Effects of the different treatments of the explants on primary culture
After 3-5 days, the seeds started their germination. The results showed that by soaking only with 75% ethanol and 3% sodium hypochlorite, the contamination rate was as high as 90% and the germination rate was only 22%. The removal of the black leathery exotesta reduced the contamination rate to 18%, and the germination rate was significantly increased to 70%. While removing the black leathery exotesta and brown villous endotesta, the contamination rate was significantly reduced to 11%, and the germination rate was significantly higher than that of other groups 98% (Fig. 1a-b). Therefore, removing the inner and outer seed coats during aseptic germination can significantly reduce the pollution rate and significantly increase the germination rate.
Induction and subculture
After one month of culture, the callus started formation (Fig. 1c), which was significantly associated with 6-BA concentration, and apparently low concentrations of 6-BA slowed callus formation while NAA had less significant effect on callus formation Table 1. For the callus proliferation, 2,4-D was added to the subculture medium, which showed that the addition of 2,4-D simultaneously optimized and promoted the proliferation of the callus. The initially induced callus was mostly yellow and calloused, but became soft, pale yellow as the callus proliferation grew (Fig. 1d). The optimum MS medium for callus induction was determined to be 3.5 mg∙L-1 6-BA and 0.1 mg∙L-1 NAA according to the induction of yellowish pure, loose granular and large callus and the proliferation medium was determined to be 4.0 mg∙L-1 6-BA, 0.1 mg∙L-1 NAA and 0.1 mg∙L-1 2,4-D.
Table1 Number of callus tissues induced by seed explants.
6-BA(mg∙L-1)
|
|
NAA(mg∙L-1)
|
|
0.1
|
0.2
|
0.3
|
1.5
|
11.33 ± 1.53 ef
|
13.67 ± 2.52 de
|
8.67 ± 2.08 f
|
2.0
|
15.67 ± 3.06 de
|
15.33 ± 1.53 de
|
12.67 ± 1.53 ef
|
2.5
|
15.67 ± 3.21 de
|
18.33 ± 1.53 cd
|
14.67 ± 3.21 de
|
3.0
|
26.33 ± 1.53 ab
|
23.67 ± 2.31 ab
|
21.00 ± 3.00 bc
|
3.5
|
27.00 ± 1.00 a
|
25.67 ± 1.53 ab
|
22.67 ± 2.08 abc
|
4.0
|
25.33 ± 1.53 ab
|
24.00 ± 2.00 ab
|
21.00 ± 2.65 bc
|
Means with only the same letters are not signifcantly diferent at the 5% level based on Duncan’s multiple range test (p≤0.05).
Differentiation and rooting
Callus isolated into small pieces, who was cultured on MS medium with different concentrations of 6-BA (Fig. 1e), developed budlets after 14 days. When the concentration of NAA was 0.2 mg∙L-1, the shoots would grow faster as 6-BA concentration decreased from 3.0 to 1.0 mg∙L-1, but the speed of plant growth would slow down when 6-BA concentration belove 0.5 mg∙L-1 (Fig. 1j). The MS medium with 0.2 mg∙L-1 NAA and 1.0 mg∙L-1 6-BA could not only stimulate shoot growth, but also promote formation and proliferation of small shoots. Therefore, the MS medium with 0.2 mg∙L-1 NAA and 1.0 mg∙L-1 6-BA were demonstrated to be the best medium formulation for shoot growth (Fig. 1f-g). Roots began to generate on the 15th day. After 30 days, roots developed to 3-5 cm in length and 4 or 5 in number (Fig. 1h). The seedings were transferred to the folwerpot after proper training (Fig. 1i), and the survival rate of transplanting reached 100%.
Genetic transformation
Five levels of the Agrobacterium tumefaciens concentrations OD600 (0.3, 0.4, 0.5, 0.6 and 0.7) were measured. Calluses with good growth status were selected as the Agrobacterium-mediated transformation receptor (Fig. 3a-c). During the period of continuous slight shock, it is necessary to ensure that the bacterial solution fully into the transformation receptor but also to avoid damage to it due to excessive shock. The infected callus is sucked dry of the surface Agrobacterium solution and transferred to the co-media (Fig. 3d-f). The results show that when the density of the bacteria (OD600) increased from 0.3 to 0.6, the transformation rate increased and the callus was in good condition. However, when the density of the bacteria (OD600) > 0.6, most of the infection callus receptsor showed browning and death, which may be caused by the excessive concentration of the infection solution (Fig. 4a).
The effect of AS concentration on the transformation efficiency
Many studies demonstrated that small phenolic inducer molecules AS can induce the expression of the vir genes, in the cause of promoting the successful transformation of the Agrobacterium Ti plasmid into the host cells (Krishnamohan et al. 2001; Veluthambi et al. 1989). Therefore, the effect of AS concentration on the transformation efficiency was investigated. Among the tested concentrations of AS, the transformation rate enhanced with the increasing of AS concentration up to 100 uM, while the higher concentrations of AS (>100 uM) decreases the transformation rate. The size of the AS concentration did not significantly affect the transformation rate, but the transformation rate was better when the concentration was 100 uM (Fig. 4b). Higher concentrations of AS can affect the normal growth of receptor tissues, such as causing the overgrowth of Agrobacterium and the browning of explants.
Optimization of Timentin concentration
In this study, Timentin was selected as the antibiotic for inhibiting the growth of Agrobacterium. The co-cultured calluses were desterilized on media supplemented with different concentrations of Timentin. In this process, all the infected explants in the control group (0 concentration of Timentin) showed a large area of Agrobacterium growth circle on one side of the culture medium, resulting in the browning and death of the explants. When the concentration of Timentin increased by 250 mg∙L-1 and 300 mg∙L-1, the growth of Agrobacterium was significantly inhibited and the contamination rate was significantly reduced (Fig. 4c). However, When the concentration of Timentin was > 300 mg∙L-1, the explants showed serious yellowing and were on the verge of death. That is, the critical concentration of Timentin bacteriostatic is selected to be 300 mg∙L-1.
Hygromycin tolerance assay
A sensitivity assessment was performed in order to find the optimal concentration of hygromycin, since it varies from species to species. Appropriate hygromycin concentrations can promote the growth of positively transformed plants and inhibit non-transformed plants. Transformation receptors after five days of sterilizing culture were evenly tiled on differentiation media containing Timentin 300 mg∙L-1 and different concentrations of hygromycin. In the control group (0 concentration of hygromycin), all explants grown normally and were light yellow. When the concentration of hygromycin increased to 9 mg∙mL-1, some explants stopped growing and their tissues turned brown within 30 days, speculated to be receptors not successfully transferred into genes, but most survived and in good condition may be positive lines. However, when the hygromycin concentration > 9.0 mg∙L-1, the condition of the explants was poor, and they gradually turned white and stopped growing after ten days, indicating that excessive concentrations of hygromycin had a lethal effect on explants (Fig. 4d). Therefore, a concentration of 9 mg∙L-1 is the effective level of hygromycin to select a recognized transgenic plant.
Confirmation of transgenic plants
For preliminary identification of transgenic plants, GUS staining of co-cultured transformation receptors showed that GUS staining (Fig. 5b-f) was detected at the protein expression level of the successful transformation receptor, while no obvious GUS staining was observed in non-overexpressed gene GUS receptors. However, some of the non-transformed receptors still show slight blue spots (Fig. 5a), presumably due to too long staining time and insufficient decolorization treatment.
Numerous regenerative buds were formed during the regeneration of the same transformation receptor in Hemerocallis fulva‘Kanai’and separated into single plants for subculture. The leaf tissue DNA from the fused T0 transgenic plants was extracted by CTAB, and the transgenic status of the regenerated buds was further examined by PCR amplification using primers specific for the FT and GUS gene. In three stability tests, PCR product of FT gene (801bp) and GUS gene (300bp) were amplified from strains 1-1, 1-2, 1-3, 7-1, 7-2, 8-1, 8-2, 11-2, 13-2 and 13-3 in 1, 7, 8, 11, 13, and were named #1-10, respectively (Fig. 5g-h), had a transformatin rate of up to 12%. The results showed that transformation did not occur throughout the receptor and that protein expression was detected only in a fraction of it.