Determining nitrate and ammonium requirements for optimal in vitro
response of diverse pear species
Wada, S., Niedz, R. P., & Reed, B. M. (2015). Determining nitrate and ammonium
requirements for optimal in vitro response of diverse pear species. In Vitro Cellular
& Developmental Biology - Plant, 51(1), 19-27. doi:10.1007/s11627-015-9662-4
10.1007/s11627-015-9662-4
Springer
Version of Record
http://cdss.library.oregonstate.edu/sa-termsofuse
In Vitro Cell.Dev.Biol.—Plant (2015) 51:19–27
DOI 10.1007/s11627-015-9662-4
MICROPROPAGATION
Determining nitrate and ammonium requirements for optimal
in vitro response of diverse pear species
Sugae Wada & Randall P. Niedz & Barbara M. Reed
Received: 28 March 2014 / Accepted: 7 January 2015 / Published online: 23 January 2015 / Editor: John Finer
# The Society for In Vitro Biology 2015
Abstract Inorganic nitrate (NO3−) and ammonium (NH4+) are
the two major components in nitrogen (N) nutrition of typical
tissue culture growth media, and the total amounts and ratios
influence both shoot induction and differentiation. This study
was designed to determine the optimal N requirements and
interactions of NH4+ ×NO3− to complete the optimization of a
pear shoot culture medium. Pyrus communis ‘Horner 51’ and
‘OH× F 87’, P. cordata, P. pyrifolia ‘Sion Szu Mi’, and
P. ussuriensis ‘Hang Pa Li’ from the pear germplasm collection
of the US Department of Agriculture, National Clonal
Germplasm Repository–Corvallis (NCGR) were evaluated.
Response surface design was used to create and analyze treatment combinations of NH4+, K+, and NO3−. Cultures were
evaluated for overall quality, shoot length, multiplication, leaf
color and size, leaf spotting and necrosis, and callus production.
Significant improvement was observed in multiplication and
length for most genotypes. Reduced callus amounts were seen
in two genotypes, and greener leaves were also seen in two
genotypes. Each species had a distinct response, and the N form
could be manipulated to produce longer shoots, more shoots, or
less callus. For the best-quality shoots, both P. communis
S. Wada (*)
Department of Horticulture, Oregon State University,
4017 Agriculture & Life Science Bldg, Corvallis,
OR 97331-7304, USA
e-mail: Sugae.Wada@Oregonstate.edu
R. P. Niedz
US Department of Agriculture, Agricultural Research Service,
US Horticultural Research Laboratory, 2001 South Rock Road,
Ft. Pierce, FL 34945-3030, USA
e-mail: Randall.Niedz@ars.usda.gov
B. M. Reed
US Department of Agriculture, Agricultural Research Service,
National Clonal Germplasm Repository, 33447 Peoria Rd.,
Corvallis, OR 97333-2521, USA
e-mail: Barbara.Reed@ars.usda.gov
cultivars required high NO3− and low to moderate NH4+,
P. cordata quality was best with high NO3− and NH4+,
P. pyrifolia ‘Sion Szu Mi’ quality improved with moderate
NO3− and high NH4+, and P. ussuriensis ‘Hang Pa Li’ required
low NO3− and high NH4+. This study illustrates that optimizing
the N components of a growth medium is very important when
working with diverse plant germplasm.
Keywords Growth medium . Micropropagation . Mineral
nutrition . Nitrogen . Pyrus . Response surface design
Introduction
Nitrogen (N) is a key macronutrient for plant growth and
development for the production of proteins, nucleic acids,
and secondary metabolites (Engelsberger and Schulze 2012).
Nitrate (NO3−) and ammonium (NH4+) are two major components in the N pool of typical growth media, and total amounts
of N and ratios of NH4+ to NO3− influence induction and
differentiation depending on the genotype (Ramage and
Williams 2002). Some plant species show a strong preference
for uptake of specific N ions (Forde et al. 1999). Availability
and uptake of N greatly affects pear tree growth and productivity in the field as well (Mitcham and Elkins 2007).
The amounts and ratios of NO3− to NH4+ influence the
initiation, growth, and differentiation of plant cell cultures
(Grimes and Hodges 1990; Leblay et al. 1991; Ramage and
Williams 2002; Niedz and Evens 2008). Responses of plant
tissues, in terms of secondary metabolites, proteins, organic
acids, and plant hormones, are greatly influenced by the forms
of N in growth media (Preece 1995), and these changes influence somatic embryo growth and development as well
(Mordhorst and Lörz 1993). Additionally, N nutrition affects
a wide range of physiological processes such as photosynthesis, electron transport, and anthocyanin production (Guidi
20
WADA ET AL.
et al. 1997; Jain et al. 1999; Sotiropoulos et al. 2005). Distinct
physiological processes may change depending on the N form
available to the plant. Many processes in Arabidopsis seedlings are influenced by the concentration of NO3− or NH4+ in
the growth medium (Engelsberger and Schulze 2012).
Nitrate is the major form of N for most plant tissue cultures.
Tobacco was cultured using NO3− as the only source of N for
growth and morphogenesis; NH4+ as the only N source resulted
in negative effects (Cousson and Van 1993). Nitrate controls the
expression of genes involved in carbon metabolism of
Nicotiana plumbaginifolia L. (Scheible et al. 1997; Vincentz
et al. 2011). Some species, such as rice (Oryza sativa L;
Grimes and Hodges 1990) and common heather (Calluna
vulgaris L.; Troelstra et al. 1995), utilize NH4+ as well as
NO3−. Ammonium can also be used as the only N source for
Ipomoea and soybean (Glycine max (L.) Merr.; Martin et al.
1977) or wild carrot (Daucus carota L.; Dougall and
Weyrauch 1980) if the medium pH is adjusted during the culture
period.
Our laboratory began to utilize response surface methodology (Niedz and Evens 2007) for pear medium development and
identified the mesos (CaCl2, MgSO4, and KH2PO4) components of Murashige and Skoog (MS) medium (Murashige and
Skoog 1962) as the most important driving factor for optimal
in vitro pear growth; N was the second most important factor
(Reed et al. 2013b). Modification of the three mesos components (Wada et al. 2013), as well as increases in the total amount
of these compounds, greatly impacted the growth and quality of
a diverse group of pear germplasm in the collection of the
National Clonal Germplasm Repository–Corvallis (NCGR).
Nitrogen may be more important for improving the quality
and growth of the species that had a limited response to the
mesos components. In order to complete optimization of mineral nutrients for culture of a wide range of pear species, the final
step was to determine optimal N content and the correct
amounts of NH4+ and NO3− for shoot cultures grown on the
partially optimized Pear 1 medium (MS with 1.5× mesos components; Reed et al. 2013a). This study was designed to elucidate N requirements in pear medium for a wide range of pear
germplasm utilizing a response surface approach.
Materials and Methods
Culture conditions. Five representative genotypes were studied from four species in the NCGR pear germplasm collection
(Table 1). Shoot cultures were grown in GA-7 containers
(Magenta Corp., Chicago, IL) with 40 ml medium per container. The base medium was Pear 1 medium (Reed et al.
2013a) composed of MS (Murashige and Skoog 1962) mineral salts with 1.5× mesos (CaCl2, MgSO4, and KH2PO4),
selected from an earlier experiment (Reed et al. 2013a), and
with the following per liter: 25 mg thiamine, 250 mg inositol,
Table 1. Plant introduction (PI) number (US National Plant Germplasm
System), local identifying number, genotype, and species of five pear shoot
cultures evaluated for response to NO3− and NH4+ in the growth medium
PI number
Local
Genotype
Species
657931
2933.001
Horner 51
P. communis L.
541415
1345.002
OH×F 87
P. communis L.
541591
1589.001
P. cordata
P. cordata Desv.
289525
532.002
Sion Szu Mi
P. pyrifolia Burm.
315064
268.001
Hang Pa Li
P. ussuriensis M.
30 g sucrose, 4.4 μM N6-benzyladenine (PhytoTechnology
Laboratories, Shawnee Mission, KS), 0.3% agar (Phytotech
A111, PhytoTechnology Labs), and 0.17% Gelrite (Culture
Gel Type I—BioTech Grade, PhytoTechnology Labs) at pH
5.7 and autoclaved (20 min, 121°C). Shoots were transferred
to new medium every 3 wk. Cultures were grown at 25°C
under a 16-h photoperiod with an average 80 μM m−2 s−1
irradiance provided by a combination of cool- and warmwhite fluorescent bulbs.
Experimental approach. The study was designed to determine the effects and optimal combinations and concentrations
of the N-salt nutrients (NH4+, NO3−, and K+) in two stock
solutions (NH 4 NO 3 and KNO 3 ) of Pear 1 medium
Table 2. Response surface design treatments for testing pear shoot
cultures for optimum NH4+: K+ and NO3− in Pear 1, a high mesos
(1.5×) MS-based medium
Treatment
NH4+ mM (ratio)
K+ mM (ratio)
NO3− (mM)
1
2
29.17 (0.58)
15.00 (0.75)
20.83 (0.42)
5.00 (0.25)
50.00
20.00
3
4
5
6
7
8
9
10
11x
12
13
14
15
16
17
18
19
10.00 (0.50)
10.00 (0.50)
15.00 (0.25)
15.00 (0.25)
5.00 (0.25)
15.00 (0.75)
45.00 (0.75)
30.00 (0.50)
20.00 (0.50)
12.50 (0.42)
10.00 (0.50)
30.00 (0.75)
22.50 (0.75)
45.00 (0.75)
20.83 (0.42)
12.50 (0.25)
5.00 (0.25)
10.00 (0.50)
30.00 (0.75)
45.00 (0.75)
45.00 (0.75)
15.00 (0.75)
5.00 (0.25)
15.00 (0.25)
30.00 (0.50)
20.00 (0.50)
17.50 (0.58)
10.00 (0.50)
10.00 (0.25)
7.50 (0.25)
15.00 (0.25)
29.17 (0.58)
37.50 (0.75)
15.00 (0.75)
20.00
40.00
60.00
60.00
20.00
20.00
60.00
60.00
40.00
30.00
20.00
40.00
30.00
60.00
50.00
50.00
20.00
x
Control, MS concentrations of NH4+ : K+ and NO3− in Pear 1 medium
NITROGEN REQUIREMENTS FOR IN VITRO PEARS
(Table 2). The factors were set up as a two-component
mixture-amount study of the proportions of NH4+ and
K+ and the mM concentration of NO3−. Inclusion of the
K+ ion is required as it is an integral part of the salt
being tested. Sufficient points were selected for quadratic modeling (Table 2). The amount of NH4+ plus K+ (in
mM) was equal to the amount of NO3− for each treatment so that the pH was equivalent across the design
space tested. The experiment was designed with 19
treatment design points with internal replication. ARSMedia software was used to remove any ion confounding (Niedz and Evens 2006; http://www.ars.usda.gov/
services/software/download.htm?softwareid=148).
Treatment points were designed to sample the range of
possible treatment combinations for modeling responses. For
each genotype five shoots, each with two nodes (approximately 10 mm), with the apical section removed, was planted in
duplicate GA-7 containers (n=10). Five replicated points
(Treatments [Trt] 2 and 8, 3 and 13, 5 and 6, 7 and 19, and 9
and 16) were included as a second set of duplicate GA-7
containers, each containing five shoots (Table 2). The control
had the same N composition as MS (20 mM NH 4 + ,
20 mM K+, and 40 mM NO3−) but in the Pear 1 medium
(Trt 11; Table 2). The amounts of each component were calculated as follows with Trt 2 as an example: 15 mM NH4+
(0.75×20 mM), 5 mM K+ (0.25×20 mM), and 20 mM NO3−.
Culture containers were assigned random numbers for arrangement on the growth room shelf. Each group was
Table 3.
21
transferred to a fresh box of the same treatment at 3-wk intervals. Shoots were harvested after three culture periods of 3 wk
each (9 wk total).
Data included. Factors evaluated included a subjective rating
of quality (1 = poor quality, 2 = acceptable quality, 3 = good
quality; Niedz and Evens 2007), shoot length (longest shoot
measured in mm), shoot multiplication (number of shoots
counted), leaf color (1 = healthy green, 2 = pale green or
yellow, 3 = pink-edged, 4 = red or brown), leaf spotting/
necrosis (1 = absent, 2 = minor, 3 = major), callus (1 = absent,
2 = callus ≤3 mm diameter, 3 = callus >3 mm diameter) and
leaf size (1 = small, 2 = medium, 3 = large). Three shoots were
sampled from each box in a predetermined pattern (two from
the corners and one center plant on a diagonal from the label)
to avoid subjective selection (n=6). The remainder of the
shoots were photographed (n=4). Design-Expert software optimization criteria were set as follows: quality and shoot
length=maximum; shoot number=3; leaf size=2; and callus,
leaf spot, and leaf color = minimum.
Statistical analysis. Experimental design and point selection,
polynomial equations, analysis of variance (ANOVA), and
graphical displays were calculated using Design-Expert 8 software (Design-Expert 2010). The experimental design was a
response surface design with a two-component mixture.
Design points that were selected were suitable for fitting a
quadratic polynomial (Niedz and Evens 2006, 2007; Evens
P values for models and factors involved in the significant responses (P≤0.05) of five pear genotypes to medium nitrogen components
Genotype
Factor
Quality
Shoot number
Shoot length
Callus
Leaf size
Leaf spots
Leaf color
‘Horner 51’
(P. communis)
Model
NH4+: K+
NH4+ ×NO3−
K+ ×NO3−
Model
NH4+:K+
NH4+ ×NO3−
K+ ×NO3−
Model
NH4+:K+
NH4+ ×NO3−
K+ ×NO3−
Model
0.0100
NS
NS
NS
0.0116
NS
NS
0.0047
0.0055
NS
0.0144
NS
0.0045
0.0339
NS
NS
NS
0.0001
0.0018
NS
0.0026
0.0154
NS
NS
NS
<0.0001
0.0036
NS
0.0361
0.0043
0.0005
NS
NS
0.0006
<0.0001
NS
0.0005
0.0271
<0.0001
0.0273
NS
0.0047
NS
0.0014
NS
NS
NS
0.0004
NS
0.0039
NS
0.0003
0.0036
NS
NS
0.0072
<0.0001
NS
NS
NS
NS
NS
NS
NS
0.0049
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.0014
NS
NS
NS
NS
0.0052
NS
NS
NS
NS
NS
NS
NS
<0.0001
NH4+:K+
NH4+ ×NO3−
K+ ×NO3−
Model
NH4+: K+
NH4+ ×NO3−
K+ ×NO3−
NS
NS
NS
0.0066
NS
0.0008
NS
NS
NS
<0.0001
<0.0001
NS
0.0014
<0.0001
NS
<0.0001
NS
0.0038
0.0480
0.0009
NS
NS
NS
<0.0001
NS
NS
NS
NS
NS
NS
NS
0.0398
NS
NS
0.0137
0.0151
NS
0.0091
NS
NS
NS
NS
0.0114
0.0011
0.0008
0.0006
NS
NS
<0.0001
‘OH×F 87’
(P. communis)
P. cordata
‘Sion Szu Mi’
(P. pyrifolia)
‘Hang Pa Li’
(P. ussuriensis)
22
WADA ET AL.
and Niedz 2008). Results are shown as the ratios between
NH4+ and K+ as well as the interactions between NO3− and
NH4+ or K+.
Results
NH4NO3 and KNO3 had an impact on pear shoot growth, and
specific NH4+ and NO3− amounts were required for optimal
growth of the diverse species. The impact of N type and the
overall interactions for the five genotypes varied by species.
The models were significant for most of the evaluated responses even though many of the individual factors were not
significant (Table 3). The models for quality, shoot number, and
shoot length were significant for all five genotypes (P≤0.05).
Figure 1. Graphs of significant
responses of pear shoot quality to
the growth medium concentration
of NO3− (mM) and the NH4+:K+
ratio. Rating scale of 1=low
quality (dark blue) to 3=high
quality (red) is indicated on the
graphs. Design points are noted
with a red dot if present in the
design space represented by the
graph.
‘Sion Szu Mi’ had significant models for all of the responses.
Significant responses for quality (P≤0.05) and highly significant models for most of the other responses (P≤0.001) are
presented (Figs. 1, 2, 3, and 4).
P. communis ‘Horner 51’. None of the models were highly
significant (P≤0.001) for ‘Horner 51’ (Table 3). The quality
model graph indicated the best quality when total NO3− was
high (52 to 60 mM) with low to medium NH4+ (Fig. 1A).
Shoots grown on control medium, Trt 11 (MS nitrogen), were
smaller and less vigorous than those on the best N-medium,
Trt 10 (30 mM NH4+, 30 mM K+, and 60 mM NO3−; Fig. 2).
Shoot length involved significant interactions of K+ ×NO3−,
and shoot numbers were highest (3.6 shoots) with low NH4+
and high K+ regardless of NO3− (data not shown). Shoot
lengths were greatest (55 mm) with moderate NO3− and varied
NITROGEN REQUIREMENTS FOR IN VITRO PEARS
Figure 2. Growth responses of
in vitro pear shoots grown on Pear
1 medium (control, Trt 11) (left)
and on the best N-treatment
combination (right) for each
genotype. Replicated treatments
are shown in parentheses.
Horner 51
Trt 10
OH×F 87
Trt 5 (6)
P. cordata
Trt 9 (16)
Sion Szu Mi
Trt 14
Hang Pa Li
23
Trt 8 (2)
only slightly with the NH4+:K+ ratio. Medium-size leaves
were produced with ≤30 mM NO3−, and there was a significant K+ ×NO3− interaction.
(Fig. 3A). Shoot length increased to 50 mm with high NO3−
and low NH4+ (Fig. 3D). Optimal leaf size (rating of 2) was
produced at moderate NH4+ and moderate NO3 (Fig. 4C).
P. c o m m u n i s ‘ O l d H o m e × F a r m i n g d a l e ( O H × F
87)’. Models were highly significant for shoot number, shoot
length, and leaf size, and the K+ ×NO3− interaction was highly
significant for shoot length (Table 3). The best-quality shoots
were produced when total NO3− was high (52 to 60 mM) with
low NH4+ (Fig. 1B). Shoots grown on control medium, Trt 11
(MS nitrogen), were shorter and less vigorous than those
grown on the best N-medium, Trt 5 and 6 (15 mM NH4+,
45 mM K+, and 60 mM NO3−; Fig. 2). Shoot numbers were
best (eight shoots) with moderate to low NO3− and low NH4+
P. cordata. Models were highly significant for shoot length
and callus production (Table 3). Quality graphs indicated the
best ratings with high NO3− and high NH4+ (Fig. 1C). Shoots
grown on control medium, Trt 11 (MS nitrogen), were slightly
shorter and less vigorous than the shoots grown on the best Nmedium, Trt 9 and 16 (45 mM NH4+, 15 mM K+, and 60 mM
NO3−; Fig. 2). Shoot numbers were best (six shoots) with
moderate NO3− and low to moderate NH4+ (data not shown).
Shoot length increased to 40 mm with medium to high NO3
and NH4+ (Fig. 3E). Callus amount significantly increased at
24
WADA ET AL.
Figure 3. Graphs of significant
responses of pear shoot number
and length to the growth medium
concentration of NO3− (mM) and
the NH4+:K+ ratio. (A–C) Shoot
multiplication (number of
shoots); (D–F) shoot length
(longest shoot measured in mm).
Low (dark blue) to high (red)
values are indicated on the
graphs. Design points are noted
with a red dot if present in the
design space represented by the
graph.
low to medium NO3− (≤40 mM) and medium to high NH4+
and was absent at lower NH4+ concentrations (Fig. 4A).
P. pyrifolia ‘Sion Szu Mi’. All the models except quality,
leaf size, and leaf spots were highly significant at P ≤
0.001 (Table 3). Quality was the best with moderate
NO3− and high NH4+ (Fig. 1D). Shoots grown on control
medium, Trt 11 (MS nitrogen), were smaller and less vigorous than shoots grown on the best N-medium, Trt 14
(30 mM NH4+, 10 mM K+, and 40 mM NO3−; Fig. 2).
Shoot number increased to five shoots with low NO3− and
low NH4+, and the K+ ×NO3− interaction was highly significant (Fig. 3B; Table 3). Shoot length was greatest
(40 mm) on medium with low NO3 and high NH4+, and
the NH 4 + × NO 3 − interaction was highly significant
(Fig. 3F; Table 3). Less callus was produced (Fig. 4B)
with high levels of either NO3− or NH4+, and fewer leaf
spots (Fig. 4D) were evident at low high NO3− and moderate to high NH4+. The K+ ×NO3− interaction was highly
significant for callus production and leaf color; lower K+
and higher NO3− produced the best responses (Table 3;
Fig. 4B, E).
P. ussuriensis ‘Hang Pa Li’. Models were highly significant
for shoot number and leaf color (P≤0.001; Table 3). Quality
was the best with low NO3− and high NH4+, with a highly
significant NH4+ ×NO3− interaction (Fig. 1E; Table 3). Shoots
grown on control medium, Trt 11 (MS nitrogen), were less
vigorous and shorter than shoots grown on the best N-medium, Trt 2 and 8 (15 mM NH4+, 5 mM K+, and 20 mM NO3−;
Fig. 2). Shoot number was best (five shoots) with low NO3−
and low to medium NH4+ (Fig. 3C), and the K+ ×NO3− interaction was highly significant. Shoot length was good (30 mm)
over a wide range of NH4+ with low to medium NO3− (data
NITROGEN REQUIREMENTS FOR IN VITRO PEARS
25
Figure 4. Graphs of significant
responses of pear shoot callus
formation, leaf size, and leaf color
to the growth medium
concentration of NO3− (mM) and
the NH4+:K+ ratio: (A–B) callus
(1=no callus), (C) leaf size
(1=small), (D) leaf spot/necrosis
(1=no spots) and (E–F) leaf color
(1=green). Scales of 1
(fewest symptoms, best treatment,
dark blue) to 3 (most symptoms,
poorest treatment, red) are
indicated on the graphs. Design
points are noted with a red dot if
present in the design space
represented by the graph.
not shown). Moderate to high NO3− and NH4+ produced the
best leaf color (Fig. 4F); a medium-size leaf (2 rating) was
produced with medium to low NO3− and NH4+.
Discussion
Successful micropropagation is reliant on the chemical composition of the culture medium to provide adequate nutrition
for the cultures. There are numerous ways to accomplish optimizations including salt- or ion-based experiments. The type
of salt-based experiment used in our previous study, where the
factors are salts, was useful for determining which mineral
nutrients were important in the growth response, but the effects of specific ions were not determined (Reed et al. 2013b).
Once the main salts that had growth-related effects were
identified, the optimal concentrations of each one could be
optimized (Niedz et al. 2014). Our initial study showed that
the nitrogen compounds (KNO3 and NH4NO3) were important in shoot growth responses and required further study for
optimization (Reed et al. 2013b). In the current study, the ions
involved were varied to determine the relative requirements
for each of the pears tested, as a mixture-amount design without ion confounding (Niedz and Evens 2008).
Through this study, clear differences among the pear species were evident in the total N amounts, forms, and ratios for
producing high-quality shoots (Table 3; Fig. 1). The two
P. communis cultivars required high NO3− with low to medium NH4+, while P. ussuriensis ‘Hang Pa Li’ grew best with
low NO3− and high NH4+. Unique N requirements were also
apparent for the two other species: P. cordata required high
NO3− and NH4+, while ‘Sion Szu Mi’ (P. pyrifolia) produced
the best quality with moderate NO3−, and high NH4+. The
26
WADA ET AL.
K+ ×NO3− interaction and NH4+:K+ ratio were significant in
some cases (Table 3), but the responses affected varied by
genotype. Since K+ was also present in other medium salts,
it was difficult to quantify the effect of total K+ in this study.
In our study, the two P. communis cultivars showed the
same trend for NH4+:NO3− ratios (1:2 for ‘Horner 51’ and
1:4 for ‘OH×F 87′) as reported by Abu-Qaoud et al. (1991)
and Leblay et al. (1991); however, the optimal ratios for
P. cordata, P. pyrifolia, and P. ussuriensis were 3:4, with large
differences in the total N (Table 2). These differences may
reflect the diverse origins and genetic backgrounds of the pear
germplasm tested. This diversity is confirmed by a study of
genome structure of highly conserved pear chloroplast DNA
that categorized 21 Pyrus species into three distinctive groups.
The type A group was mainly P. ussuriensis; type B was
composed mostly of pears native to East and South Asia
(P. pyrifolia); and type C was mainly P. communis wild relatives native to Europe, West and Central Asia, Russia, and
Africa, including P. cordata (Katayama et al. 2012).
In a study of shoot regeneration, the best regeneration from
leaves of two P. communis cultivars was obtained when the
NH4+:NO3− ratio was either 1:2 or 1:3, regardless of the total
N amount (Abu-Qaoud et al. 1991). Similarly, Leblay et al.
(1991) reported that NH4+ and total N played an essential role
in regenerating pear leaves, and the ratio of NH4+:NO3− (1:3)
was a critical factor for inducing optimal shoot regeneration
from four P. communis cultivars. The effect of nitrogen is also
seen in embryogenesis and regeneration of barley microspores
with varying N supplies: the highest embryogenesis was obtained with 20–35 mM total N and a 1:9 ratio of NH4+:NO3−
(Mordhorst and Lörz 1993).
Optimal shoot quality (Fig. 1) for two P. communis genotypes and P. cordata required high NO3− (60 mM); for
P. pyrifolia, moderate NO3− (40 mM); and for P. ussuriensis,
moderate to low NO3− (≤20 mM). The two P. communis genotypes required low or moderate NH4+ (15 or 30 mM); the
other three species grew best with high NH4+ (≥45 mM).
Many of the initial deficiency symptoms were resolved in
the Pear 1 medium by increasing the mesos concentration to
1.5× the MS amount, so many genotypes already showed
substantial improvement before this N study (Fig. 2, left).
However, some of the pear species were more affected by N
nutrients than others. The greatest effects were on overall
quality, shoot number, and shoot length (Table 3). Some effects of N were also seen for callus production, leaf color, and
leaf size (Table 3). In our earlier study of mesos components,
we found that the increased mesos present in Pear 1 medium
produced greatly improved leaf characteristics, such as darker
green color and medium-size leaves, and eliminated most negative leaf symptoms (necrosis, spots, hyperhydricity; Wada
et al. 2013).
Callus was not produced by some of the cultivars studied
and was highly significantly affected by nitrogen compounds
for only two genotypes (Fig. 4A, B). Low-quality shoot cultures usually exhibit symptoms such as chlorosis, leaf spots,
excessive callus, and/or leaf and shoot tip necrosis that indicate a suboptimal growth medium. High levels of NH4+ in
growth media are noted as the cause of hyperhydricity in a
range of studies (Paques 1991; Ziv 1991; Tsay and Drew
1998; Ivanova and Van Staden 2009). Our earlier study with
MS medium found that NH4+ was a significant factor for
hyperhydricity of pear species other than P. communis (Reed
et al. 2013c). We did not observe hyperhydricity on pear
shoots grown on high NH4+ concentrations when combined
with the higher mesos (1.5×) components found in Pear 1
medium (data not shown).
This study confirms that optimizing the entire range of
mineral nutrients, rather than just one component of a growth
medium, is important when working with diverse plant germplasm. The study also supports testing protocols or new medium formulations on a range of genotypes with diverse genetic backgrounds. Testing one or two genotypes, and/or multiple genotypes in one species, is not as conclusive and would
not provide broadly useful protocols for a diverse genus such
as Pyrus. The final step for this study is testing N nutrients on
a greater range of pear germplasm collected from additional
diverse genetic backgrounds.
Acknowledgments We thank the NCGR lab personnel for the assistance with data collection. This project was funded by a grant from the
Oregon Association of Nurseries and the Oregon Department of Agriculture and by US Department of Agriculture, Agricultural Research Service
CRIS project 5358-21000-038-00D.
References
Abu-Qaoud H, Skirvin RM, Below FE (1991) Influence of nitrogen form
and NH4-N/:NO-N ratios on adventitious shoot formation from pear
(Pyrus communis) leaf explants in vitro. Plant Cell Tissue Organ
Cult 27:315–319
Cousson A, Van KTT (1993) Influence of ionic composition of the culture medium on de novo flower formation in tobacco thin cell layers.
Can J Bot 71:506–511
Design-Expert (2010), Stat-Ease, Inc., Minneapolis, MN
Dougall DK, Weyrauch KW (1980) Abilities of organic-acids to support
growth and anthocyanin accumulation by suspension-cultures of
wild carrot cells using ammonium as the sole nitrogen-source. In
Vitro Cell Dev Biol 16:969–975
Engelsberger WR, Schulze WX (2012) Nitrate and ammonium lead to
distinct global dynamic phosphorylation patterns when resupplied to
nitrogen starved Arabidopsis seedlings. Plant J 69:978–995
Evens TJ, Niedz RP ARS-Media: Ion Solution Calculator (2008), U.S.
Horticultural Research Laboratory, Ft. Pierce, FL 34945 USA
Forde BG, Clarkson DT, Callow JA (1999) Nitrate and ammonium nutrition of plants: physiological and molecular perspectives. In:
Advances in Botanical Research. Academic Press, pp 1–90
Grimes H, Hodges T (1990) The inorganic NO3-: NH4+ ratio influences
plant regeneration and auxin sensitivity in primary callus derived
from immature embryos of indica rice (Oryza sativa L.). J Plant
Physiol 136:362–367
NITROGEN REQUIREMENTS FOR IN VITRO PEARS
Guidi L, Lorefice G, Pardossi A, Malorgio F, Tognoni F, Soldatini G
(1997) Growth and photosynthesis of Lycopersicon esculentum
(L.) plants as affected by nitrogen deficiency. Biol Plant 40:235–244
Ivanova M, Van Staden J (2009) Nitrogen source, concentration, and NH4+:
NO3− ratio influence shoot regeneration and hyperhydricity in tissue
cultured Aloe polyphylla. Plant Cell Tissue Organ Cult 99:167–174
Jain V, Pal M, Lakkineni K, Abrol Y (1999) Photosynthetic characteristics in two wheat genotypes as affected by nitrogen nutrition. Biol
Plant 42:217–222
Katayama H, Tachibana M, Iketani H, Zhang SL, Uematsu C (2012)
Phylogenetic utility of structural alterations found in the chloroplast
genome of pear: hypervariable regions in a highly conserved genome. Tree Genet Genomes 8:313–326
Leblay C, Chevreau E, Raboin LM (1991) Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis
L). Plant Cell Tissue Organ Cult 25:99–105
Martin SM, Rose D, Hui V (1977) Growth of plant cell suspension cultures with ammonium as the sole source of nitrogen. Can J Bot 55:
2838–2843
Mitcham EJ, Elkins RB (eds) (2007) Pear production and handling manual. University of California, Agriculture and Natural Resources,
Oakland
Mordhorst AP, Lörz H (1993) Embryogenesis and development of isolated barley (Hordeum vulgare L.) microspores are influenced by the
amount and composition of nitrogen sources in culture media. J
Plant Physiol 142:485–492
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio
assays with tobacco tissue cultures. Physiol Plant 15:473–497
Niedz RP, Evens TJ (2006) A solution to the problem of ion confounding
in experimental biology. Nat Methods 3:417
Niedz RP, Evens TJ (2007) Regulating plant tissue growth by mineral
nutrition. In Vitro Cell Dev Biol Plant 43:370–381
Niedz RP, Evens TJ (2008) The effects of nitrogen and potassium nutrition on the growth of nonembryogenic and embryogenic tissue of
sweet orange (Citrus sinensis (L.) Osbeck). BMC Plant Biol 8:126
Niedz RP, Hyndman SE, Evens TJ, Weathersbee AA III (2014) Mineral
nutrition and in vitro growth of Gerbera hybrida (Asteraceae). In
Vitro Cell Dev Biol Plant 50:458–470
Paques M (1991) Vitrification and micropropagation: causes, remedies
and prospects. Acta Hortic 289:283–290
27
Preece J (1995) Can nutrient salts partially substitute for plant growth
regulators? Plant Tissue Cult Biotechnol 1:26–37
Ramage CM, Williams RR (2002) Inorganic nitrogen requirements during shoot organogenesis in tobaco leaf discs. J Exp Bot 53:1437–
1443
Reed BM, DeNoma JS, Wada S, Postman JD (2013a) Micropropagation
of pear (Pyrus sp). In: Lambardi M, Ozudogru EA, Jain SM (eds)
Protocols for micropropagation of selected economically-important
horticultural plants. Humana Press–Springer, NY, p 554
Reed BM, Wada S, DeNoma J, Niedz RP (2013b) Improving in vitro
mineral nutrition for diverse pear germplasm. In Vitro Cell Dev
Biol Plant 49:343–355
Reed BM, Wada S, DeNoma J, Niedz RP (2013c) Mineral nutrition
influences physiological responses of pear in vitro. In Vitro Cell
Dev Biol Plant 49:699–709
Scheible W, González-Fontes A, Lauerer M, Muller-Rober B, Caboche
M, Stitta M (1997) Nitrate acts as a signal to induce organic acid
metabolism and repress starch metabolism in tobacco. Plant Cell 9:
783–798
Sotiropoulos TE, Moutharidou GN, Thomidis T, Tsirakoglou V, Dimassi
KN, Therios IN (2005) Effects of different N-sources on growth,
nutritional status, chlorophyll content, and photosynthetic parameters of shoots of the apple rootstock MM 106 cultured in vitro. Biol
Plant 49(2):297–299
Troelstra S, Wagenaar R, Smant W (1995) Nitrogen utilization by plant
species from acid heathland soils I. comparison between nitrate and
ammonium nutrition at constant low pH. Exp Bot 46:1103–1112
Tsay H, Drew R (1998) Effect of medium compositions at different
recultures on vitrification of carnation (Dianthus caryophyllus)
in vitro shoot proliferation. Acta Hortic 243–249
Vincentz M, Moureaux T, Leydecker MT, Vaucheret H, Caboche M
(2011) Regulation of nitrate and nitrite reductase expression in
Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant J 3:315–324
Wada S, Niedz RP, DeNoma J, Reed BM (2013) Mesos components
(CaCl 2 , MgSO 4 , KH 2 PO 4 ) are critical for improving pear
micropropagation. In Vitro Cell Dev Biol Plant 49:356–365
Ziv M (1991) Quality of micropropagated plants—vitrification. In Vitro
Cell Dev Biol Plant 27:64–69