Experimental Study on Factory-Scale Rapid Propagation Technology of Potato Test-Tube Plantlets

For many years, farmers' own propagation, self-breeding and seed retention have caused potato yields to decline year by year during production, quality to deteriorate, and commercial appearance to worsen. Scientists have proven through research that virus infection and its accumulation in tubers are the main causes of potato "degeneration." To solve the problem of severe degeneration and reduced yield of the main potato cultivars in our city, our institute made virus-free potato production a key experimental project and conducted experimental research on the problems existing in virus-free production, thereby utilizing resources, reducing production costs, providing a scientific theoretical basis for increasing potato yield and income, and powerfully accelerating the modernization process of virus-free potato production in our city.

1 Experimental Overview

1.1 Experimental Materials The virus-free plantlets were provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The cultivars were mainly the main cultivars of our city, Kexin No. 1 and Kexin No. 3, with a small number of introduced Kexin No. 13, Bashu No. 9, and Bashu No. 10, as well as observational trial cultivars such as Favorita and Atlantic.

1.2 Experimental Site The experiment was set up in the plant tissue culture and factory seedling workshop of the Municipal Institute of Agricultural Sciences, equipped with air conditioning to regulate temperature changes.

1.3 Experimental Dates In March 2004, virus-free plantlets were purchased; in May, after the laboratory instruments were purchased and returned, small-scale subculturing began; by the end of July, large-scale production was carried out.

2 Experimental Methods

The process flow of this experimental production was: medium preparation - bottling - sterilization - transfer - culture.

2.1 Medium Preparation The medium required for potato test-tube plantlets is MS medium. This medium is characterized by high concentrations of inorganic salts and ions, is a relatively stable ion-balanced solution, and its nutrient quantity and proportion can meet the nutritional and physiological needs of plant cells. (1) MS medium formula: includes macronutrients, micronutrients, iron salts, organic substances, and others (agar, sugar, sterilized water); (2) Stock solution preparation: To make weighing of various ingredients convenient and accurate, reduce errors and tediousness, generally various reagents are expanded 100-fold, weighed, and prepared into stock solutions for later use. Each time the medium is prepared, only 10 ml/L of stock solution needs to be measured out to make the medium. (3) Medium pH adjustment: After various ingredients are prepared, use pH test paper or a pH meter to measure the pH value. The pH required for potato test-tube plantlets is 5.8-6. Then heat the agar until it is completely dissolved.

2.2 Bottling The medium was put into 7 x 7 x 10 cm square colorless glass bottles with four screw mouths. Take care not to let the medium adhere to the mouth of the culture bottle. Generally, the amount of medium should be enough to support each stem segment. After filling, seal the mouth with breathable sealing film.

2.3 Sterilization Sterilization used a high-pressure steam sterilizer, with the temperature controlled at 121°C (tolerance ±1°C) and the time set at 18-20 minutes. After sterilization, slowly release air to normal pressure, or allow natural cooling. Open the lid and place the sterilized culture bottles on the workbench to cool before use.

2.4 Transfer This was carried out on the ultra-clean workbench in the inoculation room. The transfer material was introduced virus-free pre-basic seed. The transfer tools included scissors, tweezers, an alcohol lamp, 75% alcohol, etc., and the procedure was carried out completely in accordance with aseptic operating procedures. During transfer, first cut the seedling material into stem segments 8-10 mm long with 1-2 axillary buds. Insert the stem segments vertically into the sterilized culture bottle with the medium surface according to the plant's original up-down orientation (be sure to distinguish up and down clearly), and note that the bud eye at the leaf base must not be inserted into the medium. Each bottle can evenly hold 15-25 new stem segments, with 20 plants being best, which is conducive to space utilization in the bottle and seedling growth. After insertion, seal with the original sealing film and mark the cultivar name, transfer date, transfer operator, etc., and record the original file properly in the archive record book.

2.5 Culture The conditions required for culture were: temperature generally 20-25°C; for light, fluorescent lamps were used as the light source, with 2 40 w fluorescent lamps installed on each culture rack at a distance of 30 cm from the rack surface, reaching a light intensity of 2000 lx, basically meeting the growth requirements of virus-free potato plantlets.

3 Experimental Conclusions and Analysis

3.1 Growth Conditions After the experimental materials were transferred on the ultra-clean workbench, according to observation, under conditions of 18-20°C, 2000 lx light, and sealed aseptic conditions, the apical buds began to grow after 2 days, rooting began after 3 days, and after 22-25 days they could grow to 5-6 leaves and 6-7 cm high, thus becoming plantlets. After transfer using stem segments, growth began after 3 days, rooting began after 5 days, and they could become plantlets after 25-30 days. Moreover, at 18-20°C, the plantlets were strong with large leaves. At about 25°C, generally sprouting began after 2 days, rooting began after 3 days, and they could become plantlets after 20 days, with slender and weak plantlets and small leaves. At 28-30°C, after transfer the buds began to root after one day, with few roots, and it was difficult to form plantlets, with many deformities. The stems became thicker, the apical buds necrotized, there were no leaves, and fibrous roots grew. Observation conclusion: test-tube plantlets had a high plantlet formation rate when cultured at about 20°C.

3.2 Contamination Conditions According to statistical analysis, in the early-stage subculturing and winter production of test-tube plantlet production, the contamination rate was about 4.4%-5.7%, while in the mid-stage high-temperature season of August-September, the contamination rate increased to about 15%, and most of it was bacterial contamination. The conclusion is: the production period of potato test-tube plantlets needs to avoid the high-temperature season.