÿ0.69***
n.s.
ÿ0.47*
n.s.
n.s.
ÿ0.21**
ÿ0.48***
n.s.
n.s.
n.s.
n.s.
ÿ0.21***
Total non-structural
Leaves
carbohydrates (TNC) US
BS
ÿ0.55**
n.s.
ÿ0.45*
ÿ0.41***
ÿ0.36***
ÿ0.49***
ÿ0.71***
n.s.
n.s.
ÿ0.44***
ÿ0.50***
ÿ0.52***
ÿ0.39**
n.s.
n.s.
ÿ0.40***
ÿ0.31***
ÿ0.44***
Sucrose : starch ratio
RS : starch ratio
TS : starch ratio
Leaves
Leaves
Leaves
‡0.70***
‡0.53**
‡0.67***
Nitrogen (Nt)
Nitrate
Whole cutting
Whole cutting
‡0.74***
‡0.50**
‡0.62***
n.s.
n.s.
n.s.
n.s.
n.s.
After storage
(n ˆ 162)
‡0.84***
‡0.60***
‡0.72***
‡0.71***
‡0.60***
‡0.86***
‡0.59**
At harvest
(n ˆ 54)
After storage
(n ˆ 324)
‡0.76***
‡0.71***
‡0.77***
‡0.72***
‡0.55***
‡0.48***
‡0.35**
‡0.52***
‡0.40***
TS ˆ glucose ‡ fructose ‡ sucrose; TNC ˆ total sugars ‡ starch ‡ fructans; RS ˆ glucose ‡ fructose; US, upper stem; BS, basal stem; n.s.,
non-signi®cant; ***P 5 0.001; **P 5 0.01; *P 5 0.05.
pronounced than that of starch (Rufty et al., 1988; Guidi
et al., 1998). While this data also often con¯icted, the starch
pool in leaves has repeatedly been shown to be particularly
sensitive to nitrogen. Thus, increasing nitrogen supply to
cotton plants, leading to higher leaf nitrogen contents, not
only resulted in lower starch concentrations but also in
higher sucrose levels (Reddy et al., 1996). Similar results
were found for the leaves of peach (Nii et al., 1997).
The cuttings, in particular the leaves, contained only very
low concentrations of water-soluble fructans as well as a
low-degree of polymerization fructans, as determined in the
ethanolic extracts by HPLC measurements. Low fructan
levels were also determined in leaves of other members of
the Compositae (Pollock, 1986; Ernst et al., 1995). In leaves
and stems of pot chrysanthemums, Trusty and Miller (1991)
measured substantially higher concentrations compared
with our data. Other than cultivar e€ects, the very low
levels measured in this study might have resulted from the
young plant tissues analysed.
The opposing responses of starch and sucrose levels in
leaves to increasing nitrogen resulted in a strong increase in
the sucrose : starch ratio. Because this was predominantly
the result of strongly decreasing starch levels, the other
sugar : starch ratios also reacted similarly. However, these
ratios were less strongly correlated with nitrogen, which
indicated the distribution between sucrose and starch was
particularly involved. Whereas sucrose is the main transport
form of carbon exported from photosynthetic tissues, starch
and sucrose synthesis in source leaves represent competing
sinks for photosynthetically-®xed carbon (Huber, 1983).
The changes in carbohydrate distribution were strongly
correlated to total nitrogen, rather than to the nitrate
concentrations in cuttings. Both cultivars di€ered greatly in
carbohydrate partitioning even though they had the same
internal total nitrogen and nitrate concentrations. It appears
the change in partitioning was probably caused by speci®c
events associated with N-assimilation and transport rather
than with the signal e€ects of nitrate recently found by
Scheible et al. (1997). Carbohydrate biosynthesis and
N-assimilation into amino acids are potentially competitive
processes as both involve inputs of reduced carbon and
energy (Huber and Kaiser, 1996). The strong response of the
sucrose : starch ratio to nitrogen is probably based on
changed activities of fructose 1,6-bisphosphatase and/or
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
697
T A B L E 5. Correlation coecients, calculated from di€erent data pools, between pre-rooting concentrations or ratios of
carbohydrates, total nitrogen (Nt) and nitrate in the dry matter of di€erent cutting parts as independent variables and length
of subsequently-formed adventitious roots as dependent variables
`Puma'
`Cassa'
Concentration or ratio
Organ
At harvest
(n ˆ 27)
After storage
(n ˆ 162)
Glucose
Leaves
US
BS
n.s.
ÿ0.38*
ÿ0.41*
ÿ0.46***
ÿ0.28***
ÿ0.29***
Fructose
Leaves
US
BS
‡0.44*
n.s.
n.s.
Sucrose
Leaves
US
BS
Total sugars (TS)
At harvest
(n ˆ 27)
`Puma' ‡ `Cassa'
After storage
(n ˆ 162)
At harvest
(n ˆ 54)
After storage
(n ˆ 324)
n.s.
n.s.
n.s.
ÿ0.57***
ÿ0.55***
ÿ0.55***
n.s.
ÿ0.27*
ÿ0.39**
ÿ0.47***
ÿ0.41***
ÿ0.35***
ÿ0.26***
n.s.
ÿ0.28***
n.s.
n.s.
n.s.
ÿ0.52***
ÿ0.31**
ÿ0.47***
n.s.
n.s.
n.s.
ÿ0.42***
ÿ0.18**
ÿ0.36***
‡0.54**
n.s.
ÿ0.47*
n.s.
n.s.
ÿ0.34***
‡0.52**
n.s.
n.s.
ÿ0.34***
ÿ0.26***
ÿ0.41***
‡0.47***
n.s.
ÿ0.41**
n.s.
n.s.
ÿ0.26***
Leaves
US
BS
n.s.
n.s.
ÿ0.53**
ÿ0.39***
ÿ0.25**
ÿ0.33***
n.s.
n.s.
n.s.
ÿ0.55***
ÿ0.52***
ÿ0.56***
n.s.
n.s.
ÿ0.42**
ÿ0.38***
ÿ0.36***
ÿ0.37***
Starch
Leaves
US
BS
ÿ0.58**
ÿ0.42*
n.s.
ÿ0.24**
n.s.
ÿ0.23**
ÿ0.80***
ÿ0.56**
n.s.
ÿ0.23**
ÿ0.26***
ÿ0.37***
ÿ0.64***
n.s.
n.s.
ÿ0.19***
ÿ0.13*
ÿ0.18**
Fructans
Leaves
US
BS
ÿ0.59**
n.s.
ÿ0.42*
n.s.
n.s.
ÿ0.16*
ÿ0.56**
n.s.
n.s.
Total non-structural
carbohydrates (TNC)
Leaves
US
BS
ÿ0.51**
n.s.
ÿ0.48*
ÿ0.36***
ÿ0.24**
ÿ0.34***
ÿ0.69***
n.s.
n.s.
Sucrose : starch ratio
RS : starch ratio
TS : starch ratio
Leaves
Leaves
Leaves
‡0.65***
‡0.39*
‡0.55**
Nitrogen (Nt)
Nitrate
Whole cutting
Whole cutting
‡0.78***
‡0.54**
ÿ0.54***
n.s.
ÿ0.31*
n.s.
n.s.
n.s.
ÿ0.53***
ÿ0.51***
ÿ0.57***
‡0.79***
‡0.63***
‡0.73***
‡0.63***
‡0.41***
‡0.81***
‡0.55**
ÿ0.59***
ÿ0.28*
ÿ0.41**
n.s.
n.s.
n.s.
ÿ0.36***
ÿ0.35***
ÿ0.37***
‡0.61***
‡0.36**
‡0.47***
‡0.72***
‡0.42***
‡0.79***
‡0.54***
‡0.64***
‡0.40***
TS ˆ glucose ‡ fructose ‡ sucrose; TNC ˆ total sugars ‡ starch ‡ fructans; RS ˆ glucose ‡ fructose; US, upper stem; BS, basal stem; n.s.,
non-signi®cant; ***P 5 0.001; **P 5 0.01; *P 5 0.05.
T A B L E 6. Sucrose : starch ratios in leaves at harvest and rooting response of cuttings as in¯uenced by harvest dates
Unstored cuttings
Harvest date
21 April
2 June
14 July
Sucrose : starch ratio
0.41a
0.20b
0.22b
Number of roots
21.1a
15.1b
16.0b
Stored cuttings
Root length (cm)
1.24a
0.92b
1.01b
Number of roots
16.9a
15.3b
14.3c
Root length (cm)
1.79a
1.30b
1.37b
Di€erent superscripts indicate signi®cant di€erences (P 4 0.05) between di€erent harvest dates.
Average of two cultivars and three nitrogen treatments.
sucrose phosphate synthase, since sucrose synthesis vs.
starch synthesis in leaves is mainly controlled by these two
enzymes strongly responding to carbohydrate demand (Stitt
and Quick, 1989; Huber and Huber, 1992). Ahmad and
Marshall (1997) demonstrated that young vegetative
branches of chrysanthemum, equivalent to cuttings before
excision, mainly behaved as sources even 1 week after
beginning their outgrowth, and that a substantial proportion of exported assimilates was directed towards the
root system. Ammonium is assimilated mainly in the roots,
thereby requiring a sucient supply of carbon skeletons
(Schortemeyer et al., 1997; Wiesler, 1997). Thus, even
allowing for nitri®cation of a signi®cant portion of nitrogen
supplied as ammonium nitrate in the present study, the
root-derived sink in particular may have been involved in
the changed carbohydrate partitioning re¯ected by the
changed sucrose : starch ratio. In addition, hormonal signals
of the root system strongly responding to nitrogen supply
(Druege, 2000) may have caused the changed carbohydrate
partitioning (Daie, 1986; Cheik and Brenner, 1992).
698
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
40
35
30
25
30
25
20
15
15
10
10
Number per cutting
20
5
0
40
B Number of roots, stored cuttings
35
both cultivars: r = 0.52
‘Puma’: r = 0.71
‘Cassa’: r = 0.72
30
25
0
40
B Number of roots, stored cuttings
35
30
25
20
15
15
10
10
5
5
0
0
both cultivars: r = 0.62
‘Puma’: r = 0.65
‘Cassa’: r = 0.53
2.5
3.5
C Root length, unstored cuttings
3.0
both cultivars: r = 0.79
‘Puma’: r = 0.78
‘Cassa’: r = 0.81
2.5
2.0
1.5
Length per root (cm)
both cultivars: r = 0.76
‘Puma’: r = 0.70
‘Cassa’: r = 0.84
5
20
1.0
0.5
0
1.5
1.0
both cultivars: r = 0.64
‘Puma’: r = 0.63
‘Cassa’: r = 0.72
2.5
2.0
1.0
‘Puma’
‘Cassa’
0.5
3
4
0
0.2
0.4
0.6
0.8
1.0
F I G . 6. Linear regressions between sucrose : starch ratios in leaves at
harvest and number of roots subsequently formed by unstored (A) and
stored (B) cuttings and root length (C) of unstored cuttings (unstored
cuttings: n ˆ 27, stored cuttings: n ˆ 162 per cultivar).
1.5
2
both cultivars: r = 0.61
‘Puma’: r = 0.65
‘Cassa’: r = 0.79
0.5
Sucrose:starch ratio in leaves at harvest
D Root length, stored cuttings
3.0
C Root length, unstored cuttings
2.0
0
3.5
0
A Number of roots, unstored cuttings
35
Length per root (cm)
Number of roots per cutting
40
A Number of roots, unstored cuttings
both cultivars: r = 0.48
‘Puma’: r = 0.74
‘Cassa’: r = 0.86
5
6
7
Nitrogen concentration (% of dry matter)
F I G . 5. Linear regressions between pre-rooting nitrogen concentrations
in cuttings and number (A, B) or length (C, D) of roots subsequently
formed by unstored (A, C) or stored (B, D) cuttings (unstored cuttings:
n ˆ 27, stored cuttings: n ˆ 162 per cultivar).
The main e€ect of cold-storage observed in the present
study was a decrease in carbohydrate concentrations in the
cutting parts, which agrees with observations on cuttings
and on young plants of other species (Davies and Potter,
1985; Behrens, 1988; Kubota et al., 1997). Low temperatures
during dark-storage slow down metabolic processes including respiration (Behrens, 1988), but there is still a need for
energy and organic compounds, and respiration is not
completely prevented (Kubota et al., 1997; Wilson et al.,
1998). Starch was the carbohydrate fraction most sensitive
to cold-storage. This agrees with observations made on
conifer cuttings (Behrens, 1988), but not with those
obtained on young chrysanthemum plants at similar storage
temperatures (Rajapakse and Kelly, 1995). In that study, a
more pronounced decline in sugar concentrations was
observed during cold-storage, whereas starch was less
responsive. These di€erences indicate that changes in
carbohydrate composition in plant tissues in response to
cold-storage also depend on either the connection to other
plant parts or on the integration within a whole plant. In this
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
connection very little is known about the temperature
sensitivity of individual enzymes or of transport processes
(HaÈllgren and OÈquist, 1990). In the present study, a smaller
increase in sucrose concentrations was observed in stems of
`Puma' during 2 weeks of storage (increase by 108 and 33%
in upper and basal stems, respectively) compared with
stronger e€ects previously determined for whole cuttings of
the same cultivar (Druege et al., 1998). These di€erences
may be related to the di€erent growing conditions of the
stock plants discussed above. In most tissues, but particularly in the leaves, sugar concentrations decreased during
storage. The stronger decrease for the cultivar `Cassa' and
the more pronounced decrease after increased nitrogen
supply, both associated with lower starch levels at harvest,
indicate that initial starch levels a€ected the sugar responses
to storage.
Con®rming the results obtained in a previous study
(Druege et al., 1998), adventitious root formation of
chrysanthemum cuttings was principally promoted by an
increasing nitrogen supply and a corresponding increase in
internal nitrogen concentrations. This was regardless of the
cultivar and whether or not cuttings received cold-storage.
The linear relationship held true over a broad range of
internal nitrogen concentrations, even though nitrogen
supply necessary for maximum growth was exceeded and
substantial accumulation of nitrate was observed in the root
zone of stock plants (Zerche et al., 1999). The correlations
found are evidence that the initial ( pre-rooting) concentrations of individual and total carbohydrates in the
di€erent cutting parts did not impede adventitious rooting
to such an extent that the positive e€ect of increased
nitrogen supply was signi®cantly inhibited. Even when
carbohydrate reserves had been substantially reduced after
cold-storage, particularly in nitrogen-rich cuttings, the
positive in¯uence of nitrogen still predominated. These
results are contrary to the correlations found by Roeber and
Reuther (1982) for chrysanthemum cuttings rooted at low
irradiance. The importance of current photosynthesis to
actual carbohydrate balance and to adventitious rooting has
been stressed repeatedly (Haissig, 1986; Davis, 1988;
Veierskov, 1988; Druege et al., 1998). Recently, Hegewald
et al. (1999) demonstrated that chrysanthemum cuttings
have a high photosynthetic capacity within a day after
excision, allowing for high photosynthetic rates comparable
to those of intact young shoots of stock plants. These
®ndings strongly support the conclusion that under the
adequate light conditions of our study, carbohydrate
reserves per se did not limit root formation because the
carbohydrate demand was covered by current photosynthesis.
The higher root numbers produced with increasing
nitrogen supply can be interpreted as re¯ecting root initiation, while increased root length can be the consequence of
both accelerated root initiation and development. The
stronger correlations calculated between Nt and both root
numbers and length, when compared with nitrate, indicate
that the promotion of rooting was associated with N-assimilation. Increased rooting of nitrogen-rich cuttings was
associated with higher sucrose : starch ratios in leaves,
re¯ecting increased carbohydrate partitioning towards
699
assimilate export (Galtier et al., 1993). The cultivar that
showed the stronger response of sucrose : starch ratio also
revealed a stronger rooting response, and di€erences in
rooting among harvest dates was associated with parallel
di€erences in carbohydrate partitioning. The correlations
found strongly support the conclusion that enhanced
carbohydrate export in cuttings manifested at the time of
excision was causally involved in the changed rooting
response observed. The increased export activity may lead
to an accelerated subsequent export, transport and supply of
carbohydrates to the region of root regeneration, where they
can promote root initiation and development by diverse
mechanisms (Haissig, 1986; Veierskov, 1988; Koch, 1996).
However, increased basipetal transport of sugars in cuttings
would also allow for accelerated co-transport of amino acids
within the phloem along with sucrose (Riens et al., 1991;
Winter et al., 1992). Even though total nitrogen concentration did not increase in basal stems during root formation
of chrysanthemum (Good and Tukey, 1967), certain amino
acids or other nitrogenous compounds in particular may
limit root initiation and development (Suzuki, 1982;
Haissig, 1986; Gaspar et al., 1997). In addition, increased
export of assimilates favours increased basipetal transport
of auxins, which also move in the phloem following the
direction of assimilates (Goldsmith et al., 1974; Lomax et al.,
1995). Substantial evidence supports the conclusion that an
early rise in auxin level, particularly of indole-3-acetic acid
(IAA), is causally related to the initiation of adventitious
roots (Blakesley, 1994; Gaspar et al., 1997).
The stronger nitrogen response of rooting of `Cassa'
compared with `Puma', associated with lower starch levels
and higher sucrose : starch ratios, was partially lost during
cold-storage, and coincided with a stronger decrease in
initially higher sugar concentrations. In addition, an increase
in root length after cold-storage, indicating that the
biochemical events of root formation had occurred already
during this low temperature phase, was much more pronounced in the case of `Puma' than `Cassa'. These results
indicate that increased partitioning of carbohydrates
towards carbohydrate export can only favour subsequent
rooting if carbohydrate depletion of the supplying tissue
becomes the crucial factor. Nevertheless, taking into account
the positive relationship found between the partitioning
towards sucrose and root formation, carbohydrate ¯uxes,
and not carbohydrate reserves per se, should be the crucial
factor for determining the rooting of chrysanthemum
cuttings. This also assumes the high capacity of the cuttings
to photosynthesize immediately, at least under the greenhouse conditions of a Middle-European spring and summer.
There are still many questions that need to be answered,
particularly with regard to the true causal events responsible
for the associations found between nitrogen status, carbohydrate partitioning in leaves, and adventitious rooting. In
addition to carbohydrates, the in¯uence of nitrogen supply
to stock plants on ¯uxes of nitrogenous compounds as well
as of auxins should be followed during root formation. The
association between sucrose : starch ratio in leaves and
subsequent adventitious rooting, focusing on its role as a
potential predictor of rooting success, should be further
700
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
studied and should include di€erent cultivars and other
stock plant factors.
AC K N OW L E D G E M E N T S
This work was supported by the Ministries of Nutrition,
Agriculture and Forestry of the state Brandenburg, the free
state of Thuringia and the Federal Republic of Germany.
We greatly appreciate the skilful and accurate technical
assistance of Baerbel Broszies, Sabine Czekalla and Joerg
Pfotenhauer, and thank Dr J. Hansen, Research Institute
for Agricultural Science, Aarslev, Denmark for critically
reviewing the manuscript.
L I T E R AT U R E C I T E D
Ahmad J, Marshall C. 1997. The pattern of 14C-assimilate distribution
in chrysanthemum cv. Red Delano with particular reference to
branch interrelations. Journal of Horticultural Science 72:
931±940.
Behrens V. 1988. Storage of unrooted cuttings. In: Davis TD, Haissig
BE, Sankhla N, eds. Adventitious root formation in cuttings.
Portland, Oregon: Dioscorides Press, 235±247.
Blakesley D. 1994. Auxin metabolism and adventitious root initiation.
In: Davis TD, Haissig BE, eds. Biology of adventitious root
formation. New York: Plenum Press, 143±154.
Blazich FA. 1988. Mineral nutrition and adventitious rooting. In:
Davis TD, Haissig BE, Sankhla N, eds. Adventitious root formation in cuttings. Portland, Oregon: Dioscorides Press, 61±69.
Cammann K, Galster H. 1996. Das Arbeiten mit ionenselektiven
Elektroden; Eine EinfuÈhrung fuÈr Praktiker; Dritte Au¯age. Berlin,
Heidelberg: Springer-Verlag.
Chatterton NJ, Harrison PA, Thornley WR, Bennett JH. 1989.
Puri®cation and quanti®cation of kestoses ( fructosylsucrose) by
gel permeation and anion exchange chromatography. Plant
Physiology and Biochemistry 27: 289±295.
Cheikh N, Brenner ML. 1992. Regulation of key enzymes of sucrose
biosynthesis in soybean leaves. Plant Physiology 100: 1230±1237.
Daie J. 1986. Hormone-mediated enzyme activity in source leaves.
Plant Growth Regulation 4: 287±291.
Davis TD. 1988. Photosynthesis during adventitious rooting. In: Davis
TD, Haissig BE, Sankhla N, eds. Adventitious root formation in
cuttings. Portland, Oregon: Dioscorides Press, 79±87.
Davis TD, Potter JR. 1985. Carbohydrates, water potential, and
rooting of stored Rhododendron cuttings. HortScience 20:
292±293.
Dick JMcP, Dewar RC. 1992. A mechanistic model of carbohydrate
dynamics during adventitious root development in leafy cuttings.
Annals of Botany 70: 371±377.
Druege U. 1997. Proline level in leaves and yield of cuttings and ¯owers
of chrysanthemum as in¯uenced by di€erent root zone conditions.
Scientia Horticulturae 68: 171±180.
Druege U. 2000. In¯uence of pre-harvest nitrogen supply on postharvest behavior of ornamentals: Importance of carbohydrate
status, photosynthesis and plant hormones. Gartenbauwissenschaft
(in press).
Druege U, Zerche S, Kadner R. 1998. Relation between nitrogen and
soluble carbohydrate concentrations and subsequent rooting of
Chrysanthemum cuttings. Advances in Horticultural Science 12:
78±84.
Ehrenberger F. 1991. Quantitative organische Elemantaranalyse. Weinheim: VCH.
Ernst M, Chatterton JN, Harrison PA. 1995. Carbohydrate changes in
chicory (Chichorium intybus L. var. foliosum) during growth and
storage. Scientia Horticulturae 63: 251±261.
Ernst M, Chatterton NJ, Harrison PA. 1996. Puri®cation and
characterization of a new fructan series from species of Asteraceae.
New Phytologist 132: 63±66.
Friend AL, Coleman MD, Isebrands JG. 1994. Carbon allocation to
root and shoot systems of woody plants. In: Davis TD, Haissig
BE, eds. Biology of adventitious root formation. New York: Plenum
Press, 245±273.
Galtier N, Foyer CH, Huber J, Voelker TA, Huber SC. 1993. E€ects of
elevated sucrose-phosphate synthase activity on photosynthesis,
assimilate partitioning, and growth in tomato (Lycopersicon
esculentum var UC82B). Plant Physiology 101: 535±543.
Gaspar T, Kevers C, Hausman JF. 1997. Indissociable chief factors in
the inductive phase of adventitious rooting. In: Altman A, Waisel
Y, eds. Biology of root formation and development. New York:
Plenum Press, 55±63.
Goldsmith MHM, Cataldo DA, Karn J, Brenneman T, Trip P. 1974.
The rapid non-polar transport of auxin in the phloem of intact
Coleus plants. Planta 116: 301±317.
Good GL, Tukey HB Jr. 1967. Redistribution of mineral nutrients in
Chrysanthemum morifolium during propagation. Proceedings of
the American Society for Horticultural Science 90: 384±388.
Guidi L, Lore®ce G, Pardossi A, Malorgio F, Tognoni F, Soldatini GF.
1998. Growth and photosynthesis of Lycopersicon esculentum (L.)
plants as a€ected by nitrogen de®ciency. Biologia Plantarum 40:
235±244.
Haissig BE. 1984. Carbohydrate accumulation and partitioning in
Pinus banksiana seedlings and seedling cuttings. Physiologia
Plantarum 61: 13±19.
Haissig BE. 1986. Metabolic processes in adventitious rooting of
cuttings. In: Jackson MB, ed. New root formation in plants and
cuttings. Dordrecht, Boston, Lancaster: Martinus Nijho€ Publishers, 141±189.
HaÈllgren J-E, OÈquist G. 1990. Adaptation to low temperatures. In:
Alscher RG, ed. Stress responses in plants: adaptation and
acclimation mechanisms. Wiley-Liss, Inc., 265±293.
Hansen J, Stroemquist LH, Ericsson A. 1978. In¯uence of the
irradiance on carbohydrate content and rooting of cuttings of
pine seedlings (Pinus sylvestris L.). Plant Physiology 61: 975±979.
Hegewald J, Kadner R, Druege U, Zerche S. 1999. Photosyntheserate
von Chrysanthemenstecklingen waÈhrend der Bewurzelung unter
dem Ein¯uû unterschiedlicher Sticksto€versorgung der Mutterpfanzen und Lichtbedingungen waÈhrend der Vermehrung. BDGLSchriftenreihe 17: 159.
Heisterrueber D, Schulte P, Moerschbacher BM. 1994. Soluble carbohydrates and invertase activity in stem-rust-infected, resistent and
susceptible near-isogenic wheat leaves. Physiological and Molecular Plant Pathology 44: 111±123.
Hendrix DL. 1993. Rapid extraction and analysis of nonstructural
carbohydrates in plant tissues. Crop Science 33: 1306±1311.
Henry PH, Blazich FA, Hinesley LE. 1992. Nitrogen nutrition of
containerized eastern red cedar. II. In¯uence of stock plant
fertility on adventitious rooting of stem cuttings. Journal of the
American Society for Horticultural Science 117: 568±570.
Huber SC. 1983. Relation between photosynthetic starch formation
and dry-weight partitioning between the shoot and root. Canadian
Journal of Botany 61: 2709±2716.
Huber SC, Huber JL. 1992. Role of sucrose-phosphate synthase in
sucrose metabolism in leaves. Plant Physiology 99: 1275±1278.
Huber SC, Kaiser WM. 1996. Regulation of C/N interactions in higher
plants by protein phosphorylation. In: Verma DPS, ed. Signal
transduction in plant growth and development. Wien, New York:
Springer-Verlag, 87±112.
Kaiser WM. 1997. Regulatory interaction of carbon- and nitrogen
metabolism. In: Behnke HD, LuÈttge U, Esser K, Kadereit JW,
Runge M, eds. Progress in botany, Vol. 58. Berlin, Heidelberg:
Springer-Verlag, 150±163.
Koch KE. 1996. Carbohydrate-modulated gene expression in plants.
Annual Review of Plant Physiology and Plant Molecular Biology
47: 509±540.
Kolbe H, Mueller K. 1984. UÈber die quantitative Bestimmung von
Nitrat mit Hilfe einer nitratsensitiven Elektrode ( fuÈr Serienanalysen, aufgezeigt am Beispiel von Karto€elknollen). Landwirtschaftliche Forschung 37: 434±444.
Kraus EJ, Kraybill HR. 1918. Vegetation and reproduction with special
reference to Tomato. Oregon Agricultural Experimental Station
Bulletin 149.
Druege et al.ÐNitrogen Status, Carbohydrate Distribution and Adventitious Rooting
Kubota C, Rajapakse NC, Young RE. 1997. Carbohydrate status and
transplant quality of micropropagated broccoli plantlets stored
under di€erent light environments. Postharvest Biology and
Technology 12: 165±173.
Leakey RRB. 1983. Stockplant factors a€ecting root initiation in
cuttings of Triplochiton scleroxylon K. Schum., an indigenous
hardwood of West Africa. Journal of Horticultural Science 58:
277±290.
Leakey RRB, Storeton-West R. 1992. The rooting ability of Triplochiton scleroxylon cuttings: the interactions between stock plant
irradiance, light quality and nutrients. Forest Ecology and
Management 49: 133±150.
Lomax TL, Muday GK, Rubery PH. 1995. Auxin transport. In: Davies
PJ, ed. Plant hormones. Dordrecht, Boston, London: Kluwer
Academic Publishers, 509±530.
McRae JC. 1971. Quantitative measurement of starch in very small
amounts of leaf tissues. Planta 96: 101±108.
Meier H, Reid JSG. 1982. Reserve polysaccharides other than starch in
higher plants. In: Loewus FA, Tanner W, eds. Plant carbohydrates
I. Intracellular carbohydrates. Berlin, Heidelberg, New York:
Springer-Verlag, 420±462.
Nii N, Yamaguchi K, Nishimura M. 1997. Changes in carbohydrate and
ribulose biphosphate carboxylase-oxygenase contents in peach
leaves after applications of di€erent amounts of nitrogen fertilizer.
Journal of the Japanese Society for Horticultural Science 66:
505±511.
Okoro OO, Grace J. 1976. The physiology of rooting Populus cuttings.
I. Carbohydrate and photosynthesis. Physiologia Plantarum 36:
133±138.
Pollock CJ. 1986. Fructans and the metabolism of sucrose in vascular
plants. New Phytologist 104: 1±24.
Rajapakse NC, Kelly JW. 1995. Cultivar di€erences with respect to
storage potential and carbohydrate status of rooted chrysanthemum cuttings. Acta Horticulturae 405: 427±434.
Reddy AR, Reddy KR, Padjung R, Hodges HF. 1996. Nitrogen
nutrition and photosynthesis in leaves of Pima cotton. Journal of
Plant Nutrition 19: 755±770.
Riens B, Lohaus G, Heinecke D, Heldt HW. 1991. Amino acid and
sucrose content determined in the cytosolic, chloroplastic, and
vacuolar compartments and in the phloem sap of spinach leaves.
Plant Physiology 97: 227±233.
Roeber R, Reuther G. 1982. Der Ein¯uss unterschiedlicher N-Formen
und -Konzentrationen auf den Ertrag und die QualitaÈt von
Chrysanthemen-Stecklingen. Gartenbauwissenschaft 47: 182±188.
701
Rufty TW Jr, Huber SC, Volk RJ. 1988. Alterations in leaf carbohydrate metabolism in response to nitrogen stress. Plant
Physiology 88: 725±730.
Scheible W-R, Lauerer M, Schulze E-D, Caboche M, Stitt M. 1997.
Accumulation of nitrate in the shoot acts as a signal to regulate
shoot-root allocation in tobacco. The Plant Journal 11: 671±691.
Schortemeyer M, Stamp P, Feil B. 1997. Ammonium tolerance and
carbohydrate status in maize cultivars. Annals of Botany 79:
25±30.
Statsoft, Inc.. 1995. Statistica for Windows [Computer program
manual]. Tulsa, OK: Statsoft, Inc., 2325 East 13th Street, Tulsa,
OK 74104, USA.
Stitt M, Quick WP. 1989. Photosynthetic carbon partitioning: its
regulation and possibilities for manipulation. Physiologia Plantarum 77: 633±641.
Suzuki T. 1982. Changes in total nitrogen and free amino acids in stem
cuttings of mulberry (Morus alba L.). Journal of Experimental
Botany 33: 21±28.
Trusty SE, Miller WB. 1991. Postproduction carbohydrate levels in pot
chrysanthemums. Journal of the American Society for Horticultural Science 116: 1013±1018.
Tschaplinski TJ, Blake TJ. 1989. Correlation between early root
production, carbohydrate metabolism, and subsequent biomass
production in hybrid poplar. Canadian Journal of Botany 67:
2168±2174.
Veierskov B. 1988. Relations between carbohydrates and adventitious
root formation. In: Davis TD, Haissig BE, Sankhla N, eds.
Adventitious root formation in cuttings. Portland, Oregon:
Dioscorides Press, 70±78.
Wiesler F. 1997. Agronomical and physiological aspects of ammonium
and nitrate nutrition of plants. Zeitschrift fuÈr P¯anzenernaÈhrung
und Bodenkunde 160: 227±238.
Wilson SB, Iwabuchi K, Rajapakse NC, Young RE. 1998. Responses of
broccoli seedlings to light quality during low-temperature storage
in vitro: II sugar content and photosynthetic eciency. HortScience 33: 1258±1261.
Winter H, Lohaus G, Heldt HW. 1992. Phloem transport of amino
acids in relation to their cytosolic levels in barley leaves. Plant
Physiology 99: 996±1004.
Zerche S, Kadner R, Druege U. 1999. E€ect of cultivar, nitrogen
nutrition and cultivating system of chrysanthemum mother plants
on cutting yield, nitrogen concentration and subsequent rooting of
cuttings. Gartenbauwissenschaft 64: 272±278.