Plant material
After anthesis for 30 days, the immature fruit was taken from the germplasm resources garden of the canna lily at Guizhou University from July to October 2021. First, the fruits were washed with liquid detergent for 10 minutes and rinsed with distilled water 5 times. Next, the immature seeds were taken from the fruits on the ultra-clean workbench and placed in a beaker. Then the fruits were treated with 75% ethanol for 30 s, rinsed 5–6 times using sterile distilled water, disinfected with 0.1% (w/v) mercuric chloride for 7 min, rinsed 3–5 times with sterile distilled water, and dried on a sterile filter paper. After that, the immature zygotic embryo were isolated from intact seeds for embryogenic callus induction.
Embryogenic callus induction
MS medium was added with 30 g L − 1 sucrose, and 7 g L − 1 agar was used as the basic media (pH 5.8). Cultivation was conducted with different concentration plant growth regulators, namely, 6-BA (2.0, 4.0, 6.0 mg L − 1), NAA (0.1, 0.2, 0.5 mg L − 1), and TDZ (1.0, 1.5, 2.0 mg L − 1). Nine treatments were selected for the three-factor and three-level orthogonal experimental design. The sterilized young embryo was a 0.5 mm thin slice with 30 explants per treatment, replicated three times. In the second week after inoculation, the explants were transferred to a fresh induction medium daily and then transferred to a fresh medium every two weeks. The growth of embryogenic callus was observed weekly, and embryogenic callus induction was counted for 30 days respectively.
On the 15th day of induction, two kinds of calli of different shapes and forms were selected to observe the occurrence of embryonic callus microscopically. First, the MS medium on the surface was removed by gently washing it with sterile distilled water. The samples were then sucked dry, observed, and photographed with an optical microscope (DM 750 LED), and the embryonic callus with distinct characteristics was selected for paraffin section observation.
Somatic Embryogenesis Induction
Six treatments by orthogonal experimental design were set up through supplement with 6-BA (0.0, 1.0, 1.5, 2.0, 2.5, 3 mg L − 1) and TDZ (0.0, 0.5, 1, 1.5, 2, 2.5 mg L − 1). The embryogenic callus with the same growth condition was cut into 0.5 cm × 0.5 cm in size and inoculated on the medium, 10 bottles for each treatment, replicated three times. The growth status of embryoid induction was counted for 30 days, respectively.
In order to observe somatic embryogenesis microscopically, samples were collected from MS medium. When the callus showed obvious somatic embryo characteristics, samples of different somatic embryo development stages induced by embryogenic callus were taken every 5 days and then gently washed with sterile distilled water to remove MS medium on the surface. The samples were then sucked dry, viewed, and photographed using a light microscope (LEICA ICC50W).
Somatic embryo proliferation culture
The embryoids with the same growth after subculture were inoculated on the embryoid proliferation medium with different combinations of plant growth regulators. The experiments were carried out with 6-BA (1.0, 3.0, 6.0 mg L− 1), NAA (1.0, 1.5 mg L− 1), and TDZ (0.2, 0.5 mg L− 1), respectively. Each treatment of 10 bottles was replicated three times, and the growth status of embryoid proliferation was counted for 30 days.
Somatic embryo germination and conversion
The obtained white or green normal somatic embryos with a diameter of 1 cm and regular shape were inoculated onto the designed somatic embryo maturation medium (Table 4) to induce somatic embryo maturation. First, 10 bottles of each medium formula were inoculated, each treated material was inoculated with 25 individual cell embryos, and each treatment was replicated three times. Next, the embryogenesis of somatic cells was measured after 15 days of culture, then inoculating the mature somatic embryos into a designed somatic embryo germination culture medium, inducing the mature somatic embryos to regenerate, observing and counting the plant regeneration condition after 30 days.
Table 3
Effect of plant growth regulators (PGRs) on the Somatic Embryo Proliferation from Canna × generalis
Concentrations of PGRs (mg/L)
|
Somatic embryo proliferation rate (%)
|
Proliferation times
|
6-BA
|
NAA
|
TDZ
|
1
|
1
|
0.2
|
0.00 ± 0.00d
|
0.00 ± 0.00d
|
1
|
1.5
|
0.5
|
0.00 ± 0.00d
|
0.00 ± 0.00d
|
3
|
1
|
0.2
|
14.00 ± 2.00c
|
0.12 ± 0.03d
|
3
|
1.5
|
0.5
|
34.33 ± 2.96b
|
2.99 ± 0.18c
|
6
|
1
|
0.2
|
46.33 ± 2.03a
|
7.83 ± 0.16a
|
6
|
1.5
|
0.5
|
37.33 ± 2.96b
|
5.53 ± 0.13b
|
*Data in the table represent mean ± standard error, and different letters (a, b,…) in the same column represent statistically significant differences at p < 0.05 (Duncan’s test) |
Table 4
Effects of different medium formulations on seedling culture of Canna × generalis embryoids
culture medium
|
Number of embryos
|
Germination rate (%)
|
Seedling rate (%)
|
Seedling growth status
|
MS
|
25
|
94.67 ± 3.53a
|
88.00 ± 4.62a
|
green and grow robustly
|
B5
|
20
|
23.33 ± 1.66c
|
13.33 ± 1.67b
|
green and grow weakly
|
1/2MS
|
22
|
52.66 ± 4.09b
|
24.00 ± 7.62b
|
green and grow robustly
|
*Data in the table represent mean ± standard error, and different letters (a, b,…) in the same column represent statistically significant differences at p < 0.05 (Duncan’s test) |
Acclimation and transplantation
Vigorous embryoid sterile seedlings were selected and moved to the greenhouse for 1 day to adapt to the greenhouse environment. Next, the seedlings were uncovered and refined for 2 days. Then the germinated seedlings were transplanted to the peat screened by the research group in the early stage: organic fertilizer: soil = 1:1:1 for strong seedling culture.
Histomorphology observation
Two kinds of calli of different shapes and somatic embryos of different stages of good growth were fixed overnight with 75% FAA (formaldehyde, glacial acetic acid, 75% ethanol 1:1:18). After discarding the fixing solution, the somatic embryos were treated with 70, 80, 95 and 100% ethanol successively, and soaked in anhydrous ethanol twice to ensure complete dehydration. It is then treated with xylene for transparency. After paraffin embedding, the wax blocks were divided according to the material placement position, and the divided wax blocks were trimmed into trapezoids. It was placed at 4°C for 24 h, and paraffin sections were prepared. The embedded wax blocks were cut into 8–10 µm slices using a microtome (Erma, Japan), and the slides were dried in a slide dryer at 38 ℃. The sections were observed and photographed under an optical microscope (LEICA ICC50W) after dewaxing, ethanol remixing, zorubin green staining, and neutral gum seal. The different stages of SE were recorded, and the dynamic changes in cells and their origin during SE were observed.
Culture conditions
The basic medium used in this experiment was MS medium, and the reagents were purchased from Beijing Solarbio Biological Company. The medium pH of all experiments was 5.8 ± 0.2, sterilized at 121℃ for 25 minutes; unless otherwise specified, MS solid medium was supplemented with 7 g·L− 1 agar as a coagulant and 30 g·L− 1 sucrose. All plants were cultured under a light intensity of 25–35 µ mol·m− 2·s− 1 at room temperature of (25 ± 2) ℃.
Statistical analysis
Excel software (Excel 2017) was used for statistical data. SPSS software (SPSS 20.0) was used for data processing and one-way analysis of variance (ANOVA), and Duncan’s multiple tests was performed. All test treatments were set with three biological replicates.