Effects of different genotype on adventitious bud regeneration
As shown in Table 2, the rate of callus formation of the three varieties exhibited a consistent trend; as the concentration of ZT increased, the rate of callus formation decreased for all three varieties; however, when ZT was maintained at a high concentration, thereby increasing the concentration of NAA, the rate of callus formation once again exhibited an increasing trend. However, the germination rates of the three varieties exhibited different trends; the germination rates of 'Wiki' and 'Purple Sadova' increased with increasing ZT concentrations, 'Wiki' had the maximum germination rate of 23.67% on 3 mg·L− 1 ZT + 0 mg·L− 1 NAA and 'Purple Sadova' had the maximum germination rate of 36.67% on 3 mg·L− 1 ZT + 0.1 mg·L− 1 NAA. The germination rate of Red September decreased with increasing ZT concentration, with the highest value reaching 16.67% at 1 mg·L− 1 ZT + 0.1 mg·L− 1 NAA. As illustrated in Fig. 1, the genotypes of A. arguta generated a range of adventitious shoots of varying quality, with 'Wiki' and’ 'Purple Sadova' outperforming "Red September" in yield. ‘Red September’ has the ability to regenerate a single bud, and a single callus results in a single indefinite bud. ‘Wiki’ and ‘Purple Sadova’ have the potential to regenerate cluster buds, and a single callus can result in numerous indefinite buds. Therefore, it can be concluded that the regeneration rate of 'Purple Sadova' leaves was highest under the medium of 3 mg·L− 1 ZT + 0.1 mg·L− 1 NAA and the best regeneration of adventitious shoots, so 'Purple Sadova' and this hormone ratio were chosen for the subsequent study.
Table 2
Effects of different genotype on adventitious bud regeneration
ZT(mg·L− 1) | NAA(mg·L− 1) | Rate of callus formation(%) | Germination rate(%) |
‘Wiki’ | ‘Purple Sadova’ | ‘Red September’ | ‘Wiki’ | ‘Purple Sadova’ | ‘Red September’ |
1 | 0 | 100 | 66.67 | 80 | 0 f | 6.67 def | 10 cde |
1 | 0.1 | 100 | 100 | 100 | 0 f | 0 f | 16.67 bc |
1 | 0.2 | 66.67 | 100 | 86.67 | 10 cde | 0 f | 0 f |
2 | 0 | 40 | 50 | 80 | 13.33 cd | 13.33 cd | 6.67 def |
2 | 0.1 | 100 | 100 | 100 | 0 f | 13.33 cd | 3.33 ef |
2 | 0.2 | 10 | 100 | 100 | 13.33 cd | 3.33 ef | 0 f |
3 | 0 | 33.33 | 13.33 | 26.67 | 23.67 b | 16.67 bc | 0 f |
3 | 0.1 | 20 | 53.33 | 36.67 | 3.33 ef | 36.67 a | 0 f |
3 | 0.2 | 50 | 60 | 100 | 0 f | 0 f | 0 f |
Note: Different lowercase letters at the end of data in the same column indicate significant difference (P < 0.05). The following table is the same.
The leaves were inoculated on regeneration medium for about 15 d (Fig. 2, a), the leaves began to expand and curl, and there was a small amount of callus at the edge of the leaves, after 30 d of inoculation (Fig. 2, b), a large amount of white callus was observed, the texture of the callus was fluffy, then it was transferred to light culture and cultured for 7 d in the light (Fig. 2, c ~ f), the texture of the callus became compact from fluffy, and the color changed to bright green, meanwhile, a After 25 d of light culture (Fig. 2, f), adventitious buds could be observed, and with the extension of culture time, the buds gradually developed into a cluster of small plants, and after 45 d of light culture, the adventitious buds were no longer produced and some of the callus began to brown.
Effect of different parts of leaf on adventitious shoots regeneration of 'Purple Sadova' leaves
As can be seen from Table 3, as the physiological state of the explants became older, the rate of callus formation, germination rate and average number of shoots were decreasing, with the highest germination rate and average number of shoots at the base of the leaf blade, 80.00% and 16, respectively, and the lowest germination rate and average number of shoots at the tip of the leaf blade, 36.67% and 5, respectively. The difference in germination rate between the base of the leaf and the middle of the leaf was not significant, but the difference between the germination rate of these two parts and the germination rate of the tip of the leaf both reached a significant level, and the average number of germination of these two parts was 3 times and 2 times of the average number of germination of the tip of the leaf, respectively. In the experiment, it was also observed that the germination time of different leaf parts was different, and the buds could be seen at the base of the leaf in 7 d of light culture, while the middle of the leaf took about 10 d and the tip of the leaf took 15 d. Meanwhile, it was found that the quality of regenerated buds was different in different parts, and the regenerated buds at the base of the leaf had the highest quality, and most of the regenerated buds could grow into robust seedlings in a short time, while the regenerated buds at the tip of the leaf had the worst quality. Most of the buds observed stop growing and then die, although they have an intact structure. Therefore, the base of the leaf is the most desirable explant for the in vitro regeneration of 'Purple Sadova' leaves.
Table 3
Effect of different parts of the leaf on the regeneration of adventitious shoots of 'Purple Sadova' leaves
Leaf parts | The number of inoculation | Rate of callus formation | Germination rate | Average number of buds |
Blade base | 30 | 93.33% | 80.00% a | 16 |
Middle of the blade | 30 | 90.00% | 70.00% a | 11.6 |
Blade tip | 30 | 86.67% | 36.67% b | 5 |
Effect of different dark culture times on adventitious bud regeneration of 'purple Sadova' leaves
At 7 d after inoculation, it was observed that there was a majority of browning and death of the explants without dark culture; meanwhile, it was found that early light culture did not affect the germination time. As shown in Table 4, the dark culture of explants had an effect on the healing rate, germination rate and average germination number of explants, and the germination rate and average germination number increased with the extension of dark culture time, and the highest germination rate was 80.00% at 30 d of dark culture time, and the difference with other treatments reached a significant level. Therefore, the optimal dark culture time was 30 d.
Table 4
Effect of different dark culture times on adventitious bud regeneration of 'purple Sadova' leaves
Dark culture time/d | The number of inoculation | Rate of callus formation | Germination rate | Average number of buds |
0 | 30 | 43.33% | 13.33% d | 2.6 |
7 | 30 | 73.33% | 23.33% cd | 2 |
14 | 30 | 100.00% | 33.33% c | 5.4 |
21 | 30 | 80.00% | 60.00% b | 11.4 |
30 | 30 | 93.33% | 80.00% a | 16 |
Effect of 6-BA and NAA on the proliferation of adventitious shoots of 'Purple Sadowa'
The axillary bud tips at the base of the seedlings developed after 12 days of inoculation, but no new seedlings sprouted after 20 days. During the same experiment, it was noticed that adventitious shoots of A. arguta were beginning to grow from the axillary buds (Fig. 4a). According to Table 5, treatments 5 and 6 had considerably greater fresh weights than treatments 1 through 4. This is likely owing to a substantial amount of callus formation, which encased the original seedlings, inhibiting their growth and resulting in fewer shoots proliferating. Meanwhile, Some of the shoots in treatments 5 (Fig. 3e) and 6 (Fig. 3f) displayed malformed leaves with narrow leaves and sharply serrated margins, and they grew excessively long and thin. In treatment 3 (Fig. 3c), a 1:1 ratio of NAA to 6-BA was detected, which is optimum for callus growth but detrimental for shoot proliferation. The difference between the overall proliferation coefficients of treatments 1, 2, and 4 was not significant, although treatment 4 (Fig. 3d) resulted in shorter adventitious shoots than the other two treatments. Additionally, the proliferation coefficient of treatment 4 with plant height > 2 cm was significantly different from treatments 1 (Fig. 3a) and 2 (Fig. 3b), whereas the difference between treatments 1 and 2 was not significant, and the seedlings proliferated from both treatments grew vigorously, with taller plants and larger leaves. Therefore, both treatments 1 and 2 are acceptable for adventitious shoot proliferation.
Table 5
Effects of 6-BA and NAA on adventitious shoot proliferation of 'Purple Sadowa'
Processing number | The number of inoculation | The total fresh weight/g | The total dry weight/g | Total proliferation number | The proliferation number with a plant height > 2cm | Total proliferation coefficient | Proliferative coefficient of plant height > 2cm |
1 | 30 | 27.99 | 2.41 | 101 | 74 | 3.37 ± 0.25a | 2.46 ± 0.32 a |
2 | 30 | 28.78 | 2.66 | 107 | 70 | 3.57 ± 0.23a | 2.33 ± 0.25 ab |
3 | 30 | 28.83 | 2.81 | 76 | 32 | 2.53 ± 0.45b | 1.11 ± 0.19 d |
4 | 30 | 26.23 | 2.44 | 110 | 45 | 3.67 ± 0.23a | 1.50 ± 0.36 c |
5 | 30 | 56.00 | 5.09 | 86 | 69 | 2.87 ± 0.33b | 2.16 ± 0.37 b |
6 | 30 | 65.92 | 6.04 | 78 | 44 | 2.60 ± 0.31b | 1.47 ± 0.27 c |
Effect of IBA on the rooting of 'Purple Sadowa' seedlings
At 4 days after inoculation, the seedlings began to generate callus at the base, a few seedlings began to root at 7 days, the majority of seedlings began to root around 10 days, and after 20 days, no new roots emerged, the roots only became longer (Fig. 4b and c). As the concentration of IBA grew, the amount of callus at the seedlings' bases increased. As can be seen from Table 4, IBA promoted the rooting of seedlings, and the rooting rate of medium with IBA was higher than that of medium without IBA, and the average number of roots increased with the increase of IBA concentration, and the average number of roots differed significantly between treatments with different IBA concentrations, and the rooting rate and average number of roots of medium without IBA were the lowest, 83% and 3.96, respectively; When IBA was 1.0 mg·L− 1, the maximum number of roots was 17.60, but the rooting rate was only 89%; when IBA was 0.5 mg·L− 1 and 0.7 mg·L− 1, the rooting rate reached 100%, and the average number of roots was 7.60 and 14.50, respectively, and the average number of roots of the latter was significantly higher than that of the former. The total fresh weight of seedlings also increased with the increase of IBA concentration, while in the treatment of IBA at 1.0 mg·L− 1 (Fig. 5e), a large number of callus grew at the base of seedlings, resulting in a much higher total fresh weight than the other treatments. Considering the rooting rate, average root number and average root length, the optimal IBA rooting concentration was 0.7 mg·L− 1.
Table 6
Effects of IBA on rooting of 'purple Sadowa' seedlings
IBA/(mg·L− 1) | The number of inoculation | The number of rooting trees | Rooting rate | The average plant height/cm | The average number of roots | The average root length /cm | The total fresh weight/g | The total dry weight/g |
0 | 30 | 25 | 83% | 4.32 ± 0.63 | 3.96 ± 1.23e | 3.29 ± 0.38ab | 9.86 | 1.02 |
0.3 | 30 | 30 | 100% | 4.11 ± 0.69 | 5.90 ± 1.30d | 3.07 ± 0.61b | 14.22 | 1.45 |
0.5 | 30 | 27 | 89% | 4.21 ± 0.74 | 7.60 ± 1.74c | 2.94 ± 0.85bc | 17.45 | 1.79 |
0.7 | 30 | 30 | 100% | 4.00 ± 0.78 | 14.50 ± 1.69b | 2.81 ± 0.42c | 17.85 | 1.98 |
1.0 | 30 | 27 | 89% | 4.08 ± 0.57 | 17.60 ± 1.56a | 3.40 ± 0.53a | 28.72 | 2.94 |
Transplanting
After acclimatization, the seedlings were withdrawn from the vials, the root media rinsed, and the seedlings were transplanted into a substrate consisting of 7:3 coconut coir: perlite. The pots were properly watered with tap water for the first time, trays with a 1000-fold diluted nutrient solution were placed beneath the pots, and the pots were covered with a moisturizing lid to keep them hydrated. All of the seedlings survived and thrived (Fig. 4d).