**Effects of Crop Protection Unmanned Aerial System Flight Speed, Height on Effective Spraying Width, Droplet Deposition and Penetration Rate, and Control Effect Analysis on Wheat Aphids, Powdery Mildew, and Head Blight**

**Songchao Zhang 1,2,3, Baijing Qiu 1,2,\*, Xinyu Xue 3,\*, Tao Sun 3, Wei Gu 3, Fuliang Zhou <sup>4</sup> and Xiangdong Sun <sup>5</sup>**

	- <sup>5</sup> Wuxi Hanhe Aviation Technology Co., Ltd., Wuxi 214135, China; tongxm@hanhe-aviation.com
	- **\*** Correspondence: qbj@ujs.edu.cn (B.Q.); xuexinyu@caas.cn (X.X.); Tel.: +86-511-8879-7338 (B.Q.); +86-25-8434-6243 (X.X.)

**Abstract:** As a new type of crop protection machinery, the Crop Protection Unmanned Aerial System (CPUAS) has developed rapidly and been widely used in China; currently, how to use the CPUAS scientifically has become a top priority. However, the relationships between the operating parameters of the CPUAS and the effective spraying width (ESW), droplet distribution characteristics, and control effects of insect pests and diseases are not clear yet. Therefore, three levels of flight speed (FS) as 3, 4, and 5 m/s, three levels of flight height (FH) as 1.5, 2.0, and 2.5 m, and spraying volume 2.0 L/min experiments were carried out to investigate the effects of FS and FH on the ESW, droplet deposition uniformity (DDU), and droplet penetration rate (DPR) by using an electric single-rotor CPUAS CE20. Based on the obtained results, combined with the insect pests and diseases occurrence agronomic laws, the optimal operation parameters of the CPUAS were selected to control the wheat aphids, powdery mildew, and head blight. The results showed that the ESW of CE20 was not consistent, the maximum value was 5.78 m, and the minimum one was 2.51 m. The FS had a highly significant impact on ESW (*p* = 0.0033 < 0.01), while the FH and the interaction between FS and FH had no significant impact on ESW. The coefficients of variation (*CV*) of the droplet deposition were between 23.3% and 34.4%, which meant good deposition uniformity. The FH (*p* = 0.0019) and the interaction between FS and FH (*p* = 0.02) had significant impacts on the DDU. The control effects on aphids were 78.71% (1 day), 84.88% (3 days), and 90.42% (7 days), the control effects on powdery mildew were 77.17% (7 days) and 82.83% (14 days), and the control effect on head blight was 88.32% (20 days). This study proved that by the optimization of parameters and the combination of agronomy, good control effects for insect pests and diseases could be achieved by the CPUAS. The research results would provide some technical supports for CPUAS application.

**Keywords:** crop protection UAS; operation parameters; wheat agronomy; droplet distribution; aphid; powdery mildew; head blight; control effect

### **1. Introduction**

Wheat (*Triticum aestivum*) is one of the four major food crops in China, which is also one of the most important food crops around the world. The high yield of wheat is of great significance for solving the problems of poverty and hunger. The aphid (*Aphidoidea*) [1,2], powdery mildew (*Blumeria graminis*) [3,4], and head blight (*Fusarium graminearum Schw.*) [5]

**Citation:** Zhang, S.; Qiu, B.; Xue, X.; Sun, T.; Gu, W.; Zhou, F.; Sun, X. Effects of Crop Protection Unmanned Aerial System Flight Speed, Height on Effective Spraying Width, Droplet Deposition and Penetration Rate, and Control Effect Analysis on Wheat Aphids, Powdery Mildew, and Head Blight. *Appl. Sci.* **2021**, *11*, 712. https://doi.org/10.3390/app11020712

Received: 20 December 2020 Accepted: 10 January 2021 Published: 13 January 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are the major insect pest and diseases threatening the high quality and yield of wheat, which could occur from the turning green stage to the flowering stage. According to the forecast report from the National Agro-Tech Extension and Service Center of China on 13 November 2020, only the head blight would occur in an area of 6 million hectares in China in 2021, and 133.33 million hectares need to be prevented and controlled totally. How to effectively and quickly control insect pests and diseases of wheat, especially aphids, powdery mildew, and head blight is an urgent problem that farmers need to address.

In recent years, the Crop Protection Unmanned Aerial System (CPUAS) has developed rapidly in China [6–8], not only the technical level but also the application area have been already the first around the world [9,10]. With the real-time kinematic highprecision positioning technology and flight control technology, almost all the CPUASs have achieved fixed altitude and speed. Furthermore, the application of obstacle avoidance technology and terrain-following technology has improved the safety and accuracy of the CPUASs [7,11–13]. The high single pesticide application efficiency of CPUASs with an average of about 0.8–2.8 hectares is its obvious advantage [14]. In addition, the operators could be separated from the pesticide tanks on the CPUASs, which would prevent the pesticide poisonings to the operators.

Researchers have carried out many studies on how to make good use of the CPUASs for improving the pesticide utilization rate and achieving effective control of the crop diseases and insect pests. Al-Heidary et al. [15] studied the influences on the aerial spraying drift from the perspective of the droplets (size, velocity, evaporation, diameter distribution), which provided certain reference significance for this research. Qiu et al. [16] studied the effects of flight height (FH) and flight speed (FS) on the droplet deposition uniformity, and the results showed that the two factors and the interaction between them all affected the deposition and uniformity; the relationship model between deposition uniformity and FH/FS has been established for guiding the actual production application. For a multi-rotor CPUAS, Zhang et al. [17] investigated the effects of FS and FH on the effective spraying width (ESW) and droplet penetration rate (DPR) and reported that the FS had significant impacts on the ESWs, and the impacts of both FS and FH on the DPRs were highly significant. This research involved the effects of operating parameters on the droplet deposition of aerial spraying, which had great reference value for the studies of this article. For different crops, insect pests, and diseases, some scholars had also studied the effects of the aerial application parameters on the droplet deposition characters and control effects. Qin et al. [18] explored the effects of CPUAS spraying height and speed on droplet penetration and deposition uniformity on the rice. Xiao et al. [19] reported that the CPUAS had a poor droplet coverage rate, droplet density, and deposition uniformity, leading to a slightly lower control effect on pepper comparing with the electric air-pressure knapsack sprayers. Lou et al. [20] reported the good control effects of aphids and spider mites of 63.7% and 61.3% when the FH were 1.5 and 2 m above cotton. Chen et al. [21] and Wang et al. [22] reported that when the FS was 5 m/s and the FH was 1.5 m, the maximum deposition volume could be obtained in the lower layer of rice canopy. Wang et al. [23] suggested coarse droplet size and higher spray volume on wheat pests and diseases for better control effects with CPUAS. Chen et al. [24] suggested small particle sizes droplets to improve the control effect of rice plant hoppers for CPUAS.

The above studies focus on the selection of CPUAS application parameters, and the results have proved the feasibility of CPUAS for insect pests and diseases control of crops and promoted the popularization and application of CPUAS [7,16,25]. However, one factor is easy to be overlooked during the CPUAS application. As a kind of agricultural crop protection machinery with the inevitable trend for the intelligent development of green agriculture [26,27], its application should fully combine with the agronomy, so it is critical to analyze and evaluate the application with agronomies for achieving ideal and expected control effects. The occurrence time and the position on the plant of the insect pests and diseases as well as the plant height and density should be considered into the actual pesticide application. As a result of different insect pests and diseases occurrence laws, the pesticide liquid droplet deposition should be targeted to achieve better control effects. Meanwhile, the operation parameters should be changed with different plant physiological characteristics in the field, correspondingly affecting the droplet deposition characteristics. This study aims to investigate the effects of the CPUAS operation parameters on the ESW and droplet deposition characteristics on the wheat canopy. Based on the investigations, the optimized parameters have been chosen to control the aphids, powdery mildew, and head blight combined with the occurrence laws and the wheat plant characteristics. The control effects have been evaluated. The studies would provide some references for the scientific application of the CPUAS.

### **2. Materials and Methods**

### *2.1. Experimental Site, Wheat Characteristics, and Weather Conditions*

The experiment site was located in the wheat field of Sihong agricultural demonstration base (33.3636◦ N, 118.2599◦ E) in Suqian City, Jiangsu Province, China. The trials were out carried on 11 March, 14 April, and 16 May 2019, with the crop at the Turning green, Heading, and Blooming stages, respectively, and in correspondence with the local timings for controlling wheat aphids, powdery mildew, and head blight

The wheat variety is Qianmai 33, and it was sowed in the field (60 m × 120 m) with 225 kg/ha seed density. The leaf area index was measured by the canopy analyzer LAI-2200C (LI-COR company, Lincoln, NE, USA). The main characters of the wheat and the weather conditions are shown in Table 1.



### *2.2. CPUAS and Experimental Materials*

The tested CPUAS of CE20 (Wuxi Hanhe Aviation Technology Co., Ltd. Wuxi, China, as shown in Figure 1) is an electric single-rotor CPUAS with real-time kinematic Global Positioning System (RTK-GPS). It is fully autonomous flying with the routes planned by the mobile app, and the FS, FH, and the spraying volume can be also set on the mobile app with the accuracy controlled within 0.30 m, 0.30 m/s, and 0.05 L/min, respectively. The main technical parameters are shown in Table 2.

**Figure 1.** The Crop Protection Unmanned Aerial System (CPUAS) CE20.


**Table 2.** The main technical parameters of CE20.

Water-sensitive paper (WSP) was used to collect the droplets during the experiments. High-concentration insecticide and fungicide were used for aerial spraying to control aphids, powdery mildew, and head blight.

### *2.3. Experimental Treatments*

### 2.3.1. Experiment Design

According to the actual applications, the FS was set three levels as 3, 4, and 5 m/s, the FH was set three levels as 1.5, 2.0, and 2.5 m, and the spraying volume was set as 2.0 L/min during the experiments. The CE20 flew from the acceleration area to the stop spraying area along the center line of the sampling area with autonomous mode [28]. A total of nine treatments are shown in Table 3 with the treatment parameters.


**Table 3.** The experiment treatment designs.

### 2.3.2. Sampling Point Arrangements

The whole experimental area was divided into flight acceleration area, sampling area, and stop spraying area. The flight acceleration area and the stop spraying area were both 50 m long in order to ensure that the CPUAS could accelerate to a predetermined speed and stop in a timely manner. The sampling points were arranged along the vertical direction of the CPUAS flight route symmetrically with three repetitions with a 10 m interval. Twentyone sampling points were arranged symmetrically on both sides of the flight route for each repetition line. The sampling points were labeled S1 to S21 from left to right; the central one was S11. The interval distance between sampling points S10 and S11 was 1.0 m, while that between sampling points S9 and S10 was 0.50 m, and that between sampling points S9 and S1 was 0.25 m. The right and left sampling points are distributed symmetrically. The layout of sampling is shown in Figure 2.

**Figure 2.** The field sampling layout (top view).

The WSPs were fixed horizontally on the upper (the Turning green stage) or on the upper and lower layers (Heading and Blooming stages) at each sampling point without overlapping as Figure 3 shows, and there was a 15 cm vertical distance both to the top canopy of the wheat and to the ground. The bandwidths were measured by collecting droplets from the upper layer WSPs during the Turning stage, and the penetration rates were calculated by the collecting droplets from both the upper and layer WSPs during the Blooming stage.

**Figure 3.** Upper and lower layers of water-sensitive papers (WSPs) on the sampling point.

The control check (CK) area was reserved for checking the control effect of insect pests and diseases.

### *2.4. Evaluation Method of ESW and Droplet Deposition*

The WSPs were scanned to 600 dpi digital JPG images after each test in the lab and analyzed by the DepositScan (DS) [29]. The droplet deposition density, uniformity, and the DPR were analyzed further based on the droplet deposition JPG images.

According to the standard 'Technical specification of quality evaluation for crop protection UAS' (NY/T3213-2018) [30], the first sampling point of droplet quantity not less than 15 droplets per square centimeters (cm2) was judged as the boundary of the ESW each line. In this paper, the average bandwidth value analyzed of the three lines was the ESW of each flight for accuracy.

The droplet deposition uniformity was evaluated with the coefficient of variation (*CV*, %) of coverage rates [31] on the WSPs calculated from the DS within the ESW. The *CV* calculation Equation [18] is as follows.

$$CV = \frac{S}{\overline{X}} \times 100\% \tag{1}$$

$$S = \sqrt{\sum\_{i=1}^{n} \frac{\left(X\_i - \overline{X}\right)^2}{\left(n - 1\right)}}\tag{2}$$

where *S* is the standard deviation, *Xi* is the number of droplets per unit zone in the sampling card, *X* is the average number of droplets per unit zone in the sampling card, and *n* is the total number of sampling cards in reach repetition.

The droplet penetrability into the canopies was expressed by (DPR, %) and calculated by the follow formula.

$$DPR = \frac{y\_I}{y\_H} \times 100\% \tag{3}$$

(SC), 375 g a.i./ha

where *yl*, *yu* are the coverage rates of the lower layer and the upper WSP of each sampling point within the ESW range, respectively.

Analysis of variance (ANOVA) was conducted for investigating the significances of FS and FH on the ESW, DPR, and droplet deposition uniformity (DDU), taking the FS and FH as independent variables, and the ESW, DPR, and DDU as dependent variables [8,20].

### *2.5. Control Effect Survey of Aphids, Powdery Mildew, and Head Blight*

The pesticides recommended by the local crop protection station were used to control wheat diseases and insect pests. The information of aerial spraying date, major pest and disease, pesticides, and dosage, is shown in Table 4.


**Table 4.** The major wheat pests, diseases, and pesticide applications.

The control effect survey of aphids was evaluated by the live aphid quantity before and after application according to the standard 'Rules for the investigation and forecast of wheat aphids' [32]. The assessment was made by sampling five locations for wheat aphids. The aphid quantity of 10 plots of wheat per location was investigated before spraying, and

the wheat plants were labeled. One day, 3 days, and 7 days after application, the quantity of live aphids in the same location and plant was investigated again [33,34]. The control effect of aphids was calculated according to Equations (4) and (5). The control effect of powdery mildew was evaluated by the disease index (*DI*) changing according to the standard 'Rules for the investigation and forecast of wheat powdery mildew' [35]. The wheat plant *DIs* of 9 plots were investigated randomly, and the wheat plants were labeled. Seven days and 14 days after application, the *DIs* of the same plants were investigated again [36]. The control effect of powdery mildew was calculated according to Equations (6) and (7). The control effect of head blight was evaluated by the *DI* changing according to the standard 'Rules for monitoring and forecast of the wheat head blight' [37]. The same as the powdery mildew *DIs* investigations, the plant *DIs* of head blight were investigated again [38]. The control effect of head blight was calculated according to Equations (8) and (9). The wheat plants of the *CK* area were used as the reference during the evaluations.

$$
\eta\_d = \frac{n\_b - n\_d}{n\_b} \times 100\% \tag{4}
$$

$$CE\_y = \frac{\eta\_{dT} - \eta\_{d\bar{C}K}}{1 - \eta\_{d\bar{C}K}} \times 100\% \tag{5}$$

$$DI\_w = F \times \frac{\sum (d\_i \times l\_i)}{L} \times 100\tag{6}$$

$$CE\_{\rm w} = \frac{DI\_{\rm wCK} - DI\_{\rm wT}}{DI\_{\rm wCK}} \times 100\% \tag{7}$$

$$DI\_{\mathbb{C}} = \frac{\sum (h\_i \times i)}{H \times 4} \times 100\tag{8}$$

$$CE\_{\rm c} = \frac{DI\_{\rm cCK} - DI\_{\rm cT}}{DI\_{\rm cCK}} \times 100\% \tag{9}$$

where *η<sup>d</sup>* is the aphid dropping rate, *nb* is the quantity of live aphids per hundred plants of wheat before spraying application, *na* is the quantity of live aphids per hundred plants of wheat after spraying application, *CEy* is the aphid control effect, *η*dT is the aphid dropping rate in the treatment area, *ηdCK* is the aphid dropping rate in the *CK* area; *D Iw* is the disease index of powdery mildew, *F* is the diseased leaf rate of powdery mildew, *di* is the powdery mildew severity levels, *li* is the number of each diseased leaves of powdery mildew, *L* is the total number of diseased leaves in the powdery mildew survey, *CEw* is the powdery mildew control effect, *D IwCK* is the disease index of the powdery mildew in the *CK* area, *D IwT* is the disease index of powdery mildew in the treated area; *D Ic* is the disease index of head blight, *hi* is the number of each diseased ear of head blight, *i* is the head blight severity levels, *H* is the total number of diseased ears in the head blight survey, *CEc* is the head blight control effect, *D IcCK* is the disease index of the head blight in the *CK* area, *D IcT* is the disease index of the head blight in the treated area.

### **3. Results**

### *3.1. Test Data Statistics*

The average bandwidth of each treatment was as the ESW. The DDUs (coefficient of variation (*CV*), %) were calculated according to Equation (1). The test result data are shown in the Table 5.

From Table 5, it could be seen that the ESW of CE20 was not consistent and decreased as the FS increased overall, the ESW were among 2.51 to 5.78 m, and the maximum value was 5.78 m (T2). The *CV* represents the DDU, which means the smaller the *CV*, the more uniform the distribution of the droplet deposition. The *CVs* were all not exceeding 35%, of which the minimum one was 23.30% (T1 and T4) and the maximum one was 34.40% (T6), which meant good deposition uniformity within the ESWs.


**Table 5.** Test result data of bandwidths, droplet deposition uniformities, and penetration rates.

### *3.2. The ESW Analyses*

The ESW increased first and then decreased with the same FS under the FH of 3, 4, and 5 m, respectively (Figure 4). This change trend was most significant at the FS of 4.0 m/s, the ESW increase rate was 63.82% comparing T4 (ESW = 3.51 m) with T5 (ESW = 5.75 m), and the ESW decrease rate was 27.48% comparing T5 (ESW = 5.75 m) with T6 (ESW = 4.17 m). From Figure 4, the ESWs under an FS of 3.0 m/s were larger than those of same FH at 4.0 or 5.0 m/s. The ESW showed a monotonous downward trend with the same FH (Figure 5) obviously under the FS of 1.5, 2.0, and 2.5 m/s, respectively. This trend was obvious at the FH of 1.5 and 2.5 m, and the ESW maximum decrease rate was 52.40% comparing T1 (ESW = 5.42 m) with T7 (ESW = 2.58 m). From Figure 5, the ESWs under an FH of 2.0 m were larger than those of the same FS at 1.5 or 2.5 m. Therefore, it could be considered that the FS and the FH affected the ESWs. Comparing Figure 4 with Figure 5, it could be seen that FS had a larger effect on EWS than FH.

**Figure 4.** Effective spraying width (ESW) changes under different speeds. Note: ESW1.5, ESW2.0, ESW2.5 represent the ESW when the flight height (FH) was 1.5, 2.0, and 2.5 m, respectively.

The two-way ANOVA was conducted to verify the significance effect of FS and FH on ESW at the *p*-value = 0.05 level, and the results are shown in Table 6. The FS has a highly significant impact on ESW (*p* = 0.0033 < 0.01), the FH has no significant impact on the ESW (*p* = 0.136 > 0.05), and the interaction between FS and FH also has no significant impact on ESW (*p* = 0.906 > 0.05).

**Figure 5.** ESW changes under different heights. Note: ESW3, ESW4, and ESW5 represent the ESW when the flight speeds (FS) were 3.0, 4.0 m/s, and 5.0 m/s, respectively.


1,2: *p* means the significance level of the factor affecting the result, *p* < 0.01 (\*\* represents factors that are highly significant on the test result), *p* < 0.05 (\* represents factors that have a significant impact on the test result), NS (NS represents factors that have no significant impact on the test result).

### *3.3. The Deposition Uniformity Analyses*

The two-way ANOVA results (Table 7) indicated that the FH as well as the interaction between FS and FH have significant impacts on the DDU. This law is also shown in Figure 6. The *CV* of droplet deposition tended to became larger with the increase of FH, which meant that the DDU becomes worse.


**Table 7.** Two-way analysis of variance for droplet deposition uniformity (DDUs).

1,2: *p* means the significance level of the factor affecting the result, *p* < 0.01 (\*\* represents factors that have a highly significant impact on the test result), *p* < 0.05 (\* represents factors that have a significant impact on the test result), NS (NS represents factors that have no significant impact on the test result).

The droplet deposition uniformity was an important indicator to evaluate the aerial spraying quality. The average *CV* of the droplet deposition was 27.35% for the nine treatments, the maximum one was 34.4% (T6), and the minimum one was 23.3% (T1, T4). Figure 6 showed the droplet deposition uniformity of each treatment by *CVs*.

**Figure 6.** Droplet deposition uniformity of each treatment by coefficients of variation (*CVs*).

### *3.4. The DPR Analyses*

The results showed that the DPRs of the nine treatments had no obvious correlation with the changes of the FS or FH. The larger the ESW, the higher the spraying efficiency, so in this study, the DPRs under several treatments (T1, T2, T5) with larger ESW were calculated according to Equation (3), which were 60.1%, 54.6%, and 52.7%, respectively.

### *3.5. The Control Effect Analysis*

The aerial spraying efficiency was given priority with T5 operation parameters to control the aphids, the T1 operation parameters were selected to control the powdery mildew and head blight with larger ESW and good DPR. The wheat plants infected aphids, powdery mildew and head blight were shown in Figure 7.

**Figure 7.** Wheat aphids, powdery mildew, and head blight occurrence in the test field.

The control effect of aphids is shown in Figure 8. The quantity of aphids per hundred plants of wheat declined to 172 on the first day, 128 on the third day, and 97 on the seventh day in the treated area after application; the decline rate was obvious, while the quantity rose to 645, 678, and 803 from 587 on the corresponding dates in the CK area. The aphid control effect was 78.71% on the first day, 84.88% on the third day, and 90.41% on the seventh day, respectively.

**Figure 8.** Wheat aphids control effect.

The control effects of powdery mildew and head blight are shown in Table 8. For the powdery mildew, the control effect was 77.17 ± 1.15% on the seventh day and 82.83 ± 2.98% on the fourteenth day after application, and for the head blight, the control effect was 88.32 ± 1.50% on the twentieth day after application.

**Table 8.** Control effects of powdery mildew and head blight.


### **4. Discussion**

As a new type of crop protection machinery, some details of the CPUAS application need to be clarified. The ESW, DDU, and DPR are the most important indicators for evaluating qualities of the spraying by CPUAS, and the operation parameters FH and FS, which can be controlled manually, affected ESW, DDU, and DPR. In this article, the results showed that the ESW of CE20 was not consistent and changed as the FS and FH changed, which were consistent with the existing findings [16,17]. The FS had a highly significant impact (*p* = 0.0033 < 0.01) on the ESW, while the FH had no significant impact (*p* = 0.136 > 0.05) on it, and the ESW value was negatively correlated as the FS varies, which is consistent with the conclusions of Zhang et al. [17]. Through comparison from the treatments, it could be seen that the spraying DDU of CE20 was very good with *CVs* among 23.3% to 34.4%. The two-way ANOVA results showed that the FH (*p* = 0.019 < 0.05) and the interaction between FS and FH (*p* = 0.032 < 0.05) both had significant impacts on the DDU, and the DDU could be improved by appropriately reducing FH and FS. This conclusion was slightly different with some existing conclusions. According to Qiu et al. [16], both the FS and FH had highly significant impacts on the DDU, and the interaction between FS and FH had significant impacts on the DDU. The reason may be the different type of CPUAS and the spraying volume, which needed further research. For the DPR, although results showed that it had no obvious correlation with the changes of the FS or FH, the slower the FS, the lower the FH, and the larger the spraying volume, the more droplets deposited in

the lower layer of the crop canopy according to Chen et al. [21] and Wang et al. [22], so a larger spraying volume was suggested if permitted. Based on the test results, optimized parameters of T5 and T1 were selected for aerial spraying operations to control wheat aphids, powdery mildew, and head blight. The control effects could satisfy the actual application. The existing research [23] showed that better control of wheat diseases and insect pests was achieved when using a coarse droplet size and higher spray volume. The control effect of powdery mildew was lower than that of head blight, although the height and canopy density of the wheat during the Heading stage were both lower than those during the Blooming stage (see Table 1); the reason may be related to disease characteristics. The powdery mildew mainly occurs in the middle and lower layers of the wheat plants, while the head blight occurs mainly in the upper layers (ears), so the pesticide droplets would be more likely to contact the leaves infected with head blight.

### **5. Conclusions**

The research results in this study show that the ESW, DDU, DPR, and even the spraying efficiency are closely related to the operation parameters. The above results reflected that with the aerial spraying parameters optimized, the obtained control effects of aphids, powdery mildew, and head blight could meet the actual requirements in general. Therefore, it is considered that the combined operation parameters used in actual application is inappropriate. It should be combined with the agronomic requirements to select the appropriate parameters based on the crop types, the growth period, the pests and diseases characteristics, and even the environmental conditions to achieve good prevention and control effects.

For wheat, stripe rust, powdery mildew, and head blight are the major diseases that are harmful to the wheat yield [3–5,39,40], and different requirements are required in chemical crop protection applications, respectively. The stripe rust would occur from the wheat Tillering stage to the Filling stage [40], the powdery mildew mainly occurs between the Heading stage and the Milky stage of wheat [40], and the head blight mainly occurs from the Heading stage to the Filling stage [41]. For the disease occurrence parts, the stripe rust occurs in the middle and lower parts of the wheat, the powdery mildew occurs and develops from the bottom to the top layer, and the head blight is concentrated on the upper layer (ears) of wheat. According to the results of this study, when controlling the head blight, priority should be given to increase the ESW, and the FS is appropriately reduced, taking into account the aerial spraying efficiency. When controlling the powdery mildew, the penetration of droplets is an effective consideration, and the FH should be reduced possibly. When controlling the stripe rust, the ESW and the penetration should be both considered for the parameter optimization combined with the growth period of wheat.

In this study, the CPUAS of CE20 was tested, and the conclusions above were only applicable to it. Different type CPUASs may have different spraying and droplet deposition characteristics, such as single-rotor and multi-rotor CPUAS; thus, systematic experiments should be carried out to determine the optimal parameter combinations before application. Future research should be focused on the relationship among the parameters [8,16,17], the canopy structure [42], and the wind field [43,44] on the droplet deposition effect, and establish the correlation of them to achieve the best management practice and control effect.

**Author Contributions:** S.Z., B.Q., and X.X. conceived the idea of the experiments. S.Z., T.S., W.G., F.Z., and X.S. performed the experiments and analyzed the data. S.Z., B.Q., and X.X. wrote and revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China (Grant No. 2017YFD0701000), the National Natural Science Foundation of China (Grant No.31701327), the earmarked fund for China Agriculture Research System (CARS-12), the Agricultural Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences, Crop Protection Machinery Team (Grant No. CAAS-ASTIP-CPMT), the Science and Technology Development Plan of Suzhou, Jiangsu Province (Grant No. SNG2020042), the Jiangsu Science and Technology Development Plan (BE2019305).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data used to support the findings of this study are available from the first author or the corresponding author upon request.

**Acknowledgments:** The authors are very grateful to Sihong agricultural demonstration base for proving the test field and the pesticides, and to the help of Sijun Yang and Jilin Du during the tests.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**


### *Review* **Cryobiotechnology of Plants: A Hot Topic Not Only for Gene Banks**

### **Petra Jiroutová \* and Jiˇrí Sedlák**

Research and Breeding Institute of Pomology Holovousy Ltd., Holovousy 129, 508 01 Hoˇrice, Czech Republic; jiri.sedlak@vsuo.cz

**\*** Correspondence: petra.jiroutova@vsuo.cz

Received: 8 June 2020; Accepted: 6 July 2020; Published: 7 July 2020

**Abstract:** Agriculture has always been an important part of human evolution. Traditionally, farming is changing and developing with regard to challenges it faces. The major challenges of modern agriculture are food and nutrition safety for the growing world population. Promoting species and genetic diversity in agriculture appears to be an important approach to dealing with those challenges. Gene banks all around the world play a crucial role in preserving plant genetic resources for future crop improvements. The plant germplasm can be preserved in different ways, depending on the species or form of stored plant tissue. This review focuses on a special preservation method—cryopreservation. Cryopreservation is an effective technique for storing living systems at ultra-low temperatures, usually in liquid nitrogen or its vapor phase. This conservation method is crucial for plants that do not produce seeds or that produce non germinating seeds, as well as for plants that propagate vegetatively. Moreover, based on the cryopreservation method, a novel plant biotechnology tool for pathogen eradication called cryotherapy has been developed. The use of liquid nitrogen eliminates plant pathogens such as viruses, phytoplasmas, and bacteria. Our article reviews recent advances in cryo-biotechnologies such as cryopreservation and cryotherapy, with special focus on studies concerning fruit plants.

**Keywords:** cryopreservation; cryotherapy; food safety; liquid nitrogen; biotechnology; fruit plants

### **1. Introduction**

Plant genetic resources are highly important for the maintenance of agro-biodiversity and for food safety. These genetic resources, as donors of valuable traits, can be used to breed new, more productive crops with better resistance to biotic or abiotic stresses [1]. Seed storage is one of the most convenient methods for long-term conservation of plant genetic resources. However, a large number of plant species are not suitable for seed banking because they are highly heterozygous or have recalcitrant seeds that cannot be desiccated. These species are usually conserved in field collections. This type of collection is beneficial because it provides immediate access to plant material during all phenophases. However, plants in field collections are exposed to many environmental threats such as pests, diseases, and adverse weather conditions. Maintenance of this type of collection is also labor-intensive and expensive [2].

In vitro gene banks, where plants are vegetatively propagated and grown on a medium under sterile conditions, are an alternative to seed or field banking. In vitro-grown plants are stored in growing chambers that significantly save storage space and protect plants against harmful environmental factors. However, because the plantlets require periodical subcultivation on fresh medium, this method is not ideal for long-term storage [3].

Cryopreservation is the most valuable method for long-term conservation of plant germplasm. It is based on the storage of biological material at ultra-low temperatures in liquid nitrogen (−196 ◦C)

or in its vapor phase (−150 ◦C). This temperature suspends cell division and metabolic and biochemical activities, thus preventing genetic alteration during long-term storage [4].

More than 60 years ago, the first work on plant cryopreservation was published. In 1956, Akira Sakai reported the successful survival of cold hardened and prefrozen mulberry twigs after exposure to liquid nitrogen [5]. The next challenging step in the field of cryopreservation was to freeze fully hydrated tissues such as callus and suspension cells, where there is a high risk of the formation of lethal intracellular ice-crystals. For this purpose, slow freezing protocols were developed. However, this method was not sufficient for cryopreservation of organized tissues (e.g., meristems), which led to the development of fast freezing protocols (encapsulation-dehydration, droplet vitrification, etc.) [6]. To date, several methods and techniques of cryopreservation have been reported. Nevertheless, the nub is always the same. Plant tissue is first physically or osmotically dehydrated to remove all freezable water, and to avoid water crystallization and lethal injuries during the following freezing in liquid nitrogen [7].

Ultra-low temperatures can also be successfully used for pathogen annihilation. This novel progressive technique is called cryotherapy and is based on protocols that have been originally established for cryopreservation. In the process of cryotherapy, infected plant cells are eliminated using the fatal efficacy of the liquid nitrogen (the ultra-low temperature) in combination with subsequent warming [8]. One of the major advantages of using ultra-low temperature treatment for pathogen eradication is that there is no need for special equipment other than that which is used in a basic plant tissue culture laboratory. This means that cryotherapy can be efficiently incorporated into the cryopreservation methods already used in gene banks [9].

In this review article, we present updated and comprehensive information concerning the development and progress of plant cryopreservation and cryotherapy, with a focus on horticultural crops and a special focus on apples, which belong to one of the most extensively studied species in this field.

### **2. Cryopreservation of Plants**

Originally, plant cryopreservation was based on studies of the basic biology of freezing [10]. Gradual improvements in the technology and intensive research work over the past decades have resulted in great progress in the field of ultra-low temperature preservation of plant germplasm [11]. The development and improvement of cryopreservation techniques and their application to new plant species remains the center of attention of many cryogenic labs and gene banks around the world.

### *2.1. Plant Cryopreservation Methods*

Nowadays, several methods of cryopreservation exist, including both classical and new techniques (Figure 1). Advantages and disadvantages of each method, as well as other factors such as available facilities, plant species, and the type of stored germplasm, have to be considered during the process of method selection (Table 1). Often more than a single cryoprotocol is suitable for successful plant cryopreservation, considering some modifications of established methods [12]. The first step in cryopreservation is removing freezable water from tissues by dehydration. A water content of less than 0.25 g H2O g/dm (dm; dry mass) is often termed 'unfreezable' water, and plant cells containing 0.25–0.4 g H2O g/dm usually survive the liquid nitrogen exposure [13]. Proper dehydration can be achieved osmotically by treatment with highly concentrated solutions, in which case the driving force for dehydration is the concentration gradient between the solution and the intracellular liquid. The second widely used method of dehydration is air-drying, where the water is removed by the air flow [14]. In any case, the process of dehydration is essential for successful cryopreservation to avoid intracellular freezing and irreversible injury of cells caused by the formation of ice crystals [1].

**Figure 1.** Schematic diagram of major methods and steps of cryopreservation of shoot tips.

### 2.1.1. Classical Cryopreservation Methods

Classical cryopreservation methods were developed more than 40 years ago, and two main techniques are traditionally used: (i) slow freezing, and (ii) simple one-step freezing [7]. Slow freezing (also known as the two-step freezing method) includes pretreatment of samples with cryoprotectants such as dimethylsulfoxide (DMSO), glycerol, ethylene glycol, and sucrose. The samples are then slowly cooled at a controlled rate of 0.3–0.5 ◦C/min to −40 ◦C, and then rapidly immersed in liquid nitrogen. This method requires a programmable freezer [15]. On the other hand, no special equipment is required for simple one-step freezing. Samples pretreated with cryoprotectants are simply frozen at −30 ◦C for dehydration, and then immersed directly in liquid nitrogen [16]. Although these classical methods have been successfully applied to a range of plant materials, new and gentler cryogenic methods such as vitrification, encapsulation, and cryo-plates have been developed recently.

### 2.1.2. Vitrification

This approach is one of the most widely applied plant cryopreservation methods because it is relatively easy to carry out, no special equipment is needed, and it usually displays a high percentage of recovery. Vitrification is based on the formation of an amorphous glassy structure from intracellular solutes [1]. The plant material is treated with a highly concentrated vitrification solution that removes most or all freezable water from cells. The dehydrated material is then ultra-rapidly frozen by immersion in liquid nitrogen. The combination of dehydration and rapid freezing of cells causes residual water to solidify without crystallization, which could injure the living cells [17].

Three main glycerol-based vitrification solutions (PVS1, PVS2, and PVS3) with different compositions have been reported. PVS1 consists of 19% (w/v) glycerol, 13% (w/v) ethylene glycol, 13% (w/v) propylene glycol, and 6% (w/v) DMSO dissolved in 0.5 M sorbitol. PVS2 consists of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) DMSO dissolved in 0.4 M sucrose. PVS3 consists of 50% (w/v) glycerol and 50% (w/v) sucrose dissolved in water [18]. In addition, two glycerol-free vitrification solutions were published in the past—the Towill cocktail (containing 35% ethylene glycol, 1 M DMSO, and 10% polyethylene glycol) and the Watanabe cocktail (containing 44.5% DMSO and 18.7% sorbitol) [19,20].

Among them, PVS2 is the most widespread, because of its less toxic effect on plant cells. Nevertheless, it cannot be used without any osmoprotection, because direct dehydration by PVS2

causes damages to the cells and tissues of many plants. For successful cryopreservation, it is necessary to implement osmoprotection pretreatment with a loading solution (LS) containing 2 M glycerol and 0.4 M sucrose that induces osmo-tolerance and enables plants to achieve higher rates of recovery after cryopreservation [21,22].

Two main vitrification-based methods, namely encapsulation-vitrification and droplet vitrification, have been developed via the modification and optimization of vitrification techniques. The encapsulation-vitrification method was first reported by Matsumoto et al. (1995) [23] and combines encapsulation of plant germplasm within alginate beads and dehydration using a highly concentrated vitrification solution. The major merits of this mixed technique are better protection of encapsulated plant samples during vitrification and reduction of time needed for dehydration, compared to that of the classical encapsulation-dehydration method [22]. Using the method called droplet vitrification, shoot tips pretreated with LS and subsequently treated with vitrification solution are inserted individually into droplets of PVS2 that are placed on a strip of aluminum foil. The whole aluminum foil is then directly immersed in liquid nitrogen. The main benefit of this technique is the achievement of very high cooling/warming rates [24,25].


**Table 1.** List of various cryopreservation methods.

### 2.1.3. Encapsulation-Dehydration

This method was first reported by Fabre and Dereuddre (1990) [26] and combines the encapsulation of plant samples with alginate beads and physical dehydration carried out with silica gel or in the air flow in a laminar flow cabinet [1]. Applying this method, plant material is precultured with 0.3–0.6 M sucrose medium for one to three days and then incubated in a liquid medium supplemented with sucrose and sodium alginate. Finally, this mixture is released drop by drop into liquid medium containing calcium chloride. Alginate beads with explants encapsulated inside them are formed during this process. The bead formation is followed by culturing in highly concentrated sucrose solution to achieve the osmoprotection, subsequent physical dehydration to a water content of 20%–30%, and direct immersion in liquid nitrogen (Figure 2) [26]. The encapsulation-dehydration method is relatively simple; however, it requires more time-consuming handling of encapsulated samples. The main advantage of this method is elimination of the need for other cryoprotectants such as DMSO and ethylene glycol that could be toxic for plants and could cause genetic changes after regrowth [22].

**Figure 2.** Schematic diagram of the encapsulation-dehydration cryopreservation method.

### 2.1.4. Cryo-Plate Methods

The newest cryogenic procedures use cryo-plates. Two main cryo-plate methods, known as the V and D cryo-plate methods, can be distinguished [22] based on the dehydration process. Using the former method, explants are dehydrated on cryo-plates using PVS2 vitrification [27], whereas the latter method uses air dehydration of explants [28]. In both methods, shoot tips are placed in small wells of an aluminum cryo-plate with alginate beads and dehydrated there with PVS2 solution or with air flow in a laminar flow cabinet, depending on the particular method. Afterwards, the cryo-plate is directly immersed into liquid nitrogen. The main advantage of cryo-plate techniques is their user-friendliness, mainly due to the easy handling of samples on the aluminum plates [27,28]. Successful cryopreservation using cryo-plates methods has been reported for many plant species including strawberry [29], sugarcane [30], date palm [31], mat rush [32], and potato [33].

### **3. Pathogen-Free Plant Material**

Phytoplasmas, bacteria, viruses, and other plant pathogens cause harmful plant diseases that negatively affect crop yield, crop industry, and food safety every year [8,34]. Vegetatively propagated plants tend to accumulate pathogens that are transmitted to new plants in infected cuttings, tubers, roots, and other vegetative propagules [35]. Pathogen-free plant material is essential not only for higher productivity of agricultural and horticultural corps, but is also pivotal for long-term preservation of plant germplasm [36].

### *3.1. Convential Methods for Pathogen Eradication*

Conventional methods for pathogen eradication are based on in vitro meristem culture, as well as heat treatment (thermotherapy) and chemical treatment (chemotherapy), both of which are followed by meristem culture [37–41]. Although these traditional methods have been widely used for acquiring virus-free plants, all of them have some drawbacks.

The meristem culture method is based on the assumption that the youngest meristematic cells are free of viruses and other plant pathogens. Therefore, the extension and regeneration of small shoot tips containing the meristem (0.2–0.4 mm) and two to four leaf primordia should lead to pathogen-free plants [36,39]. The major limitations of meristem culture techniques are difficulties in excising tiny meristems and a low regeneration rate of shoot tips.

The use of heat treatment is another well-known conventional method for pathogen eradication in plants. Generally, thermotherapy is based on keeping the target in vitro cultivated plants at a temperature of 35 ◦C–42 ◦C for four to six weeks. Both temperature range and duration of thermotherapy are virus-type- and plant-species-dependent [42,43]. The need for specific and expensive laboratory equipment such as a growth chamber with precise temperature control is the major limitation of thermotherapy [44]. Moreover, this method cannot be used for infected plants that are not resistant to higher temperatures and it does not work for all viruses [45].

Another method used for plant pathogen elimination is chemotherapy. In this technique, in vitro-grown plants are treated with antiviral chemicals like ribavirin, whose positive effect on virus eradication has been reported [46,47]. In this method, optimization of the dose and duration of the treatment is crucial because a high concentration of antiviral compounds in the culture medium can negatively affect the growth of in vitro plants through their phytotoxic activity. The sensitivity of in vitro plants to antivirotics is species- and genotype-specific [45,47]. Because of the abovementioned limitations linked to traditional methods for pathogen eradication, it would be beneficial to develop some more efficient and simpler methods for obtaining pathogen-free plants. In this respect, cryotherapy, i.e., treatment with ultra-low temperatures, could be a useful biotechnological tool with great potential.

### *3.2. Cryotherapy*

Cryotherapy of shoot tips as a method for pathogen elimination from infected plants was first reported in 1997 by Brison et al. [48], who successfully eradicated Plum pox virus (PPV) from in vitro-grown infected shoot tips of interspecific *Prunus* rootstock [48]. The crux of this technique consists in the treatment of infected materials in liquid nitrogen for a short period of time [49,50]. Cryotherapy relies on plant cryopreservation protocols that are available for a wide range of vegetatively propagated and economically important plant species. Thus, cryotherapy has been successfully applied to many plant species such as potato (*Solanum tuberosum* L.) [51], sweet potato (*Ipomea batatas* L.) [52], banana (*Musa* spp.) [53], raspberry (*Rubus idaeus* L.) [54], grapevine (*Vitis vinifera* L.) [55,56], and apple (*Malus spp.*) [57–59].

To date, large numbers of pathogens (mostly viruses) have been eradicated via cryotherapy. These include, for example, two common viruses infecting sweet potatoes—sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCVS)—that interact synergistically and cause the sweet potato virus disease (SPVD). Both these viruses can be eliminated with 100% efficiency using cryotherapy [60,61]. Cryotherapy was also successfully applied to the eradication of two viruses infecting potato plants: potato leafroll virus (PLRV) and potato virus Y (PVY), which were among the most important viruses limiting sustainable and profitable potato production [51]. Cucumber mosaic virus (CMV) and banana streak virus (BSV) viruses, which infect bananas and cause diseases that are linked to a reduction in fruit yield or obstruction of breeding and germplasm dissemination, were effectively eradicated by cryotherapy [53]. Successful applications of cryotherapy was also reported for viruses infecting grapes, such as grape virus A (GVA) and grapevine leafroll-associated viruses (GLRaV), that cause economic losses in viticulture [55,56,62]. In addition, viruses infecting less common crops such as garlic and artichoke were recently eradicated using ultra-low temperature treatment [63,64]. Other pathogens such as phytoplasmas and bacteria can also be eradicated from plants via cryotherapy. For example, the sweet potato little leaf phytoplasma (SPLL), which causes heavy yield losses in infected plants, was efficiently eliminated from all sweet potato shoot tips via treatment with liquid nitrogen [60]. Another, more recent example of eradication of phytoplasma by using cryotherapy was reported in 2015. Jujube witches' broom phytoplasma (*Candidatus Phytoplasma ziziphi*) was fully eliminated from infected Chinese jujube (*Ziziphus jujuba*) plants [65]. So far, only one successful elimination of bacteria—a Gram-negative bacteria attacking *Citrus*, *Fortunella*, and *Poncirus*

species and causing citrus Huanglongbing disease (HLB) disease, also called "citrus greening"—from infected plants by cryotherapy was reported [66]. Ding et al. demonstrated that cryotherapy is a powerful tool for elimination of this bacteria from infected sweet orange (*Citrus sinensis* L.) and other citrus species such as mandarin, pomelo, and Beijing lemon.

### 3.2.1. Mechanism

Cryotherapy is mostly applied to shoot tips, because they contain a unique meristematic zone consisting of small cells with small vacuoles and a higher nucleo-cytoplasmic ratio [67]. Those cells are more resistant to dehydration, which prevents the formation of ice crystals in cells during freezing [68]. Simply, cryo-treatment with liquid nitrogen destroys the differentiated cells, while meristematic cells survive and are able to self-renew, divide, differentiate, and regenerate to new virus-free plants (Figure 3). Because of this phenomena, cryotherapy is most effective for the elimination of pathogens infecting differentiated cells such as banana streak virus (BSV), cucumber mosaic virus (CMV), grape virus A (GVA), potato leafroll virus (PLRV), and potato virus Y (PVY) [51,53,62]. On the other hand, the eradication of pathogens that are able to infect meristematic cells, such as raspberry bushy dwarf virus (RBDV), pelargonium flower break virus (PFBV), and pelargonium line pattern virus (PLPV), is significantly more complicated [67,69]. It has been reported that a combination of cryotherapy and thermotherapy led to virus-free plants. Thermotherapy first inhibits movement of the virus toward the meristematic cells of the shoot tips and at the same time causes subcellular alterations, for example, enlargement of vacuoles, which results in much fewer surviving cells after subsequent cryotherapy. The combination of these two techniques enables an enhancement in the eradication of viruses localized in the meristem [67].

**Figure 3.** Schematic diagram of shoot tip cryotherapy. HC = healthy cells, IC = infected cells, SC = survived cells, KC = killed cells.

### 3.2.2. Merits and Demerits

Compared to other methods for pathogen eradication in plants, cryotherapy of shoot tips has the following advantages: (i) easier handling with meristems due to the lack of correlation between the size of the shoot tip and pathogen eradication rate [49,70]; (ii) high efficiency of pathogen eradication [49,71]; (iii) no need for special and expensive equipment, and (iv) it is easy to handle a large number of samples [36,49]. The major limitation of the wider application of the cryotherapy to produce pathogen-free plant material is the genotype- or often also cultivar-dependent response of plants to cryo-treatment. This means that each cryoprotocol needs to be developed and optimized for every single plant species or cultivar [68,72]. Although the risk of somaclonal variation during short-term cryotherapy is minimal, it is important to verify the genetic stability of regenerated plants [72].

### **4. Cryobiotechnology of** *Malus* **(Apple)**

Many protocols used for cryopreservation of fruit plants have been reported. However, long-term germplasm storage of economically important horticultural woody plants in cryopreserved collections remains well-established, mainly for apple and pear plants [73,74]. Furthermore, among all plant species, *Malus* (apple) is one of the most extensively studied with respect to cryopreservation of the plant germplasm over the last decades. Interestingly, many protocols applied to other species originated from cryopreservation procedures that were first demonstrated with apples [73].

Traditionally, field collections and in vitro culture are used for preservation of *Malus* germplasm. Seed banking can be applicable only for the preservation of wild *Malus* species germplasm, since apple is genetically highly heterozygous [75,76]. Cryopreservation of apple seeds is possible after their drying to achieve 6%–19% moisture content [77]. It is valuable to cryopreserve pollen for immediate availability for breeding programs. Successful cryopreservation of *Malus* pollen has been reported many times. According to the published data, this pollen stored in liquid nitrogen can retain viability for at least 15 years [78–80].

The cryopreservation of shoot tips of in vitro-grown plants and cryopreservation of dormant buds are the most commonly used techniques for ultra-low temperature long-term storage of vegetatively propagated species (cultivars) [81]. Up to date, all known techniques for cryopreservation of shoot tips were used for *Malus*. The very first experiments concerning *Malus* in vitro shoot tip cryopreservation used classical two-step freezing [82,83]. The first experiments failed to enable regrowth of frozen shoot tips, suggesting that excessive cryo-injury occurred in the treated cells during the cryoprocedure. Wu et al. [84] overcame this problem in 1999 via optimization of the cryoprotocol by inserting the pretreatment of shoot tips with a cryoprotectant mixture and achieved a 66% shoot regrowth. Other early experiments were accomplished by using the PVS2 vitrification technique, with an average regrowth level of about 66% in different examined apple cultivars, rootstocks, and wild apple species [85]. Studies on the cryopreservation of in vitro shoot tips by encapsulation, linked with either dehydration or vitrification, have been reported. The first encapsulation-dehydration protocol was described in 1992 by Niino and Sakai [86] and subsequently more cryoprotocols using this or encapsulation-vitrification method were developed or optimized for a wide spectrum of *Malus* cultivars [84,87–89]. Finally, in 2010 Halmagyi et al. tested [90] the efficiency of two droplet cryopreservation techniques, droplet-vitrification and droplet-freezing, that included in vitro-grown shoot tips as materials for long-term storage. This study showed that the droplet-vitrification method allowed a high regrowth level of cryopreserved shoot tips, and consequent study [91] pointed out the effects of preculture conditions on the level of regenerated shoot tips after cryopreservation using this method.

Sakai and Nishiyama (1978) [92] were the first to mention the cryopreservation of in vivo apple dormant buds. In general, dormant bud cryopreservation procedures consist of desiccation and slow cooling to −35 ◦C prior to liquid nitrogen exposure [73]. Based on data from the National Laboratory for Genetic Resources Preservation (NLGRP), the lack of a need for aseptic cultures, high processing throughput, and approximately ten-times-lower cost of cryoprocessing compared to shoot tip cryopreservation are the major advantages of this method [74]. Most protocols used for ultra-low temperature storage of dormant apple buds are based on the original outlines by Forsline [93,94], involving: cutting of winter hardened twigs in segments containing the dormant bud, desiccation at a temperature from −4 ◦C to −5 ◦C of the segments until moisture content is reduced to about 30%, cooling in a programmable slow cooler to a final temperature of −30 ◦C, and placing the cooled segments in liquid nitrogen vapor or liquid nitrogen for long-term storage. For plant regeneration, recovered scions with dormant buds are grafted directly on pot-grown rootstocks, or it is also possible to excise shoot tips from cryopreserved buds and place them on culture medium [95]. Successful regeneration of cryopreserved buds is tightly linked with the cold hardening of these buds before cryopreservation, rather than with the phase of endodormancy [96].

Several papers in recent years focused on pathogen eradication in apples by using cryotherapy. For example, the vitrification cryotherapy technique was reported by Romadanova et al. (2016) [97]. They infected apple cultivars and rootstocks and treated them with ultra-low temperatures to eliminate four different viruses (apple chlorotic leaf spot closterovirus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple mosaic virus (ApMV)). Their therapy resulted in the production of virus-free plants for seven out of nine tested cultivars. In another study, the encapsulation-dehydration method was applied to in vitro-grown apple rootstocks M9 and M26 to eliminate ASPV and ASGV viruses. Cryotherapy was successful for ASPV but failed for ASGV. A probable explanation of this result was the different distribution of viruses in shoot tips. Although ASPV was not detected in the upper part of the apical dome and leaf primordia, ASGV was [89]. Results obtained two years later also showed that elimination of ASGV by cryotherapy had some limitations due to its localization in the shoot tip [58]. Finally, a recent study from the same research group examined the effect of another cryotherapy technique, i.e., droplet-vitrification, on the eradication of three latent apple viruses (ACLSV, ASGV, ASPV) from an infected 'Monalisa' apple cultivar. Cryotherapy was successful for the elimination of ASPV and ACLSV with 100% and 95% efficiencies, respectively. However, only 35% of regenerated plants were free of ASGV [59].

### **5. Conclusions**

Cryopreservation is a progressive method for long-term storage of plant germplasm that uses the ultra-low temperature of liquid nitrogen to minimalize the metabolism of living cells. This technique has great application potential in biotechnology, agriculture, horticulture, and breeding programs. Several methods and protocols for cryopreservation of plant germplasm were reviewed in this manuscript, and each of them has its merits and demerits, which should be considered before cryopreservation (Table 1). Cryopreservation can serve as an alternative storage solution to the traditional field collections. It also has possible applications in the conservation of endangered plant species germplasm. Ultra-low temperatures can be used not only for the storage of germplasm, but also for the eradication of plant pathogens, for which the term "cryotherapy" was coined. Cryotherapy of shoot tips is a promising method for plant pathogen eradication that can be easily used for different plant species and cultivars with available cryopreservation protocols. It can be carried out in a basically-equipped tissue culture laboratory, shows promising results in production of pathogen-free regenerants, and minimizes the risk of genetic changes during treatment compared to the classical methods. To date, more than 10,000 accessions of in vitro propagated crop plants are safely cryopreserved for the long term, and over 80% of these belong to five main crops (potato, cassava, bananas, mulberry, and garlic). In this review, we show that much of the work has already been done in the field of plant cryobiotechnology. However, some important challenges still remain and limit a more scaled applications of cryopreservation. For example, post-thaw regeneration of some important crop species (cassava, sweet potato) is still extremely low. Some plant species survive cryopreservation, but they are not able to root and stop the growth and development [6]. Altogether, for the future, the optimization and modification of the existing protocols will be required for more plant species and cultivars.

**Author Contributions:** Conceptualization, P.J. and J.S.; investigation, P.J.; writing—original draft preparation, P.J.; writing—review and editing, P.J. and J.S.; project administration, P.J.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Education, Youth and Sport of the Czech Republic; grant number LO1608 "Research Pomological Centre".

**Conflicts of Interest:** The authors declare no conflict of interest.

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