Establishment of Tissue Culture System of Actinidia deliciosa

In order to breed virus-free plantlets of the kiwifruit cultivar “Guichang,” which belongs to Actinidia deliciosa, in this study, stem segments with buds were used as explants, the establishment of a tissue culture rapid propagation systemwas carried out, and then the virus status of tissue culture plantlets was detected via the real-time reverse transcription-polymerase chain reaction (RT-qPCR) method. 'e tissue culture rapid propagation system proved that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segment of “Guichang” kiwifruit was sterilized with 70% alcohol for 30–60 s and 15% NaClO for 15min, respectively. Meanwhile, we screened the hormone concentration to get better results, and the appropriate medium for adventitious bud induction was MS + 6-BA (1.0mg/L) + IBA (0.2mg/L); for proliferation, it was MS+ 6-BA (1.0mg/L) + IBA (0.1mg/L); and for rooting, it was 1/2 MS+ IBA (0.3mg/L), and the efficiency of induction, proliferation, and rooting could reach 74.07%, 79.63%, and 85.18%, respectively. In addition, the RT-qPCR results demonstrated that the infection rate of 9 viruses: apple stem grooving virus (ASGV), cucumber mosaic virus (CMV), Actinidia virus X (AVX), cucumber necrosis virus (CNV), ribgrass mosaic virus (RMV), citrus leaf blotch virus (CLBV), Actinidia virus B (AcVB), Pelargonium zonate spot virus (PZSV), and cherry leaf roll virus (CLRV) in the “Guichang” kiwifruit tissue culture plantlets was 0.'is study could lay a foundation for the production of “Guichang” kiwifruit tissue culture seedlings, and the medium formula provided in this study was useful for the industrial rapid propagation of “Guichang” plantlets.


Introduction
Kiwifruit (Actinidia spp.), an important and economically substantial fruit in China, has long been known as "the king of fruits" because of its high vitamin C content and balanced nutritional composition of minerals and other health-promoting metabolites [1,2]. Kiwifruit cultivar "Guichang" (Actinidia deliciosa var. deliciosa), one excellent cultivar with the characteristics of excellent quality, beautiful appearance, strong resistance, and high yields from wild resources, developed by the Institute of Fruit Science, Guizhou Academy of Agricultural Sciences in 1979, has become the main cultivar in Guizhou province [3]. By 2018, the cultivation area of "Guichang" kiwifruit in Guizhou was nearly 16,000 hectares.
As most species of Actinidia are dioecious and have a long juvenile period, it is difficult to identify the gender of young plants [4,5]. Due to the limited number of branches, the traditional cutting and grafting methods make it difficult to cultivate large numbers of plantlets in a short time. erefore, the tissue culture had gradually become the main method to produce numerous plantlets rapidly, which had the advantages of stable inheritance, maintaining good traits, no time and space restrictions, and a high reproduction coefficient. Since the 1970s, tissue culture rapid propagation has become an important breeding method. Recently, more and more research studies on the establishment of kiwifruit rapid propagation systems have been performed [6][7][8][9]. However, in the process of in vitro rapid propagation of kiwifruit, serious browning and diseases of explants in primary culture have become a major obstacle [10,11]. In recent years, some literature has reported that many viruses, including apple stem grooving virus (ASGV), cucumber mosaic virus (CMV), Actinidia virus X (AVX), cucumber necrosis virus (CNV), ribgrass mosaic virus (RMV), Actinidia virus B (AcVB), Actinidia virus A (AcVA), Actinidia virus 1 (AcV-1), alfalfa mosaic virus (AMV), cherry leaf roll virus (CLRV), tomato necrotic spot associated virus (TNSaV), tomato zonate spot virus (TZSV), Pelargonium zonate spot virus (PZSV), citrus leaf blotch virus (CLBV), Actinidia chlorotic ringspot-associated virus (AcCRaV), turnip vein clearing virus (TVCV), and Actinidia seed-borne latent virus (ASbLV), can infect kiwifruit plants through pollen, grafting, or artificial inoculation [12][13][14][15][16][17][18][19]. Once infected, viruses cause a detrimental effect on the growth and development of kiwifruit plants, and the fruits will be deformed and the yields will be reduced, with huge economic loss. However, the traditional cutting and grafting methods had a good chance of spreading viruses to the other branches and thus expanding the spread of viral diseases. erefore, it is urgent to develop the rapid propagation technology of "Guichang" kiwifruit.
In order to breed virus-free plantlets of "Guichang" kiwifruit, in this study, the establishment of a tissue culture rapid propagation system was carried out, and then detected the virus status via the real-time reverse transcription-polymerase chain reaction (RT-qPCR) method. To our knowledge, this is the first report on the rapid production of "Guichang" kiwifruit tissue culture plantlets.
is study provides scientific guidance for large-scale factory production and boosts the healthy development of the kiwifruit industry in China.

Experimental Materials.
e materials were taken from the kiwifruit demonstration area (north latitude: 26.816339, east longitude: 106.492149) of Gubao township, Xiuwen county, Guizhou province, China, in June 2015. Plants with robust growth and good comprehensive performance were selected as the sampled parent plants. New shoots were removed from the tops with the base semilignified, and leaf blades that had not fully developed were used as explants for in vitro culture. e young leaves of the parent plant were hydropically cultured to accelerate budding, and the young leaves cultured by tissue culture were taken as test materials for virus detection.

Sterilization of the Explants.
For sterilization of the explants, the petioles, which were removed from the leaves, were cut into 5-6 cm long pieces, rinsed with tap water several times, soaked in detergent water for 30 min, and rinsed with tap water for 1-2 h. en, the explants were soaked with 70% alcohol for 10, 30, and 60 s, respectively, and soaked with 15% sodium hypochlorite (NaClO) for 10, 15, and 20 min, respectively. After that, the explants were washed with sterile water 3-4 times, sucked dry with sterile paper, cut into 1.5-2.0 cm segments with a single bud, and Tissue culture plantlets, with a height of more than 4 cm, 3-5 leaves, and more than 3 roots, were selected and placed into the greenhouse (23-25°C and 90% relative humidity (RH)) to harden the plantlets for 7 days, then the plantlets were transplanted into the paper cups with the matrix containing perlite, vermiculite, and peat (1 : 1:2, v/v) and then transferred the paper cups into the greenhouse with natural lighting, a temperature of 23-25°C, and RH to reduce the gradient from 90% to indoor humidity. Meanwhile, the plantlets were regularly irrigated with the 1/8 MS nutrient solution and a pesticide, such as carbendazim, that prevents young plants from rotting. e plantlets were grown for about 30 days, and then the survival rate was calculated using the following equation: survival rate (%) � (number of the transplanting survival/number of transplanting explants) × 100.

RNA Extraction and RT-qPCR Detection.
A total of 9 kinds of viruses, including ASGV, CMV, AVX, CNV, RMV, CLBV, AcVB, PZSV, and CLRV, were detected via the RT-qPCR method [18,19]. e RNA of the "Guichang" kiwifruit leaves was extracted using the total RNA extraction kit (Qiagen, Hilden, Germany). e purity (OD 260 /OD 280 ) and concentration of the extracted RNA were detected by an ultraviolet spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). RNA was reverse-transcribed using a cDNA kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. e primers used for RT-qPCR amplification are listed in Table 1. e RT-qPCR amplification was carried out according to the method reported by Huang et al. [2]. After that, the electrophoresis of the RT-qPCR products was performed with a 1.2% agarose gel (Bio Tech Corporation, Beijing, China).  Table 2 shows that, with the prolonged sterilization time of 70% alcohol and 15% NaClO, the contamination rate of explants gradually decreased while the browning rate gradually increased, and the survival rate increased first and then decreased. Meanwhile, Table 2 shows that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segments of "Guichang" kiwifruit were sterilized with 70% alcohol for 30-60 s and 15% NaClO for 15 min, respectively. Table 3, with the increase of 6-BA concentration, the germination rate of axillary buds first increased and then decreased; especially, using MS + 6-BA (1.0 mg/L) + IBA (0.2 mg/L) as the induction medium, the inducement rate reached 74.07% and the plantlets were robust with almost no callus at the base. Table 4 shows that all the tested concentrations of 6-BA could enhance the proliferation rate; especially, using MS + 6-BA (1.0 mg/L) + IBA (0.1 mg/L) as the proliferation medium, the proliferation rate and proliferation coefficient reached the highest values of 79.63% and 5.06, respectively. Meanwhile, the plants were robust with green leaves and longer internodes. Table 5 shows that all the tested concentrations of IBA could enhance the rooting rate, especially taking 1/2 MS + IBA (0.3 mg/L) as the optimum rooting medium, the rooting rate and average rooting number were 85.18% and 4.90, respectively, and the rooting number was 2-6 with more and longer fibrous roots and basically no callus. After that, the tissue culture plantlets with a height of more than 4 cm, 3-5 leaves, and more than 3 roots were selected and placed into the greenhouse. e survival rate exceeded 90%.

Virus Detection.
A total of 9 kinds of viruses in tissue culture plantlets, including ASGV, CMV, AVX, CNV, RMV, CLBV, AcVB, PZSV, and CLRV, were detected via the RT-qPCR method, and the results (Figure 1) demonstrated that the infection rate of 9 kinds of viruses in tissue culture plantlets was 0. erefore, the in vitro tissue culture rapid propagation system established in this study could obtain the virus-free plantlets of "Guichang" kiwifruit.

Discussion
China is the major source of wild kiwifruit and the world's largest producer and planting area [20]. It has an important significance to study the rapid propagation technology of kiwifruit, which is helpful to promote its development. In the sterilization process of in vitro rapid propagation, many scholars used alcohol and mercury chloride as the disinfecting agents, while only a few scholars used alcohol and NaClO [20,21]. In this study, the single bud stem segment of the "Guichang" kiwifruit was disinfected with 70% alcohol and 15% NaClO, and the results showed that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segment of the Guichang kiwifruit was sterilized with 70% alcohol for 30-60 s and 15% NaClO for 15 min, respectively.
ere are many reports on the combination of plant growth regulators in different stages of tissue culture of kiwifruit, and the selection of appropriate auxin types and concentrations is the key role of plant tissue culture. A previous study found that there are some differences in the morphology of tissue culture plantlets induced by different auxin combinations [21]. Yu et al. [22] and Long et al. [23] successfully established the kiwifruit stem culture using MS + 6-BA (1.0 mg/L) + naphthylacetic acid (NAA, 0.1 mg/ L) medium. It was also found that different concentrations of plant growth regulators could affect the growth and browning of kiwifruit, and the auxin IBA could delay the synthesis of polyphenols and reduce browning [24][25][26]. Based on the results of previous literature, this experiment studied the effects of IBA at different concentrations on the induction and proliferation of "Guichang" kiwifruit stem segments. As for the effective buds and proliferation coefficients induced in the first generation, when using MS + 6-BA (1.0 mg/L) + IBA (0.2 mg/L) as the optimum induction medium, the inducement rate was 74.07% and the plantlets were almost robust with no callus at the base (Figure 2(a)). Meanwhile, using MS + 6-BA (1.0 mg/L) + IBA (0.1 mg/L) as the optimum proliferation medium, the proliferation rate reached 79.63%, the proliferation coefficient was greater than 5, and the plants were robust with green leaves and longer internodes (Figure 2(b)). MS medium is an important substrate for plant tissue culture, and the selection of an appropriate medium type is crucial to the success or failure of plant tissue culture [27]. Regarding the root culture of kiwifruit, a large body of literature showed that 1/2 MS medium with a low salt content is more conducive to the rooting of kiwifruit tissue culture plantlets [28]. Meanwhile, the use of auxins in MS medium for in vitro rooting stage has also been reported by many other researchers [29]. In addition, it has also been reported that IBA induced lateral rooting better than IAA and NAA [20]. In this study, using 1/2 MS + IBA (0.3 mg/L) as the optimum rooting medium, the rooting rate and average rooting number reached 85.18%     (Figure 2(c)). After that, the tissue culture plantlets were transplanted into the greenhouse, and the survival rate exceeded 90% (Figure 2(d)).
Virus disease is a kind of disease with a long incubation period and a great production so hidden trouble. In recent years, many works of literature have reported that kiwifruit virus diseases, especially the pathogenic viruses CLRV and PZSV, which can be spread through pollen, grafting, or  mechanical inoculation, are common in preserved kiwifruit germplasm resources and cultivated kiwifruit plants [30,31]. Meanwhile, during the cultivation and promotion of "Guichang" kiwifruit for several decades, the virus disease in the main "Guichang" kiwifruit producing areas seriously affected the fruit appearance and commodity value, causing huge economic losses to the kiwifruit industry. In this study, a total of 9 kinds of viruses, including ASGV, CMV, AVX, CNV, RMV, CLBV, AcVB, PZSV, and CLRV, in tissue culture plantlets were detected via the RT-qPCR method, and the results demonstrated that the infection rate of 9 kinds of viruses in tissue culture plantlets was 0. erefore, this study could lay the foundation for the production of breed virus-free plantlets of "Guichang" kiwifruit.

Conclusions
In this study, the establishment of a tissue culture rapid propagation system of "Guichang" kiwifruit was carried out and then detected virus status via the RT-qPCR method. e tissue culture rapid propagation system proved that MS