Developmental, cytological and transcriptional analysis of autotetraploid Arabidopsis
Xiaodong Li, Erru Yu, Chuchuan Fan, Chunyu Zhang, Tingdong Fu, Yongming Zhou*
National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China
*Corresponding author: email ymzhou@mail.hzau.edu.cn
Tel: +86-27-87286869
Fax: +86-27-87280016
1
Supplementary 1 Identification of an autotetraploid Arabidopsis line BS
Big Seedling (BS) was isolated from a diploid Col-0 wild type (WT) population, whose organs were
dramatically larger than WT. BS was identified as an autotetraploid by chromosome counting
(Supplementary Fig. S1a-b) and flow cytometric analysis (Supplementary Fig. S1c-d). Simple
sequence length polymorphism (SSLP) analysis was also conducted to confirm the BS was in Col-0
background (Supplementary Fig. S1e).
The newly found autotetraploid BS plant was self-fertilized for two generations. Three
independent plants, BS-7, BS-9, and BS-12 from the second generation were advanced to the third
generation to produce three lines, each consisting of 20 plants for analysis of ploidy stabilization. All
the plants showed polyploid phenotype. Furthermore, six plants randomly selected from each
population were used for chromosome counting, which showed a tetraploid nature (data not shown). In
the next generation, forty seeds of BS-9-21 were germinated on 0.5×MS medium, and all of them were
transferred to nutrient composites and examined by chromosome counting, thirty six (90%) plants were
confirmed as tetraploid, the other four were aneuploid which showed small seedling and reduced
fertility phenotypes (data not show). BS-9-21 and its progenies were used for detailed growth and
development characterization and other studies.
Supplementary Materials and Methods
Determination of chromosome number
2
Young ovaries were used for the chromosome counting and the procedures were described as
previously (Li et al. 1995). Diploid and tetraploid Arabidopsis thaliana were grown in the same
condition. During bolting to flowering, materials were collected and immersed in ice water for 0-6
hours, treated with 2 mM 8-hydroxyquinoline for 1 hour, and then fixed in Carnoy’s fluid (70% ethanol
and 30% acetic acid) for 12 hours. The fixed ovaries were hydrolyzed with 1 M HCl at 65 °C for 3
minutes, washed in deion disinfection water (dd H2O) for 2 minutes, squashed in a drop of 10% carbol
fuchsin and observed under a Leica DMLB fluorescent microscope (leica, Wetzlar, Germany) equipped
with a Leica 300F CCD camera. Primers and procedures to identify BS ecotype were described
previously by using SSLP (Simple Sequence Length Polymorphism) detection (Lukowitz et al. 2000).
Protein, fatty acid and carbohydrate analysis
For protein assay, one hundred dry seeds were weighted and grinded with 200 μl ice-cold buffer S that
contains 50 mM Tris-HCl pH=8.0, 10 mM KCl, 1 mM EDTA, 2 mM DTT, 0.5 mM PMSF, 0.5 M
sucrose, incubated for 10 minutes, centrifuged at 12,000 g for 10 minutes at 4°C, the supernatant was
used to measure protein content with Quick StartTM Badford Dye Reagent (Bio-Rad, USA) under
Beckman DU730 DNA/ protein analyzer. For fatty acid assay, one hundred dry seeds were weighted
and extracted in a Teflon-lined screw capped glass tube that was thoroughly pre-rinsed with chloroform
and dried. Each tube was added 300 μl 2.5% (v/v) sulfuric acid and 0.01% (m/v) BHT (butylated
hydroxyl toluene) in methanol, 100 μl toluene as co-solvent, 20 μg C17:0 (triheptadecanoin, Sigma) as
internal standard. The mixture was vortexed and heated at 95 °C for 2 hours. After cooling to room
temperature, 300 μl ddH2O and 300 μl hexane were added to the mixture and vortexed thoroughly. The
3
supernatant was analyzed by GC (gas chromatography) with a flame ionization detector on a 30 m ×
250 μm × 0.25 μm column (19091N-133, Agilent), the program was as described previously (Li et al.
2006). For sugar assay, fifty seeds were weighted and grinded in 500 μl 80% ethanol, incubated at
70°C for 90 minutes, centrifuged at 16,000 g for 5 minutes and the supernatant was transferred to a
new tube. The sediment was extracted again and supernatants for the two extracts were pooled and
vacuumized to dry powder, and re-dissolved in 50 μl ddH2O that represented soluble sugar. The
sediment from above operation was mixed with 200 μl 0.2 M KOH and the mixture was incubated at
95°C for 1 hour. Then 35 μl 1 M acetic acid was added to the mixture and centrifuged at 16,000 g for 5
minutes. The supernatant represented starch. The concentration of soluble sugar, and starch was
determined by anthranone colorimetric procedures as described previously (Dubois et al. 1956).
Supplementary Reference
Dewitte W, Riou-Khamlichi C, Scofield S, Healy J, Jacqmard A, Kilby NJ, Murray JAH (2003) Altered
cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type
cyclin CYCD3. Plant Cell 15: 79-92
Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F (1956) Colorimetric method for determination
of sugars and related substances. Anal chem 28: 350-356
Li Y, Beisson F, Pollard M, Ohlrogge J (2006) Oil content of Arabidopsis seeds: the influence of seed
anatomy, light and plant-to-plant variation. Phytochemistry 67: 904-915
Li Z, Liu H, Luo P (1995) Production and cytogenetics of intergeneric hybrids between Brassica napus
and Orychophragmus violaceus. Theor Appl Genet 91: 131-136
4
Lukowitz W, Gillmor CS, Scheible WR (2000) Positional cloning in Arabidopsis. Why it feels good to
have a genome initiative working for you. Plant Physiol 123: 795-805
5
Supplementary Table S1 Primers used in this study
Open reading
Gene name
frame name
WEE1
AT1g02970
CYCA2;3
AT1g15570
CDKB2;2
AT1g20930
ICK5
AT1g49620
CYCD1;1
AT1g70210
CDKB2;1
AT1g76540
CYCB2;1
AT2g17620
CYCD2;1
AT2g22490
ICK1
AT2g23430
CKS1
AT2g27960
CDKB1;2
AT2g38620
DEL3
AT3g01330
CYCB1;3
AT3g11520
ICK4
AT3g19150
DEL1
AT3g48160
CDKA;1
AT3g48750
ICK2
AT3g50630
CDKB1;1
AT3g54180
CDKF;1
AT4g28980
CYCB2;2
AT4g35620
Product
Primer ID
Sequence
lxp163
ATGTTGCTTCATCGGCGTTTCAGG
lxp164
TCTGAATCAGCGGAACCCAACTGA
lxp165
TGAGGAAATCTCTGCTCCACGCAT
lxp166
AGAGGCTTGAGCTGCTCTGAGAAA
lxp167
CAAGCAAGCAATCTTTGCGGGAGA
lxp168
AGCTGTGGAGAGACTCAACGGTTT
lxp169
CAAGCATCAGCTCTGGTTGTTCCA
lxp170
TGCTGTTTCTCCCAGATTCTCGCT
lxp171
GGCGTGTGTCTCACGTGTTTGTTT
lxp172
ACCTTACACGCATGCAAACGCTAC
lxp173
GCTGAACTTGTGACCAACCAAGCA
lxp174
TCCACTGTGGGTATTCATGCCAGT
lxp175
TACGAGATGCTTCGGTTTCCACCA
lxp176
AGACTCACCAGCTTCCGTGAACAT
lxp177
ATCTTGCTTGTGGTGAAACCAGCG
lxp178
TCGTAAATCCGCCGCCATAGTTGA
lxp179
AGCTACGGAGCCGGAGAATTGTTT
lxp180
GTCGACGTTTCAGTGTCACCATCT
lxp181
ACATTACGCGGTTCATCGACCTGA
lxp182
TCTGAGCTTGATTCTCCTGCTGCT
lxp183
TGGTCTGTTGGATGCATCTTTGCC
lxp184
TGATAAGTCTTGCGGCTCCCACTT
lxp185
ACGACAAAGTCGGAAAGGGAAGGT
lxp186
TTCTTCCATGCCTCCGTGAAATGC
lxp187
TGGAAGAGGTGCTGTTGCTCTGAT
lxp188
TGCAAGAGAGCCATCTTTCAAGCC
lxp189
ATCAGCTCCGGTTGTTTCACCAGT
lxp190
TGTCGTTTCTCCCAAACCCTCACT
lxp191
ACTGCTTGGTGATGCCCACAATAC
lxp192
GCAACAAATCGCTGCTCAGTGTGA
lxp193
CGTGGAATTGCGTATTGCCACTCT
lxp194
AGCAAGCTTCAGTGAGTTTGTGCG
lxp195
TGAGACGGTGAAGGAAGCTGAGTT
lxp196
TCCTCCACCAAGTGGCTCATCTTT
lxp197
AGATGGTTCGGAGGCAAGCTCTTT
lxp198
TAGGGTAAACATGCCAGTCACGCA
lxp199
TTACGATGCTGACGAAGCAGTGGA
lxp200
TCTGCAAACACACAACCAAGCGAC
lxp201
AGAGGTTCCTCAAGGCAGCTCAAT
size (bp)
82
175
157
170
141
148
161
81
169
89
186
122
90
176
188
99
131
139
150
175
6
CYCB1;1
AT4g37490
DPa
AT5g02470
CYCB1;2
AT5g06150
CYCA2;2
AT5g11300
CYCA2;1
AT5g25380
CYCD4;1
AT5g65420
DPb
AT5g03410
PCNA1
AT1g07370
ICK6
AT5g48820
ICK7
AT2g32710
ICK3
AT3g24810
CYCD3;1
AT4g34160
CYCD3;2
AT5g67260
CYCD3;3
AT3g50070
E2Fb
AT5g22220
E2Fc
AT1g47870
E2Fa
AT2g36010
MINI3
AT1g55600
AP2
AT4g36920
SHB1
AT4g25350
KLUH
AT1g13710
ARF2
AT5g62000
lxp202
GTGCTGTTCCATTCACTGAAGCCA
lxp203
TCCGATTCGAGCTAAACCCGGAAA
lxp204
CCACCTGTGGTGGCCAAATTTCTT
lxp205
AGCTCCGCTCATTCTTCATCCAGT
lxp206
ATCAATGGATCCCGAGTTCCAGCA
lxp207
TACTCGAAGTTTCCGTGCCCAGTT
lxp208
TCTTTGGACCAAGAGCTGGAACCT
lxp209
TGAAAGGCAAAGACTCCAGCTCCT
lxp210
ATCTCCATGCTCAGCACTTCTGGT
lxp211
TCCTTCCATCCCTAATTGCCGCTT
lxp212
ACTCCTCCATTGCAAGTACCGTGT
lxp213
ATGGTGGAGAAGGAGAAGCAGCAT
lxp214
GCCTTCCAAATCCAATTGAGGGCA
lxp215
ACAACCAAACGGTCGGAACAACAG
lxp216
TCGTACATATCCAGCTGCGGTGTT
lxp217
TCGTGAGGATGCCTTCCAATGAGT
lxp218
TGCATCTTCCGGCTTGTCTACAGT
lxp226
TGGCGATATCAGCGTCATGGAAGT
lxp227
TCGGTTCAATCAGCGAAGAGGGTT
lxp228
TGGATTCGATTCGTGGTGATGGGT
lxp229
TGGATTCCCTTGTGGTCCTGTTGA
lxp230
AAGGATAAATCTCACCCACCCGCT
lxp231
AAGGAGTCTGCTTCGTCGGAATGA
TZ23
TGGGCAATCGTAGCCACTCCATAA
TZ24
CACGTAAGGGCATTCGCGACATTT
TZ19
CTCAGCTTGTTGCTGTGGCTTCTT
TZ20
AGAGATTGGAGTCACAGGGTGCAT
TZ21
TGCATCCTGTGACTCCAATCTCGT
TZ22
TGCAGTGGCTAACACAGAAGGACT
TZ25
TCTAAGCGGCAGCTTCATCCTTCT
TZ26
TCACAATCGCGTCAGAGACAACCT
TZ27
TCCCACGGTTTCAGAACCAGACAT
TZ28
CAACTTGTCGCTTGTTTCCGCACT
TZ29
AATGGGCGAAATAGCACCAACAGC
TZ30
TTGTATGGAACGCACCTGCCATTG
lxp131
AGGCCTTTCTCTTCGTTGGCTACA
lxp132
GCATCGCATTCATCGTCGGTTCAT
lxp133
GACGCACCACACCAAACACAAAGA
lxp134
AGAACTGATGGGTGACAAGTGGGT
lxp135
ACTGAGGCTCTGGCTCTGAACAAA
lxp136
ATCCAACCTCAGAAGCTGAGCCAT
lxp137
TGATGTTGAGCTCGCTGAGGTTCT
lxp138
ACATTCCTTGGAACAGCCTTGCAC
lxp139
GTTCCAGGTGCATTCGTTCTGCAA
159
84
95
144
147
116
108
177
195
174
107
142
185
164
139
191
175
139
183
139
81
88
7
TG2
AT2g37260
IKU2
AT3g19700
DA1
AT1g19270
UBQ10
AT4g05320
lxp140
ATCCGCATGTCGCCTAAGAACAGA
lxp141
AGATTCCGGTTGCAAGAGTAGCCA
lxp142
GGATACGCATTGCCTCCTACCAC
lxp143
AAGTGAACGAGAAGAGCGACGTGT
lxp144
TCACGCTCCAAACCCACATGACTA
lxp145
AACACGGCAATGTGAAGTTACCGC
lxp146
TCGGAATCCTTTGAGCCTCATCCA
UBQ10CF
GGCCTTGTATAATCCCTGATGAATAAG
UBQ10CR
AAAGAGATAACAGGAACGGAAACATAGT
192
126
118
64
8
Supplementary Table S2 Protein, fatty acid and carbohydrate analysis
Content (μg / seed)
Traits examined
Diploid (WT)
Autotetraploid (BS)
Fatty acid
4.79±0.17
Protein
Content (mass fraction)
Diploid (WT)
Autotetraploid (BS)
6.82±0.54**
28.20±0.72
26.63±1.44
3.70±0.15
6.07±0.45**
22.24±1.33
22.20±1.43
Starch
0.56±0.02
1.04±0.02**
3.16±0.09
3.38±0.13
Soluble sugar
0.75±0.02
1.20±0.09**
4.30±0.20
3.91±0.28
Nutrient substance comparisons between BS and WT plants. Data are presented as mean ± standard
deviation from at least six independently propagated BS and WT plants with three technical repeats. *
and ** indicate a significant difference between tetraploid and diploid plants at P<0.05 and P<0.01
levels, respectively, based on t-test.
9
Supplementary Table S3 Expression analyses of core cell cycle genes and seed size related genes in
tetraploid BS and diploid WT plants at the fifth generation
Normalized folder expression
Open reading
Gene name
frame name
Primer pairs
Diploid
Autotetraploid
p-value
CYCA2;1
AT5g25380
LXP211+LXP212
1.00±0.15
0.79±0.05
0.251
CYCA2;2
AT5g11300
LXP209+LXP210
1.00±0.10
1.18±0.08
0.224
CYCA2;3
AT1g15570
LXP165+LXP166
1.00±0.04
0.79±0.08
0.091
CYCB1;1
AT4g37490
LXP203+LXP204
1.00±0.32
1.52±0.58
0.474
CYCB1;2
AT5g06150
LXP207+LXP208
1.00±0.01
0.90±0.08
0.263
CYCB1;3
AT3g11520
LXP187+LXP188
1.00±0.08
0.93±0.03
0.363
CYCB2;1
AT2g17620
LXP175+LXP176
1.00±0.22
0.87±0.18
0.675
CYCB2;2
AT4g35620
LXP201+LXP202
1.00±0.15
1.42±0.48
0.550
CYCD1;1
AT1g70210
LXP171+LXP172
1.00±0.13
1.29±0.16
0.113
CYCD2;1
AT2g22490
LXP177+LXP178
1.00±0.11
0.94±0.03
0.647
CYCD3;1
AT4g34160
TZ23+TZ24
1.00±0.13
0.67±0.11
0.129
CYCD3;3
AT3g50070
TZ21+TZ21
1.00±0.03
0.85±0.12
0.307
CYCD4;1
AT5g65420
LXP213+LXP214
1.00±0.09
0.66±0.04*
0.027
CDKA;1
AT3g48750
LXP193+LXP194
1.00±0.02
0.52±0.03**
0.001
CDKB1;1
AT3g54180
LXP197+LXP198
1.00±0.23
1.59±0.24
0.145
CDKB1;2
AT2g38620
LXP183+LXP184
1.00±0.14
1.21±0.07
0.243
CDKB2;1
AT1g76540
LXP173+LXP174
1.00±0.11
0.97±0.17
0.875
CDKB2;2
AT1g20930
LXP167+LXP168
1.00±0.15
0.87±0.32
0.732
CDKF;1
AT4g28980
LXP199+LXP200
1.00±0.07
0.88±0.03
0.145
CKS1
AT2g27970
LXP181+LXP182
1.00±0.04
0.76±0.05
0.022
DEL1
AT3g48160
LXP191+LXP192
1.00±0.16
1.23±0.12
0.310
DEL3
AT3g01330
LXP185+LXP186
1.00±0.04
0.95±0.05
0.588
DPa
AT5g02470
LXP205+LXP206
1.00±0.11
1.03±0.03
0.822
DPb
AT5g03410
LXP215+LXP216
1.00±0.01
0.89±0.09
0.269
10
E2Fa
AT2g36010
TZ29+TZ30
1.00±0.07
0.78±0.07
0.104
E2Fb
AT5g22220
TZ25+TZ26
1.00±0.04
0.89±0.15
0. 500
E2Fc
AT1g47870
TZ27+TZ29
1.00±0.03
0.81±0.12
0.196
ICK1
AT2g23430
LXP179+LXP180
1.00±0.15
1.74±0.11*
0.017
ICK2
AT3g50630
LXP195+LXP196
1.00±0.23
2.12±0.20*
0.021
ICK4
AT3g19150
LXP189+LXP190
1.00±0.21
0.90±0.06
0.663
ICK5
AT1g49620
LXP169+LXP170
1.00±0.10
1.93±0.18*
0.023
WEE1
AT1g02970
LXP163+LXP164
1.00±0.03
0.94±0.06
0.408
PCNA1
AT1g07370
LXP217+LXP218
1.00±0.08
1.06±0.18
0.777
IKU2
AT3g19700
LXP143+LXP144
1.00±0.18
0.52±0.09
0.520
MINI3
AT1g55600
LXP131+LXP132
1.00±0.24
0.42±0.17
0.420
AP2
AT4g36920
LXP133+LXP134
1.00±0.05
1.13±0.16
1.130
TTG2
AT2g37260
LXP141+LXP142
1.00±0.06
0.98±0.20
0.980
ARF2
AT5g62000
LXP139+LXP140
1.00±0.33
1.99±0.20
1.99
DA1
AT1g19270
LXP145+LXP146
1.00±0.33
1.37±0.33
1.370
KLUH
AT1g13710
LXP137+LXP138
1.00±0.16
0.65±0.18
0.650
SHB1
AT4g25350
LXP135+LXP136
1.00±0.03
0.96±0.10
0.960
Fresh leaf tissues were sampled from 28 DAS plants of the fifth generation were conducted for core
cell cycle gene expression analysis. Freshly opened flowers (0-3 days after flowering) were sampled
for seed-size related gene expression analysis at the fifth generation plants. Data are collected from
three biological replicates with three technique repeats for each biological replicate and presented as
mean ± standard deviation. We calculated each gene expression change in tetraploid BS relatively to
diploid WT, expression of those genes in WT was set 100%, Ubiquitin10 was the reference; * and **
indicates a significant difference between the BS and WT plants at P<0.05 and P<0.01 levels,
respectively, based on t-test.
11
Supplementary Table S4 Expression analyses of core cell cycle genes in rosette leaf and flower
tissues in tetraploid BS and diploid WT plants at the sixth generation
Gene
Open reading
name
frame name
Normalized folder expression
Tissue
Diploid
Autotetraploid
p-value
CYCA2;2
AT5g11300
leaf
1.00±0.09
0.87±0.04
0.244
CYCD3;1
AT4g34160
leaf
1.00±0.06
0.71±0.08*
0.047
CYCD4;1
AT5g65420
leaf
1.00±0.09
0.88±0.14
0.603
CDKF;1
AT4g28980
leaf
1.00±0.08
CDKA;1
AT3g48750
leaf
1.00±0.07
0.87±0.08
0.293
ICK1
AT2g23430
leaf
1.00±0.13
1.60±0.16*
0.041
ICK2
AT3g50630
leaf
1.00±0.13
1.47±0.02*
0.026
ICK5
AT1g49620
leaf
1.00±0.32
1.92±0.05*
0.046
CYCD3;1
AT4g34160
flower
1.00±0.11
0.88±0.17
0.577
CYCD4;1
AT5g65420
flower
1.00±0.09
0.99±0.04
0.317
CDKA;1
AT3g48750
flower
1.00±0.09
1.16±0.06
0.224
ICK1
AT2g23430
flower
1.00±0.05
1.98±0.04*
0.010
ICK2
AT3g50630
flower
1.00±0.24
1.96±0.14*
0.023
ICK4
AT3g19150
flower
1.00±0.05
1.05±0.02
0.063
ICK5
AT1g49620
flower
1.00±0.12
1.62±0.13*
0.024
0.83±0.088
0.207
Fresh leaf tissues from 28 DAS plants and freshly opened flowers (0-3 days after flowering) were
sampled from BS and WT plants of the sixth generation for selected cell cycle gene expression analysis.
Data are collected from three biological replicates, each with three technique repeats, and presented as
mean ± standard deviation. We calculated each gene expression change in tetraploid BS relatively to
diploid WT, expression of those genes in WT was set 100%, Ubiquitin10 was the reference; * indicates
a significant difference between the BS and WT plants at P<0.05 levels based on t-test.
12
Supplementary Figure legend
Supplementary Fig. S1 BS was an autotetraploid Col-0.
a,b Chromosome numbers of WT (diploid) (a) and BS (autoretraploid) (b) in young ovaries before
flower opened. c,d Determination of WT (c) and BS (d) ploidy levels of true leaves of 10-day-old
seedlings examined by flow cytometric analysis. e Determination of ecotype background of BS, white
bars represent five InDel makers between Col-0 and Ler ecotypes, which located on five different
chromosomes, from left to right were ciw1, nga1126, nga162, ciw7 and ciw9. Sample on each lane
(from left to right): BS, WT (Col-0), WT (Col-0)×WT (Ler) F1 and WT (Ler). Bars 10 μm (a, b).
Supplementary Fig. S2 Rosette leaf emergence rate of BS and WT plants.
BS and WT plants were sowed in nutrient composites side by side, and visible leaf blade appearing was
treated as leaf emerged, measurements were determined with the ninth rosette leaves at 45 DAS. Data
are presented as mean ± standard deviation, n=40. * and ** indicate a significant difference between
BS and WT plants at P<0.05 and P<0.01 levels, respectively, based on t-test.
Supplementary Fig. S3 Leaf shape was changed in autotetraploid BS.
a BS had shorter but wider leaf blade than WT. b BS had a higher shape factor value than WT.
Observations were made with the ninth rosette leaf at 45 DAS, Shape factor = 4π × area / perimeter2
(Dewitte et al. 2003). Data are presented as mean ± standard deviation, n=16. ** indicate a significant
difference between BS and WT plants at P<0.01 levels, based on t-test.
13
Supplementary Fig. S4 Phenotypes of WT and autotetraploid plants under various phytohormone
treatments.
BS and WT young seedlings (3 DAS) were treated with different phytohormones. a Seedlings on
normal condition. b Seedlings on 10 μM gibberellic acid3 (GA3). c Seedlings on 5 μM abscisic acid
(ABA). d Seedlings on 10 μM indole-3-acetic acid (IAA). e Seedlings on 0.1 μM
2,4-dichlorophenoxyacetic acid (2,4-D). f Seedlings on 100 μM salicylic acid (SA). g Seedlings on
normal condition. h Seedlings on 10 μM ethylene. i Seedlings on 10 μM methyl jasmonate (MeJA). j
Seedlings on 10 μM zeatin (ZT). a-f Plants were photographed at 14 DAT, g-j Plants were
photographed at 21 DAT. Bars 1 cm (a-j).
Supplementary Fig. S5 Plants grown on mannitol-containing medium.
WT, BS and 4COL seedlings were applied with 5% manitol for 28 days. Leaves of WT (a), BS (b) and
4COL (c) seedlings were vitrified, twist and fragile. Bars 2 mm (a-c).
Supplementary Fig. S6 Epidermal cells in mature zone of BS, 4COL and WT roots.
a-c Mature roots of BS, 4COL and WT seedling treated with 0.5×MS (a), glucose (b) and mannitol (c).
Observations were determined at 5 DAT. Cells for size and number quantification in mature zoon were
indicated with *. Bars 200 μm (a-c).
Supplementary Fig. S7 Flow cytometric analysis (FCM) of autotetraploid and diploid under glucose
and mannitol treatment
a-c FCM analysis of WT (a), BS (b) and 4COL (c) seedlings grown on control medium (0.5×MS). d-f
FCM analysis of WT (d), BS (e) and 4COL (f) seedlings grown on glucose medium (0.5×MS+5%
14
glucose). g-i FCM analysis of WT (g), BS (h) and 4COL (i) seedlings grown on mannitol medium
(0.5×MS+5% mannitol). Three-day-old seedlings were transferred to control, glucose- /
mannitol-containing mediums, treated for 10 days and observed by FCM.
Supplementary Fig. S8 Relative expression level of cell cycle genes in WT, BS and 4COL plants
under glucose treatment.
Three-day-old seedlings were treated with glucose for 7 days, and total RNA was extracted from eight
to ten plants. Gene expression was analyzed by qRT-PCR. Data are presented as mean ± standard
deviation from three biological samples and three technical repeats for each sample. WTCK, BSCK and
4COLCK stand for plants grown on 0.5×MS medium, WTG, BSG and 4COLG stand for plants grown
on 0.5×MS+5% glucose medium.
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