| 摘要 |
The invention discloses a Dendrobium in vitro crossbreeding method. The method can be used to greatly shorten the maturation period of fruits, to enable a hybrid to bloom in vitro in a short period so as to observe flower shapes and colors, and to cultivate a novel variety, thus accelerating Dendrobium breeding, the first such report internationally. Since in vitro Dendrobium blooms annually, it enables crossbreeding of varieties having different inflorescences in nature. In addition, the medium used at each stage of the invention utilizes Hyponex which has a unique composition and costs little, thus allowing for a high blossoming rate and rapid fruit development. Also, only simple plant tissue culture equipment is required for implementing the invention, thus the entire breeding method is simple and low cost, and provides conditions for cultivating of high-quality Dendrobium varieties. |
| 主权项 |
1. A Dendrobium in vitro crossbreeding method, comprising the following steps:
a. aseptically sowing a plurality of seeds obtained from a first plurality of matured fruit of Dendrobium stock plants of different varieties that have been artificially pollinated comprising, rinsing the first plurality of matured fruit several times with sterilized water then cutting the first plurality of matured fruit open inoculating a yellowish-white powder-like embryo contained in the first plurality of matured fruit onto a first seed germination medium with an inoculating needle and developing under culture conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day, allowing germination and forming a plurality of first plantlets with a height of 2-3 centimes; b. in vitro flowering induction and in vitro crossbreeding comprising transferring the plurality of first plantlets with a height of 2-3 centimeters onto a first in vitro flowering induction medium and culturing the plurality of first plantlets under conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day to obtain a plurality of flowering plants, performing artificial interspecific pollination under aseptic conditions between the plurality of flowering plants of different varieties wherein after pollination the plurality of flowering plants are transferred to a first rooting and growth-promoting medium and cultured under conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day, to obtain a second plurality of matured fruit; c. aseptically sowing and in vitro flowering induction of hybrids comprising cutting open the second plurality of matured fruit, and sowing an embryo contained in the second plurality of matured fruit onto a second seed germination medium and culturing under conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day allowing germination and forming a second plantlet, and then transferring the second plantlet onto a second in vitro flowering induction medium and culturing under conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day for flowering induction; hybridized combination to be observed after the in vivo plantlet blooms; d. tissue culturing of new varieties comprising selecting a stem apex or stem node of a desired line obtained in step c and culturing on a protocorm induction and subculture/proliferation medium to induce protocorms and subculture proliferation under culture conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day, allowing protocorm-like bodies to form: transferring the protocorm-like bodies onto a second rooting and growth-promoting medium and culturing under conditions comprising a temperature of 24-28° C., illuminance of 1500-2000 1×, and an illumination period of 12-16 hours per day, to obtain in vitro plantlets; and e. transplanting of the in vitro plantlets that have grown to 3-4 centimeters in height for hardening under natural light and then transplanting the hardened plantlets onto a substrate and culturing to obtain Dendrobium seedlings, wherein the first and the second seed germination medium contains component', per liter: 2-3 grams of Hyponex No. 1, 0.5-2 grams of peptone; 50-100 milliliters of coconut milk; 15-30 grams of sucrose; vitamin elements which are the same as those in a Murashige and Skoog (MS) medium, including myo-inositol; 0.5-1 grams of activated carbon; 6˜8% of agar; and water, the first and the second seed germination medium having a pH between 5.2-5.4, wherein the first and the second in vitro flowering induction medium contains per liter: 1-1.5 grains of Hyponex No. 1; 1-1.5 grams of Hyponex No. 2; 0.1-0.3 grams of casein hydrolysate; 0.5-3.0 milligrams of 6-benzyladenine; 0.1-0.5 milligrams of naphthylacetic acid; 0.1-0.5 milligrams of paclobutrazol; 30-40 grams of sucrose; vitamin elements which are the same as those in the MS medium, including myo-inositol; 6˜8% of agar; and water, the first and the second in vitro flowering induction medium haying a pH between 5.2-5.4, wherein the first and the second rooting and growth-promoting medium contains per liter: 1-2 grams of Hyponex No. 1; 1-1.5 grams of Hyponex No. 2; 0.5-2 grams of peptone; 15-30 grams of sucrose; vitamin elements which are the same as those in the MS medium, including myo-inositol; 0.1-0.5 milligrams of naphthylacetic acid; 50-100 grams of banana juice; 0.5-1 gram of activated carbon; 6˜8% of agar; and water, the first and the second rooting and growth-promoting medium having a pH between 5.2-5.4, and wherein the protocorm induction and subculture/proliferation medium contains per liter: 1-2 grams of Hyponex No. 1; 1-1.5 grams of Hyponex No. 2; 0.5-2 grams of peptone; 50-100 milliliters of coconut milk; 20-30 grams of sucrose; vitamin elements which are the same as those in the MS medium, including myo-inositol; 0.5-2.0 milligrams of 6-benzyladenine; 6˜8% of agar; and water, the protocorm induction and subculture/proliferation medium having a pH between 5.2-5.4. |
