Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:
Tree Physiology 15, 11--18
© 1994 Heron Publishing----Victoria, Canada
Influence of environment, fertilizer and genotype on shoot morphology
and subsequent rooting of birch cuttings
MARGARETA WELANDER
Swedish University of Agricultural Sciences, Department of Horticulture, Box 55, S-230 53 Alnarp, Sweden
Received June 30, 1993
Summary Differences in rooting ability of birch (Betula
pubescens J.F. Ehrh.) cuttings were observed as a result of
differences in genotype and physiology of the stock plants. The
uniformity in response among cuttings from micropropagated
plants compared with cuttings from seed plants confirmed the
advantage of using micropropagated plants to study environmental effects. Shoot morphology of the seed stock plants was
influenced by both photoperiod and thermoperiod. A day/night
temperature of 15/25 °C reduced stem elongation compared
with a day/night temperature of 25/15 °C regardless of photoperiod, and a continuous light regime resulted in more shoots
per plant in both temperature regimes than a 16-h photoperiod.
A reduction in the supply of macronutrients did not influence
shoot morphology, but increased rooting substantially and
seemed to override the effects of environmental factors. In
cuttings of seed plants, the highest rooting percentage and
number of roots were obtained in a 16-h photoperiod with a
day/night temperature of 15/25 °C. In micropropagated stock
plants, there was a positive correlation between shoot length
and number of leaves per shoot and topographical distribution
of light within the plants, but there was no correlation between
these parameters and rooting ability of the cuttings. A rooting
temperature of 16 °C delayed the rate of root production
compared with the rate at higher temperatures, but the final
rooting percentage was the same over the range from 16 to
28 °C. Root branching increased with temperature. At all temperatures, there was a large increase in sucrose content at the
base of the cuttings during rooting, whereas the concentration
of nontranslocated sugars remained constant. The carbohydrate
content at the base of cuttings from micropropagated stock
plants was three times higher than at the base of cuttings from
seed stock plants, but the higher carbohydrate content was not
correlated with a higher rooting potential.
Keywords: Betula pubescens, hydroponic system, micropropagation, photoperiod, temperature, thermoperiod.
Introduction
Seasonal variations in rooting ability of cuttings emphasize the
importance of identifying the physiological status of the stock
plant when cuttings are taken (Nanda and Anand 1970, Anand
and Heberlein 1975, Hansen and Ernstsen 1982). The opportunity to manipulate the rooting potential of cuttings is generally much greater when favorable conditions are given to the
stock plants rather than the cuttings taken from them (Howard
and Harrison-Murray 1988, Howard 1991). The mineral nutrient status of the stock plant affects rooting; a low N supply
improves rooting (Haun and Cornell 1951, Preston et al. 1953).
Photoperiod (Bachelard and Stove 1963, Heide 1965, Nanda
et al. 1967) and irradiance (Fisher and Hansen 1977, Poulsen
and Andersen 1980) also influence the rooting ability of many
plants. Although irradiance has not always been associated
with high rooting potential (Okoro and Grace 1976, Hansen et
al. 1978, Veierskov and Andersen 1982, Veierskov et al.
1982a), it has been used to change the carbohydrate content of
cuttings. The need for carbohydrates during root initiation was
first demonstrated by Borthwick et al. (1937) and has been
verified by Bhattacharya et al. (1976). However, the amount of
carbohydrate required in this process is not known, and the
relationship between carbohydrates and adventitious root formation is unclear (Haissig 1984, Veierskov 1988).
The optimal root temperature for rooting cuttings is difficult
to determine, because the effects of temperature are often
associated with other features of the propagation environment
and with genotype (Andersen 1986).
There is substantial evidence that rooting ability is genetically controlled (Haissig and Riemenschneider 1988). Clonal
differences in rooting ability of cuttings from several forest
tree species have been reported (Dunn and Townsend 1954,
Ying and Bagley 1977, Pounders and Foster 1992). However,
it is important in genetic studies of rooting that confounding
environmental effects are separated and properly controlled.
One way to separate the nongenetic effects of the stock plant
from the genetic effects is to compare cuttings from seed stock
plants with cuttings from micropropagated stock plants grown
under controlled conditions.
I have studied the effects of photoperiod, thermoperiod and
fertilizer on shoot morphology and subsequent rooting of birch
cuttings, and genotypic differences in rooting ability by comparing seed plants with micropropagated plants. I also examined whether the topographical distribution of light within the
plants was related to shoot morphology and subsequent rooting. The influence of temperature during rooting in a hydroponic system was examined and attempts were made to
12
WELANDER
correlate rooting capacity of cuttings from cloned material and
seed plants to the amount of carbohydrate in the stem base of
the cuttings during rooting.
Material and methods
Seed plant cuttings
Seed collection and growing conditions In 1987, seeds were
collected from an early flowering genotype of Betula pubescens J.F. Ehrh., growing at the University College of Wales, and
brought to Sweden. The seeds were sown in a greenhouse and
the seedlings were planted in a field in July 1988. Seeds from
these trees were harvested in September 1989, sown in the
greenhouse on October 16, 1989, and then planted in pots
containing Hasselfors Master Soil 14 days later. The seedlings
were grown in the greenhouse in natural daylength at 18 °C
until February 2, 1990. The plants, which were then approximately 30 cm tall, were selected to serve as seed stock plants
based on uniformity and placed in four climate chambers under
the conditions described in Table 1. Irradiance, measured at
plant level, was supplied by a combination of incandescent and
cool-white fluorescent lamps. Each climate chamber contained
28 seed stock plants and half of the plants were watered with
Nutrient Solution I (macronutrients (g l −1) K2PO4 13.6, KNO3
20.6, Ca(NO3)2 ⋅ 4H2O 23.6, Fe-Na-EDTA 7.3, MgSO4 ⋅ 7H2O
25.0 (diluted 1/100); micronutrients (mg l −1) MnSO4 ⋅ 4H2O
89.0, CuSO4 ⋅ 5H2O 12.0, Na2MoO4 ⋅ 2H2O 0.2, ZnCl2 6.0,
H3BO3 24.7 (diluted 1/1000)) and the other half with Nutrient
Solution II (identical to Nutrient Solution I except that macronutrient concentrations were diluted 1/200). Time of transfer
to the climate chambers is referred to as Day 0 in the figures.
Plant height, number of shoots per seed stock plant and the
irradiance at each shoot used as a cutting were recorded at
about 7-day intervals for two months. At the end of the experiment the total irradiance that each shoot had received during
the growth period was calculated, and shoot length and number
of leaves per shoot were recorded. In April 1990, four seed
stock plants approximately 100 cm in height were randomly
selected from each climate chamber for rooting experiments.
Rooting experiments with cuttings from seed plants
Five
shoots per seed stock plant (numbered 1--5 counted from the
base) were used as cuttings. The cuttings were trimmed to a
final length of 15 cm and placed in reservoirs containing
Nutrient Solution II. Air was bubbled through the reservoirs.
Four reservoirs with temperatures of 16, 20, 24 and 28 °C were
used with 20 cuttings per temperature treatment. The reservoirs
were placed in climate chambers providing a 16-h photoperiod
and a relative air humidity (RH) of 90%. The reservoirs were
covered with plastic, white on top and black underneath to
minimize algae formation. The rooting percentage and number
of roots were recorded at about 3-day intervals for 25 days.
Micropropagated plant cuttings
Growing conditions, tissue culture and rooting Seeds were
sown in the greenhouse on September 28, 1990, and the seedlings were planted in pots containing Hasselfors Master Soil
on October 15 and grown in the greenhouse in a 16-h photoperiod (160 µmol m -2 s −1) with a day/night temperature of 15/25
°C and 70% RH (Climate B16 in Table 1). The plants were
watered with Nutrient Solution II.
Micropropagated stock plants were produced according to
the method described by Welander (1988). On August 23, the
micropropagated stock plants were transferred to a 1/1 (v/v)
mixture of peat and perlite, covered with plastic beakers and
placed in the greenhouse. After one week, the beakers were
replaced by a plastic tent and the plants were hardened off by
removing the tent for successively longer periods each day.
On September 18, the micropropagated stock plants were
transferred to pots containing Hasselfors Master Soil and
grown in the greenhouse in a 16-h photoperiod (160 µmol m −2
s −1) with a day/night temperature of 15/25 °C and 70% RH
(Climate B16 in Table 1) until November 1. Forty micropropagated stock plants were then placed in each of four climate
chambers under the conditions described in Table 1.
Plant height, number of shoots and the irradiance at each
shoot used as a cutting were recorded at about 7-day intervals.
At the end of the experiment, the total irradiance each shoot
had received during the growth period was calculated, and
shoot length and number of leaves per shoot were recorded.
Rooting of cuttings from micropropagated plants Six shoots
(numbered 1, 3 and 5 on one side, and 2, 4 and 6 on the opposite
side counted from the base) from each micropropagated stock
plant were used as cuttings. The cuttings were placed in reservoirs as described for cuttings from seed stock plants. Four
reservoirs with temperatures of 16, 20, 24 and 28 °C were used
with 20 cuttings per temperature treatment. The rooting per-
Table 1. Description of the four climate treatments to which the stock plants were exposed for two months. The relative humidity was 70% and
the irradiance was 160 µmol m −2 s −1 in all of the treatments.
Climate
Photoperiod
(h)
Thermoperiod
(h)
Day/night
temperature
(°C)
Irradiance
(µmol m −2 day −1)
Average day
temperature
(°C)
A24
B24
A16
B16
24
24
16
16
16/8
16/8
16/8
16/8
25/15
15/25
25/15
15/25
3840
3840
2560
2560
21.7
18.3
25
15
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
centage and number of roots were recorded at about 3-day
intervals for 25 days.
13
Results
Shoot morphology of stock plants
Measurement of carbohydrates
During rooting, the basal 1.0 cm of the stem from three shoots
per plant was analyzed for glucose, fructose and sucrose.
Samples (30--50 mgFW) were taken at Days 0, 7, 10 and 14. The
samples were freeze dried and extracted in buffered methanol
(0.1 M acetate buffer, pH 6.6, containing methanol, 2/8 v/v) in
a water bath at 55 °C for 30 min. The extracts were shaken
several times during the day and clarified overnight. The clear
supernatant was analyzed enzymatically for glucose, fructose
and sucrose (Boehringer Mannheim, Mannheim, Germany).
Statistical analyses
Data in Figure 1 were subjected to analysis of variance with
SAS software (SAS Institute Inc., Cary, NC, USA) and
Tukey’s studentized range test for multiple comparison of
means. In Figure 3, significance was determined by Fisher’s
exact test for percent rooting and Welch’s approximate t-test in
pairs (Zar 1984) for root number. Linear regression analysis
was used for calculations of relationships between shoot
length, number of leaves per shoot and irradiance.
Figure 1 shows the average seed plant height (A) and the
average number of shoots per seed plant (B) in four different
climates including two nutrient regimes. Seed plant height in
Climate A24 was significantly higher than in Climate B24, and
seed plants in Climate A16 were significantly taller than in
Climate B16, indicating that temperature had a greater influence on seed plant height growth than daylength. The shortest,
most compact seed plants were in Climate B16 and the tallest
seed plants with the longest internodes and longest shoots were
in Climate A24 (Figure 2). Significantly more shoots were
produced in Climates A24 and B24 than in Climates A16 and
B16, indicating that number of shoots per seed plant was
influenced more by daylength than temperature. The nutrient
treatments did not affect either seed plant height or the number
of shoots.
Micropropagated stock plants were more uniform in height
(Figure 3) and number of shoots per plant (Figure 4) than
plants derived from seeds. In micropropagated plants, there
was a positive relationship between final axillary shoot length
and light intensity (r = 0.633) and between number of leaves
per shoot and light intensity (r = 0.631). In seed plants, the
Figure 1. The influence of daylength and
temperature on average seed stock plant
height (A) and average number of shoots
per seed stock plant (B). The irradiance was
160 µmol m − 2 s − 1 and plants in each climate were given either Nutrient Solution I
or II. Nutrient Solution II contained halfstrength macronutrient concentrations.
A24 = 24 h light, thermoperiod 16/8 h at
25/15 °C; B24 = 24 h light, thermoperiod
16/8 h at 15/25 °C; A16 = 16 h light, thermoperiod 16/8 h at 25/15 °C; B16 = 16 h
light, thermoperiod 16/8 h at 15/25 °C. Day
0 represents the onset of exposure to the different climates. The vertical bars denote
LSD (P ≤ 0.05).
14
WELANDER
Figure 2. The influence of various combinations of daylength and
temperature on shoot morphology of seed stock plants. Only plants
given Nutrient Solution I are shown as there were no differences in
morphology between plants given the two nutrient solutions. Abbreviations as in Figure 1.
Figure 4. Number of shoots per plant for seed plants (A) and micropropagated plants (B). Plants were watered with Nutrient Solution II
and exposed for 47 days to a 16-h photoperiod at 160 µmol m −2 s − 1
and a thermoperiod of 16/8 h at 15/25 °C.
Figure 3. Individual plant height for seed plants (A) and micropropagated plants (B). Plants were watered with Nutrient Solution II and
exposed for 47 days to a 16-h photoperiod at 160 µmol m −2 s −1 and a
thermoperiod of 16/8 h at 15/25 °C.
correlation coefficients for final shoot length and light intensity (r = 0.470) and for number of leaves and light intensity (r
=0.367)werelower.
Rooting of cuttings
The rooting ability of cuttings taken from seed stock plants
subjected to the different climates is shown in Figure 5. Variation in rooting ability within treatments was high, probably
because of genotypic differences in rooting capacity between
individual seed stock plants, and only a few treatments differed
significantly. Rooting percentage and the number of roots was
Figure 5. Percent rooting (A) and number of roots per cutting (B) as
influenced by various combinations of daylength and temperature on
seed stock plants. Abbreviations as in Figure 1. Bars with the same
letter are not significantly different (P ≤ 0.05). Percent rooting was
analyzed by Fisher’s exact test and root number was analyzed by
Welch’s approximate t-test in pairs.
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
increased by Nutrient Solution II compared with Nutrient
Solution I, and the continuous light regime inhibited rooting of
cuttings compared with the 16-h photoperiod. The highest
rooting percentage and number of roots per cutting were obtained from seed stock plants grown in Climate B16 with
Nutrient Solution II. This stock plant treatment was chosen for
pretreating the micropropagated stock plants.
Shoot length and number of leaves per shoot were unrelated
to percent rooting or number of roots per cutting. Both root
branching and root length increased with increasing rooting
temperature (Figure 6).
Genotypic differences in the rooting ability of cuttings from
seed plants were observed at all rooting temperatures. The
genotypic variations dominated the temperature effects on
rooting percentage (Figure 7A) and number of roots per rooted
cutting (Figure 7B). A temperature of 16°C increased the time
15
required for rooting (Figure 8A), but the final rooting percentage and number of roots (Figure 8B) were the same or slightly
higher compared to those in the other temperature treatments.
No differences in rooting were observed at rooting temperatures of 20, 24 and 28 °C. Cuttings from micropropagated
plants achieved optimum rooting percentage one week earlier
than cuttings from seed plants at all temperatures except 16°C.
Carbohydrate content and rooting
The analysis of fructose, glucose and sucrose in the basal 1.0
cm of the stem during rooting showed a similar pattern in
cuttings from both micropropagated trees and seed plants
(Figures 9 and 10). The fructose and glucose contents were
constant over the rooting period, whereas there was a large
accumulation of sucrose between Days 7 and 10 in all of the
temperature treatments. The endogenous carbohydrate content
Figure 6. The photographs show increased root branching and root length of
cuttings at higher temperatures. Great
genotypic differences in the rooting capacity were observed in cuttings from seed
stock plants regardless of temperatures
used in the rooting experiments. Such diversity was not observed within the micropropagated stock plants (data not shown).
(A) Two genotypes at 16 °C, (B and C)
two genotypes at 20 °C, (D and E) two
genotypes at 24 °C, and (F) two genotypes at 28 °C.
16
WELANDER
Figure 7. Effect of rooting temperature on percent rooting (A) and
number of roots (B) of cuttings from seed stock plants. (j) 16 °C, (h)
20 °C, (r) 24 °C, (e) 28 °C.
Figure 9. The amount of extractable carbohydrates in the stem base at
various times during the rooting period of cuttings from seed stock
plants. The cuttings were subjected to a rooting temperature of 16, 20,
24 or 28 °C. Glucose (j), fructose (h), sucrose (r).
Figure 8. Effect of rooting temperature on percent rooting (A) and
number of roots (B) of cuttings from micropropagated stock plants.
(j) 16 °C, (h) 20 °C, (r) 24 °C, (e) 28 °C.
Figure 10. The amount of extractable carbohydrates in the stem base
at various times during the rooting period of cuttings from micropropagated stock plants. The cuttings were subjected to a rooting
temperature of 16, 20, 24 or 28 °C. Glucose (j), fructose (h), sucrose
(r).
was almost three times higher in the stem base of the cuttings
from micropropagated plants than from seed plants (cf. Figures 9 and 10). However, differences in rooting percentage and
number of roots were not correlated with carbohydrate content.
Discussion
Differences in rooting ability were the result of both genotypic
differences and physiology of the stock plants. In this study,
the difficulty of separating environmental effects from genetic
differences was overcome by comparing cuttings from seed
plants with cuttings from micropropagated cuttings (Burdon
and Shelbourne 1973). The large variation in rooting capacity
among the seed plants (Figure 6) exemplifies the potential for
selecting clones with improved rooting characteristics
(Fancher and Tauer 1981, Foster et al. 1984).
Plant height was influenced more by the temperature treatments than by the photoperiodic regimes. A higher night than
day temperature reduced plant height, whereas a lower night
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
than day temperature stimulated stem elongation (cf. Moe and
Heins 1990). Lateral branching is also affected by daylength
(Healy et al. 1980). Although a reduced concentration of
macronutrients did not limit plant height and number of shoots
produced per stock plant, it increased the rooting ability of the
cuttings significantly. Because gibberellins inhibit rooting in a
variety of species and gibberellin content is decreased in plants
subjected to a higher night than day temperature (Erwin et al.
1989), I hypothesized that cuttings from stock plants grown in
a higher night than day temperature would root better than
cuttings grown in a lower night than day temperature. The
highest rooting percentage and number of roots was found in
cuttings taken from stock plants grown in climate B16; however, a reduction in macronutrient supply dominated the effect
of the environment. Stock plants given a reduced supply of
macronutrients showed a higher rooting capacity under all
climatic conditions. Pearse (1943) found that cuttings from
plants grown under conditions of mineral starvation rooted
easily, and Hyndman et al. (1982) showed that rooting was
enhanced by lowering the salt concentration, which was
mainly attributed to a lower nitrogen concentration.
The effects of temperature on rooting ability of cuttings
from seed plants were difficult to interpret because of genotypic differences. In contrast, the results obtained with cloned
material were unequivocal and showed that rooting was delayed at 16 °C compared with rooting at higher temperatures,
but the final rooting percentage and number of roots were
similar within the temperature range of 16 to 28 °C. However,
root branching was greater at 20--28 °C than at 16 °C. Moore
et al. (1975) reported an increase in the time required for
rooting at low temperatures, and Takahaski et al. (1981) found
that the final number of roots is higher at higher temperatures,
which might be a consequence of a longer period available for
root initiation.
Cuttings generally accumulate soluble carbohydrates at
their bases during the early stages of root formation (Middleton et al. 1980, Veierskov et al. 1982b). The rapid accumulation
of sucrose indicates that rooting is an energy demanding process but the amount of carbohydrate required for this process is
not known. I observed a large accumulation of sucrose at the
base of the cutting before any roots were visible. In cuttings of
Pinus banksiana Lamb. (Haissig 1984) and pea (Pisum sativum L.) (Veirskov 1988), rapid accumulation of carbohydrates
was observed two days after propagation. Jarvis (1986) suggested that adventitious rooting is dependent on sugar transported from the stele and not on sugar from the surrounding
tissues. This suggestion is supported by the finding that there
were only minor changes in nontranslocatable reducing sugars
in birch cuttings in this study. The carbohydrate content of
cuttings from micropropagated plants was about three times
higher than that of cuttings from seed plants; however, the
higher carbohydrate content was not correlated with a higher
rooting potential. Based on this observation and the finding
that the increase in carbohydrate content was similar at all
temperatures although rooting was delayed in the low temperature treatment, I conclude that carbohydrates do not control
root initiation in birch cuttings. The large accumulation at the
17
base of the cuttings indicates the need for an adequate supply
of carbohydrates during rooting. Thus, sugar availability can
limit adventitious rooting in leafless or defoliated cuttings
(Fabijan et al. 1981) or if carbohydrates are depleted in the
stock plant (Veierskov et al. 1976).
Acknowledgments
The author is indebted to Lena Persson and Johnny Swahn for skillful
technical assistance and Georgios Iliadis and Jan Englund for statistical treatment of the results. The financial support of the Swedish
Council for Forestry and Agricultural Research is gratefully acknowledged.
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herbaceous cutting propagation in Chrysanthemum morifolium. I.
Influence of temperatures on the increasing rate of adventitious
root. Sci. Bull. Fac. Agr. Kyushu Univ. 35:1--4.
Veierskov, B. 1988. Relation between carbohydrates and adventitious
root formation. In Adventitous Root Formation in Cuttings. Eds.
T.D. Davis, B.E. Haissig and N. Sankhla. Dioscorides Press, Portland, Oregon, pp 70--78.
Veierskov, B. and S.A. Andersen. 1982. Dynamics of extractable
carbohydrates in Pisum sativum. III. The effect of IAA on content
and translocation of carbohydrates in pea cuttings during rooting.
Physiol. Plant. 55:179--182.
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extractable carbohydrates in Pisum sativum. I. Carbohydrate and
nitrogen content in pea plants and cuttings grown at two different
irradiances. Physiol. Plant. 55:167--173.
Veierskov, B., J. Hansen and S. Andersen. 1976. Influence of cotyledon excision and sucrose on root formation in pea cuttings. Physiol.
Plant. 36:105--109.
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1982b. Dynamics of extractable carbohydrates in Pisum sativum. II.
Carbohydrate content and photosynthesis of pea cuttings in relation
to irradiance and stockplant temperature and genotype. Physiol.
Plant. 5:174--178.
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York, London, pp 79--99.
Ying, C.C. and W.T. Bagley. 1977. Variation in rooting capability of
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© 1994 Heron Publishing----Victoria, Canada
Influence of environment, fertilizer and genotype on shoot morphology
and subsequent rooting of birch cuttings
MARGARETA WELANDER
Swedish University of Agricultural Sciences, Department of Horticulture, Box 55, S-230 53 Alnarp, Sweden
Received June 30, 1993
Summary Differences in rooting ability of birch (Betula
pubescens J.F. Ehrh.) cuttings were observed as a result of
differences in genotype and physiology of the stock plants. The
uniformity in response among cuttings from micropropagated
plants compared with cuttings from seed plants confirmed the
advantage of using micropropagated plants to study environmental effects. Shoot morphology of the seed stock plants was
influenced by both photoperiod and thermoperiod. A day/night
temperature of 15/25 °C reduced stem elongation compared
with a day/night temperature of 25/15 °C regardless of photoperiod, and a continuous light regime resulted in more shoots
per plant in both temperature regimes than a 16-h photoperiod.
A reduction in the supply of macronutrients did not influence
shoot morphology, but increased rooting substantially and
seemed to override the effects of environmental factors. In
cuttings of seed plants, the highest rooting percentage and
number of roots were obtained in a 16-h photoperiod with a
day/night temperature of 15/25 °C. In micropropagated stock
plants, there was a positive correlation between shoot length
and number of leaves per shoot and topographical distribution
of light within the plants, but there was no correlation between
these parameters and rooting ability of the cuttings. A rooting
temperature of 16 °C delayed the rate of root production
compared with the rate at higher temperatures, but the final
rooting percentage was the same over the range from 16 to
28 °C. Root branching increased with temperature. At all temperatures, there was a large increase in sucrose content at the
base of the cuttings during rooting, whereas the concentration
of nontranslocated sugars remained constant. The carbohydrate
content at the base of cuttings from micropropagated stock
plants was three times higher than at the base of cuttings from
seed stock plants, but the higher carbohydrate content was not
correlated with a higher rooting potential.
Keywords: Betula pubescens, hydroponic system, micropropagation, photoperiod, temperature, thermoperiod.
Introduction
Seasonal variations in rooting ability of cuttings emphasize the
importance of identifying the physiological status of the stock
plant when cuttings are taken (Nanda and Anand 1970, Anand
and Heberlein 1975, Hansen and Ernstsen 1982). The opportunity to manipulate the rooting potential of cuttings is generally much greater when favorable conditions are given to the
stock plants rather than the cuttings taken from them (Howard
and Harrison-Murray 1988, Howard 1991). The mineral nutrient status of the stock plant affects rooting; a low N supply
improves rooting (Haun and Cornell 1951, Preston et al. 1953).
Photoperiod (Bachelard and Stove 1963, Heide 1965, Nanda
et al. 1967) and irradiance (Fisher and Hansen 1977, Poulsen
and Andersen 1980) also influence the rooting ability of many
plants. Although irradiance has not always been associated
with high rooting potential (Okoro and Grace 1976, Hansen et
al. 1978, Veierskov and Andersen 1982, Veierskov et al.
1982a), it has been used to change the carbohydrate content of
cuttings. The need for carbohydrates during root initiation was
first demonstrated by Borthwick et al. (1937) and has been
verified by Bhattacharya et al. (1976). However, the amount of
carbohydrate required in this process is not known, and the
relationship between carbohydrates and adventitious root formation is unclear (Haissig 1984, Veierskov 1988).
The optimal root temperature for rooting cuttings is difficult
to determine, because the effects of temperature are often
associated with other features of the propagation environment
and with genotype (Andersen 1986).
There is substantial evidence that rooting ability is genetically controlled (Haissig and Riemenschneider 1988). Clonal
differences in rooting ability of cuttings from several forest
tree species have been reported (Dunn and Townsend 1954,
Ying and Bagley 1977, Pounders and Foster 1992). However,
it is important in genetic studies of rooting that confounding
environmental effects are separated and properly controlled.
One way to separate the nongenetic effects of the stock plant
from the genetic effects is to compare cuttings from seed stock
plants with cuttings from micropropagated stock plants grown
under controlled conditions.
I have studied the effects of photoperiod, thermoperiod and
fertilizer on shoot morphology and subsequent rooting of birch
cuttings, and genotypic differences in rooting ability by comparing seed plants with micropropagated plants. I also examined whether the topographical distribution of light within the
plants was related to shoot morphology and subsequent rooting. The influence of temperature during rooting in a hydroponic system was examined and attempts were made to
12
WELANDER
correlate rooting capacity of cuttings from cloned material and
seed plants to the amount of carbohydrate in the stem base of
the cuttings during rooting.
Material and methods
Seed plant cuttings
Seed collection and growing conditions In 1987, seeds were
collected from an early flowering genotype of Betula pubescens J.F. Ehrh., growing at the University College of Wales, and
brought to Sweden. The seeds were sown in a greenhouse and
the seedlings were planted in a field in July 1988. Seeds from
these trees were harvested in September 1989, sown in the
greenhouse on October 16, 1989, and then planted in pots
containing Hasselfors Master Soil 14 days later. The seedlings
were grown in the greenhouse in natural daylength at 18 °C
until February 2, 1990. The plants, which were then approximately 30 cm tall, were selected to serve as seed stock plants
based on uniformity and placed in four climate chambers under
the conditions described in Table 1. Irradiance, measured at
plant level, was supplied by a combination of incandescent and
cool-white fluorescent lamps. Each climate chamber contained
28 seed stock plants and half of the plants were watered with
Nutrient Solution I (macronutrients (g l −1) K2PO4 13.6, KNO3
20.6, Ca(NO3)2 ⋅ 4H2O 23.6, Fe-Na-EDTA 7.3, MgSO4 ⋅ 7H2O
25.0 (diluted 1/100); micronutrients (mg l −1) MnSO4 ⋅ 4H2O
89.0, CuSO4 ⋅ 5H2O 12.0, Na2MoO4 ⋅ 2H2O 0.2, ZnCl2 6.0,
H3BO3 24.7 (diluted 1/1000)) and the other half with Nutrient
Solution II (identical to Nutrient Solution I except that macronutrient concentrations were diluted 1/200). Time of transfer
to the climate chambers is referred to as Day 0 in the figures.
Plant height, number of shoots per seed stock plant and the
irradiance at each shoot used as a cutting were recorded at
about 7-day intervals for two months. At the end of the experiment the total irradiance that each shoot had received during
the growth period was calculated, and shoot length and number
of leaves per shoot were recorded. In April 1990, four seed
stock plants approximately 100 cm in height were randomly
selected from each climate chamber for rooting experiments.
Rooting experiments with cuttings from seed plants
Five
shoots per seed stock plant (numbered 1--5 counted from the
base) were used as cuttings. The cuttings were trimmed to a
final length of 15 cm and placed in reservoirs containing
Nutrient Solution II. Air was bubbled through the reservoirs.
Four reservoirs with temperatures of 16, 20, 24 and 28 °C were
used with 20 cuttings per temperature treatment. The reservoirs
were placed in climate chambers providing a 16-h photoperiod
and a relative air humidity (RH) of 90%. The reservoirs were
covered with plastic, white on top and black underneath to
minimize algae formation. The rooting percentage and number
of roots were recorded at about 3-day intervals for 25 days.
Micropropagated plant cuttings
Growing conditions, tissue culture and rooting Seeds were
sown in the greenhouse on September 28, 1990, and the seedlings were planted in pots containing Hasselfors Master Soil
on October 15 and grown in the greenhouse in a 16-h photoperiod (160 µmol m -2 s −1) with a day/night temperature of 15/25
°C and 70% RH (Climate B16 in Table 1). The plants were
watered with Nutrient Solution II.
Micropropagated stock plants were produced according to
the method described by Welander (1988). On August 23, the
micropropagated stock plants were transferred to a 1/1 (v/v)
mixture of peat and perlite, covered with plastic beakers and
placed in the greenhouse. After one week, the beakers were
replaced by a plastic tent and the plants were hardened off by
removing the tent for successively longer periods each day.
On September 18, the micropropagated stock plants were
transferred to pots containing Hasselfors Master Soil and
grown in the greenhouse in a 16-h photoperiod (160 µmol m −2
s −1) with a day/night temperature of 15/25 °C and 70% RH
(Climate B16 in Table 1) until November 1. Forty micropropagated stock plants were then placed in each of four climate
chambers under the conditions described in Table 1.
Plant height, number of shoots and the irradiance at each
shoot used as a cutting were recorded at about 7-day intervals.
At the end of the experiment, the total irradiance each shoot
had received during the growth period was calculated, and
shoot length and number of leaves per shoot were recorded.
Rooting of cuttings from micropropagated plants Six shoots
(numbered 1, 3 and 5 on one side, and 2, 4 and 6 on the opposite
side counted from the base) from each micropropagated stock
plant were used as cuttings. The cuttings were placed in reservoirs as described for cuttings from seed stock plants. Four
reservoirs with temperatures of 16, 20, 24 and 28 °C were used
with 20 cuttings per temperature treatment. The rooting per-
Table 1. Description of the four climate treatments to which the stock plants were exposed for two months. The relative humidity was 70% and
the irradiance was 160 µmol m −2 s −1 in all of the treatments.
Climate
Photoperiod
(h)
Thermoperiod
(h)
Day/night
temperature
(°C)
Irradiance
(µmol m −2 day −1)
Average day
temperature
(°C)
A24
B24
A16
B16
24
24
16
16
16/8
16/8
16/8
16/8
25/15
15/25
25/15
15/25
3840
3840
2560
2560
21.7
18.3
25
15
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
centage and number of roots were recorded at about 3-day
intervals for 25 days.
13
Results
Shoot morphology of stock plants
Measurement of carbohydrates
During rooting, the basal 1.0 cm of the stem from three shoots
per plant was analyzed for glucose, fructose and sucrose.
Samples (30--50 mgFW) were taken at Days 0, 7, 10 and 14. The
samples were freeze dried and extracted in buffered methanol
(0.1 M acetate buffer, pH 6.6, containing methanol, 2/8 v/v) in
a water bath at 55 °C for 30 min. The extracts were shaken
several times during the day and clarified overnight. The clear
supernatant was analyzed enzymatically for glucose, fructose
and sucrose (Boehringer Mannheim, Mannheim, Germany).
Statistical analyses
Data in Figure 1 were subjected to analysis of variance with
SAS software (SAS Institute Inc., Cary, NC, USA) and
Tukey’s studentized range test for multiple comparison of
means. In Figure 3, significance was determined by Fisher’s
exact test for percent rooting and Welch’s approximate t-test in
pairs (Zar 1984) for root number. Linear regression analysis
was used for calculations of relationships between shoot
length, number of leaves per shoot and irradiance.
Figure 1 shows the average seed plant height (A) and the
average number of shoots per seed plant (B) in four different
climates including two nutrient regimes. Seed plant height in
Climate A24 was significantly higher than in Climate B24, and
seed plants in Climate A16 were significantly taller than in
Climate B16, indicating that temperature had a greater influence on seed plant height growth than daylength. The shortest,
most compact seed plants were in Climate B16 and the tallest
seed plants with the longest internodes and longest shoots were
in Climate A24 (Figure 2). Significantly more shoots were
produced in Climates A24 and B24 than in Climates A16 and
B16, indicating that number of shoots per seed plant was
influenced more by daylength than temperature. The nutrient
treatments did not affect either seed plant height or the number
of shoots.
Micropropagated stock plants were more uniform in height
(Figure 3) and number of shoots per plant (Figure 4) than
plants derived from seeds. In micropropagated plants, there
was a positive relationship between final axillary shoot length
and light intensity (r = 0.633) and between number of leaves
per shoot and light intensity (r = 0.631). In seed plants, the
Figure 1. The influence of daylength and
temperature on average seed stock plant
height (A) and average number of shoots
per seed stock plant (B). The irradiance was
160 µmol m − 2 s − 1 and plants in each climate were given either Nutrient Solution I
or II. Nutrient Solution II contained halfstrength macronutrient concentrations.
A24 = 24 h light, thermoperiod 16/8 h at
25/15 °C; B24 = 24 h light, thermoperiod
16/8 h at 15/25 °C; A16 = 16 h light, thermoperiod 16/8 h at 25/15 °C; B16 = 16 h
light, thermoperiod 16/8 h at 15/25 °C. Day
0 represents the onset of exposure to the different climates. The vertical bars denote
LSD (P ≤ 0.05).
14
WELANDER
Figure 2. The influence of various combinations of daylength and
temperature on shoot morphology of seed stock plants. Only plants
given Nutrient Solution I are shown as there were no differences in
morphology between plants given the two nutrient solutions. Abbreviations as in Figure 1.
Figure 4. Number of shoots per plant for seed plants (A) and micropropagated plants (B). Plants were watered with Nutrient Solution II
and exposed for 47 days to a 16-h photoperiod at 160 µmol m −2 s − 1
and a thermoperiod of 16/8 h at 15/25 °C.
Figure 3. Individual plant height for seed plants (A) and micropropagated plants (B). Plants were watered with Nutrient Solution II and
exposed for 47 days to a 16-h photoperiod at 160 µmol m −2 s −1 and a
thermoperiod of 16/8 h at 15/25 °C.
correlation coefficients for final shoot length and light intensity (r = 0.470) and for number of leaves and light intensity (r
=0.367)werelower.
Rooting of cuttings
The rooting ability of cuttings taken from seed stock plants
subjected to the different climates is shown in Figure 5. Variation in rooting ability within treatments was high, probably
because of genotypic differences in rooting capacity between
individual seed stock plants, and only a few treatments differed
significantly. Rooting percentage and the number of roots was
Figure 5. Percent rooting (A) and number of roots per cutting (B) as
influenced by various combinations of daylength and temperature on
seed stock plants. Abbreviations as in Figure 1. Bars with the same
letter are not significantly different (P ≤ 0.05). Percent rooting was
analyzed by Fisher’s exact test and root number was analyzed by
Welch’s approximate t-test in pairs.
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
increased by Nutrient Solution II compared with Nutrient
Solution I, and the continuous light regime inhibited rooting of
cuttings compared with the 16-h photoperiod. The highest
rooting percentage and number of roots per cutting were obtained from seed stock plants grown in Climate B16 with
Nutrient Solution II. This stock plant treatment was chosen for
pretreating the micropropagated stock plants.
Shoot length and number of leaves per shoot were unrelated
to percent rooting or number of roots per cutting. Both root
branching and root length increased with increasing rooting
temperature (Figure 6).
Genotypic differences in the rooting ability of cuttings from
seed plants were observed at all rooting temperatures. The
genotypic variations dominated the temperature effects on
rooting percentage (Figure 7A) and number of roots per rooted
cutting (Figure 7B). A temperature of 16°C increased the time
15
required for rooting (Figure 8A), but the final rooting percentage and number of roots (Figure 8B) were the same or slightly
higher compared to those in the other temperature treatments.
No differences in rooting were observed at rooting temperatures of 20, 24 and 28 °C. Cuttings from micropropagated
plants achieved optimum rooting percentage one week earlier
than cuttings from seed plants at all temperatures except 16°C.
Carbohydrate content and rooting
The analysis of fructose, glucose and sucrose in the basal 1.0
cm of the stem during rooting showed a similar pattern in
cuttings from both micropropagated trees and seed plants
(Figures 9 and 10). The fructose and glucose contents were
constant over the rooting period, whereas there was a large
accumulation of sucrose between Days 7 and 10 in all of the
temperature treatments. The endogenous carbohydrate content
Figure 6. The photographs show increased root branching and root length of
cuttings at higher temperatures. Great
genotypic differences in the rooting capacity were observed in cuttings from seed
stock plants regardless of temperatures
used in the rooting experiments. Such diversity was not observed within the micropropagated stock plants (data not shown).
(A) Two genotypes at 16 °C, (B and C)
two genotypes at 20 °C, (D and E) two
genotypes at 24 °C, and (F) two genotypes at 28 °C.
16
WELANDER
Figure 7. Effect of rooting temperature on percent rooting (A) and
number of roots (B) of cuttings from seed stock plants. (j) 16 °C, (h)
20 °C, (r) 24 °C, (e) 28 °C.
Figure 9. The amount of extractable carbohydrates in the stem base at
various times during the rooting period of cuttings from seed stock
plants. The cuttings were subjected to a rooting temperature of 16, 20,
24 or 28 °C. Glucose (j), fructose (h), sucrose (r).
Figure 8. Effect of rooting temperature on percent rooting (A) and
number of roots (B) of cuttings from micropropagated stock plants.
(j) 16 °C, (h) 20 °C, (r) 24 °C, (e) 28 °C.
Figure 10. The amount of extractable carbohydrates in the stem base
at various times during the rooting period of cuttings from micropropagated stock plants. The cuttings were subjected to a rooting
temperature of 16, 20, 24 or 28 °C. Glucose (j), fructose (h), sucrose
(r).
was almost three times higher in the stem base of the cuttings
from micropropagated plants than from seed plants (cf. Figures 9 and 10). However, differences in rooting percentage and
number of roots were not correlated with carbohydrate content.
Discussion
Differences in rooting ability were the result of both genotypic
differences and physiology of the stock plants. In this study,
the difficulty of separating environmental effects from genetic
differences was overcome by comparing cuttings from seed
plants with cuttings from micropropagated cuttings (Burdon
and Shelbourne 1973). The large variation in rooting capacity
among the seed plants (Figure 6) exemplifies the potential for
selecting clones with improved rooting characteristics
(Fancher and Tauer 1981, Foster et al. 1984).
Plant height was influenced more by the temperature treatments than by the photoperiodic regimes. A higher night than
day temperature reduced plant height, whereas a lower night
GENETIC AND C EFFECTS ON SHOOT MORPHOLOGY AND ROOTING
than day temperature stimulated stem elongation (cf. Moe and
Heins 1990). Lateral branching is also affected by daylength
(Healy et al. 1980). Although a reduced concentration of
macronutrients did not limit plant height and number of shoots
produced per stock plant, it increased the rooting ability of the
cuttings significantly. Because gibberellins inhibit rooting in a
variety of species and gibberellin content is decreased in plants
subjected to a higher night than day temperature (Erwin et al.
1989), I hypothesized that cuttings from stock plants grown in
a higher night than day temperature would root better than
cuttings grown in a lower night than day temperature. The
highest rooting percentage and number of roots was found in
cuttings taken from stock plants grown in climate B16; however, a reduction in macronutrient supply dominated the effect
of the environment. Stock plants given a reduced supply of
macronutrients showed a higher rooting capacity under all
climatic conditions. Pearse (1943) found that cuttings from
plants grown under conditions of mineral starvation rooted
easily, and Hyndman et al. (1982) showed that rooting was
enhanced by lowering the salt concentration, which was
mainly attributed to a lower nitrogen concentration.
The effects of temperature on rooting ability of cuttings
from seed plants were difficult to interpret because of genotypic differences. In contrast, the results obtained with cloned
material were unequivocal and showed that rooting was delayed at 16 °C compared with rooting at higher temperatures,
but the final rooting percentage and number of roots were
similar within the temperature range of 16 to 28 °C. However,
root branching was greater at 20--28 °C than at 16 °C. Moore
et al. (1975) reported an increase in the time required for
rooting at low temperatures, and Takahaski et al. (1981) found
that the final number of roots is higher at higher temperatures,
which might be a consequence of a longer period available for
root initiation.
Cuttings generally accumulate soluble carbohydrates at
their bases during the early stages of root formation (Middleton et al. 1980, Veierskov et al. 1982b). The rapid accumulation
of sucrose indicates that rooting is an energy demanding process but the amount of carbohydrate required for this process is
not known. I observed a large accumulation of sucrose at the
base of the cutting before any roots were visible. In cuttings of
Pinus banksiana Lamb. (Haissig 1984) and pea (Pisum sativum L.) (Veirskov 1988), rapid accumulation of carbohydrates
was observed two days after propagation. Jarvis (1986) suggested that adventitious rooting is dependent on sugar transported from the stele and not on sugar from the surrounding
tissues. This suggestion is supported by the finding that there
were only minor changes in nontranslocatable reducing sugars
in birch cuttings in this study. The carbohydrate content of
cuttings from micropropagated plants was about three times
higher than that of cuttings from seed plants; however, the
higher carbohydrate content was not correlated with a higher
rooting potential. Based on this observation and the finding
that the increase in carbohydrate content was similar at all
temperatures although rooting was delayed in the low temperature treatment, I conclude that carbohydrates do not control
root initiation in birch cuttings. The large accumulation at the
17
base of the cuttings indicates the need for an adequate supply
of carbohydrates during rooting. Thus, sugar availability can
limit adventitious rooting in leafless or defoliated cuttings
(Fabijan et al. 1981) or if carbohydrates are depleted in the
stock plant (Veierskov et al. 1976).
Acknowledgments
The author is indebted to Lena Persson and Johnny Swahn for skillful
technical assistance and Georgios Iliadis and Jan Englund for statistical treatment of the results. The financial support of the Swedish
Council for Forestry and Agricultural Research is gratefully acknowledged.
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