CHEN Xi , GENG Wen-juan *, XIE Gang-gang , XIE Jun

1. Xinjiang Agricultural University, College of Forestry and Horticulture, Urumqi 830052, PRC;

2. Xinjiang Academy of Agricultural Sciences, Urumqi 830052, PRC

Abstract [Objective] To establish the callus induction and proliferation system of the petiole of the wild European plum. [Methods] The petiole of the wild European plum was used as an explant. The effects of disinfection time, type and concentration of carbon source, dark treatment time, basic medium, growth regulator and sampling time on callus induction and petiole proliferation were studied. [Results] The best sampling time was from mid-May to mid-June. The lowest pollution rate was 3.33%when they were disinfected with 75% alcohol for 45 s and 0.1% mercuric chloride for 7 min. 21 d of dark treatment was the best dark treatment time. The best formula for callus induction and proliferation was: B5+0.2 mg/L 6-BA+2 mg/L NAA+30 g/L sucrose+7 g/L agar. [Conclusion] A theoretical foundation was laid for the tissue culture and regeneration system of the wild European plum.

Key words Wild European plum; Petiole; Callus induction

1. Introduction

The wild European plum (

Prunus domestica

L.), commonly known as the wild sour plum, belongs to (

Prunus

) of the (

Rosaceae

) genus of Rosaceae. It is a shrub or small tree and is one of the rare wild plants in Xinjiang. It can survive well only on a warm and humid slope or near the water. Its resistance to extraneous intrusion is weak, and its distribution area is small. The European plum can be processed and eaten, and it has certain medicinal effects, making it widely loved by consumers at home and abroad. As a result, it has considerable market value, whereas the wild European plum has not yet been developed and utilized. At present, due to the poor living environment, overgrazing,man-made destruction, and other factors, the wild European plum population decreases year by year as the habitat shrinks and it is endangered. Therefore, it is urgent to establish a protection scheme of wild European plum resources in Xinjiang.Tissue culture, which has been widely used in recent years, uses the totipotency of cells to culture plant explants

in vitro

. This method not only has high reproduction coefficient and fast reproduction speed, but it also can be used for the detoxification and rejuvenation of varieties, the conservation of germplasm resources and the expansion of endangered plant populations.Tissue culture plays a key role in the protection of wild fruit tree resources. Its usage has been reported in the wild cherry plum, the wild loquat, the Xinjiang wild apple, the wild mountain apricot,the wild

Actinidia arguta

etc

. Most plum tissue cultures used the stem tip and leaf as explants,and there are few reports on the tissue culture of the petiole of the plum. Therefore, in this experiment,the petiole of the wild European plum in Xinjiang was taken as the research object, and the main factors affecting petiole tissue culture were screened, with the expectation of establishing a system for the induction and proliferation of the petiole callus of the wild European plum. The purpose of this study is to provide a theoretical basis for the tissue culture and regeneration of the petiole of the wild European plum.It has practical significance for the protection of wild European plum resources in Xinjiang. At the same time, it also provides a reference for the tissue culture of other wild drupe fruit trees.

2. Materials and Methods

2.1. Materials

The petiole materials used in the experiment were collected from the third team of the Xinyuan wild fruit forest improvement field in Yili, Xinjiang. We cut green branches with leaves that grew well during the growing season, wrapped them in newspapers,soaked them in water, put them in a foam box with ice bags and brought them back to the laboratory, where they were kept in the refrigerator at 4℃ during the experiment.

2.2. Testing method

2.2.1. Pre-disinfection of explants

We selected branches that were better preserved and picked the leaves off from petioles that had no worm eyes and no damage. We gently scrubbed the dust and impurities from the surface of these petioles with a soft brush and removed the blade with scissors.We then put the petioles into a 250 mL beaker filled with tap water, into which we dropped a little diluted detergent, and soaked them for 15 min, while shaking the beaker continuously. Then we covered the mouth of the beaker with a clean gauze, fixed it with a rubber band, and rinsed the petioles under slow flowing tap water for 1～1.5 h. After this pre-disinfection, we put the petioles into a clean 50 mL beaker and moved the beaker to an ultra-clean worktable after ultraviolet sterilization.

2.2.2. Screening of disinfection time treatment

Under sterile conditions, 75% alcohol and 0.1%mercuric chloride were selected as the disinfection reagents. We disinfected with 75% alcohol, rinsed with sterile water 2～3 times, and then disinfected with 0.1%mercuric chloride, and finally rinsed with sterile water 4～5 times; we disinfected with 75% alcohol for 30 s+0.1% mercuric chloride for 1, 3, 5, 7 and 9 min; we disinfected with 75% alcohol for 45 s+0.1% mercuric chloride for 1, 3, 5, 7 and 9 min; and we disinfected with 75% alcohol for 60 s+0.1% mercuric chloride for 1, 3, 5, 7 and 9 min, for a total of 15 treatments.The formula was B5+2 mg/L AA+0.2 mg/L 6-BA+30 g/L sucrose+7 g/L agar. Each treatment was used to inoculate 10 bottles, and each bottle contained 3 or 4 explants. The pollution and growth status were counted after 30 d.

2.2.3. Screening of carbon sources

Sucrose, glucose and fructose were selected as the carbon sources. The concentration gradients were 10, 20 and 30 g/L. B5+2 mg/L NAA+0.2 mg/L 6-BA was used as the formula for screening. A total of 9 groups were treated, 10 bottles of each treatment were repeated, and each bottle was inoculated with 3 or 4 explants. After 30 d, the growth status of the petiole callus was observed and counted.

2.2.4. Shading time screening

With B5+2 mg/L NAA+0.2 mg/L 6-BA as the formula, the dark treatment time was 0, 7, 14,21 and 28 d. There were 5 treatments, 10 bottles of each treatment were repeated, and each bottle was inoculated with 3 or 4 explants. After 30 d, the growth status of the petiole callus was observed and counted.

2.2.5. Screening of basic medium

Four basic mediums: MS, B5, WPM and 1/2MS were selected for screening. 2 mg/L NAA+0.2 mg/L 6-BA+30 g/L sucrose+7 g/L agar was added for culturing. There were 4 groups of treatments; each treatment was repeated in 10 bottles; and each bottle was inoculated with 3 or 4 explants. After 30 d, the growth status of the petiole callus was observed and counted.

2.2.6. Screening of growth regulators

Three basic mediums of MS, B5 and WPM were selected and combined with auxin sieve (NAA,IBA) and cytokinin (6-BA, TDZ). There were 3 combinations and 15 treatments; each treatment was repeated in 10 bottles, and each bottle was inoculated with 4 explants. After 30 d, the growth status of the petiole callus was observed and counted (Table 1).

Table 1 The ratio of 15 different combinations of growth regulators

2.2.7. Screening of the best sampling period

Samples were collected during the growth periods of May, June, July, August, September and October in 2019. A total of 6 treatments were cultured with 0.2 mg/L NAA+2 mg/L 6-BA+30 g/L sucrose+7 g/L agar. Each treatment was used to inoculate 10 bottles, and each bottle was inoculated with 3 explants. After 30 d, the growth status of the petiole callus was observed and counted, and the pollution and callus growth in different periods were analyzed.

3. Results and Analysis

3.1. Effects of different disinfection times on the contamination rate of the petiole tissue culture

As can be seen from Table 2, the calli of the petiole explants with the different disinfection times were contaminated differently, and their growth status was different. The pollution rate decreased with the increase of the disinfection time with 75%alcohol and 0.1% mercuric chloride for 30, 45 and 60 s. The lowest pollution rate was 3.33%. At the same time, the mortality rate increased. The maximum mortality rate was 43.33%. In the five groups disinfected with 75% alcohol for 30 s + 0.1% mercuric chloride for 1, 3, 5, 7 and 9 min, the pollution rate was high, the mortality rate was high, there were different molds or bacteria around the petiole, and there were more browning deaths (Fig. 1A and 1B).In the five groups disinfected with 75% alcohol for 45 s + 0.1% mercuric chloride for 1, 3, 5, 7 and 9 min, the degree of pollution was small, the mortality rate was low, and the growth condition was good(Fig. 1C). Among them, the petiole of 0.1% mercuric chloride disinfection for 7 min expanded obviously and the callus grew well. In the five groups disinfected with 75% alcohol for 60 s + 0.1% mercuric chloride for 1, 3, 5, 7 and 9 min, the contamination rate was the lowest, but the petiole differentiation callus was late, more petioles were not differentiated, the surface began browning and wrinkling, and it finally died.

Table 2 Effects of different disinfection time on the contamination rate of petiole tissue culture

Fig. 1 Petiole tissue culture

3.2. Effects of different carbon source concentrations on the callus induction of the petiole

It can be seen from Table 3 that the induction degree and growth status of the petiole callus were affected by the type and concentration of the carbon source. The induction ability of the petiole callus by the three carbon sources was sucrose>glucose>fructose, and the callus induction rate increased with the increasing concentration of the carbon source.The highest callus induction rate was 96.70% in the medium with 30 g/L sucrose. After 14 d of dark culture, the petiole began to expand from the incision and more white calli grew, the volume became larger,and the color was light green, but it grew poorly in the medium supplemented with 10 and 20 g/L sucrose.In the medium containing glucose and fructose, the petiole swelling was not obvious, and most petioles had a little white callus at the enlarged part of the cut,especially in the presence of 10 g/L fructose, while there was a little browning in the petiole cut. To sum up, the medium with 30 g/L sucrose was the best for the callus induction of the petiole.

Table 3 Effects of different carbon source concentrationson callus induction of petiole

3.3. Effects of different dark treatment times on the callus induction of the petiole

It can be seen from Table 4 that the induction degree of the petiole callus was different for different dark treatment times. As the dark treatment time increased, the callus induction rate of the petiole increased. After 0 and 7 d of dark treatment,the callus rate of the petiole was in the range of 20.00%～33.33%. A few petioles began to expand at both ends, and there were a few white calli (Fig. 2A).The callus induction rate of the petiole was higher after 14, 21 and 28 d of dark treatment, and the callus induction rate of the petiole reached 93.33% after 28 d of dark treatment. This time, the callus of the petiole expanded obviously, and the color of the callus was light yellow (Fig. 2C). The surface of the little callus browned and the texture was soft. Nevertheless,after 14 d of dark treatment, both ends of the petiole expanded obviously, the color of the callus was bright green, and the texture was moderate (Fig. 2B).After 21 d of dark treatment, the petiole completely expanded, the volume increased, the callus color was yellowish green and the texture was soft. To sum up,the growth state of the petiole callus was the best after 14 d of dark treatment.

Table 4 Effects of different basic mediums on the callusinduction of the petiole

3.4. Effects of different basic media on callus induction of petiole

Fig. 2 Effects of different dark treatment times on the callus induction of the petiole

It can be seen from Table 5 that the induction degree of the petiole callus was different in different mediums. In the four basic mediums of B5, WPM, MS and 1/2MS, the contamination rate was low, between 5.00%～10.00%, and the mortality rate of the petiole in these four mediums was B5WPM>MS>1/2MS. The highest callus induction rate was 80.00% in the B5 medium. At this time, the petiole expanded obviously,the volume increased, and the callus grew well. The effect was poor in the WPM medium, and a few calli were browned. The callus induction rate of the petiole in the MS and 1/2MS mediums was low, the petioleexpansion was not obvious, browning was obvious at the incision at both ends of the petiole, and the growth of the callus was not good. To sum up, the callus induced by the petiole in the B5 medium grew well.

Table 5 Effects of different basic media on callus induction of petiole

3.5. Effects of different concentration ratios of growth regulators on the callus induction rate of the petiole

It can be seen from Table 6 that different ratios of growth regulators had great influence on the callus induction ability of the petiole. Among the 15 treatments, the callus induction rate was in the range of 0～85.00%, and the overall callus induction rate of treatments 1, 2, 3, 4 and 5 was higher than that of other treatments. The highest callus induction rate of treatment 2 was 85.00%. In the culture process,compared with other treatments, the petiole mortality rate of these five treatments was as low as 5.00%.After about a week of dark treatment, the petiole began to expand, and there were white calli at both ends of the incision. After 30 d of culture, the callus was completely callous and the volume increased obviously. The callus was yellowish green (Fig.3A). In other treatments, most of the petioles did not differentiate after about 10 d of culture, which was the same as that before inoculation. Both ends of the petiole began to brown and died gradually. The overall mortality rate was in the range of 45.00%～75.00%.The expansion of the surviving petiole was not obvious and the callus induction rate was low. Among them, the callus induction rate of treatment 13 was 0, and the callus grew poorly after 30 d of culture.Red bacteria appeared at the bottom of calli in the treatment 6, 7, 8, 9 and 10 (Fig. 3B). To sum up,treatment 2 (B5+2.0 mg/L NAA+0.2 mg/L 6-BA) had the best effect on the callus induction and proliferation of the petiole.

Table 6 Effects of different concentration ratio of growth regulators on callus induction rate of petiole

Fig. 3 The callus induction and proliferation of the petiole

3.6. Effects of different sampling periods on the callus induction of the petiole

It can be seen from Table 7 that different sampling periods had different effects on the callus induction of the petiole. In the 6 sampling times,as the months increased, the pollution rate and mortality rate increased, and the callus induction rate decreased. Among them, the lowest pollution rate of T1 was 3.33. The highest contamination rate of T5 and T6 was 43.33%. The lowest mortality rate of T1 and T2 was 6.67% and the highest mortality rate of T6 was 50.00%. The callus induction rate was T1>T2>T3>T4>T5>T6. The callus induction rates of T1 and T2 were 90.00% and 86.67%, respectively.After 30 d of culture, the petiole was completely callous and the color was light green. The growth condition was good. The calli of T3 and T4 were yellowish green, and a few were browned. On the other hand, the calli of T5 and T6 were light yellow and browned to different degrees. To sum up, the best time for petiole sampling was from mid-May to mid-June.

4. Discussion and Conclusion

Different dark treatment times had different effects on callus induction from explants, and whether the callus grew well or not would directly affect theregeneration of adventitious buds, using the petioles of the Chinese plum ‘Nubiana’ and cherry plum testtube plantlets as explants. ZHANG L Yfound that the optimum dark treatment days for callus induction were 14 and 21 d, respectively. When the dark treatment time was more than or less than the optimum time, the growth status of the callus was also different. In this study, it was concluded that the best dark treatment time of the wild European plum petiole was 14 d. If the dark treatment time was less than 14 d, the petiole was not completely callous, which was not conducive to further redifferentiation, after dark treatment for more than 14 d, although the petiole had completely healed and was large, it began to brown after being transferred to light culture for 10 d and had no differentiation ability.

Table 7 Effects of different sampling periods on callus induction of petiole

MS was a widely used basic medium in the tissue culture of drupe fruit trees, whereas WPM, B5,F14, LP and other mediums were also used. Different tree species respond best to different culture mediums.In the reports related to the tissue culture of the plum petiole, the WPM medium was reported to be the most suitable medium for the petiole regeneration of the Chinese plum. MS basic medium had a better effect on the callus regeneration induced by the petiole of the cherry plum. The effects of B5, WPM,MS and 1/2MS on the callus induction rate of the wild European plum petiole were compared in this experiment. The results showed that B5 had the best callus induction, WPM was poor, and MS and 1/2MS were the worst. Among the previous studies of stem tissue culture, B5 was the most suitable medium for the wild European plum.

The type, concentration and ratio of growth regulators played an important role in the formation of organs in plant tissue culture. TDZ was widely used in the leaf tissue culture of drupe fruit trees,NOWAK B

et al

.showed that a suitable concentration of TDZ could promote the production of a large number of calli in leaves. In this experiment, it was found that the callus induction effect of 6-BA was better than that of TDZ, and similar results were obtained in the experiment of the leaf regeneration of the small yellow plum. The combination of different growth regulators also had different effects on explant regeneration. The results showed that the callus induction effect of 6-BA+NAA was better than that of TDZ + 2,4-D and 6-BA+IBA. FAN Q Falso obtained the optimal formula for this combination to induce a small number of adventitious buds from the petiole of the Nubiana plum. WU S S, in the multifactor experiment of leaf callus induction of European plum variety ‘Nongda 7’ in the field, it was found that the combination of 6-BA and NAA could induce 98.67% of adventitious buds, while TDZ + 2,4-D was the most suitable hormone combination for the regeneration of buds from the petiole of the Chinese plum and the European plum. This experiment did not produce buds. This may be due to the different genotypes and the different sources of the explants.Other adventitious buds were regenerated from petioles of plums using the petioles of test-tube plantlets as material. In this experiment, the wild European plum picked in the field was used as the experimental material, and the petiole regeneration of test-tube plantlets was studied. The culture environment and explant browning were key factors affecting the proliferation of the petiole callus, which needs to be optimized.

To sum up, after 14 d of dark treatment, the best formula for the callus induction and proliferation of the wild European plum petiole was B5+0.2 mg/L 6-BA+2 mg/L NAA+30 g/L sucrose + 7 g/L agar. The callus cultured with this formula had a high induction rate, light green color and moderate soft texture. In this experiment, the induction and proliferation of the petiole callus of the wild European plum were studied and the best proliferation system was selected,whereas the result of bud differentiation was not ideal and needs to be further explored.