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A pollen and tobacco technology, applied in horticultural methods, botanical equipment and methods, applications, etc., can solve the problems of difficult absorption of mutagens, small variation range, and high difficulty in screening, so as to overcome obvious and recessive obstacles and mutations The effect of large range and saving breeding time
Inactive Publication Date: 2013-08-14
TOBACCO RES INST OF HUBEI PROVINCE
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However, these mutagenesis treatments have problems such as difficult absorption of mutagen, low frequency of mutagenesis, small variation range, difficulty in offspring screening, low yield and easy generation of chimeras, etc.
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Embodiment 1
[0021] Embodiment 1: Flue-cured tobacco material 1 pollen in vitro liquid culture mutagenesis, the operation steps are as follows:
[0022] (1) Sample collection: Take healthy age-appropriate flower buds on the main inflorescence of the flue-cured tobacco plant, with a length ranging from 2.0 to 2.2 cm, and quickly store them in an ice box at 4-7°C;
[0023] (2) Disinfection treatment: Take out the flower buds in the ice box in step (1), carefully peel off the anthers in the flower buds with tweezers on the ultra-clean workbench, pack the anthers with sterile gauze and immerse them in 70% ethanol for 15 seconds, Immerse in 10% saturated calcium hypochlorite solution for 8 minutes, rinse with sterile water 3 times, 5 minutes each time;
[0024] (3) Collect pollen: Put the anthers sterilized in step (2) into a large round-bottomed test tube, add 2ml of B5-13 liquid medium (the concentration of sucrose in B5 basic medium is 13%), and then add 1-2ml of mixed enzyme solution with ...
Embodiment 2
[0031] Embodiment 2, flue-cured tobacco material 2 pollen isolated liquid culture mutagenesis, its operation steps are as follows:
[0032] (1) Sample collection: Take healthy age-appropriate flower buds on the side inflorescences of flue-cured tobacco plants at the single-nucleus edge stage, with a length range of 2.1-2.3cm, and quickly store the collected flower buds in an ice box at 4-7°C;
[0033] (2) Disinfection treatment: Take out the flower buds in the ice box in step (1), carefully peel off the anthers in the flower buds on the ultra-clean workbench, put the anthers in sterile commercial stockings and immerse them in 75% ethanol for 10 seconds , and then immersed in 10% saturated calcium hypochlorite solution for 10 minutes, rinsed twice with sterile water, 5 minutes each time;
[0034] (3) Collect pollen: transfer the anthers sterilized in step (2) into a large round-bottomed test tube, add 3ml of B5-13 liquid medium (the sucrose concentration in B5 basic medium is 13%...
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Abstract
The invention relates to an effective mutagenesis method for tobacco pollen. The method mainly comprises the following steps: acquiring the tobacco pollen under the aseptic condition; placing the tobacco pollen into a liquid culture medium; mutagenizeing the free pollen in the culture medium for 8-10 hours by utilizing an 0.1-0.2% ethyl methyl sulfomar (EMS) under the condition of keeping out of the sun at the temperature of 30-32 DEG C; configuring for removing the EMS; carrying out chromosome doubling treatment for 50-70 hours by utilizing an N16 liquid culture medium under the condition of keeping out of the sun at the temperature of 30-32 DEG C; configuring for removing colchicine; placing the pollen into an N13 liquid inducement culture medium; culturing an embryo for 14-21days under the condition of keeping out of the sun at the temperature of 24-26 DEG C; carrying out shake culturing for 6-8 days in a shaking bed at the temperature of 24-26 DEG C after the embryo emerges; placing the embryo with cotyledon into a solid culture medium for subculturing; and transplanting the embryo and transplanting the embryo into the field for identifying the mutant characters after refining the seedling management. In the method in the invention, the recessive obstacles expressed on the gene is overcome, thereby being beneficial for selecting, greatly shortening the stable period of the mutant characters, and saving the breeding time; and the method has the characteristics of high efficiency, practicality, great mutant amplitude, and high mutant frequency and the like.
Description
technical field [0001] The invention relates to the technical field of tobacco mutagenesis, in particular to a mutagenesis method for tobacco pollen. Background technique [0002] Tobacco (Nicotiana tabacum L.) belongs to Dicotyledoneae, Tubiflorae, Solanaceae Nicotiana in plant taxonomy, and is an important economic crop in many countries, among which flue-cured tobacco ( 2n=24II) is the tobacco type with the largest planting area in China and the world. [0003] For a long time, due to the excessive use of a few main parents in tobacco breeding, combined with directional selection, the genetic basis of bred varieties is narrow and the phenomenon of single varieties is becoming more and more serious, making it difficult to breed new varieties with greater breakthroughs. The embarrassing situation of "reviewing but not planting" of new varieties often occurs, which also brings great difficulties to the construction of molecular marker genetic linkage maps and gene positioni...
Claims
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