Method for regenerating isolated culture adventive bud evoked plant of larix olgensis

Abstract

A method for in vitro culturing an adventitious bud by a Korean larch to induce a plant regeneration relates to a method for in vitro culturing the adventitious bud by pine to induce the plant regeneration which solves the problems of difficult generation of the tissue culture organs of the Korean larch, low plant regeneration rate and low inducing rate by using a mature embryo, a flower bud and axillary bud, as well as a tender stem segment of the Korean larch as an explant adventitious bud currently. The method relates to: 1. Culturing and inducing a callus by a zygotic embryo; 2. Inducing to generate the adventitious bud; 3. Continuously culturing and inducing to root, namely acquiring the regeneration plant in vitro of the Korean larch. The invention induces the adventitious bud by taking a mature seed of the Korean larch as the explant and acquires the adventitious bud of high inducing rate which is improved by more than 9 times than the currently existing adventitious bud inducing technology and the inducing rate of the callus is all higher than 90 percent, which has a wide application value on the massive expanding propagation and gene engineering breading research of theKorean larch.

Description

technical field

[0001] The invention relates to a method for inducing plant regeneration by in vitro culturing adventitious buds of pine trees. Background technique

[0002] Changbai larch (Larix olgensis) is mainly distributed in my country's Changbai Mountains, Laoyeling and the adjacent northern North Korea and the Russian Far East. It is widely used in construction, decoration, and papermaking. It is one of the important timber tree species in Northeast my country. Changbai larch has a long growth cycle, and it takes a long time to breed new varieties using conventional breeding techniques, and the results are slow. At the same time, artificial breeding also has constraints such as lack of gene sources and hybrid incompatibility. Directly introducing the target gene into the recipient system and expressing it through the transgenic method can greatly shorten the breeding cycle, but the establishment of an efficient tissue culture regeneration system is one of the prerequ...

Examples

specific Embodiment approach 1

[0007] Specific embodiment one: the method for plant regeneration induced by in vitro cultured adventitious buds of Larix longbai in this embodiment is realized by the following steps: one, get the zygotic embryos of larix longbai through pretreatment and inoculate them with additional 6-benzylaminopurine (BA ), inositol, glutamine, hydrolyzed casein, agar and sucrose in the BM medium, the temperature is 24 ± 1 ° C in the environment of dark culture or light culture until the callus appears; 2, the callus Place the temperature in an environment of 24±1°C for light culture until adventitious buds grow; 3. Cut the callus with adventitious buds into pieces and add activated carbon and gibberellin (GA 3 ) in the BM medium for light culture, subculture once after 4 weeks, induce rooting, and obtain the in vitro regenerated plant of Larix longbai; the light culture in steps 1, 2 and 3 is all at 30-40 μmol / m 2 Carry out under the condition of s; the concentration of 6-benzylaminopuri...

specific Embodiment approach 2

[0020] Embodiment 2: The difference between this embodiment and Embodiment 1 is that in step 1, naphthaleneacetic acid (NAA) is added to the BM medium, and the concentration of NAA in the medium is 0.1-0.5 mg / L. Other steps and parameters are the same as those in Embodiment 1.

[0021] The effects of various culture conditions on callus induction (statistics were performed after 4 weeks of light culture in step 2) are shown in Table 2.

[0022] Table 2

[0023] nourish

[0024] illumination

[0025] dark culture

[0026] The induction rates of adventitious buds under various conditions (statistics were performed after 3 weeks of light culture in step 3) are shown in Table 3.

[0027] table 3

[0028] callus source

[0029] dark culture

[0030] The experimental data in Table 3 shows that naphthaleneacetic acid (NAA) has an inhibitory effect on the differentiation of adventitious buds, and the induction rate of adventitious bu...

technical field

[0001] The invention relates to a method for inducing plant regeneration by in vitro culturing adventitious buds of pine trees. Background technique

[0002] Changbai larch (Larix olgensis) is mainly distributed in my country's Changbai Mountains, Laoyeling and the adjacent northern North Korea and the Russian Far East. It is widely used in construction, decoration, and papermaking. It is one of the important timber tree species in Northeast my country. Changbai larch has a long growth cycle, and it takes a long time to breed new varieties using conventional breeding techniques, and the results are slow. At the same time, artificial breeding also has constraints such as lack of gene sources and hybrid incompatibility. Directly introducing the target gene into the recipient system and expressing it through the transgenic method can greatly shorten the breeding cycle, but the establishment of an efficient tissue culture regeneration system is one of the prerequ...

Examples

specific Embodiment approach 1

[0007] Specific embodiment one: the method for plant regeneration induced by in vitro cultured adventitious buds of Larix longbai in this embodiment is realized by the following steps: one, get the zygotic embryos of larix longbai through pretreatment and inoculate them with additional 6-benzylaminopurine (BA ), inositol, glutamine, hydrolyzed casein, agar and sucrose in the BM medium, the temperature is 24 ± 1 ° C in the environment of dark culture or light culture until the callus appears; 2, the callus Place the temperature in an environment of 24±1°C for light culture until adventitious buds grow; 3. Cut the callus with adventitious buds into pieces and add activated carbon and gibberellin (GA 3 ) in the BM medium for light culture, subculture once after 4 weeks, induce rooting, and obtain the in vitro regenerated plant of Larix longbai; the light culture in steps 1, 2 and 3 is all at 30-40 μmol / m 2 Carry out under the condition of s; the concentration of 6-benzylaminopuri...

specific Embodiment approach 2

[0020] Embodiment 2: The difference between this embodiment and Embodiment 1 is that in step 1, naphthaleneacetic acid (NAA) is added to the BM medium, and the concentration of NAA in the medium is 0.1-0.5 mg / L. Other steps and parameters are the same as those in Embodiment 1.

[0021] The effects of various culture conditions on callus induction (statistics were performed after 4 weeks of light culture in step 2) are shown in Table 2.

[0022] Table 2

[0023] nourish

[0024] illumination

[0025] dark culture

[0026] The induction rates of adventitious buds under various conditions (statistics were performed after 3 weeks of light culture in step 3) are shown in Table 3.

[0027] table 3

[0028] callus source

[0029] dark culture

[0030] The experimental data in Table 3 shows that naphthaleneacetic acid (NAA) has an inhibitory effect on the differentiation of adventitious buds, and the induction rate of adventitious bu...