Method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation

Abstract

The invention relates to a method for inducing adventitious buds of hybrid eucalyptus isolated organs through callus tissue differentiation. The method comprises: an axillary bud acquisition step: clipping a bud strip from hybrid eucalyptus, disinfecting the bud strip, and inoculating the disinfected bud strip into a culture medium so as to carry out germination culture to grow axillary buds; a rooting induction culture step: cutting the axillary buds, transferring the axillary buds to an MS (Murashige and Skoog) culture medium so as to carry out successive transfer culture to grow sprouts which meet requirements, cutting the sprouts and transferring the sprouts to a 1 / 2 MS culture medium so as to carry out rooting induction culture to obtain a rooted seedling; a sprout culture step: cutting off the terminal buds of the rooted seedling, reserving 1-4 axillary buds of root and base parts, inoculating the axillary buds onto the MS culture medium so as to carry out sprout culture; a callus tissue induction culture step: inoculating a cut sprout section utilized as an explant onto a callus induction culture medium so as to culture under a dark condition and then under a weak illumination condition, thus obtaining callus tissues; and an adventitious bud differentiation step: transferring the callus tissues onto a differential medium so as to carry out successive transfer culture, thus obtaining the adventitious buds. The method is based on the bud strip of clonal adult eucalyptus, thus the regeneration efficiency reaches about 60%.

Description

technical field

[0001] The invention relates to the technical field of biological tissue culture and seedling cultivation, in particular to a method for inducing hybrid eucalyptus isolated organs to differentiate into adventitious buds through callus. Background technique

[0002] Eucalyptus is a species of Eucalyptus in the family Myrtaceae (Myrtaceae) Eucalyptus) Generic name for plants. It is one of the three fast-growing tree species in the world. Widely distributed in Australia, South America, Asia, Europe and other regions. Eucalyptus is widely used in cellulose industry, pulpwood and fiberboard, and can also be used as a raw material for extracting tannin and essential oil. Eucalyptus extract is used in many fields such as chemical industry and medical treatment, and has been widely used in many countries and regions. to plant. Therefore, eucalyptus has extensive development and utilization value.

[0003] Hybrid eucalyptus plays an important role in the field of...

Examples

example 1

[0037]Select self-collected eucalyptus DH32-29 three-year-old plants, cut off the crown, and keep the 0.5-1.5 meter high stump. After 20-30 days, collect newborn bud strips without axillary buds, cut them into 2.0cm long stems with axillary buds, soak them in 70% ethanol by volume for 20 seconds, wash them twice with sterile water, and then wash them in 0.1% mercuric chloride by mass percentage. Disinfect in medium for 5 minutes, rinse with sterile water 4 times, trim into 1.0 cm long stems with axillary buds, then disinfect in 0.1% mercury chloride for 2 minutes, rinse with sterile water 6 times. After drying, inoculate in MS medium, pick 1-2 seeds per bottle, and culture in dark at 25°C for 28 days to obtain germinated axillary buds.

[0038] Cut the axillary buds about 1.0cm long, transfer to the medium of MS medium + 0.1mg / L 6-benzylpurine + 0.01mg / L naphthaleneacetic acid, transfer to the same medium every 3-4 weeks for subculture Cultivation, light 16h / day, light intens...

example 2

[0043] Select self-collected three-year-old plants of Eucalyptus Guanglin No. 9, except that the medium used in the callus induction culture step contains 1.0 mg / L N-phenyl-N-1,2,3-thiadiazole-5 -Except for the callus induction medium of urea+0.1mg / L naphthaleneacetic acid, the rest of the steps are the same as the corresponding steps of Example 1.

example 3

[0045] Choose the purchased Eucalyptus DH32-29 three-year-old plant, except that the culture medium used in the induction culture step of the callus is to contain 2.0mg / L N-phenyl-N-1,2,3-thiadiazole-5-urea Except the callus induction medium of +0.15mg / L naphthaleneacetic acid, all the other steps are the same as the corresponding steps of example 1.

[0046] The numbers of differentiated calluses obtained in the above three examples are shown in Table 1 below:

[0047] Table 1: Differentiation effects of experimental materials in examples 1-3

[0048] instance number Total number of explants total callus number of differentiated callus Regeneration efficiency (%) Example 1 598 586 358 61.09 Example 2 570 563 336 60.98 Example 3 501 501 306 61.08

[0049] It can be found from the above table that the regeneration efficiency of the present invention reaches about 60%, and it can be applied to excellent strains such as DH32-29 an...

technical field

[0001] The invention relates to the technical field of biological tissue culture and seedling cultivation, in particular to a method for inducing hybrid eucalyptus isolated organs to differentiate into adventitious buds through callus. Background technique

[0002] Eucalyptus is a species of Eucalyptus in the family Myrtaceae (Myrtaceae) Eucalyptus) Generic name for plants. It is one of the three fast-growing tree species in the world. Widely distributed in Australia, South America, Asia, Europe and other regions. Eucalyptus is widely used in cellulose industry, pulpwood and fiberboard, and can also be used as a raw material for extracting tannin and essential oil. Eucalyptus extract is used in many fields such as chemical industry and medical treatment, and has been widely used in many countries and regions. to plant. Therefore, eucalyptus has extensive development and utilization value.

[0003] Hybrid eucalyptus plays an important role in the field of...

Examples

example 1

[0037]Select self-collected eucalyptus DH32-29 three-year-old plants, cut off the crown, and keep the 0.5-1.5 meter high stump. After 20-30 days, collect newborn bud strips without axillary buds, cut them into 2.0cm long stems with axillary buds, soak them in 70% ethanol by volume for 20 seconds, wash them twice with sterile water, and then wash them in 0.1% mercuric chloride by mass percentage. Disinfect in medium for 5 minutes, rinse with sterile water 4 times, trim into 1.0 cm long stems with axillary buds, then disinfect in 0.1% mercury chloride for 2 minutes, rinse with sterile water 6 times. After drying, inoculate in MS medium, pick 1-2 seeds per bottle, and culture in dark at 25°C for 28 days to obtain germinated axillary buds.

[0038] Cut the axillary buds about 1.0cm long, transfer to the medium of MS medium + 0.1mg / L 6-benzylpurine + 0.01mg / L naphthaleneacetic acid, transfer to the same medium every 3-4 weeks for subculture Cultivation, light 16h / day, light intens...

example 2

[0043] Select self-collected three-year-old plants of Eucalyptus Guanglin No. 9, except that the medium used in the callus induction culture step contains 1.0 mg / L N-phenyl-N-1,2,3-thiadiazole-5 -Except for the callus induction medium of urea+0.1mg / L naphthaleneacetic acid, the rest of the steps are the same as the corresponding steps of Example 1.

example 3

[0045] Choose the purchased Eucalyptus DH32-29 three-year-old plant, except that the culture medium used in the induction culture step of the callus is to contain 2.0mg / L N-phenyl-N-1,2,3-thiadiazole-5-urea Except the callus induction medium of +0.15mg / L naphthaleneacetic acid, all the other steps are the same as the corresponding steps of example 1.

[0046] The numbers of differentiated calluses obtained in the above three examples are shown in Table 1 below:

[0047] Table 1: Differentiation effects of experimental materials in examples 1-3

[0048] instance number Total number of explants total callus number of differentiated callus Regeneration efficiency (%) Example 1 598 586 358 61.09 Example 2 570 563 336 60.98 Example 3 501 501 306 61.08

[0049] It can be found from the above table that the regeneration efficiency of the present invention reaches about 60%, and it can be applied to excellent strains such as DH32-29 an...