Callus induction and differentiation in different leaf parts
Figure 2. The effect of different parts of leaves on callus induction and differentiation. A. The induction rate of callus in different parts of leaves; B. Budding rate of callus in different parts of leaves
Different parts of the leaf tissue used as explants have an impact on callus induction. As shown in Figure 2A, the three types of explant set up were fully unfolded leaves, unexpanded leaves, and fully unfolded leaf margins. Among them, there was no significant difference in the induction rate of callus between fully unfolded leaves and unexpanded leaves, which were 92.41% and 90.77%, respectively, which is significantly higher than the induction rate of callus with fully unfolded leaf margins (76.79%). There was no significant difference among the three different parts of the germination rate, but the highest germination rate was found in the unexpanded leaves, which was 92.71%. The germination rates of fully unfolded leaves and leaf edges were 84.25% and 88.74%, respectively. The experiment showed that leaves with different growth times and maturities had little effect on the induction rate of calluses of Caladium bicolor in vitro, while the germination rate had an impact, but there was also no significant difference.
Callus induction and differentiation under different light intensities
Figure 3. The effect of different light intensities on callus induction and differentiation. A. Callus induction rate under different light intensities; B. Budding rate of calli under different light intensities.
The induction rate and germination rate of calli under different light intensities are shown in Figure 3. Under darkness and 0.5 μmols-1m-2, 10 μmols-1m-2, 15 μmols-1m-2, and 20 μmols-1m-2 light intensities, there were no significant differences in the callus induction rate or germination rate. The highest callus induction rates were found at 0.5 μmols-1m-2 and 10 μmols-1m-2, both of which were 94.84%, while the other three treatments were 93.75%, 86.9%, and 90.87%, respectively. The highest germination rate was 92.79% for the 15 μmols-1m-2 treatment, followed by 92.36% for the 20 μmols-1m-2 treatment and 85.42%, 86.33%, and 84.52% for the other three treatments. The results showed that light intensity had a relatively small impact on the induction and differentiation of calluses in Caladium bicolor.
Table 1. Analysis of variance (ANOVA) of explant site, light intensity, and their combination (explant site×light intensity) on the number of buds per explant.
Parameter
|
Source of variation
|
explant site
|
light intensity
|
explant site × light intensity
|
F
|
Sig.
|
F
|
Sig.
|
F
|
Sig.
|
Number of buds
|
8.883
|
0.0003
|
11.04
|
<0.0001
|
0.37
|
0.9342
|
Table 2.A simple effect analysis of the effects of different explant locations and light intensities on the number of buds per explant.
Light intensity
|
Number of buds per explant
|
Fully unfolded leaf
|
Unexpanded leaf
|
Fully unfolded leaf edge
|
Darkness
|
5.98 ± 0.84 b B
|
9.05 ± 0.43 ab A
|
6.08 ± 0.29 b B
|
0.5 μmol/s/m2
|
5.04 ± 0.45 b B
|
9.05 ± 0.79 b A
|
6.54 ± 0.87 b B
|
10 μmol/s/m2
|
9.67 ± 0.81 a A
|
11.6 ± 1.07 a A
|
9.72 ± 0.95 a A
|
15 μmol/s/m2
|
8.85 ± 0.85 a B
|
12.55 ± 1.22 a A
|
10.26 ± 1.09 a AB
|
20 μmol/s/m2
|
10.44 ± 0.49 a A
|
11.88 ± 1.16 a A
|
10.1 ± 0.85 a A
|
Note: The figure contains different capital letters indicating significant differences in the number of sprouts at different levels of light intensity at different parts of the explant (P<0.05). The figure contains different lowercase letters indicating significant differences in the number of sprouts at different levels of explants under different light intensity levels (P<0.05).
As shown in Table 1, the analysis of variance for the explant site and light intensity showed that the main effect of "explant site" was significant, and the main effect of "light intensity" was also significant. There was an interaction effect between "explant site × light intensity", but it did not reach significance. The light intensity and explant location have a significant impact on the number of buds per explant. Furthermore,, we conducted a simple effect analysis on the experimental results to gain a detailed understanding of the impact of the two on the number of sprouts. The results in Table 2 indicate that the growth of three different explants under five different light intensities of LED light sources has an impact. For fully unfolded leaves, the number of buds per explant under 10 μmols-1m-2, 15 μmols-1m-2, and 20 μmols-1m-2 light intensities was significantly higher than that under dark and 0.5 μmols-1m-2 light intensities. The response of fully unfolded leaf edges to light intensity was similar to that of fully unfolded leaves. The number of buds per explant under 10 μmols-1m-2, 15 μmols-1m-2, and 20 μmols-1m-2 light intensities was significantly higher than that under darkness and 0.5 μmols-1m-2 light intensities. For unexpanded leaves, the number of buds per explant under 10 μmols-1m-2, 15 μmols-1m-2, and 20 μmols-1m-2 light intensities was significantly higher than that under 0.5 μmols-1m-2 light intensities, but there was no significant difference in the number of buds per explant under dark conditions compared to other treatments.
There are differences in the effects of lighting intensities of LED light sources on three different parts (Table 2). Under dark conditions, the number of buds per explant of unexpanded leaves was significantly higher than that of fully unfolded leaves and fully unfolded leaf margins, reaching 9.05 buds; at 0.5 μmols-1m-2 light intensity, the performance was similar to that under dark conditions. Under 10 μmols-1m-2conditions, there was no significant difference in the number of sprouts between different explant parts, but the highest number of sprouts was still unexpanded leaves. Under 15 μmols-1m-2 conditions, the number of buds from unexpanded leaves was significantly higher than that of fully unfolded leaves, reaching 12.55 buds, with an average of 3.7 buds more than fully unfolded leaves. However, there was no significant difference in the number of buds from each explant of fully unfolded leaf edges compared to both fully unfolded and unexpanded leaves. Under 20 μmols-1m-2 conditions, there was no significant difference in the number of buds per explant among the three leaf explants. Therefore, the number of buds per explant of Caladium bicolor in different parts is affected by light intensity. When the light intensity reaches 20 μmols-1m-2, the differences in low light intensity between different leaf parts lose their effect.
Callus induction and differentiation at different light exposure times
Figure 4. The effect of different light exposure times on callus induction, differentiation, and the number of buds per explant. A. Callus induction rates at different light exposure times; B. The germination rate of calli at different light exposure times; C. The number of buds per explant at different light exposure times.
The growth differences at different light exposure times are shown in Figure 4A. On the 0th, 15th, 30th, 45th, 60th, 75th, and 90th days, the explant bottle seedlings were placed under light intensity. There was no significant difference in callus induction rates among the 7 treatments, all reaching over 80%, with the highest being 75 days, reaching 95.83%. There was no significant difference in the germination rate of calli among the 7 treatments (Figure 4B), all reaching over 80%, with a 15 day growth rate of over 90%. There were differences in the number of buds per explant among the 7 treatments, but they were not significant (Figure 4C), reaching an average of over 9 buds, with the highest being 90 days, reaching an average of 10.87 buds. Therefore, under the conditions of this experiment, different light exposure times had little effect on the tissue culture of Caladium bicolor.
Callus differentiation with different light wavelengths
Figure 5. Differentiation of calli under different wavelengths of light. A. LED White; B. LED Red; C. LED Blue; D. LED-Complex 1; E. LED-Complex 2; F. LED-Complex 3; G. Fluorescence; H. Darkness..
Table 3. The effect of different wavelengths of light on callus differentiation.
Light wave
|
Callus differentiation rate
|
LED-White
|
94.44% ± 5.56 a
|
LED-Red
|
86.11% ± 6.05 a
|
LED-Blue
|
90.3% ± 5.8 a
|
LED-Complex 1
|
100% ± 0 a
|
LED-Complex 2
|
94.44% ± 5.56 a
|
LED-Complex 3
|
94.44% ± 5.56 a
|
Fluorescent
|
96.88% ± 2.76 a
|
The different wavelengths of light affected the differentiation of Caladium bicolor calli (Figure 5). Compared with dark conditions, all seven light treatments can cause pigment accumulation in the callus. According to Table 3, different light waves have different effects on the germination rate. Among them, the highest germination rate was observed for LED-Complex 1, which reached 100%. The lowest germination rate was observed for LED-Red, which was 86.11%. The germination rate of other wavelengths of light was above 90%, but there was no significant difference between the seven treatments. Therefore, different light qualities have little effect on the differentiation of callus tissue in Caladium bicolor.
Growth and development of different light wavelengths
Figure 6. Appearance of tissue-cultured seedlings under different wavelengths of light. A. LED-White; B. LED-Red; C. LED-Blue; D. LED-Complex 1; E. LED-Complex 2; F. LED-Complex 3; G. Fluorescence; Scale bar=5 cm.
Figure 7. The effect of different wavelengths of light on the growth of tissue-cultured seedlings. A. Plant height with different light wavelengths; B. Root length of different light wavelengths; C. The number of elements with different light wavelengths.
Different wavelengths of light have an impact on the appearance and morphology (Figure 6). The impact on plant height is shown in Figure 7A, and there were differences among different treatments, ranging from large to small LED-White > LED-Blue > LED-Complex 1 > LED-Complex 3 > Fluorescent > LED-Complex 2 > LED-Red. The LED-White treatment had the highest plant height of 6.1 cm, which was significantly higher than the LED-Red, LED-Complex 1, LED-Complex 2, LED-Complex 3, and Florescent treatments. There was no significant difference compared to the LED-Blue treatment, and the LED-Red with the lowest plant height was only 4.28 cm. Pure red light cultivation significantly reduced the plant height of Caladium bicolor compared to white light.
The root growth of tissue-cultured seedlings is influenced by different light wavelengths. As shown in Figure 7B, the experimental results show that the root lengths of the LED-White, LED-Complex 1 and Fluorescent treatments are significantly higher than those of the LED-Blue. The root lengths of LED-Red, LED-Complex 2, and LED-Complex 3 are greater than those of LED-Blue, but there is no significant difference. The largest root length is Florescent, which is 7.48 cm, and the smallest is LED-Blue, which is only 4.94 cm, with an average root length of 2.54 cm less than that of the Florescent treatment. As shown in Figure 7C, there was no significant difference in root length between LED-White, LED-Red, LED-Complex 1, LED-Complex 2, LED-Complex 3, and Florescent, and the root numbers of the six treatments were significantly higher than those of LED-Blue. The LED-White had the highest number of roots, with an average of 22.5, while the LED-Blue had the lowest number, at only 16.63. Therefore, LED-Blue is not conducive to the growth of root systems in tissue culture seedlings.
Figure 8. Effect of different wavelengths of light on leaf growth in tissue culture seedlings. A. Petiole thickness under different light wavelengths; B. Leaf thickness with different light wavelengths; C. Leaf area of different light wavelengths; D. Leaf width with different light wavelengths; E. Leaf length with different light wavelengths; F. Leaf aspect ratio of different light wavelengths.
Different light qualities have different effects on the leaves of tissue-cultured seedlings. As shown in Figure 8A, the petiole thickness of the LED-Red treatment was significantly higher than that of the LED-White, LED-Complex 1, LED-Complex 2, LED-Complex 3, and Florescent treatments and was higher than that of the LED-Blue treatment but did not reach significance. The petiole thickness of the LED-Red treatment reached 1.7 mm, with the lowest petiole thickness being only 1.1 mm for the LED-Complex 3 and Florescent treatments. There was no significant difference between LED-Blue and the other treatments. From the perspective of leaf thickness, as shown in Figure 8B, the arrangement of leaf thickness from large to small is LED-Complex 3 > LED-Complex 1 > Florescent > LED-Red > LED-Blue > LED-Complex 2 > LED-White. The leaf thickness of the LED-Complex 3 treatment was significantly higher than that of the other treatments, with a value of 1.8 μm. The blade thickness treated with LED-White is the lowest, at 1.3 μm.
From the perspective of leaf area (Figure 8C), tissue-cultured seedlings showed significant differences, with LED-White significantly higher than LED-Red, LED-Blue, LED-Complex 2, LED-Complex 3, and Florescent, reaching 5.0 cm2, followed by LED-Complex 1 at 4.3 cm2. Among them, the LED-Blue treatment had the smallest leaf area, only 2.2 cm2, which was more than half smaller than that of LED-White. As shown in Figure 8D-E, the trends of leaf length and width among the different treatments were similar. The leaf width and length of the LED-Blue treatment were significantly lower than those of the other treatments, at 1.68 cm and 1.65 cm, respectively. The leaf length and width of the LED-White treatment were greater than those of the other treatments, at 2.50 cm and 2.50 cm, respectively. From the aspect ratio perspective (Figure 8F), there was no significant difference among the treatments.
Pigment content of different light wavelengths
Figure 9. The effect of different wavelengths of light on the pigment content in the leaves of tissue cultured seedlings. A. The effect of different light waves on chlorophyll a; B. The effect of different light wavelengths on chlorophyll b; C. The effect of different light wavelengths on total chlorophyll content; D. The effect of different light wavelengths on carotenoids; E. The effect of different light wavelengths on relative anthocyanin content.
Different wavelengths of light treatment affect the accumulation of pigments in tissue-cultured seedlings. As shown in Figure 9A, the chlorophyll a content is arranged in descending order: Fluorescent > LED-Blue > LED -White > LED-complex 3 > LED-complex 1 > LED-complex 2 > LED-Red. Compared with Fluorescent, there was no significant difference in the chlorophyll a content of LED-Blue, while the other five treatments significantly decreased. Among them, LED-Red had the lowest chlorophyll a content, only 1.41 mg/g FW. From the perspective of chlorophyll b (Figure 9B), the results are similar to chlorophyll a, with the exception of LED-Blue, the chlorophyll b content of all treatments being significantly lower than that of Fluorescent. The results of total chlorophyll content were similar to those of chlorophyll a and chlorophyll b (Figure 9C). The total chlorophyll content of Fluorescent and LED-Blue was higher than that of the other treatments, at 2.82 mg/g FW and 2.46 mg/g FW, respectively. The lowest was LED-Red, at 1.87 mg/g FW. From the perspective of carotenoid content (Figure 9D), the order of carotenoids from large to small is Fluorescent > LED-White > LED-Complex 3 >LED-Blue > LED-Complex 2 > LED-Complex 1 > LED-Red. Compared with Fluorescent, there was no significant difference in carotenoid content between LED-White, LED-Blue, LED-complex 1, LED-complex 2, and LED-complex 3. The carotenoid content of LED-Red was significantly lower than that of Fluorescent, at only 0.72 mg/g FW.
From the relative content of anthocyanins, LED-Blue was significantly higher than the other treatments, reaching 1.6 U/g FW. Next were LED-Complex 2, LED-Complex 3, and Fluorescent, with a relative anthocyanin content of 0.6 U/g FW, 0.7 U/g FW, and 0.7 U/g FW, respectively. The relative anthocyanin content of these three treatments was significantly higher than that of LED-White and LED-Red; the lowest was that of LED-Red, which was only 0.3 U/g FW, which is 81.25% less than LED-Blue. Therefore, LED-Blue is beneficial for the accumulation of anthocyanins in tissue culture seedlings of Caladium bicolor, while LED-Red is not conducive to the accumulation of anthocyanins in tissue culture seedlings.
Number of seedlings with different light wavelengths
Figure 10. The effect of different wavelengths of light on the number of tissue-cultured seedlings. Different lowercase letters represent differences in the total number of seedlings, and different capital letters represent differences in the number of transplantable seedlings (P<0.05).
Table 4. The Effect of Different Wavelengths of Light on the Number of Transplantable Tissue Culture Seedlings.
Treatments
|
Proportion of transplantable seedlings (%)
|
Increased ratio of transplantable seedlings (%)
|
coefficient of variation (%)
|
Transplantation survival rate (%)
|
LED-White
|
23.17
|
84.75
|
43.29
|
100
|
LED-Red
|
10.64
|
-23.73
|
41.83
|
100
|
LED-Blue
|
25.19
|
94.92
|
44.88
|
100
|
LED-Complex 1
|
19.46
|
48.31
|
35.36
|
100
|
LED-Complex 2
|
15.77
|
36.44
|
84.71
|
100
|
LED-Complex 3
|
19.96
|
68.64
|
78.38
|
100
|
Fluorescent
|
15.33
|
0
|
64.32
|
100
|
The effect of different wavelengths of light on the total number of seedlings and the number of transplantable seedlings in tissue culture is shown in Figure 10. The average number of seedlings per callus of LED-White, LED-Red, LED-Blue, LED-Complex 1, LED-Complex 2, and LED-Complex 3 was higher than that of Fluorescent, with 13.1, 11.7, 12.7, 12.5, 14.1, and 13.8 plants, respectively. Among them, the average number of seedlings per callus of LED-Complex 2 and LED-Complex 3 was significantly higher than that of Fluorescent's 10.7 plants. From the number of seedlings reaching 3 cm, that of LED-White, LED-Blue and LED-Complex 3 was significantly higher than that of LED-Red. Among them, LED-Blue had the highest number of seedlings above 3 cm and was significantly higher than the LED-Red and Fluorescent treatments, with 3.19 plants, accounting for 25.16% of the total number of seedlings. The lowest among them was LED-Red, with an average of only 1.25 plants reaching 3 cm, accounting for only 10.65% of the total number of seedlings. The coefficient of variation of LED-Red, LED-Blue, and LED-Complex 1 is lower than that of Florescent (Table 4), and the number of seedlings reaching transplanting height is more stable. The survival rate of transplanted tissue culture seedlings treated with different wavelengths was 100%. Therefore, compared to fluorescence, LED-Blue is beneficial for increasing the number of Caladium bicolor seedlings reaching the transplanting level, and the number is more stable, while red light is not conducive to reaching the transplanting level, even if the number is equally stable.