Induction medium: ms+6-ba1mg/l+NAA 0.1mg/l; Proliferation medium: ms+6-ba1mg/l+NAA 0.1mg/l; Rooting medium:1/2 ms+IBA 0.2 mg/L.
Medium treatment: MS medium contains 3% sucrose and 0.7% agar, and its pH is pH5.8. Culture conditions: temperature (25 65438 0)℃, sunlight 65438±04h, illumination intensity 2000Lx.
Selection and sterilization of explants
In tissue culture, we usually choose dynamic meristems such as shoot tips, buds, tender shoots and stem segments as explants, but we should judge the specific parts according to the characteristics of different plants. Because the young shoot tips and leaves of seedlings are easy to pick and post-treat, and callus is easy to form, we choose seedling leaves for tissue culture when selecting tissue culture materials.
Take the leaves or buds of robust and disease-free seedlings as materials. After cutting the material, rinse it with tap water for 1 ~ 2 hours, then soak it in saturated washing solution for 5- 10 minutes, rinse it with sterile water for 1 0-0/5 minutes, remove the surface residue, then soak it in 70% alcohol for 20 seconds in sterile state and rinse it with sterile water. Finally, the explants were washed with sterile water for 4-5 times, 65,438 0-2 min each time, and the water on the surface of the explants was absorbed by absorbent paper, and the leaves were cut into 5mm×5mm sizes, inoculated on the induction medium, and cultured under aseptic conditions. The culture temperature is 24℃ 2℃, the light intensity is 1500lx, and the light intensity is about 10 hour every day.
3. Primary culture of seedlings
It has been known that only under the most suitable conditions can the cultured seedling leaves develop better and germinate faster. I know that under the optimum composition and concentration of culture medium, seedling leaves are more likely to form callus and induce buds; No matter the concentration of medium, it will affect the development of callus and bud of seedlings, and even lead to the death of explants in serious cases.
Therefore, according to the actual situation, we inoculated sterilized leaves or axillary buds on 0.7% agar-solidified MS medium, supplemented with NAA0. 1 mg/L, BA0. 1 mg/L, glucose or sucrose 3%, and adjusted 3% to pH 5.8. Cultured in a culture room at 25 ~ 30℃ with light intensity 1500 ~ 4000 LX and daily light 14 hours. The explants inoculated on the induction medium began to germinate in about 3 weeks. After 1 month, the explants of leaves or axillary buds expanded, and then a large number of callus and buds were formed, and each explant could produce 30 ~ 40 adventitious buds on average.
4. Subculture of seedlings
Primary culture will produce a large number of rootless seedlings, so we should always observe the growth of seedlings. When the plant is full, we need to cut off the seedlings differentiated in the primary culture from the middle and lower parts, and select the well-grown shoot tip tissue for subsequent culture. In this process, firstly, the seedlings of love grass should be extracted and cultivated from test tubes, and the aged leaves and stems should be removed with tweezers. The selecte robust seedling leaves are cut off again, put into subculture medium, and put back into tissue culture room for further culture. We choose 25 days as a cycle, so that a large number of seedlings can be obtained by repeated subculture several times.
We always observe that when 2-3cm aseptic buds are induced to grow, the larger clustered buds are cut off from the primary cultured callus and transferred to the subculture medium, or the smaller clustered buds are directly transferred to the subculture medium. Cluster buds were subcultured with MS basic medium+1 ppm 6-BA as subculture medium. When the buds grow up, they are transferred to MS medium supplemented with NAA0. 1 mg/L and BA0. 1 mg/L, and the buds can further grow into seedlings. In this way, after subculture for 1 month, the clustered buds grow at a speed of 20 ~ 30 times, and a large number of high-quality tissue culture seedlings can be obtained after half a year.
Seedling rooting culture
Because in the process of subculture, the callus of seedlings can only differentiate into adventitious buds and lateral buds, but can not induce the differentiation of roots, and the formation of roots must be induced on rooting medium. Therefore, we should observe the growth of seedlings at any time. When the seedlings in subculture grow 3-5 leaflets, the rootless seedlings should be cut off from the base of the seedlings, taking care not to damage the seedlings, and the seedlings should be transferred to the rooting medium on the aseptic operation table.
Under certain conditions, tissue culture seedlings can be directly transplanted into the substrate, and roots can grow in about 20 days. However, in order to obtain large and robust tissue culture seedlings, rootless tissue culture seedlings can be transplanted into 1/2 ms medium supplemented with 0. 1ppmNAA, and after two weeks, when small roots grow at the base. Such seedlings grow better and faster than the former.
6 hardening and preparation of tissue culture seedlings before transplanting
When the test-tube seedlings are 2 ~ 3 cm high, with 3 ~ 5 leaves and developed roots, they should be transferred to the domestication room and the bottle mouth should be closed for hardening. During this period, the temperature and light in the greenhouse can be properly enhanced. After 7 days of sealing, the bottle cap should be opened to adapt to the external environment, and the bottle should be opened for hardening seedlings. When seedlings are used for training, they are not allowed to open at once. When the seedlings are tempered by the open method, one third of them should be opened on the first day, and one half should gradually adapt to the external humidity after two days. Then open the bottle on the sixth day, and it takes longer to open the bottle and refine the seedlings than to close the bottle. Culture 1 ~ 2 weeks at about 20℃ under the condition of properly reducing air humidity. Two days before transplanting, open the bottle cap, add a little water to soften the agar, then take out the test-tube seedlings and wash off the agar at the base of the test-tube seedlings to avoid breeding mixed bacteria and causing root rot.
Transplanted test-tube seedlings like fertile and loose substrates. The selected matrix formula is that the ratio of peat, mushroom mud and sawdust is 2: 1: 1, and perlite can be added appropriately. Before transplanting, sterilize the substrate in the autoclave for 65438 05 minutes, select a planting tray with the length, width and height of 48×30×5cm, spread the substrate in it, water it and leave it for a period of time, and then plant tissue culture seedlings in it.
7. Transplanting after hardening seedlings
The temperature of transplanted seedlings should be kept at 18℃ ~ 25℃, and the relative air temperature should be about 80%. The survival rate of transplanted seedlings can reach more than 90%. There is no need to fertilize the seedlings at the initial stage of transplanting, and water should be sprayed every 3-5 days. After 20 days of transplanting, the seedlings began to grow rapidly, and at this time, nutrient solution was poured once a week. When the seedlings are long enough to be squeezed into the planting tray, they can be planted in the pot, and the substrate is still selected. In order to fix the root system better, a certain proportion of river sand is mixed into the substrate. The diameter of the selected flowerpot is 6 cm. When the seedlings grow up and the roots are full of flowerpots, replacing flowerpots with the diameter of 10cm is more conducive to the growth and early flowering of seedlings.
8 abstract
Seedling is a popular indoor potted flower in the world. Usually, seeds and leaves with petioles are used for propagation, which not only leads to character separation, seedling degradation and low propagation coefficient, but also takes a long time to cultivate a large number of neat and consistent seedlings. Tissue culture of seedlings is convenient and economical, and successful test-tube seedlings can be obtained in a short time. In this study, the feasible scheme of rapid propagation of seedlings by tissue culture technology was discussed, which provided scientific basis for expanding seedling propagation and obtaining a large number of excellent tissue culture seedlings in a short time. Tissue culture technology of seedlings has unlimited development opportunities. Rational utilization can not only promote regional economic and social development, but also promote the leap-forward development of China's flower industry. The culture conditions we explored laid the foundation for the industrialized production of tissue culture seedlings in the future.