2.1 Plant materials and bacteria
Leaf explants from 20-d to 30-d aseptic seedlings (Fig. 1) of P. auriculata obtained via seeds aseptic germination, which were used for hairy roots induction at the Biotechnology Laboratory.
Three A. rhizogenes strains A4, ATCC 15834 and LBA 9402, which were used to transfect purchased from the ATCC (American Type Culture Collection, Rockville, Maryland, USA), and stored at − 80℃. The bacteria was activated on yeast mannitol broth (YMB, yeast extract 1 g·L− 1, mannitol 10.0 g·L− 1, K2HPO4 0.5 g·L− 1, MgSO4·7H2O 0.2 gL− 1, NaCl 0.1 g·L− 1, agar 12 g·L− 1, pH 7.2) media at 28 ℃ for three times as it was described previously protocol. In the last time of liquid culture of bacteria, the liquid YMB media was supplemented with acetosyringone (AS, 100µmol· L− 1) to increase the frequency of hairy roots induction.
2.2 Different species of A. rhizogenes-mediated genetic transformation of hairy roots
In order to facilitate the description of hairy root systems induced by three different A. rhizogenes A4, ATCC 15834 and LBA 9402, they were named PAHR 4, PAHR 15834 and PAHR 9402 respectively in this paper, while the roots of the control group were named PAR TK.
The hairy root induction formula can be seen as follows: (1) immerse about 1cm2 of sterile leaves in active bacteria (OD600 about 0.5 to 0.8) for 25 to 30 minutes, and shake gently during this period; (2) Take out the explants and absorb the water with sterile filter paper. Then it was transferred to the medium added with 1/2 MS + AS 100 µ mol·L– 1, and incubated at 28 ℃ in complete darkness for 2–5 days; (3) After obvious plaques appeared around the explants, they were transferred to 1/2 MS + Cef medium containing cefotaxime in complete darkness at 25 ± 1 ℃ until they were completely sterilized. Cef concentration changes were adjusted to 500, 400, 300, 200, 100 and 0 mg·L– 1/week. The control group was replaced with sterile water, and the biological experiment was repeated three times.
2.3 Identification of hairy roots
Morphological identification.
Morphological characteristics of hairy roots and untransformed roots (namely sterile tissue culture roots, as the control) were observed, including initial state, color, thickness, presence or absence of branches, villi, and geotropism.
Organization structural identification.
Hairy roots (30 d) and tissue culture roots (30 d, as control) took for paraffin section analysis. The steps were as follows: (i) Picking and fixing: Samples that were collected and placed in FAA fixative (70% ethanol: glacial acetic acid: formalin was 18:1:1), and fixed at 4℃ for more than 24h. (ii) Dehydration and transparency༚Samples dehydrated with 75% alcohol for 4 h, 85% alcohol for 2 h, 90% alcohol for 2 h, 95% alcohol for 1 h, absolute ethanol I for 30 min, then absolute ethanol II for another 30 min. Then material was then transparented for 5 ~ 10 min for alcohol-benzene, 5 ~ 10 min for xylene I and 5 ~ 10 min for xylene II. (iii) Wax dipping and embedding༚Samples was placed in a beaker with melted wax powder and dry 1 h at 60°C, and this step needs to repeat three times. Then samples conduct of embedding. (iv)Sectioning and gluing༚ Samples slice to a thickness of 3 µm and stick to a glass slide with one drop of 10 g·L− 1 gelatin solution. (v) Dewaxing and staining༚The slices were placed in xylene for 20 min and another 20 min, absolute ethanol for 5 min and another 5 min, 75% alcohol for 5 min. Then staining with 5 g·L− 1 sarranine solutions for 1–2 h .Then slices were placed in 50%, 70%, and 80% gradient alcohol for 3–8 s each.Then put it into 5 g·L− 1 fast green staining solution for 30–60 s, and dehydrate with absolute ethanol. (vi) Cover slides and microscopy: The slices need to be sealed with neutral gum and dried for 12 h at 40°C. Finally, we carry out microscopic examination and analysis.
PCR molecular identification.
Appropriate hairy roots and un-transformed roots were collected randomly, Using the modified CTBA method (štorchova et al., 2000; Nathar & Gadge, 2013) extracted genomic DNA from each root system, SanPrep column plasmid DNA small volume extraction kit was used to extract plasmid genomic DNA from A. rhizogenes. The rolA, rolB, rolC genes on the T-DNA of A. rhizogenes and the virD gene outside the T-DNA fragment were detected, and the forward and reverse primers used for amplification are shown in Table 3. The known nucleotide sequence was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.
Table 1
Statistics for dynamic parameters and morphological characteristics of hairy roots and tissue culture roots of P. auriculata. PAR C was the logogram of P. auriculata roots of control and PAHR was the logogram of P. auriculata hairy roots. Induction rate (%) = number of rooting explants / number of inoculation explants × 100%. Browning rate (%) = number of browning explants / number of inoculation explants × 100%. Callus rate (%) = number of callus explants / number of inoculation explants × 100%.Pollution rate (%) = number of pollution explants / number of inoculation explants × 100%.Mortality rate (%) = number of mortality explants / number of inoculation explants × 100%. Rooting time (d) represented the first root was starting from the first day of co-culture. Slender (or thick) represented that diameter of roots was between 0.5 to 1.0 cm. −, there was no root grew; +, roots grew well; ++, roots grew very well. ND, not detected. Control group were conducted in sterile water. Data represented standard deviations (SDs) of the mean (n = 3). Different lowercase letters indicated that mean are significantly different among the treatments at P < 0.05 according to Duncan’s test. For description conveniently, hairy roots induced by A4, ATCC 15834 and LBA 9402 were named as PAHR 4, PAHR 15834 and PAHR 9402, and the tissue culture roots (the control) was named as PAR TC. The ND is an abbreviation for no detected, indicating that it is not detected; "-" means no growth; "+" means average growth; "++" means good growth; "+++" means good growth; The values in the table are mean ± standard error, and different lower case letters in the same column indicate significant difference between treatments (P < 0.05).
Strains
|
Naming
|
Co-culture
(d)
|
Induction rate
(%)
|
Browning rate
(%)
|
Callus rate
(%)
|
Pollution rate
(%)
|
Mortality rate
(%)
|
Rooting time
(d)
|
Colour
|
Thick/Slender
|
Branches
|
Tomentum
|
Geotropism
|
Others
|
|
Control
|
PAR TC
|
3ཞ4
|
0.00 ± 0.00 a
|
23.17 ± 1.48 d
|
4.95 ± 0.21 d
|
0.12 ± 0.03 a
|
68.01 ± 0.43 d
|
ND
|
ND
|
ND
|
ND
|
ND
|
ND
|
-
|
|
A4
|
PAHR 4
|
3ཞ4
|
46.18 ± 1.62 b
|
12.08 ± 2.11 c
|
3.99 ± 0.12 c
|
0.1 ± 0.01 a
|
15.8 ± 0.43 c
|
14.33 ± 0.58 c
|
White
|
Minuteness
|
D
|
D
|
ND
|
+
|
|
15834
|
PAHR 15834
|
3ཞ4
|
86.78 ± 0.74 d
|
2.07 ± 0.32 a
|
2.04 ± 0.08 a
|
0.11 ± 0.02 a
|
5.43 ± 0.69 a
|
8.33 ± 0.58 a
|
White
|
Minuteness
|
D
|
D
|
ND
|
+++
|
|
9402
|
PAHR 9402
|
2ཞ3
|
54.23 ± 2.34 c
|
8.71 ± 0.63 b
|
3.57 ± 0.40 b
|
0.12 ± 0.01 a
|
14.06 ± 0.58 b
|
11.67 ± 0.58 b
|
White
|
Minuteness
|
D
|
D
|
ND
|
+
|
|
Table 2
Plumbagin content in different roots of P. auriculata. DW meant dry.
Roots
|
Actual injection volume
(µL)
|
Retention time
(min)
|
Peak area
(mAUs)
|
Content
(mg/g)
|
Percentage content
(%)
|
PAR TC
PAR NC
|
20
20
|
21.55
21.55
|
26.15
422.59
|
0.54
9.84
|
0.05
0.98
|
PAHR 4
|
20
|
21.54
|
1043.28
|
24.72
|
2.47
|
PAHR 15834
|
20
|
21.55
|
1436.61
|
38.95
|
3.90
|
PAHR 9402
|
20
|
21.55
|
1135.46
|
24.90
|
2.49
|
Table 3
Amplication primer of gene rolA, rolB, rolC and virD
Num.
|
Gene names
|
Forward/Rreverse
|
Primer sequences
|
Length (bp)
|
References
|
A
|
rolA
|
F
|
5′-CGT TGT CGG AAT GGC CCA GAC C-3′
|
258
|
Hu J et al., 2016.
|
R
|
5′-CGT AGG TCT GAA TAT TCC GGT CC-3′
|
B
|
rolB
|
F
|
5′-ATG GAT CCG AAA TTG CTA TTC CTT CCA CGA-3′
|
780
|
Gangopadhyay et al., 2008.
|
R
|
3′-TTA GGC TTC TTT CTT CAG GTT TAC TGC AGC-5′
|
C
|
rolC
|
F
|
5′-ATG GCT GAA GAC GAC CTG TGT-3′
|
550
|
Thiruvengadam et al., 2014.
|
R
|
5′-TTA GCC GAT TGC AAA CTT GCA-3′
|
D
|
virD
|
F
|
5′-ATG TCG CAA GGC AGT AAG CCC-3′
|
438
|
Mohammad et al., 2016.
|
R
|
5′- GGA GTC TTT CAG CAG GAG CAA − 3′
|
The reaction system (25 µL) included Buffer (2.5 µL), dNTPs (2.0 µL), forward primer (1.0 µL), reverse primer (1.0 µL), Taq enzyme (0.35 µL), ddH2O (16.2 µL), and template DNA (2.0 µL). After vortex mixing and centrifugation, PCR reaction: predenaturation at 95 ℃ for 5 min, (denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s) repeated for 34 cycles, extension at 72 ℃ for 5 min; 4℃ terminal extension ∞. DL 2000 DNA Marker, plasmid DNA and un-transformed roots DNA amplification products used as reference, positive and negative control, respectively, and electrophoresed on a 1% agarose gel electrophoresis. Then gel stained with ethidium bromide (EB) for 10 min and scanned by UV light scanning on a gel imaging system (UV 260 nm).
2.4 Plumbagin content quantification of hairy roots
This experiment was performed by high-pressure liquid chromatography (HPLC) .
Chromatographic condition. The mobile phase was 0.1% H3PO4 : methanol ( v : v, vacuum filtered through a 0.45 µm filter before use), and the elution gradient was 0 min ( 0 : 40), 21 min ( 25 : 75), 23 min ( 10 : 90), 30 min ( 10 : 90), the flow rate was 1 mL·min–1, the detection wavelength was 254 nm, the column temperature was 40 ℃, the detection temperature was room temperature, the sensitivity was 0.16 AUFS, the injection volume was 20 µL, and the retention time was about 21 min. The operating temperature was kept at room temperature.
Standards and Sample Preparation. The following are the specific steps for preparing the standard: (1) In the first step, we need to weigh 5.1mg of the standard plumbagin and place it in a 10mL volumetric flask. Then, add an appropriate amount of methanol into the bottle to dissolve and fix the volume. Finally, shake it well to obtain standard stock solution A (0.510 mg·mL–1). (2) First, we need to take 2mL of standard stock solution A and put it into a 10mL volumetric flask. Then, add an appropriate amount of methanol to the bottle to constant volume. Finally, shake it well to obtain standard stock solution B (0.102 mg·mL–1). (3) First, we need to take 1mL of standard stock solution B and put it into a 25mL volumetric flask. Then, add an appropriate amount of methanol to the bottle to constant volume. Finally, shake it well to obtain standard stock solution C (0.004 mg·mL–1). The standard stock solution of each concentration shall be stored at 4 ℃ for standby. The following are the specific steps of sample preparation: (1) In the first step, we need to randomly select an appropriate amount of roots from different sources PAHR 4, PAHR 15834, PAHR 9402, tissue culture seedling root system (PAR TC) and seedling root system (PAR NG) in triplicate. It is dried in an oven at 65 ℃ for 24 hours. Then it is dried to constant weight at 45 ℃, and then it is fully ground and crushed into coarse powder, which is then sieved through a 40 mesh sieve. (2) In the first step, 0.10 (± 0.02) g of dried powder of each root system needs to be weighed, dissolved in methanol solution and fixed to 25 mL. After the fresh-keeping film is sealed and placed in the ultrasonic cleaner for ultrasonic extraction at room temperature for 30 min, the sample solution is replenished to 25mL with methanol solution again. (3) It is necessary to mix three solutions from the same source with 0.22 µM water soluble filter membrane is filtered and stored in 10mL EP tube.
For the determination of the sample plumbagin, it is necessary to draw an appropriate amount of solution from each EP tube to an Agilent bottle (1.5mL), and then make use of the machine for HPLC analysis with the injection volume of 20 µL.
2.5 Statistics and Analysis
All data were analyzed using SPSS Statistics 22 (SPSS Inc., Chicago, USA) software statistical analysis, one-way ANOVA and least significant difference test (LSD), Excel 2016 and Origin 9.0 (OriginLab Inc., Massachusetts, USA) Software tabulation. The significance level was set to α = 0.05.