In order to obtain a simple, efficient and low-pollution disinfection method for industrial hemp seeds, industrial hemp seeds of fiber type (Jinma No. 1), medicinal type (Jinma No. 4) and seed type (Fenma No. 3) were selected as experimental materials, and 2 sodium hypochlorite concentrations (5%, 10%) and 5 treatment times (5, 10, 15, 20, 25 min) were set. The effects of sodium hypochlorite on seed germination of industrial hemp were studied with germination rate, germination potential and contamination rate as indicators, and the optimal treatment concentration and time of sodium hypochlorite were determined, thus laying a foundation for the cultivation of bacteria-free industrial hemp seedlings.
Industrial Cannabis (Cannabis sativa L.), also known as hemp, hemp, etc., is a 1-year old upright herb belonging to hemp family, which can be divided into three types according to its uses: fiber type, seed type and medicinal type. At present, fiber type and seed type industrial cannabis are widely used. Industrial hemp fiber is hard, slender, hygroscopic and breathable, and is a textile material with high commercial value. Hemp oil produced from hemp seeds is rich in unsaturated fatty acids (linoleic acid, linolenic acid, oleic acid, etc.), epalin globulin and albumin, which are easily absorbed by human body, and 8 kinds of essential amino acids, and has high edible value. The stem, leaves and inflorescence of medicinal industrial hemp can be used as raw materials for the production of hypnotics and sedatives, especially cannabidiol (CBD) in inflorescence plays an important role in anti-epilepsy, treatment of mental disorders, analgesia and so on. At present, the production and application of industrial hemp in industry are severely restricted due to the weak topsoil ability of the sprouts, inconsistent seed emergence time and uneven size. Based on this, the cultivation of industrial hemp seedlings by tissue culture and rapid propagation technology is helpful to solve the problems of difficult and weak emergence in hemp breeding, so as to accelerate the pace of high yield, high efficiency and high quality of industrial hemp. As a low toxicity and high efficiency disinfectant, sodium hypochlorite not only maintains the disinfection effect, but also does not affect the normal growth of plant tissue because of its low toxicity. Through the continuous exploration and practice of predecessors, the combined sterilization method of ethanol and sodium hypochlorite has been applied in rice, potato, corn, millet, soybean and other crops, and the appropriate concentration and treatment time are combined and matched according to the differences of different species and tissues. In this study, the seeds of three different types of industrial hemp varieties were selected as the research object. Based on previous studies, different combinations of sodium hypochlorite concentration and treatment time were designed, and the optimal conditions for disinfection treatment of the seeds of three industrial hemp varieties were obtained through comparison of germination rate and pollution degree.
Materials and methods
Experimental materials: Industrial hemp seeds such as Jinma No. 1 (fiber type), Jinma No. 4 (medicinal type) and Fenma No. 3 (seed type) were provided by the Cash Crop Research Institute of Shanxi Agricultural University. Sodium hypochlorite and ethanol were supplied by BLUEWAV.
Test methods: Industrial hemp seeds of similar size, full and consistent color were selected for rubbing and washing, and the seeds were laid flat in a dry and ventilated place to dry. 3 300 seeds of each variety were stored in glass bottles away from light. Place the selected seeds in 3 beakers according to variety, add an appropriate amount of 75% ethanol to disinfect them for 2 to 3 min, then rinse them off and set aside. The seeds of various industrial hemp varieties were then disinfected (Table 1).
The disinfection test was set up with 2 sodium hypochlorite concentrations (5%, 10%) and 5 treatment times (5, 10, 15, 20, 25 min), each treating 100 seeds, repeated for 3 times. After disinfection is complete, wash the seeds with sterile water for 3 to 4 times to wash the sodium hypochlorite on the surface. The sterilized seeds were evenly placed in a covered germinating box with 3 layers of wet filter paper (the filter paper and germinating box have been sterilized), and a layer of wet filter paper was covered over the seeds, and the germinating box was placed in a constant temperature light incubator at 25 ℃ with a light intensity of 1 000 lx and 12 h light /12 h darkness. The number of germination and the number of contaminated seeds were recorded every day during the culture period, and the filter paper was kept moist and the contaminated seeds were removed in time. Change the filter paper every 3 days.
Determination indexes and methods: After sowing, observe the germination of seeds and the degree of pollution every day, and count the number of seed germination and pollution. The standard for germination is the germination of the seed radicle, the length of which is twice the length of the seed itself. The third day of the germination rate reaching 50% was used as the first count time; The germination rate reached the highest, and the last count time was the 7th day when no seed germination appeared.
Results: Different sodium hypochlorite concentration and treatment time had effects on germination rate, germination potential and contamination rate of Jinma No. 1 seed. Under 5% sodium hypochlorite concentration, the germination rate and germination potential of seeds increased first and then decreased with the prolongation of treatment time. Under the condition of 10% sodium hypochlorite concentration, the germination rate and germination potential of seeds decreased gradually with the extension of treatment time. Under the same treatment time, the effect of 10% sodium hypochlorite on seed germination was more obvious than that of 5% sodium hypochlorite. The germination rate and germination potential of Jinma No. 4 seed increased gradually with the increase of sodium hypochlorite concentration and the prolongation of treatment time. On the contrary, the higher the concentration of sodium hypochlorite, the longer the treatment time, the lower the pollution rate. When 10% sodium hypochlorite was treated for 25 min (D5 treatment), the germination rate and germination potential of Jinma No. 4 seeds were the highest. When the concentration was 5%, the germination rate and germination potential of Fenma No. 3 seed increased first and then decreased with the extension of treatment time. When the concentration of sodium hypochlorite was 10%, the germination rate and germination potential showed a gradually decreasing trend. At 5% and 10% sodium hypochlorite concentration, the pollution rate decreased slowly with the extension of treatment time.
As an important disinfectant, sodium hypochlorite treatment of seeds is not only the process of disinfection, but also the process of seeds being stimulated by the outside world, which is bound to have an important impact on seed germination and subsequent growth of bacteria-free vaccine, and the growth of bacteria-free vaccine directly affects the transformation of exogenous genes. Therefore, it is necessary to study the effect of sodium hypochlorite on seed germination and bacteria-free vaccine growth. The BLUEWAV sodium hypochlorite generator allows you to use it for energy efficient industrial production, not just hemp seed germination. Generally speaking, a longer disinfection time can reduce the contamination rate, but it will cause a certain degree of damage to the seeds and affect the viability of the seeds. Therefore, to select the appropriate sodium hypochlorite concentration and treatment time, on the one hand to reduce the bacterial infection rate, on the other hand to prevent sodium hypochlorite disinfection time is too long, inhibit seed germination, reduce germination rate. It is particularly important to select a moderate sterilization condition between the rate of infection and the rate of germination. In this study, it was found that the germination rate of Fenma 3 seed for seed use was the highest, Jinma 4 seed for medicinal use was the lowest, and Jinma 1 seed was in the middle. Therefore, the reason for the result may be related to the characteristics of hemp seeds themselves. When sodium hypochlorite concentration and treatment time reached a certain level, the toxic effect of sodium hypochlorite on seeds began to appear, and the germination rate and germination potential of seeds were inhibited. In this study, it was found that long-term high concentration of sodium hypochlorite promoted the germination of Jinma No. 4, or that the seeds of Jinma No. 4 showed significantly different resistance to the external stimulation of high concentration of sodium hypochlorite for a long time than Fenma No. 3 and Jinma No. 1, and the reasons for the resistance need to be further studied. In this process, the seed coat color of three different types of industrial hemp seeds appeared to be pale, especially the medicinal variety Jinma 4, which may be due to the fading effect of sodium hypochlorite on the seed coat.
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The article is from the Journal of Shanxi Agricultural Sciences