Autotetraploid induction and identification results
Figure 1 displays the results of tetraploid induction from leaves and tubers treated with colchicine. As colchicine concentration and treatment time increased, the number of tubers produced per leaf in the leaf treatment group decreased substantially, and the growth rate was also inhibited. The number of sprouted tubers per bottle of leaves decreased by 30% when treated with 0.05% colchicine for 72 h. When leaves were treated with 0.1% colchicine for 108 h, only half as many tubers sprouted per bottle as in the blank group. In the tuber treatment group, treated tubers began to expand and deform after approximately 10 days of incubation (Fig. 2A), transformed to a pseudo-dead state after 30 days, and after 3 months, new plants emerged from the brown tubers (Fig. 2B). The number of chromosomes in diploid plants was determined to be 2n = 2x = 66 by observing temporary slides of root tip chromosomes from the blank group (Fig. 2C). The chromosome number of tetraploid plants in each treatment group was determined to be 2n = 4x = 132 (Fig. 2D). Meanwhile, the DNA content of tetraploid (4x) was twice that of diploid (2x) by flow cytometry (Fig. 3).
In addition, under the same treatment conditions, the tetraploid induction rate of leaves was significantly higher than that of tubers; the highest tetraploid induction rate of 29.49% was achieved in leaves at 0.05% colchicine treatment for 96 h, whereas the highest tetraploid induction rate of 21.56% was achieved in tubers at 0.1% colchicine treatment for 108 h. However, chimeras were found in both materials across all treatment groups. Comparing the two materials, the leaves were more suitable for low concentration and prolonged colchicine treatment to induction tetraploid, whereas the tubers induced tetraploid are more effective under high concentration and prolonged treatment. The interval between treatment and identification of tubers was nearly double that of leaves, and the rate of tetraploid induction in tubers was lower than that of leaves. The optimal treatment for inducing autotetraploidy in ‘Pearl’ P. ternata was the 96-hours treatment of leaves with a colchicine solution containing 0.05% colchicine.
Establishment of an autotetraploid plant regeneration system
The results of the orthogonal experiments were shown in Table 1, and the extreme difference in proliferation coefficients showed that R6-BA>RKT>RNAA>RError, indicating that all three factors were effective for the proliferation culture of autotetraploid P. ternate. It was evident from the ANOVA results (Table 2) that 6-BA had a significant effect on the leaf proliferation coefficient (P<0.05), whereas KT and NAA did not. Duncan's test for the three levels of 6-BA (Table 3) showed that the most suitable concentration for the proliferation of P. ternata was level 2 (0.1 mg·L-1), followed by level 3 (0.5 mg·L-1) and level 1 (0.05 mg·L-1). The mean analysis showed that the best hormone combination for the simultaneous culture of proliferation and rooting of ‘Pearl’ P. ternata was MS + 0.1 mg·L-1 6-BA + 0.1 mg·L-1 NAA + 1.0 mg·L-1 KT.
Table 1 The L9 (34) orthogonal test results of autotetraploid P. ternata one-step seedling formation.
Factors
|
source
|
Type III sum of squares
|
DOF
|
mean square
|
f-value
|
significance
|
Proliferation coefficient
|
A
|
697.707
|
2.00
|
348.853
|
37.218
|
P<0.05
|
B
|
67.947
|
2.00
|
33.973
|
3.624
|
P>0.05
|
C
|
68.240
|
2.00
|
34.120
|
3.62
|
P>0.05
|
Error
|
18.747
|
2.00
|
9.373
|
|
|
Table 2 Analysis of variance for proliferation coefficient.
NO.
|
Factors (mg·L-1)
|
Proliferation coefficient
|
A(6-BA)
|
B(NAA)
|
C(KT)
|
Error
|
1
|
1
|
1
|
1
|
1
|
65.20±0.37
|
2
|
1
|
2
|
2
|
2
|
69.80±0.37
|
3
|
1
|
3
|
3
|
3
|
73.40±0.25
|
4
|
2
|
1
|
2
|
3
|
84.00±0.71
|
5
|
2
|
2
|
3
|
1
|
99.00±0.32
|
6
|
2
|
3
|
1
|
2
|
89.80±0.37
|
7
|
3
|
1
|
3
|
2
|
78.00±0.32
|
8
|
3
|
2
|
1
|
3
|
76.80±0.37
|
9
|
3
|
3
|
2
|
1
|
80.40±0.25
|
Proliferation coefficient
|
K1
|
69.47
|
75.73
|
77.27
|
81.53
|
|
K2
|
90.93
|
81.87
|
78.07
|
79.20
|
|
K3
|
78.40
|
81.20
|
83.47
|
78.07
|
|
R
|
21.46
|
6.14
|
6.2
|
3.46
|
|
Note: K: mean; R: range (R = Kmax - Kmin)
Table 3 Duncan’s test in three levels of 6-BA.
factor
|
Levels
|
Mean
|
0.05 Level
|
6-BA
|
2
|
90.93
|
a
|
3
|
78.40
|
b
|
1
|
69.47
|
b
|
Note: Different letters in the table indicate significant differences in the 5% level; ± Standard error.
The leaves of ‘Pearl’ P. ternata plants were inoculated with the optimal medium described above. After 15 days, small yellow-green tubers formed on the leaf surface, and leaf buds and adventitious roots emerged on the tubers (Fig. 4A). After 25 days of cultivation, the leaves were almost entirely covered by small tubers, new leaves were emerging on all tubers, and adventitious roots were evident (Fig. 4B). After 35 days, the small tubers grew in size, and the leaves became dark green (Fig. 4C). After 45 days, the regenerated plants flourished in growth, and adventitious roots covered the entire substrate (Fig. 4D). 15 days after inoculation, petioles exhibit apical expansion (Fig. 5A). After 25 days in culture, small tubers formed at the base of the petiole, and pearl buds with adventitious roots and leaves formed at the apical expansion (Fig. 5B). After 35 days, all the pearl buds on the petiole’s apex produced new leaves (Fig. 5C). Both the pearl buds at the apex of the petiole and the small tubers at the base of the petiole regenerated into vigorously growing plants after 45 days (Fig. 5D). 10 days after inoculation, tubers developed a few leaf buds (Fig. 6A). After 20 days, the number of leaf buds on the tubers increased, and adventitious roots became visible (Fig. 6B). After 30 days, the leaves expanded swiftly, the petioles lengthened, and the adventitious roots multiplied (Fig. 6C). After 40 days, the petioles and leaves grew vigorously, the tubers enlarged, and the adventitious roots were robust (Fig. 6D). At this time, the proliferation coefficient was calculated by dividing the three materials of leaves, petioles, and tubers, and it could reach 99.0.
The result of acclimatization and transplanting
After acclimatization in the bottle, the petioles, leaves, and roots of the plants were removed, leaving only the tubers (Fig. 7A). The tubers were transferred to a foam box to keep warm and moist, and after 10 days, some of the tubers produced new leaves with curled leaves and short petioles (Fig. 7B). After 20 days, all tubers had successfully sprouted with spreading leaves and elongated petioles (Fig. 7C). After 30 days, the leaves continued to increase, and the plants grew better (Fig. 7D). ‘Pearl’ P. ternata tetraploid plants all grew normally after transplanting and were more likely to survive and grow more robustly than diploid plants, with a acclimatization survival rate of 100%.
Morphological and anatomical comparison results
The growth of autotetraploid plantlets of P. ternata differed significantly from that of diploid plantlets. After 15 days of leaf culture, most of the tetraploid leaves had formed small tubers with adventitious roots, while the diploid leaves formed only a few small tubers on the surface (Figs. 8A and B; top right). Observations revealed that the tetraploid plantlets grew quicker and that the tetraploid leaves produced a greater number of small tubers than the diploid leaves. However, after 30 days of incubation, the development of the tetraploid plantlets was inhibited, and the height of the plants at 45 days of growth was not significantly different from the height at 30 days. At this time, the diploid plants grew faster than the tetraploid plants and their height exceeded that of the tetraploid plants (Figs. 8A and B). There was no difference in the time required for tetraploid and diploid petioles to produce pearl buds. However, pearl buds on tetraploid petioles produced leaves more quickly than those on diploid petioles, and tetraploid pearl buds developed into more robust plantlets (Figs. 8C and D). The tubers of both tetraploid and diploid plants produced leaf buds simultaneously, but the diploid tubers produced more leaf buds and grew more quickly; after 40 days of growth, the diploid plants were taller and had more leaves than the tetraploid plants (Figs. 8E and F).
The P. ternate autotetraploid differed significantly from the diploids in morphology and cytoarchitecture, as shown in Table 4. The tetraploid plantlets measured approximately 8.5 cm in height, while the diploid plantlets measured approximately 11 cm (Fig. 9A). Compared to diploids, autotetraploid had wider leaves, darker leaf coloration, and a more rounded leaf shape (Fig. 9B). The petioles of tetraploid plants were stronger but shorter than those of diploid plants (Fig. 9C). There was no significant difference in the size and number of roots between tetraploid and diploid tubers, but the tetraploid tubers produced roots that were thicker and shorter (Fig. 9D). It was discovered that the cell and tissue thickness of tetraploid leaves was greater than that of diploid leaves (Figs. 10A and B). Under a microscope, the stomata of the lower epidermis of tetraploid and diploid leaves were observed. The results demonstrated that the stomata of tetraploid leaves were larger than those of diploid leaves, but the density of stomata was substantially lower than that of diploid leaves, which was only half that of diploid leaves (Figs. 10C, D, and E). In terms of morphology and cellular tissue structure, the P. ternate tetraploid showed typical polyploid characteristics and differed significantly from the diploid.
Table 4 Comparison of morphology and stomata of diploid and autotetraploid plants of P. ternate.
Ploidy
|
NLBT
|
Plant height
(cm)
|
Petiole diameter
(mm)
|
Leaf length
(cm)
|
Leaf wide
(cm)
|
Stomatal length
(mm)
|
Stomatal frequency
|
2X
|
4.21±0.88a
|
11.19±0.24a
|
1.53±0.59b
|
3.14±0.78b
|
2.14±0.46b
|
30.15±0.58b
|
5.28±0.45a
|
4X
|
1.22±0.78b
|
8.56±0.56b
|
2.80±0.98a
|
4.18±0.37a
|
3.45±0.56a
|
50.13±0.98a
|
3.18±0.46b
|