Tissue culture rapid propagation technology
1. Introduction of Mosaic Rock
1.1 Plant name Trachelospermurnjasmlnoides F1ame. Also known as Shilongteng, it is an evergreen vine of the family Apocynaceae. It has long roots and often grows on trees, rocks, and walls; in early summer, white flowers are created in the shape of “million†and have aroma. . Chalcopyrite, with white or creamy yellow spots on the leaves, with a blush. The stems have inconspicuous lenticels, the branches and leaves, and the lower petiole are pubescent, and the old branches and leaves are glabrous. Leaf leathery, elliptic to hatchy with round or wide inverted shape, length 2-6cm, width 1-3cm. Old is near green or light green, the first round of new leaves is red, the majority of each branch is a pair, a small number of 2-3 pairs of pink leaves, the second to third pairs of pure white leaves, between pure white leaves and old green leaves There are several pairs of mosaics, most of the veins are white, with irregular white patches on the leaves, and the white components from the new leaves to the old leaves are gradually reduced.
1.2 Cultivated areas The quarries are naturally distributed in the southeast of China and are distributed in the south of the Yellow River. From the current trials of planting and potting trials in Jinhua and Xiaoshan, Zhejiang Province, the cultivation of colorful ground cover plants and selected plants in the wild can be carried out within their original species. As an indoor potted ornamental plant, it can be used in any area, especially in areas with better lighting conditions.
1.3 Ornamental value and garden use The ornamental value of the karst karst stone lies in its three levels of leaf color, a group of colors consisting of red, pink, pure white, and white and green leaves. It resembles a blooming bunch of flowers. Colorful, colorful. Due to the color rather than the color of the new leaf, its viewing season is all year round, especially in the spring, summer and autumn. Relatively speaking, due to the lack of new leaves in the winter, the ornamental effect is worse. In order to achieve the best color effect, strength trimming is needed to promote sprouting, form compact plant clusters, and promote new leaf growth. In the garden, it is an extremely beautiful ground cover plant, which can be used as a perennial “flowering†plant for a variety of flower paths. At the same time, it is an excellent potted plant material that can replace the current park, modern facilities, potted plants, Overcome the shortcomings of potted flowers, often with high cost disadvantages. In addition, it is also an excellent plant that can be used as a home potted plant.
1.4 The survival rate of the introduction of karstic morifolium was 65%. After experimental breeding, it was found that the kaedo stone can be propagated in any season of the year. The rooting rate in spring and autumn can reach more than 80%. Some of the branches that grow too young are perishable, but those that reach semi-woody branches can survive. However, after multi-generation breeding through vegetative propagation, leaf color degradation is prone to occur, and the color of seedlings is not stable. Through the tissue culture method of propagating Hualeluo Stone technology, mosaic leaves can appear during the tissue culture and the hereditary traits are stable.
2 Material categories New shoots in the year.
3 Culture conditions The basic medium is HS. (1) Induced dormant bud germination medium MS+NAA 0.05mg/L-1 (unit same below) +6-BA2.0; (2) Bud proliferation medium: MS+IAA0.2+6-BA1.5; (3) Long Miao medium: MS+NAA0.5+6-BA0.5; (4) Rooting medium: l/2MS+NAA0.5+IBA0.2. The above medium is supplemented with 20g/L-1 sucrose, 7g-1 agar, PH6.0-6.2; Illuminance 2000-2500lx, light 14h per day; incubation temperature is 25 to 30°C during the day and 15-20 at night °C.
4 Growth and differentiation
4.1 Obtainment of Sterile Materials One-year-old seedlings were selected for growth; the parent strains without disease and insect pests were cut from the tip of spring shoots that had germinated during the year, the leaves were removed, the petioles were kept, and the shoots were cut into 2–3 cm segments. When disinfected, soak in a 10% detergent solution for 15 minutes, then use running water; wash for 30 minutes. Sterilize with 75% ethanol solution under aseptic conditions for 30 seconds, and sterilize with 0.1% mercury solution plus Tween-20 for 3, 5, 10, and 15 min. The sterilizing effect of 0.1% mercury-upgrading solution increases with the prolonging of the sterilization time. The sterilization rate is less than 13.5% if the sterilization time is less than 10 minutes, and the rate of sterile seedlings is 23.7% when the sterilization time is 15 minutes. The body mortality rate reached 33.3%.With the prolongation of the sterilization time, the toxic effect of mercury on plant materials was intensified, and the sterilization time of 0.1% mercury-mercury solution was suitable for 10-15min.Use sterile water; After 5 times, inoculate on MS solid medium.
4.2 Induction of shoots 20 days after inoculation, the aseptic seedlings were transferred to the medium (1). The shoot tips started to germinate on the 5-7th day, buds sprouted on the 10th day, the buds gradually grew, and the shoots continued to grow for 20 days. Germination into lateral branches. Collateral growth is normal, dark green leaves, less callus at the base of the stem, yellow-green.
4.3 Propagation of test-tube seedlings Lateral shoots of test-tube seedlings were divided into 2 to 3 buds and were inoculated on medium (2). After 10 days, the buds of the stem segments sprouted, and they grew into a large number of lateral branches after 30 days, and a large number of twin and triple branches were produced. The inoculated stem segments formed clustered shoots with 5-8 lateral branches with an average proliferation of 5.8 times. The internodes of test-tube seedlings were obviously elongated. The stems of the young leaves were all reddish, the leaves were large, the stems were thick, the tissues were not full, and the callus of the stem base was large. Subsequent subcultures every 30 days, vitrification occurred after 3-4 subcultures.
4.4 Strong Seedling Culture The cultured plantlets are transferred to medium (3) three to four times after each passage. After 40 days, a large number of lateral branches were formed, with an average of 4 to 6 lateral branches per stem. The average length of lateral branches was 1.8 cm, with an average proliferation of 3.1 times. Basically no twin branches or three branches. Lateral branches grow well; D, tissues are full, dark green leaves, small base callus, yellow-green, no vitrification.
4.5 Induction of Roots Lateral shoots with robust growth and length greater than 1.2 cm were divided and transferred to medium (4). Increase the illumination to 2500-3000 lx and extend the illumination time to 16h per day. After 15 days of cultivation, the formation of root primordium began, and the root primordium was formed and the seedlings could be cultivated. The rooting rate was surveyed on the 20th to 25th days. The rooting rate was 73%, and the average number of rooting per plant was 1-2.
4.6 Transplantation of the hardening seedlings The rooting test tube seedlings are brought to the greenhouse for cultivation for 1 week. When the root length 0.5-1 omb inch, open the cap 3-5 days, remove the rooted shoots, wash the root medium, planted in the nursery plug. With black mud as the substrate, the temperature is controlled between 20-25 °C, and the humidity is about 85%-95%. It can survive 20 days and the survival rate is over 73%.
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