The Establishment of Rapid Propagation System of ‘RED SUN’ Phalaenopsis aphrodite

Abstract

Phalaenopsis aphrodite Rchb. F. is a plant of great ornamental and economic value, and its growth has strict requirements in terms of environment. In order to establish the rapid propagation of Phalaenopsis aphrodite Rchb. F., this study used the popular large-flowered variety ‘RED SUN’, which is found on the market, as the material, and studied the effects of pedicel taking time, site, plant growth regulators, and organic substances on the establishment of a regeneration system. The results indicated that the best combination of initiation culture was collecting peduncles in January + upper position of peduncles + 0.1% HgCl₂ sterilization for 8 min. The best recipe for inducing adventitious buds was MS + TDZ 0.4 mg/L + NAA 0.5 mg/L. The optimal combination for adventitious bud propagation was MS + TDZ 0.2 mg/L + NAA 0.4 mg/L. The best recipe for strong seedling from aseptic seedlings was MS + NAA 0.4 mg/L + banana powder 20 g/L + tryptone 2 g. The best recipe for rooting for aseptic seedlings was 1/2 MS + IBA 0.5 mg/L + charcoal 2 g/L. The best combination of acclimation and transplanting for Phalaenopsis was using sphagnum in the training seedling room, which produced a seedling survival rate of 97%. Our findings, therefore, demonstrate the methods of rapid breeding of the Phalaenopsis orchid, which provide a scientific basis for the conservation of this species.

1. Introduction

Global climate change has affected many ecosystems and biological groups worldwide. Future climate change may accelerate the loss of biodiversity and threaten the current distribution of species and their protected areas [1]. There is growing observational evidence that the biosphere is already responding to the recent rapid warming with shifting phenology, species distributions, and genetic population structures, and that vegetation dynamics are also responding to other global change factors. Plant communities play a key role in global biogeochemical cycles of carbon, oxygen, water, and nitrogen, with feedback to the oceans, atmosphere, and climate. The distribution of animals on land is often influenced by the distribution of vegetation, and therefore, plant community dynamics affect biodiversity. Broadly speaking, we have a vested interest in understanding how rapid global change effects terrestrial plant communities [2].

Phalaenopsis aphrodite Rchb. F., also known as the butterfly orchid, is an epiphytic single-stemmed orchid of the monocotyledonous family, which was discovered in 1750. More than 70 native species have been found so far [3]. Because of its beautiful flower shape, rich color, the colorful butterfly-shaped flower blooms, and the long flowering period, which can reach more than 2 months, it is known as a high-end flower and is sought after by people in many parts of the world. Moreover, it has a high ornamental and economic value and a wide market potential [4]. Phalaenopsis orchids were introduced from abroad in the 1980s into coastal areas of China [5]. At present, there are nearly 20,000 registered Phalaenopsis hybrids, and each year, hundreds of new Phalaenopsis hybrids are registered with the RHS in the United Kingdom alone. Phalaenopsis likes high temperatures and a high humidity. It is not cold tolerant and likes ventilated semi-shaded environments. Because of the increase in the price, scholars in various countries have been actively exploring various rapid propagation methods for Phalaenopsis.

The propagation of Phalaenopsis can generally be achieved by both sexual and asexual propagation [6]. At present, seed propagation alone cannot meet the requirements of factory production, and Phalaenopsis orchids are mainly propagated by tissue culture in production. In addition, the Phalaenopsis orchid is more difficult to reproduce asexually than other orchid species due to it rarely developing lateral buds. Plant tissue culture comprises a variety of in vitro techniques, methods, and strategies that are used to improve many economically important plants. Rapid multiplication by tissue culture could be an alternative method for the mass propagation of the plant [7]. This would allow the grower to obtain a large number of propagules originating from a small number of elite plants within a short period [8].

Studies show that the best growth of in vitro Phalaenopsis seedlings was achieved by adding activated carbon and potato juice to the modified VW medium and adjusting the pH value to 5.1–5.3 [9]. The newly developed root tip of Phalaenopsis was cut at about 2–3 cm and inoculated on the medium. Then, the root end of the incision began to expand after 2 weeks of shading, producing light green verrucous healing tissue, before the buds gradually formed at around 4 weeks and then differentiated into shoots [10]. The leaves of pedicel shoots induced by Phalaenopsis blossoms were used as explants and were grown on Ms + 6-BA + 5 mg/L + NAA1 mg/L + cW (coconut water) 100 mL/L, with an induction rate of about 32.8% [11]. Sterile buds could be obtained by axillary bud germination of pedicels, and then the resulting shoot stem segments and leaves were used as proliferating material for in vitro culture, with a large number of adventitious buds being obtained after 2–3 months [12]. The leaves were cut into 1.0 cm × 0.7 cm pieces and placed flat on Hyponex basic medium with 10 mg/L 6-BA + 2 mg/L NAA + 20% coconut juice for induction [13]. 6-BA plays a decisive role in facilitating the tissue culture process of Phalaenopsis [14], and 2,4-D only plays a supporting role [15]. The stem tips of sterile seedlings obtained from young Phalaenopsis pedicels were used as explants, and the optimal medium formulations were screened by adding different mass concentrations of 6-BA, NAA, and 2,4-D to MS and 1/2 MS medium to initially establish the tissue culture regeneration system of Phalaenopsis [16]. Under the same conditions, there were significant differences in adventitious bud induction rates and multiplication (differentiation) coefficients among different Phalaenopsis species, and the species with high pedicel bud induction rates and multiplication coefficients also had higher adventitious bud induction rates and differentiation coefficients of leaves [17]. MS medium + 3 mg/L 6-BA had the most significant promotion effect on shoot differentiation, with an axillary bud differentiation rate of 96% [18]. Using Phalaenopsis fruit pod seed embryos and leaves and stem tips from histoculture seedlings as explants, a large number of primary bulbs could be obtained in the presence of 6-BA, NAA, and banana puree [19]. Phalaenopsis orchids are typical single-stemmed orchids with limited lateral branch development, and the reproduction multiplicity via their own division is low, making it difficult to achieve conventional asexual reproduction. The seeds of Phalaenopsis are exceptionally small, but aseptic sowing after surface sterilization can produce live seedlings, which is a simple and easy operation but is prone to segregation of traits [20]. Foreign scholars have obtained intact Phalaenopsis plants by applying plant tissue culture techniques to both pedicels and stem tips [21,22]. The appropriate light condition for the tissue culture of Phalaenopsis was 12 h, the temperature was 22–28 °C, and the appropriate inoculum density was 6–8 plants with a medium of Ms + BA5 mg/L + NAA0.5 mg/L + Ad5 mg/L + Vc4 mg/L, producing a proliferation coefficient of more than 4 [23]. The proliferation of Phalaenopsis indeterminate buds was 3 on MS + BA12.5 mg/L + NAA0.05 mg/L [24]. The proliferation factor was 7.24 on Flora 1 3 g/L + 6-BA 10 mg/L + NAA 0.2 mg/L + adenine 10 mg/L + inositol 100 mg/L + coconut water 100 mL/L [25] In this experiment, KC+ TDZ 0.20 mg/L + coconut water 200 mL/L + sucrose 14 g/L + citric acid 30 mg/L achieved a better proliferation effect, with a multiplication rate of 12.0 [26]. Sucrose, CO₂, and light intensity in the microenvironment of histoponic culture had significant effects on the growth of Phalaenopsis histoponic seedlings at the rooting stage [27]. The optimal rooting medium was KC + IAA 0.8 mg/L + sugar 10 g/L + agar 10 g/L + activated charcoal 2 g/L pH 5.4 [28]. This method was more favorable for transplanting and produced a survival rate of 90.0% [29]. The embryo culture of Phalaenopsis is easier to sterilize without opening the pods, and the sowing medium is modified KC with 5% banana juice [30]. The economic benefits of the industrialized production of Phalaenopsis are much higher than those of other agricultural products, and it is a high-input, high-return industry with a yield of 2.72%, an investment profit rate of 17.5%, and a payback period of 4 years [31]. Therefore, how to optimize rapid tissue culture propagation technology for Phalaenopsis production is a matter of great significance [32].

The flowers of ‘RED SUN’ Phalaenopsis aphrodite are like butterflies; they are beautiful in shape, rich in color, and have a long in flowering period, which is why they are one of the most popular orchids. They have a high commercial value in conjunction with a high ornamental value. Regrettably, to date, limited research has been focused on tissue cultures of this variety. In this study, ‘RED SUN’ Phalaenopsis aphrodite was chosen to explore the effects of pedicel taking time, site, plant growth regulators, and organic substances on the velocity of regeneration. Ultimately, a tissue culture system for the rapid propagation of Phalaenopsis by pedicel was established.

The Phalaenopsis germplasm is the material basis for the independent development of Phalaenopsis in China and is an important guarantee for the healthy and sustainable development of the phalaenopsis industry. Some studies showed that Phalaenopsis germplasm resources were composed of more than 70 native species and nearly 31,818 hybrid species, among which the hybrid species included intermediate hybrids and intergeneric hybrids [33]. In 1975, Henshaw and Morel first proposed the strategy of in vitro conservation of plant germplasm. In vitro preservation has overcome the shortcomings of field preservation due to its characteristics of aseptic culture, low space and labor requirements, low maintenance costs, and easy international exchange, thus attracting attention from all over the world. Some international organizations and many countries have established in vitro plant germplasm gene banks [34]. Through the efforts of many scientists, the growth of preserved materials was slowed down by modifying the tissue culture conditions, mainly by adjusting the medium formula and lowering the culture temperature. The application of ultra-low temperature technology can even completely inhibit the growth of preserved materials, thus prolonging the subculture cycle, controlling the generation of variation, and reducing the variation to an acceptable minimum level. Great progress has been made. According to the technical means of preservation, in vitro preservation methods of plant germplasm resources can be divided into general preservation, slow growth preservation, and cryopreservation. In this study, we mainly preserved Phalaenopsis through in vivo preservation and tissue culture in vitro preservation.

2. Materials and Methods

The experimental materials were sourced from the Yangtze River Institute of Rare Plants in Yichang, Hubei Province, and the experimental base was a culture room and intelligent greenhouse, 4000 square meters in size, on the first floor of the Yangtze River Institute of Rare Plants. On three occasions in January, March, and May 2019, the whole pedicels extracted from Phalaenopsis plants were selected in a greenhouse and cut off from the base. Then, the upper, middle, and lower parts of the pedicels were cut with scissors into segments with nodes of about 6 cm, wrapped with gauze, soaked in a beaker containing laundry powder for 5 min, and then washed with running water for 45 min for later use. After the outdoor pretreatment, they were taken to a clean work table and soaked in medical ethanol with the concentration of 75% for 30 s. After pouring out the ethanol solution, they were immediately washed once with sterile water. Then, they were transferred to 0.1% HgCl₂, 5 drops of Tween 80 was added, and they were soaked for 8 min, 10 min, 12 min, respectively. Finally, sterile filter paper was used to blot the water in the internode of the pedicel. The damaged discoloration was excised before inoculation because of the damage to the incision tissue during both the sterilization and flushing of the explants. The materials for the Phalaenopsis induction test were 12 cm adult seedlings of the ‘RED SUN’ variety purchased from Guangzhou market and planted in the greenhouse of Three Gorges Nursery Research Center. The medium for the stages of primary culture (MS + NAA 0.5 mg/L + TDZ 0.4 mg/L), proliferation culture (MS + TDZ 0.2 mg/L + NAA 0.4 mg/L), and robust culture (MS+ NAA 0.4 mg/L + banana meal 20 g/L + tryptone 2 g) was based on the optimal medium screened by the experiment, with additional agar 7.0 g/L and sucrose 30 g/L; after the sterile seedlings completed the proliferation culture stage, they entered the rooting culture stage. The basic medium for rooting culture needed to be screened again for the experiment according to the specific situation (1/2 MS + IBA 0.5 mg/L + activated carbon 2 g), with the same additional agar 7.0 g/L and sucrose 30 g/L being added while establishing the medium. The pH value in the medium was between 5.8 and 6.0, the light intensity was 1500–2000 Lux, and the photoperiod was 12 h of artificial spectral light culture plus 12 h of dark culture.

An orthogonal design with three factors and three levels was used in the initial experiment, and NAA and TDZ were used as growth regulators to induce adventitious buds in the pedicel. A two-factor experimental design was used for adventitious bud proliferation. The test data were analyzed by SAS statistical software. The percentage data were first converted by arcsine and then analyzed by variance and multiple comparisons.

Germplasm was selected according to the sequence of seeds, vegetative propagules, including branches, buds, roots, and pollen, and genetic material (DNA). Seed and pollen samples were collected in accordance with GB 4401, and branches, buds, roots, and DNA samples were collected in accordance with

Table 1. Germplasm sampling.

Sequence Number	Germplasm Types	Quantity
1	branch	≥10
2	root	≥10
3	bud	≥50
4	DNA	≥6

. In vitro preservation materials were preferentially obtained from ex situ conservation bases. Materials that could not be provided by the base without near-ground protection or ex situ protection were collected from natural populations. For the preservation of Phalaenopsis in vivo, we adopted the germplasm resource nursery method.

3. Results

3.1. Effect of Sampling, Sterilization Time, and Material Site on the Contamination Rate and Germination Rate of Explants

There are studies that indicate that Phalaenopsis pedicels increase in tissue hardness, coarseness, and maturity with time, and the contamination rate also increases [35]. From the analysis of variance (ANOVA), it was shown that the three factors, time of sampling, sterilization time, and material site, all reached highly significant effects on the contamination rate of Phalaenopsis pedicels. Further multiple comparisons showed that the three different times of sampling produced high and low contamination rates in Phalaenopsis pedicels in the following order: May > March > January, with contamination rates reaching 54.2%, 20.2%, and 10.5%, respectively, i.e., as the monthly age of the pedicels increased, the contamination rate increased in an extremely significant manner and the more difficult it was to sterilize the surface, with the highest contamination rate being 54.2% in May. This may be due to the fact that the longer the explants grow outdoors, the more bacteria they carry, resulting in incomplete sterilization of sterilized surfaces. The three levels of sterilization time had the highest influence on the contamination rate of Phalaenopsis pedicels in the order of 8 min > 10 min and 12 min, i.e., there was no significant difference between the two levels of 10 min and 12 min. The contamination rates were 19.0%, 30.5%, and 35.4%, respectively, i.e., the contamination rates increased with the increase in tissue hardness, thickness, and maturity of the pedicels. Theoretically, the combination with the lowest contamination rate was January collection + sterilization time of 10 min or 12 min + upper part of the pedicel. This is also consistent with previous research findings.

The three factors, time of sampling, time of sterilization of the surface of the explants, and the part of the pedicels, had different effects on explants germination, which could be induced to the maximum extent by different combinations. From the analysis of variance, it was shown that both the factors of time of extraction and material site had highly significant effects on the germination rate of Phalaenopsis pedicels, but the sterilization time had no significant effect on the germination rate of Phalaenopsis pedicels. Further multiple comparisons showed that the germination rate of Phalaenopsis pedicels was 60.6%, 50.2%, and 45.2% in the order of January > March > May for the three levels of fungicidal time, i.e., the germination rate decreased significantly as the age of the pedicels increased. The three levels of sterilization time affected the germination rate of Phalaenopsis pedicels in the order of 8 min > 12 min > 10 min, and there was no significant difference between the three levels. The germination rate of Phalaenopsis pedicels was 58.9%, 54.9%, and 42.2% in the order of the three levels of influence on the germination rate of the upper part > lower part > middle part, and the upper part and lower part were higher than the middle part, probably due to the apical dominance in the upper part and the accumulation of growth regulators in the lower part, but only in the middle transport site. Theoretically, the combination with the highest germination rate was January collection + sterilization time of 8 min, 10 min, or 12 min + upper part of the pedicel.

This experiment did not consider the interaction between factors; however, a role between the factors cannot be ruled out. The purpose of this experiment was to establish the best combination, and a further comparison between treatment combinations showed that the lowest pollution rate of only 1.1% was achieved for the combination of January + upper pedicel + fungicidal time 8 min, which was significantly lower than the other eight combinations. The highest contamination rate was 85.6% for the combination May + lower pedicel + sterilization time of 8 min. If we want to consider different sampling times, the lowest contamination rates were 5.6% and 41.1% for March + upper part of pedicel + sterilization time 12 min and May + upper part of pedicel + sterilization time 10 min, respectively. The combinations with the highest sprouting rates of 87.8% and 81.1% were January + upper pedicel + sterilization time 8 min and January + lower pedicel + sterilization time 12 min, respectively, and these two combinations were significantly lower than the other six combinations. The combination with the lowest sprouting rate of 35.6% was May + middle pedicel + sterilization time 12 min. If we want to consider different sampling times, the highest germination rates were observed for March + upper pedicel + fungicidal time 12 min, March + lower pedicel + fungicidal time 10 min, and May + upper pedicel + fungicidal time 10 min, reaching 67.8%, 63.3%, and 61.1%, respectively.

3.2. Induction of Adventitious Buds in Butterfly Pedicels

TDZ is a synthetic compound that has a very obvious effect on plant induction and is a type of cytokinin. A large number of scientific studies have shown that it has a magical effect in inducing the proliferation of adventitious buds or axillary buds in woody plants [36]. The analysis of variance (ANOVA) showed that different concentrations of NAA and TDZ had significant effects on the induction of adventitious buds of Phalaenopsis pedicels, among which TDZ had a highly significant effect on the induction of adventitious buds of Phalaenopsis pedicels, and NAA had a significant effect on the induction of adventitious buds of Phalaenopsis pedicels. Further multiple comparisons showed that the order of significant differences between the three levels of basic media of different compositions on the induction of adventitious buds of Phalaenopsis pedicels was MS > Hua Bao I > B5, and the induction rates reached 70.9%, 44.8%, and 38.8%, respectively. The order of significant differences between the three levels of NAA on the induction of adventitious buds of Phalaenopsis pedicels was 0.5 mg/L& gt, 1.0 mg/L, 1.5 mg/L, and the induction rates reached 53.9%, 51.1%, and 49.6%, respectively. The order of significant differences between the three levels of TDZ on the induction of adventitious buds of Phalaenopsis pedicels were 0.4 mg/L, 0.6 mg/L > 0.2 mg/L, and the induction rates reached 53.7%, 53.6%, and 47.2%, respectively. It was theoretically introduced that the combination with the highest germination rate was: MS + NAA0.5 mg/L + TDZ0.4 mg/L or 0.6 mg/L, this is also consistent with the results of Chevreau et al. [37].

The multiple comparisons among the combinations in Table 1 show that the combination MS + NAA0.5 mg/L + TDZ0.4 mg/L had the most significant effect on the induction of infructescence buds of Phalaenopsis pedicels, with the average induction rate reaching 95.6%, which was significantly higher than the other eight combinations (Figure 1b), while the combination B5 + NAA0.5 mg/L + TDZ0.2 mg/L had the lowest induction rate of infructescence buds of Phalaenopsis pedicels, with an induction rate of 33.3%. The induction rate of infructescence buds of Phalaenopsis pedicels was the lowest, with only 33.3%, and the difference between the two combinations was 62.3%. The induction rate of infructescence buds for the MS + NAA1.0 mg/L + TDZ0.6 mg/L combination was the second highest, reaching 87.8%. There was no significant difference between the combination of Flower Power No.1 + NAA0.5 mg/L + TDZ0.6 and Flower Power No.1 + NAA1.5 mg/L + TDZ0.4. There was no significant difference between the combinations of Flower Power I + NAA1.0 mg/L + TDZ0.2 and B5 + NAA1.5 mg/L + TDZ0.6. It can be concluded that the best combination was MS + NAA0.5 mg/L + TDZ0.4 mg/L for the Phalaenopsis pedicel explant adventitious shoot induction test.

3.3. Proliferation Culture of Adventitious Buds Induced by Phalaenopsis Pedicels

The analysis of variance (ANOVA) showed that different concentrations of TDZ and the interaction between TDZ and NAA had a highly significant effect on the proliferation rate of Phalaenopsis adventitious shoots, and naphthalene acetic acid had a significant effect on the proliferation rate of Phalaenopsis adventitious shoots. Further multiple comparisons showed that the order of significant differences in the effect of TDZ on the proliferation rate of Phalaenopsis indeterminate buds in the three levels was 0.2 mg/L > 0.3 mg/L > 0.1 mg/L, and the proliferation rates reached 73%, 35.2%, and 30.1%, respectively. The order of significant differences in the effect of NAA on the proliferation rate of Phalaenopsis indeterminate buds in the three levels was 0.4 mg/L. The proliferation rates reached 45.9%, 42.5%, and 41.1%, respectively. The analysis of variance (ANOVA) showed that the different concentrations of TDZ and NAA and their interactions in the proliferation of Phalaenopsis adventitious shoots were highly significant. Further multiple comparisons showed that the order of significant differences in the effects of three levels of TDZ on the proliferation rate of Phalaenopsis indeterminate buds was 0.2 mg/L > 0.3 mg/L > 0.1 mg/L, and the proliferation rates reached 6.0, 3.9, and 3.8, respectively. The order of significant differences in the effects of three levels of NAA on the proliferation rate of Phalaenopsis indeterminate buds was 0.4, 0.3, and 0.3 mg/L, respectively. The order of significant differences in the three levels of NAA on the proliferation rate of Phalaenopsis adventitious buds was 0.4 mg/L > 0.6 mg/L and 0.2 mg/L, and the proliferation folds reached 5.5, 4.1, and 4.1, respectively. The analysis of variance (ANOVA) in

Table 2. The multiple comparison of the effect of a combination of NAA and TDZ on the induction of adventitious bud from pedicels in Phalaenopsis aphrodite.

No.	NAA (ml/L)	TDZ (ml/L)	Inoculation Amount	Induction Rate (%)
1	0.5	0.2	30	33.3 ± 3.3 G
2	1.0	0.2	30	37.8 ± 1.9 FG
3	1.5	0.2	30	46.7 ± 3.3 DE
4	0.5	0.4	30	95.6 ± 1.9 A
5	1.0	0.4	30	87.8 ± 1.9 B
6	1.5	0.4	30	82.2 ± 1.9 B
7	0.5	0.6	30	55.6 ± 1.9 C
8	1.0	0.6	30	43.3 ± 3.3 EF
9	1.5	0.6	30	50.0 ± 3.3 CD

Significant differences between groups are represented by different capital letters.

showed that when the concentration of TDZ was 0.2 mg/L and the concentration of NAA was 0.4 mg/L, the proliferation rate and proliferation multiplicity of Phalaenopsis adventitious buds were the highest, reaching 87.78% and 8.1, respectively (Figure 1c,d). The proliferation rate of Phalaenopsis adventitious buds was the lowest when the concentration of TDZ was 0.1 mg/L and the concentration of NAA was 0.2 mg/L at only 28.9%. The proliferation rate of Phalaenopsis adventitious buds was the lowest at 28.9% with a TDZ concentration of 0.1 mg/L and an NAA concentration of 0.2 mg/L, and the lowest of 3.5 was observed with a TDZ concentration of 0.3 mg/L and an NAA concentration of 0.2 mg/L.

3.4. Culture of Adventitious Buds and Strong Seedlings Induced by Phalaenopsis Pedicels

In the Phalaenopsis orchid pedicel explant adventitious sprouting test, in addition to the effect of growth regulators, some natural organic substances also had a good promotion effect on the Phalaenopsis orchid pedicel adventitious sprouting.

The analysis of variance (ANOVA) showed that different concentrations of banana powder, tryptone, and the interaction between them all had a highly significant effect on the height of Phalaenopsis sterile seedlings. The interaction between tryptone and naphthalene acetic acid had a significant effect on the stem thickness of Phalaenopsis seedlings, tryptone had a highly significant effect on the number of leaves of Phalaenopsis seedlings, and banana powder had a significant effect on the number of leaves of Phalaenopsis seedlings.

Further multiple comparisons shows that the order of highly significant differences in the effects of banana powder on plant height of sterile seedlings in the three levels was 20 g/L > 30 g/L, 10 g/L, and plant height reached 2.5 cm, 1.9 cm, and 1.5 cm, respectively. The result of significant differences in the effects of tryptone on the plant height of sterile seedlings in the three levels was comparisons between banana flour and tryptone showed that the combination of banana flour 20 g/L and tryptone 2 g/L had a significantly higher effect on the plant height of Phalaenopsis sterile seedlings than all other combinations. The effect of banana flour on the stem thickness of sterile seedlings in the three levels was significantly higher than that of all other combinations. The order of significant difference between the three levels of banana powder on the plant stem thickness of sterile seedlings was 20 g, 30 g > 10 g, and the plant stem thickness reached 1.04 cm, 1.02 cm, and 0.8 cm respectively. The order of significant difference between the three levels of tryptone on the plant stem thickness of sterile seedlings was 2 g/L > 3 g/L > 1 g/L, and the plant stem thickness reached 1.2 cm, 1.0 cm, and 0.7 cm respectively. The order of significant differences between the three levels of banana powder on the number of leaves of sterile seedlings was 20 g/L > 30 g/L and 10 g/L, and the number of leaves reached 3.3, 2.8, and 2.7, respectively. The order of significant differences between the three levels of tryptone on the number of leaves of sterile seedlings was 2 g/L > 3 g/L, 1 g/L and 2.7, respectively. The order of significant differences between the three levels of tryptone on the number of leaves of sterile seedlings was 2 g/L > 3 g/L and 1 g/L, respectively, and the number of leaves reached 3.9, 2.6, and 2.3, respectively. The results of multiple comparisons between banana powder and tryptone combinations showed that the combination of banana powder 20 g/L and tryptone 2 g/L had a significantly higher effect on the number of leaves of Phalaenopsis sterile seedlings than all other combinations.

The ANOVA in

Table 3. The multiple comparison of the effect of different growth regulators and the level of regulators on the propagation fold of adventitious bud.

No.	TDZ (mg/L)	NAA (mg/L)	Inoculation Amount	Proliferation Rates (%)	Proliferation Fold
1	0.1	0.2	30	28.9 ± 1.9 Cd	3.6 ± 0.1 Cd
2	0.1	0.4	30	64.4 ± 5.1 Bb	5.0 ± 0.1 Bb
3	0.1	0.6	30	36.7 ± 3.3 Cc	3.8 ± 0.3 Ccd
4	0.2	0.2	30	30.0 ± 3.3 Ccd	4.2 ± 0.2 Cc
5	0.2	0.4	30	87.8 ± 5.1 Aa	8.1 ± 0.3 Aa
6	0.2	0.6	30	32.2 ± 3.8 Ccd	4.2 ± 0.5 Cc
7	0.3	0.2	30	33.3 ± 3.3 Ccd	3.5 ± 0.2 Cd
8	0.3	0.4	30	66.7 ± 3.3 Bb	5.0 ± 0.0 Bb
9	0.3	0.6	30	36.7 ± 3.3 Cc	3.8 ± 0.2 Ccd

Different letters represent significant differences, while the same letter has no significant differences.

showed that the most significant effect on the seedlings of Phalaenopsis in terms of strength was observed when the concentration of banana powder and tryptone was 20 g/L and 2 g/L, respectively, with the same basic medium and growth factor concentration. The highest number of plant leaves averaged 4.3, the highest plant height averaged 5.2 cm, the thickest plant stem thickness averaged 1.3 cm, and the plant height/stem thickness value reached 4.13, which is more desirable. The number of leaves of Phalaenopsis sterile seedlings reached 3.67 at 10 g/L and 30 g/L and 3 g/L and 30 g/L with a combination of banana powder and tryptone, which was also superior. The plant height of Phalaenopsis sterile seedlings reached 3.4 cm and 3.2 cm for the combinations of banana powder and tryptone at 3 g/L vs. 30 g/L and 10 g/L vs. 30 g/L, which were significantly lower than the best combinations of 20 g/L and 2 g/L, but also significantly higher than the other combinations. The stem thickness of Phalaenopsis sterile seedling plants reached 1.2 cm at concentrations of 20 g/L, 10 g/L, 20 g/L, and 3 g/L, and 30 g/L and 3 g/L for banana powder and tryptone combinations, respectively, which were slightly lower than the optimal combinations.

3.5. Effect of Medium, IBA, and Activated Carbon on Rooting of Sterile Seedlings

Phalaenopsis is a plant that roots easily, and the medium can be used with more or without less growth regulators at the rooting stage [38]. The addition of activated charcoal to the rooting medium is beneficial to the formation and growth of roots and to obtain rooted seedlings with better root systems, and the results of this experiment were consistent with both. Analysis of variance showed that medium, IBA, and activated charcoal all had highly significant effects on the number of rooted sterile seedlings. Further multiple comparisons showed that the three levels of medium affected the rooting number of sterile seedlings in the following order: 1/2 MS > 1/4 MS, MS, with 58.8, 34.3, and 30.6 roots, respectively, and there was no significant difference between the levels of 1/4 MS and MS. The three levels of IBA affected the rooting number of sterile seedlings in the following order 0.5 mg/L > 1.0 mg/L and 1.5 mg/L, and there was no significant difference between the 1.0 mg/L and 1.5 mg/L levels. The number of roots was 61.8, 31.7, and 30.2, respectively. The three levels of activated carbon affected the number of roots of sterile seedlings in the following order: 2 g/L > 1 g/L and 3 g/L, with 64.3, 30.9, and 28.4 roots, respectively, and there was no significant difference between the two levels of 1 g/L and 3 g/L. Theoretically, the combination that produced the highest rooting number was 1/2 MS + IBA0.5 mg/L + activated carbon 2 g/L.

The ANOVA showed that medium, IBA, and activated carbon all had a highly significant effect on the rooting rate of sterile seedlings. Further multiple comparisons showed that the three levels of medium affected the rooting rate of sterile seedlings in the following order: 1/2 MS > 1/4 MS, MS, and the rooting rate was 56.8%, 41.4%, and 37.6%, respectively; there was no significant difference between the 1/4 MS and MS levels. The three levels of IBA affected the rooting rate of sterile seedlings in the following order: 0.5 mg/L >1.0 mg/L and 1.5 mg/L, and the number of roots was 61.8, 31.7 and 30.2, respectively; there was no significant difference between the 1.0 mg/L and 1.5 mg/L levels. The three levels of activated carbon affected the rooting number of sterile seedlings in the following order: 2 g/L > 1 g/L, 3 g/L, and the number of roots was 64.3, 30.9, and 28.4, respectively; there was no significant difference between the 1 g/L and 3 g/L levels. Theoretically, the combination that produced the highest rooting rate was 1/2 MS + IBA0.5 mg/L + activated carbon 2 g/L.

As the purpose of this experiment was to find the best combination, further comparisons between treatment combinations were performed. As shown in

Table 4. The multiple comparison of the effect of banana powder and tryptone on the culture of sterile healthy seedlings.

No.	Banana Powder (g)	Tryptone (g)	Inoculation Amount	Leaf Number	Plant Height (cm)	Stem Diameter (cm)	Height/ Diameter
1	10	1	30	2.0 ± 0.0 Cd	0.6 ± 0.10 F e	0.5 ± 0.1 Fe	1.20
2	10	2	30	2.7 ± 0.6 BCcd	0.8 ± 0.1 EF de	0.9 ± 0.1 CDbc	0.96
3	10	3	30	2.3 ± 0.6 BCcd	1.3 ± 0.2 CD c	0.6 ± 0.1 EFde	2.11
4	20	1	30	3.7 ± 0.6 ABab	3.2 ± 0.1 B b	1.2 ± 0.1 ABa	2.67
5	20	2	30	4.3 ± 0.6 Aa	5.2 ± 0.2 A a	1.3 ± 0.2 Aa	4.13
6	20	3	30	3.7 ± 0.6 ABab	3.4 ± 0.10 B b	1.2 ± 0.1 Aa	2.76
7	30	1	30	2.3 ± 0.6 BCcd	0.8 ± 0.2 EF e	0.7 ± 0.1 DEcd	1.05
8	30	2	30	3.0 ± 0.0 BCcd	1.4 ± 0.2 C c	1.0 ± 0.1 BCb	1.43
9	30	3	30	2.3 ± 0.6 BCcd	1.1 ± 0.2 DE d	1.2 ± 0.1 ABa	0.89

Different letters represent significant differences, while the same letter has no significant differences.

, the combination with the highest number of roots was 1/2 MS + IBA0.5 mg/L + activated carbon 2 g/L, with 111 roots, which was significantly higher than the other eight combinations (Figure 1f). If we were to consider different media, the higher number of roots was observed for the 1/4 MS + IBA1.5 mg/L + activated charcoal 2 g/L combination with 45.3 roots. The combination with the highest rooting rate was 1/4 MS + IBA1.5 mg/L + activated charcoal 2 g/L, with a 47.6% rooting rate.

3.6. Grading and Maintenance of Sterile Seedling Transplants of Phalaenopsis

The analysis of variance (ANOVA) showed that the substrate had a highly significant effect on the survival rate of sterile seedling transplants, while the interaction between the seedling environment and substrate did not have a significant effect on the survival rate of sterile seedling transplants. Further multiple comparisons in

Table 5. The multiple comparison of the effect of medium, IBA, and activated carbon on the rate of rooting of sterile seedlings.

Medium	IBA	Activated Carbon	Explant Number	Roots Number	Rooting Rate
MS	0.5	1	30	39.7 ± 5.7 BCd	41.8 ± 2.9 BCbc
MS	1.0	2	30	36.7 ± 3.5 BCd	40.5 ± 3.0 BCcd
MS	1.5	3	30	15.3 ± 3.1 Ee	30.3 ± 3.4 Ef
1/2 MS	0.5	2	30	111.0 ± 3.6 Aa	76.6 ± 2.6 Aa
1/2 MS	1.0	3	30	35.3 ± 3.1 BCb	50.2 ± 3.0 BCcd
1/2 MS	1.5	1	30	30.0 ± 4.4 CDcd	43.7 ± 4.0 CDd
1/4 MS	0.5	3	30	34.7 ± 3.5 Cd	38.6 ± 3.0 Ccd
1/4 MS	1.0	1	30	23.0 ± 2.6 DEd	37.9 ± 3.0 Dee
1/4 MS	1.5	2	30	45.3 ± 5.5 Bbc	47.6 ± 2.9 Bb

Significant differences between groups are represented by different capital letters.

show that the two levels of substrate had a higher and lower effect on the survival rate of sterile seedling transplants in the order of water moss > peat, with survival rates of 80.8% and 40.6%, respectively. The two levels of seedling refinement environment influenced the survival rate of sterile seedlings in the order of glasshouse > greenhouse, with a 65% and 61.4% survival rate, respectively. Theoretically, the combination with the highest survival rate was seedling refinement in the refinement room and transplanting with water moss. A control group involving clear water substrate was established along with the experiments with seedlings in both water moss and peat substrates in which the rooting survival rate reached 90%.

As the purpose of this experiment was to find the best combination, further comparisons were made between the treatment combinations. The results in

Table 6. The table of various factors of transplanting of sterile seedlings.

No.	Substrate	Environment	Leaf Size	Plant Height	Survival Rate	Growth Potential
1	moss	Glasshouse	5.3 ± 1.0 Aa	1.7 ± 0.0 Aa	1.0 ± 0.1 Aa	rapid and strong
2	moss	Greenhouse	3.5 ± 0.1 Bb	0.9 ± 0.1 Cc	0.5 ± 0.1 Bb	slow
3	peat	Glasshouse	4.1 ± 0.1 ABb	1.2 ± 0.1 Bb	0.9 ± 0.1 Aa	slow and yellow
4	peat	Greenhouse	3.2 ± 0.0 Bb	0.7 ± 0.0 Dd	0.5 ± 0.1 Bb	slow

Different letters represent significant differences, while the same letter has no significant differences.

show that the largest leaf size was 5.3 cm for the combination of water moss + seedling room and 4.1 cm in the combination of peat + seedling room, and there was no significant difference between them. The highest plant height was 1.7 cm for the combination of water moss + seedling refinery, and the lowest was 0.7 cm for the combination of peat + greenhouse. Moreover, the highest survival rates of 100% and 90% were for the combination of water moss + seedling refinery and peat + seedling refinery, but the growth potential and plant strength were higher for the combination of water moss + seedling refinery than for the combination of peat + seedling refinery.

3.7. Preservation of Phalaenopsis Germplasm

In this study, the in vivo preservation of Phalaenopsis was explored in a greenhouse in the scientific research base of the Yangtze River Rare Plant Research Institute. At present, 34 species of Phalaenopsis are preserved in vivo and in vitro by tissue culture (Figure 2).

4. Discussion

This experiment established a rapid histoponic propagation system for Phalaenopsis pedicels, but there are still many technical contents and details that can be improved in follow-up work.

In terms of explant types, only Phalaenopsis pedicel parts were used to induce differentiation abilities and multiplication rates, and no targeted tests were conducted on Phalaenopsis leaves, stem tips, stem segments, root tips, or pollen explants.

In terms of explant sites, they were limited to the upper, middle, and lower parts of Phalaenopsis pedicels, and no detailed comparative tests were conducted on the upper, middle, or lower parts of the leaves and the basal, middle, or apical parts of the root system.

In terms of the explant surface area, only one internode part of the pedicel with buds was used. Therefore, there was still scope for testing the distance between the upper part of the pedicel internode and the lower part of the pedicel internode, and the number of internodes contained in the pedicel.

With regard to the polarity of explant inoculation, only the inoculation of pedicels in the direction of natural vertical growth was carried out, and no comparative tests were conducted on pedicels with inclined angles and horizontal contact with the medium. In the subsequent perfection tests, various inoculation methods, such as positive and negative inoculation of leaves, vertical inoculation, inoculation with certain inclined angles, and vertical inoculation of roots with certain inclined angles, should be considered.

In terms of the relationship between the contact between the explants and the medium, only one method of inserting the flower roots into the medium was tested in this part of the experiment, and the depth of the pedicel inserted into the medium or placed on the surface was not tested. Furthermore, several other explant materials, and the relationship between the contact between the leaf, stem tip, stem segment, root, and pollen should be considered in subsequent experiments, as should the relationship between the pedicel explants and the medium.

In terms of the timing of explant collection, only the months of January, March, and May were sampled in our experiment, while the other nine months of February, April, June, July, August, September, October, November, and December were not sampled, and the effects are not known.

In terms of disinfection of explants, only one disinfection solution was used, and it was a heavy metal solution. Although the test waste solution was handed over for specialized solid waste and hazardous material pollution-free treatment, there is still a risk of contamination of soil and crops in the interim; thus, future disinfection tests should consider sodium hypochlorite, neosporin, hydrogen peroxide, and new environmentally friendly pollution-free disinfection and sterilization solutions to reduce the chance of pollution.

In the Phalaenopsis test species, only one species, i.e., ‘RED SUN’, was used as the test material; however, other popular ornamental species and color lines, such as “Full Sky Red”, “Maple Leaf “, “Jui Bao gold brick”, “Garfield”, “perfect girl”, “golden armor “, “New Century Red”, “Venus”, “Fuller Sunset”, “Big Dragon King “, “Panda”, “Green Apple”, “Pink Butterfly”, “Scented Flower“, and other large-flowered and velvet-flowered species, are of great significance in terms commercial production and the preservation of germplasm resources.

Although this experiment established the stages of the rapid histoponic breeding system for Phalaenopsis orchids, further exploration is needed in terms of standardized production and cost saving to improve economic efficiency.

5. Conclusions

This study explored the effects of pedicel taking time, site, plant growth regulators, and organic substances on the establishment of a regeneration system and to establish the rapid propagation of Phalaenopsis aphrodite Rchb. F. for its germplasm conservation and commercial cultivation.