Key Technology for Virus-free Seed Potatoes Pre-elite Breeding

Jie XU1*, Li SUN2, Wenting ZHU

1. Zaozhuang Academy of Agricultural Sciences, Zaozhuang 277800, China; 2. Zaozhuang Cotton Original Breeding Farm, Zaozhuang 277300, China; 3. Zaozhuang Denghai Detai Seed Industry Co., Ltd., Zaozhuang 277100, China

Abstract [Objectives] To establish a breeding system for pre-elite seed potatoes. [Methods] Shoot-tip seedlings were cultured with different medium, and then transplanted in different substrates. They were sprayed with nutrient solutions of different formulas. Potato minitubers were induced. [Results] The medium of MS+2.0 mg/L 6-BA, 1.0 mg/L NAA+3% sugar was preferable for potato shoot tip culture. After virus detection, the virus-free potato seedlings were propagated. The medium composed of vermiculite, peat and perlite (1∶3∶1) was recommended for transplanting potato tissue culture seedlings in greenhouse. The three-dimensional spray method was used to produce pre-elite seed potatoes. The full-amount MS nutrient solution could increase the tuber number per plant to 55. Induction of minitubers is a better breeding technology for pre-elite seed potatoes. The optimal medium screened for induction of potato minitubers was MS+(5-7) mg/L 6-BA, with a dark culture duration of about 20 d. [Conclusions] The breeding technology established is suitable for the two-season cultivation areas in Zaozhuang.

Key words Potato, Pre-elite seed, Breeding, Spray method

1 Introduction

Potato is annual herb with edible tubers. It is the fourth most important food crop in the world, after wheat, rice and maize.

Zaozhuang City is one of the important potato producing areas in Shandong Province, and it is also the largest two-season potato production area in China, with a cultivation area of more than 44 700 ha in spring and more than 10 000 ha in autumn, of which facility cultivation area is more than 28 000 ha. The yield of potatoes in Zaozhuang ever set the highest record for the two-season potato production in the Central Plains of China, with the maximum yield of 88.5 t/ha and average yield of 43.5 t/ha. The main cultivation areas are in Tengzhou City, Yicheng District, Xuecheng District, Taierzhuang District, Shanting District,etc.In Zaozhuang area, the temperature rises rapidly in spring, and the occurrence of aphids and other insect mediators is large, which provides convenient conditions for the spread of viral diseases, resulting in the degeneration of potato varieties, which can lead to a 20%-50% reduction in yield. In autumn, if potato is sown too early, the variety will degenerate; and if potato is sown too late, its yield will be affected. Therefore, the seeds of potato used in Zaozhuang area are mostly transported from the northeast. In recent years, due to continuous planting in old bases such as Hebei and Inner Mongolia, coupled with unclear breeding generations of virus-free potato seedlings and uneven quality of seed potatoes, seed-borne diseases, including verticillium wilt, fusarium wilt, scab, powdery scab, black stem disease and ring rot, occur from time to time, increasing introduction risk. Therefore, it is urgent to establish a breeding system and key breeding technology for pre-elite seed potatoes suitable for the two-season cultivation areas in Zaozhuang.

2 Materials and methods

2.1 MaterialsThe experimental material was Luyin No.1.

2.2 Methods

2.2.1Induction of shoot tip seedlings. A plant with vigorous growth was chosen, and at the top of the main stem, a segment of 1 cm length was taken. After cutting off the large leaves, the segment was rinsed in tap water for 30 min. Subsequently, it was transferred to an ultra-clean workbench, disinfected with 75% alcohol for 30 s and 0.1% mercuric chloride for 4-5 minutes, and rinsed with sterile water for 8-10 times successively. The shoot tip, about 0.1-0.3 mm long, was peeled off under a dissecting microscope, and then inoculated in the medium composed of full-amount MS, 2.0 mg/L 6-BA and 1.0 mg/L NAA. The medium was pre-sterilized at 121 ℃ for 22 min. Culture was performed under the conditions with temperature of 20-25 ℃, illumination of 12-16 h, and light intensity of 1 200-2 000 lx. A total of 100 flasks inoculated with shoot tips were prepared, one shoot tip per flask. The induction of shoot tip seedlings was observed and recorded 60, 70, 80 and 90 d after the inoculation, respectively[1].

2.2.2Virus detection. Virus detection of shoot tip seedlings is a key link in cultivating virus-free seedlings. There are many kinds of potato viruses, such as X virus, Y virus, A virus, S virus and M virus. At present, visual inspection method, indicator plant detection method, reagent strip detection method and serum detection method are commonly used for detection. Visual inspection method is to retain the shoot-tip seedlings with fast growth, dense leaf color, spread leaves and robust growth and discard those with weak growth, pale leaves, and chlorotic spots on leaves. In indicator plant detection, the plant with suspicious symptoms was used as scion and grafted to the indicator plant globe amaranth, and after two weeks of inoculation, if disease spots appear on the leaves of the indicator plant, the corresponding shoot tip should be discarded. In reagent strip detection method, five potato tissue culture seedlings are taken and ground into juice, which is then tested with various virus disease reagent strips. If the reagent strips show one bar, it indicates that there is no virus infection, and if they show two bars, it indicates that the plants are infected with virus disease and should be eliminated. The serum detection method is to add a drop of sap of the plant to be tested to several different antisera, and if precipitation appears in certain antiserum (antisera), it proves that the plant is infected with corresponding virus (viruses).

2.2.3Subculture. After virus detection, vigorous virus-free shoot-tip seedlings were selected. On the ultra-clean workbench, segments of about 1 cm with one bud on each of them were cut off. They were placed on sterile filter paper in a petri dish. According to the polarity direction, the segments were inoculated into the medium. After each operation, tweezers and other instruments were sterilized for the next use. In each flask, 10-15 single-bud stem-segments were inoculated. After each flask was inoculated, the flask mouth was turned around on the flame, then sealed, and finally labeled. After inoculation, the workbench was cleaned and sterilized with UV lamp for 30 min.

The culture flasks were placed in the environment with temperature of (25±2) ℃, light intensity of 1 200-2 000 lx, and illumination of 12-16 h. They were observed once a week, and culture flask was discarded once contaminated.

2.2.4Transplanting test-tube seedlings. The seedlings were transplanted into greenhouse (net house) when their roots grew to 3-5 cm long. Before transplanting, the seedlings were hardened by natural scattered light for 6-7 d, weeds in the greenhouse (net house) were removed, the field was sprayed with pesticide to kill aphids, and the substrates were sterilized. The seedlings to be transplanted were first washed off the medium, then transplanted into different substrates, and finally covered with plastic film to keep humidity. After 7-10 d when the seedlings survived, the plastic film was removed, and the seedlings were subjected to normal management.

The substrates used included vermiculite, peat, river sand, and vermiculite+peat+perlite (1∶3∶1). There were 100 plants in each plot. Three replications were arranged for each treatment. The substrate of river sand was used as the control. The survival and growth status of the seedlings were observed and recorded 10, 20 and 30 d after transplanting. The effects of different substrates on the survival rate of potato tissue culture seedlings transplanted in the field were compared to screen out the optimal one.

2.2.5Production of pre-elite seed potatoes with three-dimensional spray method. The three-dimensional spray system is composed of a regular spray device and a cultivation tray, in which a nutrient pot, in diameter of 15 cm, was placed for cultivating tissue culture seedlings. The nutrient pots were filled with different substrates for cultivating tissue culture seedings. The cultivation tray had a hole every 15 cm, and a nutrient pot was placed in each hole. The spray device was installed under the tray, and the nutrient solution was sucked by the suction pump, pressurized into spray, and sprayed on the potato roots. Potato roots were covered with black film, which encouraged the potato to form mini-tubers in the dark. A time controller was used to perform spraying regularly. During the day, spraying was performed for 1 min every 10 min, and at night, spraying was performed for 1 min every 15 min. The temperature of the nutrient solution was controlled at 16-20 ℃, and the temperature of the cultivation room was controlled at 16-25 ℃[2].

The tissue culture seedlings were sprayed with nutrient solutions of different formulas. On day 50 and 70 after transplanting, 30 plants were taken to investigate the average tuber number per plant and average single tuber weight and compare the effects of different nutrient solutions on tuber number per plant and single tuber weight.

The nutrient solutions included compound fertilizer (15-15-15) solution (2 kg∶1 000 L), full-amount MS formula nutrient solution, half-amount MS formula nutrient solution, potassium compound fertilizer (18-9-18) solution (2 kg∶1 000 L). The compound fertilizer (15-15-15) liquid (2 kg∶1 000 L) was used as the control.

2.2.6Induction of potato minitubers. In the two-season cropping area, the temperature in summer is high, and if test-tube seedlings are directly transplanted, they will be difficult to grow tubers, and their survival rate will be low. Inducing minitubers, as a replacement of transplanting test-tube seedlings, is a suitable breeding technique for pre-elite seed potatoes.

MS medium was used as the basic medium, which was added with medium with different concentration of hormone. Minitubers were induced in the dark for different lengths of time. There were 10 seedlings in each flask. The medium used was liquid medium that was added with high-concentration sugar. Culture was performed in the conditions with temperature of (25±2) ℃, light intensity of 2 000-2 500 lx, and illumination duration of 12-16 h. After the seedlings grow to about 10 cm high (about four weeks), induction medium sterilized was poured into the flasks. Then, the culture was performed in a dark room with scattered light at 18-20 ℃. After 5-6 weeks, the minitubers were harvested. The tuber number per flask and single tuber weight were measured to screen out the optimal medium.

In the basic medium of MS, different concentrations of hormone were added. Culture was performed in dark for 10 and 20 d, respectively. After about 6 weeks, 30 flasks were taken to investigate the tuber number per flask and average single tuber weight.

3 Results and analysis

3.1 Induction of shoot tip seedlingsAfter observation, 42% of the shoot tips peeled formed induced seedlings on day 60, 48% formed induced seedlings on day 70, 55% formed induced seedlings on day 80, and 59% formed induced seedlings on day 90.

3.2 Virus detection of tissue culture seedlingsOn the basis of visual inspection and indicator plant grafting detection, reagent strip detection method was further used to detect virus. A total of five plants were taken from each treatment, and ground into juice, which was tested by various reagent strips. The virus-free seedlings were retained, while the infected were eliminated.

3.3 Subculture of tissue culture seedlingsSubculture was carried out using 1/2 MS medium. One generation could be bred every 20-30 d, and the multiplication of each generation was 5-10 times.

3.4 Transplanting of test tube seedlingsThe results (Table 1) show that the substrate of vermiculite+peat+perlite (1∶3∶1) had the best effect for transplanting potato tissue culture seedlings. After 10 d of transplanting, the survival rate was 100%. After 30 d of transplanting, the survival rate was 98%. The survival rate of potato tissue culture seedlings transplanted in vermiculite and peat ranked the second. The survival rate of potato tissue culture seedlings transplanted in river sand was the lowest, and management should be strengthened.

3.5 Production of pre-elite seed potatoes with three-dimensional spray methodTable 2 shows that the tuber number per plant of full-amount MS formula nutrient solution was 15 more than that of the control after 50 d of culture, and the average single tuber weight was increased by 2.9 g. After 70 d of culture, the tuber number per plant was increased by 16, and the average single tuber weight was increased by 6.5 g compared with the control. Half-amount MS nutrient solution and potassium compound fertilizer ranked the second and third, and their tuber number per plant and single tuber weight were both lower than those of full-amount MS nutrient solution. As the differences in tuber number per plant and average single tuber weight were not big between full-amount MS nutrient solution and half-amount MS nutrient solution, considering the cost, half-amount MS nutrient solution is recommended in production, in order to save costs, while producing high-quality pre-elite seed potatoes.

Table 1 Effects of different substrates on transplanting of potato tissue culture seedlings

Table 2 Effects of different nutrient solutions on tuber number per plant and single tuber weight

3.6 Induction of minitubersThe duration of dark culture had a greater impact on the induction of potato minitubers. The tuber number per flask on day 20 of dark culture was obviously greater than that on day 10 of dark culture. The use of 6-BA hormone also had a great influence on the induction of potato minitubers. With the increase of hormone concentration, the number of tubers per flask increased significantly, and the single tuber weight decreased slightly, but the difference was not significant (Table 3). Based on the above data analysis, the optimal medium for induction of potato minitubers was MS plus 5-7 mg/L 6-BA. The tuber number per flask was 13.5 and 14.6, respectively, and the single tuber weight was 0.26 and 0.25 g, respectively. The duration of dark culture was about 20 d.

Table 3 Effects of different media on tuber number per flask and single tuber weight

4 Conclusions and discussion

(i) Potato shoot tips were peeled off, and inoculated into medium composed of MS as the basic medium, 2.0 mg/L 6-BA and 1.0 mg/L NAA, and after 90 d of culture, 59% of the shoot tips grew into seedlings.

(ii) After virus detection, virus-free potato tissue culture seedlings were taken for subculture. The multiplication multiple of each generation was 5-10 times. After breeding a large number of virus-free tissue culture seedlings, they were transplanted into the field. The substrate of vermiculite+peat+perlite (1∶3∶1) had the best effect on transplanting potato tissue culture seedlings, and the survival rate of potato tissue culture seedlings transplanted in vermiculite and peat ranked the second. The survival rate of potato tissue culture seedlings transplanted in sand was high in the early stage and low in the later stage, so management should be strengthened.

(iii) The three-dimensional spray method was adopted to produce pre-elite seed potatoes, and it increased the tuber number per plant, up to 55, and reduced viral disease infection. The effects of full-amount MS formula nutrient solution and half-amount MS formula nutrient solution on the production of pre-elite seed potatoes were not too different. Considering the cost factor, half-amount MS formula nutrient solution is recommended in production, in order to producing high-quality pre-elite seed potatoes, while saving costs.

(iv) Inducing minitubers, as a replacement of test tube seedlings, is a better breeding technique for pre-elite seed potatoes. The optimal induction medium of potato minitubers is MS plus 5-7 mg/L 6-BA, with a dark culture duration of about 20 d.