Mutation induction of EMS and 60Co γ irradiation in vitro cultured seedlings of red pulp pitaya (Stenocereus) and ISSR analyzing of mutant

Renju Deng (  15362220@qq.com ) Guizhou Provincial Academy of Agricultural Sciences Jianxin Fan Institute of Subtropical Crops, Guizhou Provincial Academy of Agricultural Sciences Yongqing Wang College of Horticulture, Sichuan Agricultural University Tao Liu Institute of Fruit Research, Guizhou Provincial Academy of Agricultural Sciences Jifen Jin Institute of Fruit Research, Guizhou Provincial Academy of Agricultural Sciences


Conclusions
It concluded that pitaya mutant might be produced regardless of the mutagenic dose high or low, and a higher dose could increase the frequency of variation. In this study, critical dose and half lethal dose can be used as the appropriate dose for mutation breeding of pitaya. Additional, molecular analysis showed that there was a certain difference between the mutant plants and the control. It needs to be further veri ed whether this difference was a physiological variation or a real variation due to the randomness of mutation, so as to lay a foundation for better carrying out mutation breeding of pitaya.

Background
Mutation breeding is one of the effective methods of screening new varieties. In particular, the combination of mutation technology and culture technology in vitro, which can broaden mutation spectrum, improve mutation frequency, and keep clone variation (Predieri, 2001;Shang et al. 2014).
Moreover, the whole process is carried out under sterile conditions, thus opening up a greater space for mutation screening. According to the data on the o cial website of FAO/IAEA in 2016 (Wu et al. 2016), breeders in all countries have cultivated 55 new varieties of deciduous fruit trees by mutation technology, including varieties of 2 jujubes, 6 peaches, 13 apples, 1 apricot, 1 plum, 8 pears, 1 grape, 21 cherries and 2 pomegranates.
Pitaya, a member of cactus family, is a perennial, climbing and succulent plant, and also a new type of tropical and subtropical fruit tree developed widely in recent years in China (Deng et al. 2019). But there are very few reports on breeding of pitaya at home and abroad (Deng et al. 2011). Therefore, the study took seedlings in vitro of red pulp pitaya as materials, which treated with different dose of EMS and 60 Co γ, respectively, then screened the optimal mutagen dosage and mutant plants, which would lay the foundation for screening mutants with bene cial characters and cultivating new resources of pitaya fruit with excellent comprehensive characters.

Results
Effect of the EMS concentration and treatment time on the survival of seedlings It can be seen from table 1, the survival rate of seedlings decreased with the increase of EMS concentration and treatment time. When the time was constant, a straight line regression equation could be tted according to the relationship between EMS concentration and the survival rate. The lowest correlation coe cient (R 2 = 0.7542) among the EMS concentration and survival rate occurred at the treatment time of 3 h, while the highest correlation (R 2 = 0.9916) occurred at treatment time of 9 h.
According to the linear regression equation Y = 205.44x + 41.902, when the EMS concentration was 3.71%, 50% of the pitaya seedlings survived, while the survival rate decreased to 40% at an EMS concentration of 3.95% (Table 2). when the EMS concentration was constant, the data could be tted by a straight line regression equation according to the relationship between the treatment time and the survival rates. the lower EMS concentration, the lower correlation between the treatment times and survival rates. The highest correlation coe cient (R 2 = 0.9873) was obtained at EMS concentration of 3.8%. According to the linear regression equation Y = −9.4300x + 127.90, the survival rate reached 50% at the treatment time of 8.26 h, while it decreased to 40% at the treatment time of 9.32 h (Table 3). It concluded that the suitable EMS concentration for pitaya seedlings ranged from 3.71-3.95%, and the appropriate treatment time ranged from 8.26-9.32 h.
Effect of subculture time on the survival of seedlings treated by EMS It can be seen from the table 4, the survival rate of seedlings was the lowest when they were directly treated by EMS after being separated from their mother plants. When seedlings subcultured for 15-30 d, the average survival rate was marked increase. Thus, it concluded that the resistance of seedlings to EMS increased with the extension of subculture time, and if the transgenerational adaptation time was too short, EMS might have more damaging effect on the seedlings, which makes recovery growth more di cult.
Effect of EMS concentration and treatment time on the differentiation of seedlings As shown in gure 1, the differentiation ability of seedlings degraded signi cantly with the increase of EMS concentration and treatment time. When EMS concentration was 3.5% and the treatment time was 9 h, the differentiation rate of seedlings was only 46.6%. When EMS concentration was greater than or equal to 3.8% and the treatment time was more than 12 h, the differentiation capacity was very weak (4.7%). When EMS concentration equaled to 4.0%, buds cannot be differentiated.
Effect of 60 Co γ irradiation on the survival and differentiation of seedlings As seen from table 5, it had no effect on survival rate, and little effect on differentiation rate when the radiation dose less than or equal to 10 Gy. The survival and differentiation rate of seedlings decreased signi cantly with the increase of radiation intensity when the dose was higher than 15 Gy. According to the relations between radiation doses and survival rate, which tted the linear regression equation as shown in gure 2, and then calculated that the half lethal dose (LD50) of pitaya seedling in vitro was 38.5Gy, and the critical lethal dose (LD40) was 42.4Gy.

Screening and ISSR analyzing of mutant plants
By observing the morphological characteristics of seedlings treated with EMS and 60 Co γ, 14 mutants were screened out, materials with non-mutagen as control. The speci c characteristics can be seen from table 6 and gure 3. According to the ISSR analysis, 9 primers (Table 7), with good polymorphism and stable ampli cation, were screened out from 38 primers to amplify mutant materials, and a total of 67 bands were ampli ed, 48 of which were polymorphic bands, and the proportion of polymorphic bands was 71.6%, which indicated that the variation of test materials was relatively rich. Each primer can amplify 3-14 bands, with an average of 5.33 polymorphic bands (Table 8). Among them, primer M08 ampli ed the most number of bands with 14, and primer M866 ampli ed the highest ratio of polymorphic bands with 100%, and the lowest ratio of polymorphic bands was ampli ed by M06, with 45.5%, but a speci c band was found on electrophoresis channel 5 ( Figure 4). The ampli ed bands ranged from 260 bp to 2000 bp, most of which were from 500 bp to 1200 bp ( Figure 5 and Figure 6).

Genetic distance and cluster analysis
Based on the ISSR data analysis, the genetic distance (GD) of mutant plants ranged from 0.0120-0.4169 (Table 9). Among them, the smallest genetic distance was between No.1 and No.3, GD = 0.012, which indicated that the genetic background of the two was similar, and the variation of No.3 was smaller compared with the control (No.1). The largest genetic distance was No.2 and No.5, GD = 0.4169, which could be seen from that there was a large variation between them. Cluster analysis (Figure 7) shows that the control and 14 mutants can be divided into 6 groups, when GD = 0.11, among which No. 5, 6, 10, 12 and 14 materials can be separated into one group, which showed that there was a great variation among No.5, 6, 10, 12 and 14 in compared with the control.

Discussion
Different plant species and different parts of the same plant have different sensitivity to mutagens. In general, stem tips, blades and calluses can be used as mutagenic material in vitro, but it is very important that the mutagen can penetrate the tissues adequately and the mutagenic material can continue to grow and develop to form a new individual eventually (Ahloowalia and Maluszynski, 2001;Stefano, 2001;Wang et al. 2011;Serrat et al. 2014). The appropriate dose is one of the key factors for the success of mutation breeding whether it is chemical or physical mutation (Cui et al. 2011). Increasing the dose in a certain range can improve the mutation rate and expand the mutation spectrum of plant materials, but it can also reduce the survival rate when using too much higher dose, resulting in the increase of bad mutation characters. Critical dose and half lethal dose are generally used to determine the appropriate dose, but there is also a trend to select two more doses within the range of 20% difference between the upper and lower half lethal doses as the appropriate dose to produce more bene cial mutations (Li et al. 1999).
Before this study, there was only one report on the mutation of pitaya using stems and buds in eld.
However, it was di cult to control the eld conditions and there was also no continuous observation and study. In addition, the leaves of pitaya degenerate into spines, and the variation of morphological characteristics is less than other plants, so there are fewer morphological types that can be marked (Peng, 2007;Liu et al. 2010). On the basis of morphological screening, molecular markers can more objectively re ect the differences between different mutants. Studies have shown that it is di cult to identify point mutations or deletions of minimal fragments by RAPD (Gustavo and Peter, 1998). ISSR primers are repetitive sequences, which are not affected by natural selection or less, and it is the fastest in the genome, and the variation is easy to retain. Therefore, ISSR markers with high polymorphism can effectively reveal the differences between individuals with very similar genetic relationship.

Conclusion
The study took red pulp pitaya 'Zi Honglong' seedlings in vitro as experimental materials, and selected the suitable EMS concentration and the optimum dose of 60 Co γ irradiation. The conclusion was very different from the result of mutagenesis using stems or buds of adult pitaya plant (Deng et al., 2011), which may be related to the sedlings in vitro were more sensitive to the mutagens and were more likely to mutate. ISSR molecular detection showed that a total of 67 bands were ampli ed with 9 better primers from 15 samples, 48 of which were polymorphic bands, the proportion of polymorphic bands was 71.64%. it was signi cantly higher than the results of RAPD and ISSR labeling pitaya resources by Junqueira et al. (2010), Zhang et al. (2013), Wang et al. (2013). From the PCR results of all primers, M08 ampli ed the most bands, and m866 had the highest polymorphism ratio (100%), while M06, although the polymorphism ratio was relatively low, found a speci c band around 380 bp in Lane 5, and its functional characteristics needed further study and veri cation. What's more, from the genetic distance and cluster analysis, we can see that the average genetic distance of 15 resources of Pitaya is 0.2836, which showed that the mutation rate was high. However, the genetic distance between No. 3 and No. 1 (the control) was the smallest, which indicated that the variation of No. 3 mutant was the smallest, which was consistent with the result of cluster analysis.
Although the mutants were screened by EMS and radiation mutagenesis in the study, due to the randomness of mutation breeding, the difference between the mutant individuals and the control may be physiological or real variation. Therefore, it is necessary to further study, observing the growth, eld performance and stability of mutant individuals in the future, so as to lay a foundation for better carrying out mutation breeding of pitaya.

Methods
The original materials were from the pitaya variety 'Zihonglong', which was independently cultivated by my institution. The sampling point was located in the demonstration park of Longping Town, Luodian county (106°45' E, 25°26' N, a.s.l 385 m). Then seedlings in vitro were obtained by stem tissue culture in the College of Horticulture, Sichuan Agricultural University (102°58' E,29°58' N a.s.l 630 m).
EMS treatment. Referring to the methods of Deng et al. (2011) andCui et al. (2011) and making some improvements. Mother liquors containing different EMS concentrations (2.8%, 3.0%, 3.2%, 3.5%, 3.8%, 4.0%) were prepared, and lter sterilized under aseptic conditions. Phosphate buffer [0.01M, pH 7.0] was prepared by autoclaved sterilization and then cooled to room temperature The EMS mother liquors were transfer into a sterile cylindrical bottles and bring to volume by phosphate buffer to prepare different concentration gradient EMS working liquid under aseptic condition. (EMS has a half-life of 25.9 h at 30 ℃). According to the times of subculture in the transfer medium (0, 7, 15, and 30 d, respectively), the seedlings were divided into four groups. Then, the seedlings were placed into sterilized solutions containing different concentration of EMS and shaken for 3, 6, 9, and 12 h, respectively. After the treatment, washing the seedlings with sterile water at least ve times, and drying the surface water by blotting, then culturing on the subculture medium MS + 6-BA 1.0 mg/L + NAA 0.2 mg/L + AC 0.05%+ CCC 0.5 mg/L for 45 d to promote growth and differentiation, Seedlings without EMS treatment took as control. 60 Co γ irradiation treatment. According to the radiation related researches of other plant seedlings in vitro (Liu et al. 2010;Li et al. 2014) and pitaya plant (Deng et al. 2011), the seedlings of red pulp pitaya cultured on subculture medium for 30 days were irradiated with 60 Co γ in Jinnong Irradiation Center of Guizhou Academy of Agricultural Sciences. The dosage was set as 5 Gy,10 Gy,15 Gy,20 Gy,25 Gy,30 Gy,35 Gy,40 Gy and 45 Gy. The dose rate was 1 Gy/min, and the corresponding irradiation time was materials, mutants would be screened out. Seedlings without mutation were used as control. Molecular detection of mutants was analyzed by inter simple sequence repeats (ISSR), referring to the established ISSR marking system by Yuan et al. (2013) and Zhang et al. (2013)

Availability of data and material
All data generated or analyzed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests. In this study, the rst funding body provide nancial support for independent innovation and training of high-level talents; and we applied for the project from the second funding body according to the actual needs of industrial development, and we put forward research ideas, solutions and expected results, etc. The project had no doubt through expert review, and we were given nancial support.

Authors Contributions
R.J.D. and J. X. F. performed the experiments, analyzed the data, and drafted the manuscript. Y.Q.W. conceived the study, designed experiment, retouched and revised the paper. T. L. and J.F.J helped with sampling, participated in its design and coordination, and helped draft the manuscript. All authors have read and approved the nal manuscript.
12 180 10 5.6dF Note: Lowercase and uppercase letters within a column denote significant differences (P <0.05) in the survival rate at EMS concentrations and treatment times.  Note: ** indicates a statistically significant difference at the 0.01 level.  Treated with 3.0% EMS for 12 h. New bud with red tip and morphological variation of stem.

10
Treated with 3.5% EMS for 9 h. New bud with red tip and morphological variation of stem.

11
Treated with 2.8% EMS for 12 h. Morphological variation of stem.

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Treated with 35Gy 60 Co γ irradiation. Morphological variation of stem 14 Treated with 35Gy 60 Co γ irradiation. New bud with red tip and morphological variation of stem.

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Treated with 38.5Gy 60 Co γ irradiation. New bud with red tip and morphological variation of stem.  Figure 1 The effects of the EMS concentration on the differentiation of seedlings Regression analysis between radiation intensity and survival rate Electrophoresis pattern with primer M06 Lane M. DL2000 Marker; Abbr. of samples of other lanes seen in Table 7. The same below.

Figure 6
Electrophoresis pattern with primer 845 Figure 7 ISSR cluster analysis of 14 mutants and 1 control sample