Influence of Rhizosphere Temperature and Humidity Regulation on Rooting, Mortality, and Transplant Survival of Aeroponically Rapid Growth Mulberry Cutting

Abstract

This study explores the impact of different temperature and humidity conditions on Mulberry cutting rooting and transplanting survival rates in an aeroponically rapid propagation system. It investigates the relationship between droplet adhesion and mortality based on mildew and rot distribution in cuttings. The regulating strategies were divided into three groups: humidity, temperature, and combined humidity and temperature. The humidity group included a fixed spray frequency (H3) and fixed-range humidity conditions (H1: 90% ≤ Humidity ≤ 100% and H2: 95% ≤ Humidity ≤ 100%). The temperature group comprised room temperature (T2) and fixed-range temperature circumstances (T1: 25 °C < Temperature < 27 °C). The non-regulation group (THCK) made no particular modifications, whereas the combined temperature and humidity group (TH) maintained both temperature and humidity within a set range (90% ≤ Humidity ≤ 100% and 25 °C < Temperature < 27 °C). Moreover, the humidity control group (HCK) and the temperature control group (TCK) integrated a soil cultivation system. Then, the transplantation experiment and the droplet adhesion amount experiment were carried out. The results demonstrate that faster callus formation and rooting with aeroponic propagation, temperature and humidity regulation significantly improves root growth and survival rate. The temperature and humidity regulation group outperforms, increased callus rate, rooting rate, average root length, average root number, fresh weight, and dry weight by 30%, 25%, 4.54 mm, 1.09, 0.12 g, and 0.012 g, while reducing mortality by 20%. Conversely, soil culture showed no callus formation or rooting throughout the same timeframe. Significant (p < 0.01) differences between regulation and non-regulation groups exist in growth parameters, mortality, and transplant survival rates. All groups achieved 80–100% transplant survival, with temperature regulation enhancing hardening effects. Excessive droplet adhesion heightens the risk of mortality, with an optimal adhesion threshold of approximately 0.06444 g. This study offers valuable insights into aeroponically rapid propagation and intelligent nurseries.

1. Introduction

Agricultural production is poising to encounter significant challenges arising from the dual pressures of a swiftly escalating demand for food and the progressively dwindling availability of resources. According to the United Nations Development Program projections, the world population is expected to reach 9.8 billion by 2050. In light of this, food production would need to increase by 60–70%, accompanied by an estimated 40% increase in water consumption for food production [1]. Meanwhile, over 35% of the world’s agricultural land has suffered varying degrees of degradation, with approximately 25% labeled as barren and unsuitable for agricultural activities due to a combination of natural and human-induced factors. These factors include inadequate management practices, rapid urbanization, and soil erosion [2]. The situation could worsen if the Food and Agriculture Organization’s (FAO) prediction of needing an additional 69 million hectares of farmland between 2005 and 2050 materializes [3]. On the other hand, the availability of low-cost freshwater resources such as rivers, groundwater, and natural rainwater is also facing uncertainty in the future due to issues like pollution, overexploitation, and the impacts of climate change. An estimated 3.2 billion people worldwide will live in chronically water-scarce areas by 2050 [4]. Notably, about 80–90% of available water resources are directed towards agricultural irrigation, putting additional pressure on water availability for human consumption. Consequently, agricultural production is also faced with a significant freshwater crisis [5]. To address these challenges, nations must prioritize efficiently utilizing limited resources, particularly land and freshwater. This necessity is compounded by the need for targeted interventions. These interventions might include management model innovation, resource protection, policy reinforcement, and the adoption of modern technological advancements [6,7].

In the realm of modern technology, aeroponic cultivation plays a significant role (Figure 1). It is a soilless plant growth method that utilizes suspended roots and nutrient-rich sprays to foster rapid, controlled growth, mitigating the impacts of climate change and pests. This resource-efficient system significantly reduces water and nutrient consumption compared to traditional soil-based cultivation, making it a pivotal technology for ensuring future food security [8,9,10]. In this unique setup, plants can recycle the nutrient-rich solution in the aeroponic system, utilizing very little water per unit of planting area. Precisely, it reduces 90–95% of water consumption, 50% of nutrient consumption, and 45% of cultivation time [11]. As a robust and technologically advanced solution, the aeroponic system holds great promise in safeguarding future food security. Its efficacy has led to its endorsement and widespread adoption by numerous countries [12,13,14].

Aeroponically rapid propagation, a fusion of aeroponics and asexual propagation, holds promise in accelerating plant reproduction [15]. This method, characterized by efficient resource utilization and controlled environments, has shown success in enhancing root growth potential, ensuring uniform temperature and humidity, and breaking propagation bottlenecks for various plant species [16,17]. Successful applications in forestry, vegetables, medicinal materials, and horticulture underscore its potential to propagate valuable plants effectively and sustainably. These successful instances serve as valuable references for employing aeroponically rapid propagation to cultivate more valuable plants for human benefit. The success of cuttings’ asexual propagation depends on the ability of the base to develop adventitious roots, with temperature and humidity being important factors affecting the rooting of cuttings [18,19]. There are obvious differences in the rooting speed and rooting rate of cuttings in different temperature and humidity environments [20,21].

While traditional practices involve manual control of temperature and humidity based on experience, advancements in environmental monitoring technology can potentially reduce labor, time, and skill requirements associated with this process. Environmental monitoring and control are crucial in optimizing crop growth conditions by regulating temperature, humidity, light, and CO₂ levels, such as greenhouse environmental monitoring and control [22] and soilless cultivation irrigation management systems [23]. However, there’s a lack of studies applying advanced control to aeroponically rapid propagation, posing a pressing need for sophisticated control systems in line with the technology’s growth.

The Mulberry tree exhibits rapid growth and high adaptability to various environments, serving multiple ecological roles such as soil improvement, wind and sand control, water conservation, and air purification. Leaves of Mulberry are vital for silkworm rearing and silk production, while its roots, bark, stems, and fruits contain valuable bioactive compounds for food, health products, and cosmetics [24,25,26]. Given its manifold benefits, the Mulberry holds significant value for humanity. Despite this, there’s a lack of information on aeroponically rapid propagation of Mulberry cutting. Thus, this study focuses on investigating the propagation of Mulberry through aeroponic cuttings.

Consequently, this study aims to employ an aeroponically rapid propagation system with integrated temperature and humidity regulation to foster the cultivation of Mulberry cutting. The investigation seeks to elucidate the impact of temperature and humidity modulation on cutting rooting parameters and post-transplant survival rates. Additionally, the study addresses the challenge of cutting mortality attributed to mildew and rot by analyzing droplet adhesion effects.

2. Materials and Methods

2.1. Design of Aeroponically Rapid Propagation Experimental Test Bench

The experimental setup composes multiple autonomous aeroponically rapid propagation systems meticulously designed to accommodate subsequent comparative experiments and facilitate precise environmental factor control within the cultivation apparatus. Each aeroponically rapid propagation system functions autonomously without interfering with others. The study employs a total of three aeroponically rapid propagation systems.

The configuration of the aeroponically rapid propagation experimental setup is illustrated in Figure 2a. Each system encompasses six drive boards (1). The nutrient solution box (2) and accommodates the required liquid for spraying. The aeroponic box (3) is located above the nutrient solution box (2) and matches the size of the nutrient solution box (2). Each system integrates two fixed covers: one above the aeroponic box (3) seals it, while the other above the nutrient solution box (2) separates the two. The fixed cover (4) features apertures for securing the planting basket (8) with 20 evenly distributed planting holes (5). A sponge rod (6) absorbs the nutrient solution, extending from the atomization sheet (7) into the liquid. The atomization sheet, sponge rod, and drive board constitute an atomizing nozzle, delivering atomized liquid from the nutrient solution box (2) into the aeroponic box (3) for cutting absorption. Each aeroponic system comprises 26 planting baskets, with 20 fixed baskets on the aeroponic box’s fixed cover (4) to secure cuttings and the remainder serving as air vents. Six fixed baskets on the fixed cover (4) above the nutrient solution box (2), six fixed baskets fix atomizing nozzles, and the rest remain open. A temperature and humidity sensor (9) are placed within the aeroponic box (3) compartment to prevent interference with temperature and humidity data detection caused by droplet accumulation. The liquid (10) in the nutrient solution box (2) is typically water or nutrient solution, adjustable for different stages. Two device controllers (11, A and B) serve distinct functions: (11, A) connects the smart sensor and wireless communication module, relaying collected data to the core controller, while (11, B) receives commands from the core controller to manage actuator switches. A cooling fan (12) within the aeroponic box (3) maintains airflow directionality to prevent mist droplets from escaping. A 5V DC power supply (13) powers atomizing sheets and drive boards across each system. The table (14) provides equipment placement.

The experiment setup features both manual and automatic control modes. In the manual mode, environmental data can be observed through the remote-control terminal, enabling manual activation and deactivation of the atomizer, cooling fan, and other equipment. The automatic mode allows for preset control ranges of environmental factors, with the system autonomously managing the atomizer, cooling fan, and other equipment based on the defined range. Excess droplets adhering to cutting roots are channeled back into the nutrient solution box via holes on the fixed cover. The louvers open during axial flow fan operation, accelerating air circulation in the aeroponic box for temperature reduction and closing once the fan stops.

2.2. Temperature and Humidity Monitoring and Control System

The system is designed to monitor and control temperature and humidity within the growth box. The core controller is developed based on STM32F103 (chip model: STM32F103ZET6), integrated with TFT LCD capacitive touch screen, DL-20 wireless serial port transparent transmission module, ESP8266 IoT module, and SD memory card. The device controllers are developed based on STC89C52 single-chip microcomputer and integrate DL-20 wireless serial port transparent transmission module. Actuators include: AM2301 protected temperature and humidity sensor, cooling fan, atomizing nozzle, and LED light. The device controller is connected to the AM2301 sensor (Guangzhou Aosong Electronics Co. Ltd., Shenzhen, China), reads the environmental factor data packet at a frequency of 0.5 s/time, and then transmits the data to the UCOS-III2.1 message queue through the serial port. The EMWIN task displays real-time data and generates graphical representations. Data storage consists of STM32’s FLASH, SD card, and FatFs file management system. The device controller receives instruction data packets from the core controller, enabling control of relevant actuators.

Moreover, the core controller communicates with a cloud server through the ESP8266 IoT module, allowing remote control and receiving instructions from the cloud server to manage electrical appliances. Sensors are connected to controllers via jumpers, and a 6-way 5V optocoupler isolation relay links to each actuator. Adapters are provided for both controllers and actuators. The hardware architecture is depicted in Figure 2b.

2.3. Experimental Site and Plant Material

The experiment occurred from June to September 2022 at the 107 Laboratory of Agricultural Equipment Engineering, Jiangsu University, located in Jiangsu Province, China (coordinates: 32°12′23.57″ N, 119°30′55.11″ E, altitude 17 m). The laboratory is adequately sealed, and an air-conditioning system is in place to maintain a consistent indoor temperature. Moreover, Mulberry was selected as a test crop for this experimental study. Explants of Mulberry were selected from strong mother plants to obtain better experiment results. The semi-lignified branches, with a diameter of approximately 10.00 mm, exhibited robust growth on the Mulberry mother plant and remained unaffected by diseases and insect pests. These branches were meticulously transformed into cuttings, each measuring around 150.00 mm in length. For each cutting, 2 to 3 side buds were deliberately retained, and a precise, smooth oblique incision, angled at approximately 30 degrees, was meticulously made at the lower end. Subsequently, the treated cuttings underwent a thorough rinse with clean water before being positioned in a shaded area for drying. After the cuttings were completely dried, they were submerged in a solution containing the rooting powder ABT1 (indole-naphthalene acetic acid). ABT1 contains 20% naphthalene acetic acid and 30% indole acetic acid, which were used to promote the rooting of the scion. The lower ends of the cuttings were submerged in the solution for 6 h. Following the soaking process, the cuttings were again placed in the shade to allow for adequate air drying. It is worth noting that microorganisms often adhere to the exterior surface of the cuttings. The cuttings underwent thorough sterilization utilizing a 75% alcohol solution. Each group of cuttings was subsequently enveloped with square sponge clips and organized into planting baskets, ensuring that approximately 50.00 mm of the cuttings remained exposed. These cuttings were then divided into groups and randomized into distinct aeroponic containers or soil-based cultivation environments.

During the initial four days following the cuttings’ planting, a clean water solution was employed for spraying purposes. After this period, the nutrient solution utilized was the Hoagland Formula A/B solution (Henan Caiju Dongli Agricultural Technology Co. Ltd., Luoyang, China). Given the elevated temperatures experienced during the summer months and the relatively sluggish flow characteristics of the nutrient solution, it was imperative to replace the nutrient solution every five days. The arrangement was strategically designed to mitigate the effects of external light factors [27]. The methodology of cultivating the cuttings was aligned with the configuration depicted in Figure 2c. Once the cuttings had developed leaves, light was gradually introduced. For the present study, LED plant growth lights were employed. These lights maintained a red-to-blue ratio of 4:1 and were administered for 8 h each day, effectively contributing to the growth and development of the cuttings.

2.4. Experimental Arrangement

The experiment was separated into three stages: humidity regulation, temperature regulation, and temperature and humidity co-regulation of scion rooting, to examine the impacts of diverse variables.

The experimental timeline spanned from 24 June to 14 July 2022. Throughout this duration, the average temperatures within the indoor environment exhibited consistent values of 28.45 °C ± 1.15 °C during the daytime and 27.34 °C ± 1.02 °C during the nighttime. Concurrently, the average humidity levels within the indoor space were recorded at 45.51% ± 5.69% and 50.68% ± 2.03%, respectively, for day and night periods.

The experimental framework encompassed distinct groups: the aeroponically rapid propagation groups, denoted as H1, H2, and H3, and the soil culture group (HCK). The fluctuations in humidity within the three distinct aeroponic box groups were diligently documented on the 5th, 10th, and 15th days, respectively. Each experiment cycle, delineated from the commencement of spraying to the initiation of the subsequent spraying event, served as the timeframe for recording humidity variations within each aeroponic box.

The experimental timeframe encompassed the period from 19 July to 8 August 2022. Throughout this interval, the indoor environment maintained average temperatures of 28.76 °C ± 1.15 °C during the day and 27.38 °C ± 1.12 °C during the night. Additionally, the average indoor humidity levels were measured at 54.42% ± 4.99% for daytime and 52.53% ± 1.83% for nighttime. The experiment comprised several distinctive groups, namely the aeroponically rapid propagation groups designated as T1 and T2 and the soil culture group labeled TCK.

The designated experimental duration spanned from 8 August 2022 to 28 August 2022. Throughout this defined period, the indoor environment exhibited consistent average temperatures of 30.35 °C ± 1.76 °C during the daytime and 28.16 °C ± 1.34 °C during the nighttime. Furthermore, the average indoor humidity levels were recorded at 60.51% ± 5.69% during the day and 69.68% ± 2.03% at night. The experiment featured distinct configurations, primarily encompassing the aeroponically rapid propagation group classified as TH and the THCK subgroup within it. Specifically, the TH group served as a two-factor regulation experimental subgroup. Detailed descriptions of the specific configurations for each group are systematically provided in

Table 1. Regulation experiment group.

Groups	Type	Condition
Humidity regulation experiment
H1		90% ≤ Humidity ≤ 100%
H2	Aeroponic rapid cultivation	95% ≤ Humidity ≤ 100%
H3		Spray for 2 min every 30 min
HCK	Soil cultivation	Regular watering
Temperature regulation experiment
T1	Aeroponic rapid cultivation	Spray for 2 min every 30 min, 25 °C < Temperature < 27 °C
T2		Spray for 2 min every 30 min, There is no temperature Regulation, Room temperature
TCK	Soil cultivation	Regular watering, Room temperature
Temperature and humidity regulation experiment
TH	Aeroponic rapid cultivation	90% ≤ Humidity ≤ 100% 25 °C < Temperature < 27 °C
THCK		Spray for 2 min every 30 min, There is no temperature Regulation, Room temperature

.

2.5. Experimental Indices

2.5.1. Indices of Cultivation

To compare the impact of regulated temperature and humidity on aeroponically rapid propagation, conventional aeroponically rapid propagation, and soil culture on Mulberry cutting, we monitored and recorded significant growth parameters reflecting root development every five days. These parameters included callus rate, rooting rate, average root length, average root number, mortality rate, and root fresh-dry weight. Observations of cutting growth were conducted, and data were obtained through counting and measurement.

• The computation methodologies for callus rooting and mortality rates remained consistent across treatment groups. In each treatment group, callus formation at the incision site, root system development at the base, and the count of deceased cuttings were compared to calculate the callus, rooting, and mortality rates.
• Root length was non-invasively measured using a vernier caliper, and root count was recorded directly. The subsequent determination of average root length and average root number followed.
• Upon experiment completion, five randomly selected rooted plants underwent root clipping. The fresh weight of the roots was assessed using an electronic balance model JM-B6002 (Max = 600 g, d = 0.01 g, e = 10 d). After measuring the fresh weight, the roots were placed in an envelope and dried for three hours in an 85 °C dryer. The dry weight was measured using a high-precision electronic balance, Sartorius model BS223S (Max 220 g, d = 0.001 g).

Photographic documentation was conducted at five-day intervals throughout the experiment. A random selection from each experimental group was chosen for photography and subsequent recording.

2.5.2. Transplanting Survival Rate

Following each experiment, five rooted cuttings were randomly chosen from each aeroponically rapid propagation system group and transplanted into a soil environment. The soil seedbed was sterilized before transplanting the cuttings, and regular watering was carried out daily after transplantation. After seven days, the cuttings were extracted from the soil to assess the survival condition of their root systems, and the survival rate was calculated and documented.

2.5.3. Droplet Adhesion Amount

In this approach, the weight of the cuttings was measured both before and after atomization. The difference between the weight of the cuttings before and after atomization was then calculated, representing the droplet adhesion amount on the cuttings. The calculation process is illustrated in Equation (1).

$$\Delta W=W_{\mathrm{F}}-W_{\mathrm{L}}$$

(1)

The experimental setup consists of an aeroponically rapid propagation experiment bench and a high-precision electronic balance (Guangzhou Aosong Electronics Co., Ltd., Shenzhen, China), with a 0 to 120 g range and precision of 0.01 mg). Material selection criteria refer to Section 2.3; 20 cuttings were prepared following the earlier selection criteria. The cuttings were weighed before the experiment commenced, and their weights (designated as W_F) were recorded. The cuttings were then strategically positioned within the aeroponic box and marked accordingly. The spraying duration for the measurement experiment was set at 120 s, 73 s, and 56 s for the three stages, respectively. Following each spraying session, the cuttings were removed and weighed again, resulting in a new weight measurement (denoted as W_L). Using Equation (1), the difference in weight (ΔW) was calculated to represent the droplet adhesion amount on the cuttings. Each stage’s measurement was repeated three times to minimize random errors. The average droplet adhesion amount on the cuttings at various positions was determined.

2.6. Statistical Analyses

SPSS Statistics 27.0 and Microsoft Excel 2016 were used to analyze the data. The results were subjected to a mean ± standard error (S.E.). The data of the analysis, as determined by ANOVA, underscore significant differences (p < 0.01) in the callus rate, rooting rate, average root length, average root number, fresh and dry weight, as well as the mortality rate of Mulberry cutting when subjected to varying humidity regulation strategies. Differences between means were compared using Duncan’s new complex difference test at p < 0.05.

3. Results and Discussion

3.1. Rapid Propagation and Transplanting Results

a.

Effect of humidity regulation on rooting of cuttings

Humidity regulation results at different growth stages are shown in Figure 3a–c. The humidity data analysis reveals distinct patterns. Within the H3 group, humidity consistently maintained at 97% or higher levels. Moreover, in the two humidity-regulated spray experiment groups (H1 and H2), conditions were successfully achieved as per the predefined humidity settings. Furthermore, it is worth noting that the duration of the spray cycle exhibited variations corresponding to the growth stage. On the fifth day, H1 and H2 took approximately 75 min and 50 min, respectively, to reach the desired humidity conditions, with average spray durations of 73 s and 56 s, respectively. By the 10th and 15th days, the H1 and H2 groups attained the target humidity levels in roughly 60 min and 45 min, respectively, maintaining average spray durations consistent at approximately 73 and 56 s, respectively.

The growth parameter records and results observed throughout the 20-day experiment are graphically presented in Figure 4a. Statistical analysis unveils notable trends within the data. Firstly, the callus rate followed the order of H1 > H2 = H3 > HCK, with H1 demonstrating a callus rate approximately 10% higher than H3. The rooting rate exhibited a similar trend across the treatments, with H1 > H2 = H3 > HCK. H1 recorded a rooting rate 5% higher than that of H3. Moving on to average root length, the order of treatments was H3 > H1 > H2 > HCK. H3 displayed an average root length of 3.55 mm and 7.83 mm greater than H1 and H2, respectively. The average root number, too, displayed a trend with H3 > H1 > H2 > HCK. H3 exhibited an average root number of 0.72 and 1.60, higher than H1 and H2, respectively. Regarding fresh and dry weight, both followed an H3 > H1 > H2 > HCK pattern. Compared to H1 and H2, H3 displayed an increased fresh weight of 0.06 g and 0.18 g and a dry weight increase of 0.003 g and 0.011 g, respectively. The trend was HCK > H3 > H1 > H2 when considering the mortality rate. Notably, H2 exhibited a mortality rate 10% lower than H3. Significantly, no callus formation or rooting of cuttings was observed in the soil culture environment during the humidity regulation experiment.

Humidity regulation exhibits distinct impacts on the growth parameters of cuttings. Notably, the experimental findings highlight specific outcomes for each treatment group: (a) H1 demonstrated the highest rooting rate among the treatments. (b) H3 fostered the most robust root growth in cuttings. (c) H2, on the other hand, yielded the lowest mortality rate among the cuttings.

However, it is worth mentioning that no significant differences (p > 0.01) were observed in the rooting rate and callus rate of cuttings when subjected to humidity regulation within the range of 95% to 100%, particularly in the context of interval spraying within the H2 treatment, as detailed in the statistical results of the humidity regulation experiment in

Table 2. Effect of different regulation on cutting propagation and growth.

Groups	The Rooting Rate/%	The Callus Rate/%	The Average Root Number	The Average Root Length/mm	The Mortality Rate/%	The Fresh Weight/g	The Dry Weight/g
Humidity regulation experiment
H1	54 ± 2 a	77 ± 3 a	5.27 ± 0.08 b	72.10 ± 0.34 b	40 ± 1 b	0.722 ± 0.007 b	0.062 ± 0.002 b
H2	52 ± 2 b	71 ± 2 b	4.35 ± 0.05 c	67.30 ± 0.41 c	34 ± 2 c	0.613 ± 0.008 c	0.053 ± 0.001 c
H3	51 ± 2 c	70 ± 0.01 c	5.87 ± 0.05 a	75.70 ± 0.35 a	43 ± 2 a	0.772 ± 0.011 a	0.064 ± 0.001 a
Temperature regulation experiment
T1	43.8 ± 2.3 a	64.2 ± 1.9 a	3.183 ± 0.054 a	18.690 ± 0.265 a	44.6 ± 2.6 a	0.473 ± 0.005 a	0.033 ± 0.001 a
T2	31.7 ± 2.5 b	45.4 ± 2.6 b	3.107 ± 0.068 b	17.983 ± 0.620 b	58.3 ± 2.5 b	0.463 ± 0.006 b	0.032 ± 0.001 b
Temperature and humidity regulation experiment
TH	48.8 ± 0.023 a	68.8 ± 2.3 a	3.767 ± 0.049 a	34.877 ± 0.214 a	34.2 ± 1.9 a	0.473 ± 0.005 a	0.033 ± 0.003 a
THCK	34.6 ± 0.026 b	43.3 ± 2.5 b	2.810 ± 0.074 b	30.167 ± 0.047 b	53.3 ± 2.5 b	0.37 ± 0.009 b	0.024 ± 0.001 b

Note: Different lowercase letters within the same column of the same experimental group indicate statistically significant differences at the 0.01 level.

These findings illuminate the nuanced impact of humidity regulation on the growth dynamics of Mulberry cutting.

b.

Effect of temperature regulation on rooting of cuttings

The temperature regulation during the experiment maintained a range between 25 °C and 27 °C. We defined the time between the first stop of the fan and the next stop as a temperature recording cycle. Temperature variations were documented continuously throughout both day and night, covering regulated and normal temperature conditions, with readings taken every five minutes. The findings indicated that each temperature regulation cycle duration during the day and night was approximately 25 min and 30 min, respectively. The temperature fluctuations are illustrated in Figure 3d,e.

The statistical results indicated a mortality rate trend among the treatments, with TCK > T2 > T1. Specifically, the mortality rate in T1 was 15% lower than that in T2. Additionally, other indices followed the order of T1 > T2 > TCK. Comparing T1 with T2, the callus rate, rooting rate, average root length, average root number, fresh weight, and dry weight increased by 20%, 15%, 3.11 mm, 0.39, 0.12 g, and 0.012 g, respectively, As shown in Figure 4b. Similar to the findings in the humidity regulation experiment, the temperature regulation experiment also revealed that callus and rooting in Mulberry cutting cultivated through the aeroponically rapid propagation method occurred at a faster rate.

Using a DC axial flow fan for temperature regulation in rapid plant aeroponic propagation is entirely viable. It effectively reduced the temperature within the aeroponic chamber by 2.5–3.0 °C compared to the control group (TCK). The experimental results demonstrated that T1 exhibited the highest rooting rate, the most favorable root growth, and the lowest mortality rate among the cuttings. The ANOVA results revealed significant differences (p < 0.01) in all indices of Mulberry cuttings under various humidity regulation strategies, as outlined in the statistical results of the temperature regulation experiment in Table 2. Therefore, it can be concluded that the growth conditions of the cuttings were notably improved under the temperature regulation conditions.

c.

Effect of temperature and humidity regulation on rooting of cuttings

Impact of Temperature and Humidity Regulation on Cutting Rooting The root growth morphology of the cuttings was documented at various stages of temperature and humidity regulation (TH). The complete process is illustrated in Figure 3f–i. Figure 3f shows the initial state of the cuttings. By the fifth day, noticeable calluses had developed at the base of the cuttings, as depicted in Figure 3g. Progressing to the 10th day, numerous lateral roots of varying lengths had emerged from the base of the cuttings, as seen in Figure 3h. By the 15th day, the number and length of roots at the base of the cuttings had notably increased, as illustrated in Figure 3i.

The growth parameter records and experimental outcomes throughout the 20-day experiment are depicted in Figure 4c. The statistical analysis revealed that the mortality rate trend among the treatments was THCK > TH, with TH displaying a 20% lower mortality rate than THCK. Moreover, regarding other indices, the order was TH > THCK. Specifically, comparing H1 with H3, there were improvements in the callus rate, rooting rate, average root length, average root number, fresh weight, and dry weight by 30%, 25%, 4.54 mm, 1.09, 0.12 g, and 0.012 g, respectively.

The statistical analysis of the data presented above demonstrates that TH yielded the highest rooting rate for cuttings, the most robust root growth, and the lowest mortality rate among the cuttings. These results are visually depicted in Figure 3j,k, which summarizes the findings from both experimental groups.

The ANOVA results have shown generally significant differences (p < 0.01) in the growth parameters of Mulberry cutting between the two-factor regulation and the conventional aeroponically rapid propagation, as presented in the statistical results of the temperature and humidity regulation experiment in Table 2. Consequently, it can be concluded that the growth conditions of the cuttings were notably improved under the conditions of temperature and humidity regulation.

During the 20-day experiments involving humidity and temperature regulation, aeroponically propagated Mulberry cutting displayed callus and root formation, unlike soil-cultured cuttings. Poor soil aeration likely contributed to slower root growth. Therefore, aerosol rapid growth can enable scions to root quickly and maintain a high survival rate compared to traditional soil cultivation while also promoting a higher absorption rate of the nutrient solution under the controlled temperature and humidity condition of the fog cultivation box. These findings align with prior research indicating the superiority of aeroponics in producing rooted cuttings more quickly.

Moderate temperature and humidity regulation promoted callus and root formation while reducing mortality. Compared to the control groups, cuttings subjected to humidity regulation exhibited increased root and callus rates (1.96% to 5.88%) and decreased mortality (7.50% to 26.47%). Similarly, temperature regulation significantly increased rooting (38.17%) and callus (41.41%) rates, reducing mortality by 30.72%. The combined temperature and humidity regulation showed even more promising results, with increased rooting (41.40%) and callus (58.89%) rates and reduced mortality (55.85%). Some fogging of the root environment can be applied to keep the root system moist for optimal rooting [28]. Devi et al. studied the germination behavior of potatoes in controlled and natural indoor environments and found that germination and seedling emergence rates were significantly higher in controlled environments than in natural indoor environments. These differences were statistically significant [29].

Temperature is critical in water and nutrient uptake, enzyme activity, and plant response. Moderate water stress can also enhance cutting rooting. These findings align with research by others who have regulated temperature and humidity to improve plant growth. Maintaining specific temperature and humidity levels in aeroponic systems enhances the rooting process of scions [30].

3.2. The Impact of Aeroponically Rapid Propagation Transplanting Survival Rate Statistics

The statistical results of transplanted Mulberry cuttings are that the survival rate of the T1 and TH groups is 100%, and the survival rate of the other groups (including H1, H2, H3, T2, THK) all are 80%. In the humidity regulation group transplanting experiment, the survival rates for H1, H2, and H3 were all 80%. For the temperature regulation group’s transplanting experiment, T1 achieved a 100% survival rate, while T2 had an 80% survival rate, with T1 > T2. In the transplanting experiment of the temperature and humidity regulation group, TH and THCK both had 100% survival rates, with TH > THCK. Notably, the transplanting survival rate for the temperature regulation group (T1) and the temperature and humidity regulation group (TH) reached 100%. In both experimental groups, 12V 0.5 A DC axial flow fans were employed for temperature regulation. These fans served the purpose of temperature control and enhanced air circulation within the aeroponic box environment, contributing positively to hardening the seedlings. This, in turn, increased the activity of the root systems of the cuttings to some extent, resulting in improved overall survival rates.

The transplanting survival rate of cuttings in each experimental group was 80% to 100%. This result shows that Mulberry cuttings cultivated by aerosol rapid growth have good adaptability [31]. We successfully transferred the rooted Tamarix cuttings in the aerosol rapid growth system into plastic bags and transplanted them into the nursery. The survival rate was 95%, which is very close to our results [32].

3.3. Study the Relationship Between the Droplet Adhesion Amount, Mildew, and Rot of Cuttings

3.3.1. Distribution of Moldy and Rotten Cuttings in Aeroponically Rapid Propagation System

The outcomes of the three aforementioned aeroponically rapid propagation experiments involving Mulberry cuttings revealed varying degrees of mildew and rot among the cuttings in each group. This directly contributed to a higher mortality rate among the Mulberry cuttings and impacted the overall effectiveness of the aeroponically rapid propagation experiments. The instances of mortality among the cuttings are illustrated in Figure 5a,b.

Significant variations in the growth status of cuttings at various locations are apparent. Cuttings near the atomizing nozzle exhibit a higher likelihood of mildew and rot, whereas cuttings positioned farther away from the atomizing nozzle display a lower probability of mildew and rot. The growth disparities among cuttings at different locations are visually depicted in Figure 5c.

After each spraying session, noticeable irregularities in droplet adhesion were observed. The droplet adhesion pattern revealed that cuttings near the sprinkler had excessive adhesion. In contrast, those farther from the sprinkler had reduced adhesion, as illustrated in Figure 5d,e. A high droplet adhesion level on cuttings could lead to mildew or root rot. Conversely, when the droplet adhesion was minimal, cuttings did not receive an adequate nutrient solution, resulting in an extended rooting period. We conducted related experiments to establish a connection between mildew and the amount of droplet adhesion.

3.3.2. Result Analysis of Droplet Adhesion

The before-and-after results of the droplet adhesion amount experiment on cuttings are illustrated in Figure 5f,g. The droplet adhesion amount on cuttings at different positions before and after atomization exhibits significant variations.

Figure 5h shows that the calculated droplet adhesion amount for a spray duration of 56 s was 0.1539 g, with considerable variability in the droplet adhesion amount across different planting holes. The droplet adhesion amount at various positions can be categorized into three levels:

The first level, with larger droplet adhesion amounts, includes A1, A3, A5, D1, D3, and D5, averaging 0.3216 g.

The second level of droplet adhesion includes A2, A4, D2, and D4, with an average adhesion of 0.1234 g.

The third level, with the smallest droplet adhesion amount, encompasses other positions, averaging 0.06444 g.

The experiment results for a spray duration of 73 s show that the calculated average droplet adhesion amount was 0.1072 g. The droplet adhesion amount at different positions can also be divided into three levels:

The first level includes A1, A3, A5, D1, D3, and D5, with an average droplet adhesion amount of 0.2138 g.

The second level consists of A2, A4, D2, and D4, averaging 0.1022 g.

The third level, with the smallest droplet adhesion, pertains to other positions, averaging 0.0452 g.

In the experiment with a spray duration of 56 s, the average droplet adhesion amount was 0.0938 g. Similar to the other durations, the droplet adhesion at different positions can be classified into three levels:

The first level comprises A1, A3, A5, D1, D3, and D5, with an average droplet adhesion amount of 0.1849 g.

The second level involves A2, A4, D2, and D4, with an average adhesion of 0.0941 g.

With the smallest droplet adhesion, the third level applies to other positions, averaging 0.0390 g.

Based on the experimental results, locations where cuttings exhibited mildew and rot were generally associated with the first and second levels of droplet adhesion for all three spraying durations. It is important to note that a delay in rooting was observed in cuttings with the third level of droplet adhesion amount, but this delay was not observed in cuttings subjected to 120 s of spraying. Therefore, a droplet adhesion amount of approximately 0.06444 g appears to be more suitable for the survival and propagation of cuttings in the process of aeroponically rapid propagation.

In aeroponic systems, cuttings rely on mist droplets for nutrients and humidity [33]. Interestingly, more frequent spraying, while promoting root growth, can also lead to prolonged exposure to high humidity (>97%), increasing the risk of mold and rot. The study’s ANOVA results confirmed that simultaneous temperature and humidity regulation had a more significant impact on root growth and mortality compared to single-factor regulation [34,35,36].

The rhizosphere, a dynamic micro-ecosystem teeming with microorganisms, can create favorable conditions for harmful microorganisms in aeroponic systems, potentially leading to mold and rot in cuttings. Excessive droplet adhesion appears to be a contributing factor. Maintaining a droplet adhesion level of approximately 0.06444 g seems optimal for Mulberry cuttings’ survival and reproduction during aeroponic propagation [37,38,39].

4. Conclusions

In this study, we investigated the impact of temperature and humidity regulation on the rooting parameters and transplanting success of Mulberry cuttings in an aeroponically rapid propagation system. Our findings reveal that the aeroponically rapid propagation method outperforms traditional soil culture for Mulberry cutting propagation. Temperature and humidity regulation significantly enhanced callus formation, rooting, and root development (average root number, average root length, fresh-dry weight) and reduced mortality. In contrast, humidity regulation had a positive impact only on callus formation, rooting, and mortality. Furthermore, Mulberry cuttings propagated through the aeroponically rapid propagation method exhibited exceptionally high transplant survival rates. Based on these results, the aeroponically rapid propagation method, coupled with temperature and humidity regulation, emerges as the optimal choice for Mulberry cutting propagation. We also identified a critical droplet adhesion threshold of approximately 0.06444 g for optimal cutting growth. Currently, research on rapid plant aeroponic propagation remains limited. Future investigations in this area should focus on the following:

• Develop intelligent systems to regulate the plant’s aeroponically rapid propagation process precisely.
• Investigate temperature and humidity conditions with greater detail and specificity, narrowing down the regulation ranges. Explore other environmental factors, such as light intensity, nutrient solution composition, and concentrations.
• Address the issue of uneven droplet adhesion by redesigning aeroponically rapid propagation devices to improve cutting survival rates.