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Seeds of C. korshinskii germinated normally are very difficult to handle for explant preparation since the embryo tip is delicate, tiny, and generally quite smooth. Therefore, we pre-treated the explants: in brief, seeds that had germinated for approximately 4 d (Fig. 1b) were transferred to MSB5 pre-culture medium with 1 mg·L−1 6-BA added and had cultured for approximately 1 week, and significant differences in appearance in the embryo tip (Fig. 1f, photos in the lower panel) were observed compared to those without cytokinin addition (Fig. 1f, photos in the upper panel). The pre-cultured seeds had short and thick embryonic axes, and the apical parts were also significantly thicker and tended to be 'enlarged' (Fig. 1c−e, & g, pictures in the lower panel), possibly as a result of cytokinin inhibition on the apical bud dominance and promotion on the increased cell division size.
Embryonic tip in vitro regeneration system
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After pre-treatment (Fig. 2a), 2 mm of the 'enlarged' apical explants were removed and inoculated into the adventitious shoot induction media, and then the explants have been kept for expansion for around 5 d (Fig. 2b). Granular protrusions could be seen to form on the surface of the expanded explants at around 20 d (Fig. 2c). Next, they were sub-cultured once to successfully generate adventitious buds (Fig. 2d). The adventitious bud induction rate was calculated statistically (Table 1), and it was discovered that with 6-BA concentration increased, the adventitious bud induction rate exhibited a tendency of initially increasing and then decreasing. When the concentration of 6-BA was 3 mg·L−1, the rate of adventitious bud induction reached 78%, the average number of buds per explant was around eight, and the buds were in a healthy growing condition. When the concentration of 6-BA was 4 mg·L−1, the induction rate of adventitious buds was 63%, and the average number of buds was around five. With the concentration of 6-BA increasing further, adventitious buds differentiation was hindered, while base browning and yellowish seedlings emerged. Therefore, 3 mg·L−1 6-BA was the optimal hormone concentration for inducing adventitious buds from the 'enlarged' embryonic tip.
Table 1. Effect of different 6-BA concentrations on the induction rate of adventitious buds from embryonic tip explants.
6-BA concentration
(mg·L−1)Induction rate of adventitious buds (%) Average number of adventitious buds 0 8.89 ± 0.02e 2.72 ± 0.25e 1 30.00 ± 0.07d 3.80 ± 0.18d 2 52.22 ± 0.05c 5.14 ± 0.38c 3 77.78 ± 0.07a 8.19 ± 0.29a 4 63.33 ± 0.06b 5.75 ± 0.24b 5 48.89 ± 0.07c 4.71 ± 0.25c 6 31.11 ± 0.02d 4.18 ± 0.17d Values represent the mean ( ± standard error) of three independent experiments. Different letters of the same column indicate significant differences at P < 0.05. To improve the frequency of embryonic tip regeneration and obtain more regenerated buds, we observed the growth of adventitious buds at various stages (Supplemental Fig. S2) and noticed that after 20 d of culture, the embryonic tip had thickened, the base had enlarged, and occasionally 1−2 buds sprouted. The number of adventitious buds was between eight and 12 after 40 d of cultivation, and they formed tight clusters. After 50 d of culture, the embryonic tip expanded further, 'storm-like' adventitious buds (Fig. 2e), and the number of adventitious buds was between 26 and 31. However, as culture time prolonged further, the increasing rate of adventitious buds slowed down after 60 d, the leaves color of the bushy buds deepened, the buds felt more fragile, and the water-soaked appearance was severe. Therefore, the above-mentioned results indicate that the cluster buds required one subculture to produce 'stormy-like' adventitious buds. The local magnification images of the adventitious buds regenerated from the embryonic tips are shown in Supplemental Fig. S3.
Adventitious bud elongation requires both cytokinin and auxin, and in plant tissue culture, the predominant auxin used is naphthylacetic acid (NAA). For research on adventitious bud elongation, we induced longer shoots using a range of 6-BA and NAA combinations at various concentrations (Table 2). Except for the control, all other treatments were capable of making the adventitious shoots elongate. However, the elongation effect and growth status of the adventitious shoots varied greatly due to the various hormone levels. For example, after only one week in the medium without any added hormones, the yellow seedlings had already perished, and the adventitious buds showed no tendency to elongate. The greatest effect on elongation was achieved when 1 mg·L−1 6-BA was combined with 0.1 mg·L−1 NAA, and some of the adventitious buds started to elongate after about 4 weeks of culture (Fig. 2f), and the adventitious bud elongation rate was 68% with good growth status. After subculture, a lot of calli formed at the base, and the shoots displayed various degrees of vitrification. At higher concentrations of hormone combinations, however, the elongation rate of adventitious shoots decreased, a large number of calli formed, and the elongated shoots displayed severe vitrification. Therefore, the hormone ratio of 1 mg·L−1 6-BA and 0.1 mg·L−1 NAA were only suitable for the one-step elongation of adventitious shoots that were transferred to MSB5 medium with 0.7 mg·L−1 6-BA and 0.07 mg·L−1 NAA, the final elongated adventitious shoots were produced after 2 weeks (Fig. 2g).
Table 2. Effect of different combinations of 6-BA and NAA on the elongation of adventitious buds.
Treatments Hormones and concentrations (mg·L−1) Elongation
rate (%)6-BA NAA 1 0 0 6.03 ± 0.04d 2 0.3 0.03 30.50 ± 0.07bc 3 0.5 0.05 41.43 ± 0.06b 4 0.7 0.07 59.49 ± 0.05a 5 1.0 0.10 67.92 ± 0.08a 6 2.0 0.20 26.11 ± 0.06c Values represent the mean ( ± standard error) of three independent experiments. Different letters of the same column indicate significant differences at P < 0.05. A single shoot from the elongated buds (the shoot height > 2.5 cm) was separated and planted in an MSB5 medium with different concentrations of NAA to induce roots. The rate of adventitious root induction and the growth status of the resulting seedlings were then measured and calculated (Table 3). In this test, we found that the plant acclimatization and transferring to pots were difficult to survive if there were both massive calli growth and roots at the base of the stem. It is probably owing to the weak connection of the vascular tissue between the roots and shoots[27]. It was observed that 1 mg·L−1 NAA was the best concentration for inducing adventitious roots, and 3 cm of roots were visible after 15 d of induction. In addition, the adventitious roots developed directly from the vascular tissue when treated with this concentration of NAA. The rooted regenerating seedlings were obtained after over 30 d of culture (Fig. 2h), with a rooting induction rate of 79%. Calli developed readily at the root base when the NAA concentration was higher than the ideal one, and the induced adventitious roots were unhealthy and difficult to transfer successfully. Robust bottle seedlings were selected for nursling, and after 5 weeks of transferring, the survival rate of the rooted seedlings (Fig. 2i) was more than 95%.
Table 3. Effect of NAA concentrations on adventitious roots induction.
Treatments NAA concentration
(mg·L-1)Number of
explantsRooting rate
(%)1 0 90 0d 2 0.5 90 41.11 ± 0.18bc 3 1 90 78.89 ± 0.15a 4 2 90 55.56 ± 0.14ab 5 3 90 28.89 ± 0.08c Values represent the mean ( ± standard error) of three independent experiments. Different letters of the same column indicate significant differences at P < 0.05. Antibiotic effects on non-transformed explants growth
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Appropriate concentrations of cefalexin have been proven to decrease the harmful effects of Agrobacterium on explants and to successfully inhibit Agrobacterium growth after co-cultivation[28]. Thus, we investigated how the embryonic tip was sensitive to cefalexin when establishing the Agrobacterium-mediated genetic transformation system. The results showed that embryonic tips were more sensitive (Fig. 3a) when the concentration of cefalexin was 300 mg·L−1 or higher, and the growth of the explants was obviously inhibited, yellowish and dead seedlings appeared at high concentrations of cefalexin. When the concentration of cefalexin was 200 mg·L−1, the sprouts grew slowly but in good status and could still develop into seedlings. So 200 mg·L−1 was determined as the optimal inhibitory concentration of cefalexin.
Figure 3.
Effects of cefotaxime, kanamycin and hygromycin on growth of explants. (a) Growth status of the embryonic tips in response to different concentrations of cefotaxime. (b) Growth status of the embryonic tips after being treated with various concentrations of kanamycin. (c) Growth status of the regenerated shoots under gradient concentrations of kanamycin. (d) Growth status of the embryonic tips after being treated with various concentrations of hygromycin.
During Agrobacterium-mediated transformation, a step-by-step screening approach was used. After co-culture with A. tumefaciens, the embryonic tips should be transferred to a selection medium for culture. It is generally acknowledged that cells that survived the selective pressure would be positive transformants and that the screening antibiotics concentration should neither be too low to increase the possibility of false-positive plants, nor too high to inhibit the growth and differentiation of the transformed cells. Our results revealed that the sensitivity of different explants to kanamycin was not the same (Fig. 3b, c), and the frequency of explant regeneration decreased with the increasing kanamycin concentration. When the kanamycin concentration was 30 mg·L−1, the embryonic tip essentially stopped growing and the explants turned yellowish and died. Hence, the selection pressure of kanamycin concentration for the first step is 30 mg·L−1. To obtain more positive shoots, we increased the selection pressure in the latter stages. When the kanamycin concentration exceeded 40 mg·L−1, stem elongation was completely inhibited, and the explants perished. So the selection pressure of kanamycin concentration for the second step was determined to be 40 mg·L−1. To avoid selection escape and chimerism, 60 mg·L−1 Kan+ was used to select the fertile transgenic plants finally.
The embryonic tips were sensitive to hygromycin, and the survival rate decreased as the concentration increased. When the concentration of hygromycin was 3 mg·L−1, the growth of the explants was affected; when it was increased to 15 mg·L−1, the growth of the embryonic tips was severely inhibited and dead explants appeared; when the concentration of hygromycin reached to 25 mg·L−1, all the explants died. Therefore, 25 mg·L−1 was selected as the screening concentration of hygromycin.
Genetic transformation and plant regeneration
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The process of genetic transformation of C. korshinskii by the recombinant construct carrying CiDREB1C gene (which bears the kanamycin resistance) and plant regeneration is shown in Fig. 4. Explants were co-cultured for 2 d (Fig. 4e), then were transferred to an MSB5 recovery medium and bacteriostatic culture with 200 mg·L−1 cef for 3 d. After which, an adventitious bud induction selection culture was performed; and 4−8 weeks later, kanamycin-resistant granular protrusions were observed (Fig. 4f). After 3−6 weeks, resistant adventitious buds were effectively induced (Fig. 4g), and after further transferring to adventitious bud elongation selection medium until shoots elongated (Fig. 4h); then they were transferred to rooting medium and adventitious roots were successfully induced in roughly 5 weeks. Transgenic plants had been obtained when plants with well-developed root systems were transferred into sterile soil (Fig.4i, j). Through rigorous screening, non-kanamycin-resistant buds ceased to grow, progressively turned yellow-brownish, and ultimately perished (Supplemental Fig. S4).
Figure 4.
Schematic illustration of embryonic tip transformation system and GUS staining results in C. korshinskii. In the schematic of genetic transformation ((a)−(j), upper panel), the plumule is removed from the red line. (k) Pictures of GUS staining from the un-transformed calli and buds. (l) Pictures of GUS staining from the transformed calli and buds.
Using GUS histochemical staining that transformation efficiency was identified to demonstrate the event's reliability. The process of genetic transformation by the recombinant construct carrying the GUS gene (which bears the hygromycin resistance) and plant regeneration shown in the lower panel of Fig. 4. The histochemical verification of GUS activity was performed in presumed resistant transgenic buds, with the un-transformed buds serving as a negative control (Fig. 4k). Blue staining was observed during adventitious bud induction (Fig. 4l), indicating that the GUS gene had been stably expressed.
Molecular identification of transgenic plants
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Based on the initial success of transforming GUS reporter gene, we attempted to transform RUBY and CiDREB1C genes into C. korshinskii with an effort to set up a stable genetic transformation system. RUBY can function as a visual selection marker for transgenic events to identify transgenic-positive plants with an accumulation of reddish betalain. We achieved positive results in C. korshinskii which was consistent with that of other species[8]: the reddish color was observed in petioles and leaves (Fig. 5a) of the potential transformants, and further identification of the RUBY gene in transgenic buds were performed by extracting leaf genomic DNA for PCR amplification. The results revealed that the RUBY gene had been transferred into C. korshinskii successfully (Fig. 5c). To further test the efficacy of the genetic transformation system, the CiDREB1C gene was transformed into C. korshinskii, and primers specific to the CaMV35S promoter, the NOS terminator, and the Kan marker on the pCanG-HA vector were designed for PCR detection, and the un-transformed buds served as a negative control. The CaMV35S promoter was not amplified in the negative control, but the predicted bands were detected in the transgenic samples, as shown in Fig. 5d. The above results indicate that transgenic CiDREB1C plants can be produced with kanamycin selection, and all transgenic plants were viable following acclimatization and after transferring into the soil. Except for the dwarf phenotype of the transgenic CiDREB1C plants, there were no discernible differences compared to non-transgenic plants (Fig. 5b). Using embryonic tips as explants, our results show a stable transformation system for C. korshinski has been established. Even though the transformation efficiency is still low currently, the stable transformation system provides the technological foundation for future optimization.
Figure 5.
Accumulation of betalain in transgenic C. korshinskii buds and PCR detection of the RUBY and CiDREB1C genes. (a) Phenotype of RUBY-positive buds, as indicated by the red arrows. (b) CiDREB1C transgenic plants and the negative control (WT). (c) Eletrophoresis results of the PCR products from RUBY in the positive transgenic buds; Lane R1-R5, the PCR products from the five segments of RUBY. M, molecular weight marker (DL5000). (d) Electrophoresis results of the PCR products from CiDREB1C in the positive transgenic seedlings and wild type control; M, molecular weight marker (DL2000). Lane 1: negative control, PCR products for the Kan gene (lane 2), the NOS terminator (lane 3), and the CaMV35S promoter (lane 4).
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Using the embryonic tip as explant is an efficient method of transgenic breeding. We describe for the first time that the embryonic tip of C. korshinskii was utilized as the explant for in vitro regeneration and genetic transformation, and we established a stable and efficient Agrobacterium-mediated genetic transformation system, which has substantial advantages over previous research. The findings of this research provide a powerful technique foundation for the application of genetic engineering for the modification and improvement of C. korshinskii.
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About this article
Cite this article
Liu B, Shang X, Zhang X, Shao W, Ren L, et al. 2023. In vitro regeneration and Agrobacterium-mediated genetic transformation of Caragana korshinskii. Forestry Research 3:14 doi: 10.48130/FR-2023-0014
In vitro regeneration and Agrobacterium-mediated genetic transformation of Caragana korshinskii
- Received: 29 January 2023
- Accepted: 16 May 2023
- Published online: 31 May 2023
Abstract: Caragana korshinskii is a deciduous shrub with large eco-economic value and strong tolerance to abiotic stresses. However, the shortage of reliable genetic transformation technology severely hinders its research on stress tolerance mechanisms and stress-resistant gene mining and application. In this study, the embryonic tip of the C. korshinskii seedling was used as the initiating explant to get regenerated plant through the direct organogenesis pathway, which significantly shortened the culture cycle and set the foundation for investigation of Agrobacterium-mediated genetic transformation. Our results suggest that the embryonic tip possesses robust meristem capacity and is an efficient method for transgenic breeding. This research provides a technical basis for asexual reproduction, molecular breeding, and gene function investigation in C. korshinskii by establishing, for the first time, an effective in vitro regeneration system and an Agrobacterium-mediated stable genetic transformation system utilizing the embryonic tip of C. korshinskii as explants.