CN114097619B - In-vitro conservation method for sugarcane germplasm resources - Google Patents

In-vitro conservation method for sugarcane germplasm resources Download PDF

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CN114097619B
CN114097619B CN202111548059.0A CN202111548059A CN114097619B CN 114097619 B CN114097619 B CN 114097619B CN 202111548059 A CN202111548059 A CN 202111548059A CN 114097619 B CN114097619 B CN 114097619B
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sugarcane
culture
culture medium
stem tip
basal
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CN114097619A (en
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张保青
段维兴
黄玉新
张革民
周忠凤
周珊
杨翠芳
刘俊仙
高轶静
宋修鹏
李杨瑞
陆衫羽
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Guangxi Zhuang Nationality Autonomous Region Academy of Agricultural Sciences
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax

Abstract

The invention relates to an in vitro preservation method of sugarcane germplasm resources, which comprises the following steps: obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem; when the shoot apical meristem is cultured, IBA, IAA and TDZ are added into the culture medium, and white light illumination and red light illumination are combined, so that the proliferation rate is increased, the pollution rate can be reduced, and the quality of non-toxic seedlings for storing sugarcane germplasm resources is ensured; when the stem tips are propagated and cultured into seedlings, the GA3 and TDZ are added into the culture medium, the leaf emergence rate can be improved, and the leaf emergence rate of the green stem tip meristem is more than 95%; proper amount of NAA is added into the rooting culture medium, so that the root system of the sugarcane plantlet is developed, the sugarcane plantlet is convenient to store at low temperature, the in-vitro storage time is prolonged, and the storage time is as long as more than 300 days; and rhizome basal culture is also carried out, and the adaptability of the young seedlings to the outside can be improved after the rhizome basal culture.

Description

In-vitro conservation method for sugarcane germplasm resources
Technical Field
The invention relates to the technical field of sugarcane germplasm preservation, in particular to an in vitro preservation method of sugarcane germplasm resources.
Background
Sugarcane, sugarcane genus, perennial big solid herbs. The root is strong and developed. The height of the stalk is 3-5(-6) m. Taiwan, Fujian, Guangdong, Hainan, Guangxi, Sichuan, Yunnan and other tropical areas in south China. The sugarcane is suitable for being planted in places with fertile soil, sufficient sunlight and large temperature difference in winter and summer. Sugar cane is a temperate and tropical crop, a raw material for sucrose production, and ethanol can be extracted as an energy substitute. The sugarcane contains rich sugar and water, also contains various vitamins, fat, protein, organic acid, calcium, iron and other substances which are very beneficial to the metabolism of a human body, is mainly used for preparing sugar, and the epidermis is generally purple and green with two common colors, namely red and brown, but is rare.
The germplasm resource is genetic material which is directly transferred to offspring through germ cells or somatic cells to determine the characters of the offspring, is a material basis of biological genetic variation and biological diversity, and is a genetic material basis for new variety breeding and improvement. The preservation of germplasm resource is preferably a seed preservation method, and the method has the advantages of being most economical, capable of keeping stable inheritance and the like.
In vitro preservation is a method for storing plant tissue cultures such as single cells, protoplasts, callus, suspension cells, somatic embryos, test-tube plantlets and the like under the condition that the plant tissue cultures inhibit growth or have no growth so as to achieve the purpose of preservation.
At present, a plurality of in vitro conservation methods for sugarcane germplasm resources exist, wherein a common conservation method comprises the following steps: adopting stem tip meristem to detoxify and culture germ plasm resource to ensure that the germ plasm resource is in a juvenile state, and then storing the germ plasm resource at low temperature; the storage method has the problems that the adaptability of young seedlings to the outside is poor, shading treatment is needed during greenhouse seedling hardening, and the seedling hardening time is long.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the in vitro preservation method of the sugarcane germplasm resource, which has long preservation time, stable heredity and high germination rate.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: an in vitro conservation method of sugarcane germplasm resources comprises the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: cutting the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; washing the stem tip with disinfectant, washing the stem tip with sterile water, and sucking water on the surface of the stem tip with filter paper to obtain disinfected stem tip;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.3-0.5 mg/L IBA, 0.12-0.15 mg/L IAA, 0.08-0.12 mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 8-10 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 6-8 hours every day, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip propagation seedling culture medium is an MS basal medium added with 0.25-0.35 mg/L GA3, 0.13-0.15 mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane plantlets subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.25-0.35 mg/L NAA;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a rootstock basal medium for culture to obtain basal-cultured sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 5-8 g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of a bean curd tree;
(6) and (4) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.0-1.2 mg/L6-BA, 0.2-0.3 mg/L NAA, 0.5-0.7 mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 8-10 hours.
Further, 2.5-5 cm of the top end of the sugarcane stem tip is cut in the step (1), and the stem tip is obtained; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with Tween with the mass fraction of 0.3% as a disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip after disinfection treatment.
Further, in the step (2), a shoot apical meristem is obtained from the sterilized shoot tip, and the obtained shoot apical meristem is inoculated into a shoot apical meristem culture medium for culture until the shoot apical meristem turns green, so that a green shoot apical meristem is obtained; the stem tip meristem culture medium is an MS basal culture medium added with 0.35-0.5 mg/L IBA, 0.13-0.15 mg/L IAA, 0.09-0.12 mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 9-10 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 7-8 hours per day, and the red light illumination intensity is 1000 LX.
Further, the green shoot tip meristem in the step (2) is transferred to a shoot tip multiplication seedling culture medium for culture in the step (3), and a sugarcane seedling with the height of 2.5-3.5 cm is obtained through culture; the stem tip proliferation seedling culture medium is an MS basal medium added with 0.28-0.35 mg/L GA3, 0.14-0.15 mg/L TDZ, 10g/L sucrose and 15g/L agar.
Further, in the step (4), the sugarcane plantlets in the step (3) are transferred to a rooting culture medium for culture, so as to obtain sugarcane plantlets subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.28-0.35 mg/L NAA.
Further, 1.2-1.5 mg/L nicotinic acid is added into the rooting culture medium in the step (4).
Further, in the step (5), the sugarcane seedlings subjected to rooting culture in the step (4) are transferred to a rootstock basal medium for culture, so that basal-culture sugarcane seedlings are obtained; the rhizome base culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 6-8 g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of bean curd trees.
Further, in the step (6), the sugarcane seedlings cultured by the base part in the step (5) are transferred to a low-temperature storage culture medium for culture, so as to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.1-1.2 mg/L6-BA, 0.25-0.3 mg/L NAA, 0.6-0.7 mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 9-10 hours.
Further, the method for restoring and culturing the sugarcane seedlings stored at the low temperature in the step (6) comprises the following steps: 1) putting the sugarcane seedlings stored at low temperature into a greenhouse for hardening the seedlings for 1-3 days; 2) the roots of the sugarcane seedlings are cleaned by clean water and then transplanted to a seedbed.
Further, the method for restoring and culturing the sugarcane seedlings stored at the low temperature in the step (6) comprises the following steps: 1) putting the sugarcane seedlings stored at low temperature into a greenhouse for hardening the seedlings for 2-3 days; 2) the roots of the sugarcane seedlings are cleaned by clean water and then transplanted to a seedbed.
The invention has the beneficial effects that: the in-vitro conservation method of the sugarcane germplasm resources can effectively improve the virus detoxification rate and the seedling rate of the nontoxic seedlings for the conservation of the sugarcane germplasm resources, and has the advantages of high propagation speed, short period and stable quality of the nontoxic seedlings; when the stem tip meristem is cultured, the culture medium is added with IBA, IAA and TDZ, and simultaneously white light illumination and red light illumination are combined, so that the propagation rate is accelerated, the pollution rate can be reduced, and the quality of nontoxic seedlings for storing sugarcane germplasm resources is ensured; when the stem tips are propagated and cultured into seedlings, the GA3 and TDZ are added into the culture medium, the leaf emergence rate can be improved, and the leaf emergence rate of the green stem tip meristem is more than 95%; because the growth of the leaves causes a large amount of nutrient consumption, the growth of the root system of the sugarcane plantlets is not facilitated, the sugarcane plantlets need to be subjected to rooting culture, and a proper amount of NAA is added into a rooting culture medium, so that the root system of the sugarcane plantlets is developed, the number of the roots is more than 55, the low-temperature storage is facilitated, the in-vitro storage time is prolonged, and the storage time is as long as more than 300 days; the root and stem base culture is also carried out, partial agar is replaced by the bean curd leaf juice and the gypsum, the root and stem base of the sugarcane plantlet can be enabled to turn green, compared with the sugarcane plantlet which is not subjected to the root and stem base culture, the adaptability of the seedling to the outside can be improved after the root and stem base culture, the greenhouse seedling hardening time is shortened, shading treatment is not needed during seedling hardening, the greenhouse seedling hardening difficulty is reduced, the field planting survival rate of the seedling is improved, and meanwhile, the growth speed of the seedling can be accelerated.
According to the in-vitro preservation method of the sugarcane germplasm resources, the nicotinic acid is also added into the rooting culture medium, so that the longitudinal growth of the root system of the sugarcane plantlet can be promoted, and the root system of the sugarcane plantlet is lengthened.
Detailed Description
The following examples may help one skilled in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
The invention relates to an in vitro preservation method of sugarcane germplasm resources, which is selected from fifth generation seedlings obtained by sugarcane stem tip detoxification culture in a biological room of Guangxi sugarcane research institute.
The invention relates to an in vitro preservation method of sugarcane germplasm resources, which comprises the following culture conditions of stem tip meristem: the temperature is 25 +/-3 ℃, the daily white light illumination time is 8-10 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 6-8 hours every day, and the red light illumination intensity is 1000 LX; it should be explained that: through multiple experiments, experimental results show that the illumination effect of completely adopting white light illumination or completely adopting red light illumination is poor, and the illumination effect of combining white light illumination and red light illumination is optimal. Specifically, in every day, the time of white light illumination and the time of red light illumination are enough, and the problem of sequencing of the time of white light illumination and the time of red light illumination is not needed; namely, the white light illumination can be completed within 8-10 hours every day, and then the red light illumination can be completed within 6-8 hours; or the red light illumination can be completed firstly, and then the white light illumination can be completed; the red light illumination and the white light illumination can also be performed alternately, for example, the red light illumination is performed for one hour, then the white light illumination is performed for one hour, and the steps are repeated until the daily illumination is completed.
The invention relates to an in vitro conservation method of sugarcane germplasm resources, which is characterized in that after rooting culture, rootstock base culture is also carried out, so that the rootstock base of sugarcane seedlings turns green, and the adaptability of young seedlings to the outside is improved; for this purpose, the culture medium used for the root stock culture is changed continuously, so that the root stock of the sugarcane plantlets is changed to be green; the colloid substance formed by the interaction of the bean curd leaf juice and the gypsum can also make the root and stem base of the sugarcane plantlet green, and improve the adaptability of the young plantlet to the outside.
The invention relates to an in vitro conservation method of sugarcane germplasm resources, which is a method for restoring and culturing sugarcane seedlings stored at low temperature and comprises the following steps: 1) putting the sugarcane seedlings stored at low temperature into a greenhouse for hardening the seedlings for 1-3 days; 2) the roots of the sugarcane seedlings are cleaned by clean water and then transplanted to a seedbed.
Example 1
An in vitro conservation method of sugarcane germplasm resources comprises the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: cutting the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with tween of which the mass fraction is 0.3% as disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip subjected to disinfection treatment;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.3mg/L IBA, 0.12mg/L IAA, 0.08mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 8 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 6 hours every day, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip proliferation seedling culture medium is an MS basal culture medium added with 0.25mg/L GA3, 0.13mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane seedlings subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.25mg/L NAA and 1.2mg/L nicotinic acid;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a rootstock basal medium for culture to obtain basal-cultured sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 5g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of a bean curd tree;
(6) and (4) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.0 mg/L6-BA, 0.2mg/L NAA, 0.5mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 8 hours.
Example 2
An in vitro conservation method of sugarcane germplasm resources comprises the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: shearing the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with Tween with the mass fraction of 0.3% as a disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip after disinfection treatment;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.5mg/L IBA, 0.15mg/L IAA, 0.12mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 10 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 8 hours every day, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip proliferation seedling culture medium is an MS basal culture medium added with 0.35mg/L GA3, 0.15mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane plantlets subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.35mg/L NAA and 1.5mg/L nicotinic acid;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a rootstock basal medium for culture to obtain basal-cultured sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 8g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of a bean curd tree;
(6) and (3) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.2 mg/L6-BA, 0.3mg/L NAA, 0.7mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 10 hours.
Example 3
An in vitro conservation method of sugarcane germplasm resources comprises the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: cutting the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with Tween with the mass fraction of 0.3% as a disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip after disinfection treatment;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the shoot apical meristem culture medium is an MS basal medium added with 0.35mg/L IBA, 0.13mg/L IAA, 0.09mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 9 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 7 hours every day, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip proliferation seedling culture medium is an MS basal culture medium added with 0.28mg/L GA3, 0.14mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane plantlets subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.28mg/L NAA and 1.3mg/L nicotinic acid;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a rootstock basal medium for culture to obtain basal-cultured sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 6g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of bean curd trees;
(6) and (4) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.1 mg/L6-BA, 0.25mg/L NAA, 0.6mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 9 hours.
Example 4
An in vitro conservation method of sugarcane germplasm resources comprises the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: cutting the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with Tween with the mass fraction of 0.3% as a disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip after disinfection treatment;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.35mg/L IBA, 0.13mg/L IAA, 0.09mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 9 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 7 hours every day, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip proliferation seedling culture medium is an MS basal culture medium added with 0.28mg/L GA3, 0.14mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane plantlets subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.28mg/L NAA;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a rootstock basal medium for culture to obtain basal-cultured sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 6g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of a bean curd tree;
(6) and (4) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.1 mg/L6-BA, 0.25mg/L NAA, 0.6mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 9 hours.
Comparative example 1
Compared with the example 3, the stem tip meristem culture medium in the step (2) of the sugarcane germplasm resource in-vitro preservation method in the comparative example 1 is an MS basal culture medium added with 0.35mg/L IBA, 0.13mg/L IAA, 10g/L sucrose and 15g/L agar, and the steps of in-vitro preservation of the other germplasm resources are the same as the example 3.
Comparative example 2
Compared with the example 3, the stem tip meristem culture medium in the step (2) of the sugarcane germplasm resource in-vitro preservation method of the comparative example 2 is an MS basal culture medium added with 0.13mg/L IAA, 0.09mg/L TDZ, 10g/L sucrose and 15g/L agar, and the steps of in-vitro preservation of the rest germplasm resources are the same as the example 3.
Comparative example 3
Compared with the example 3, the stem tip meristem culture medium in the step (2) of the sugarcane germplasm resource in the comparative example 3 is an MS basal medium added with 0.35mg/L IBA, 0.09mg/L TDZ, 10g/L sucrose and 15g/L agar, and the steps of in vitro preservation of other germplasm resources are the same as the example 3.
Comparative example 4
Compared with the example 3, the culture conditions of the shoot apical meristem in the step (2) of the sugarcane germplasm resource in the comparative example 4 in vitro preservation method are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 16 hours, and the white light illumination intensity is 800 LX; the procedure for the in vitro preservation of the remaining germplasm resources was the same as in example 3.
Comparative example 5
Compared with the example 3, the in vitro preservation method of the sugarcane germplasm resource in the comparative example 5 has the following culture conditions of the shoot apical meristem in the step (2): the temperature is 25 +/-3 ℃, the daily red light illumination time is 16 hours, and the red light illumination intensity is 1000 LX; the procedure for the in vitro preservation of the remaining germplasm resources was the same as in example 3.
Comparative example 6
Compared with the example 3, the culture medium for the stem tip proliferation and seedling formation in the step (3) of the sugarcane germplasm resource in vitro preservation method of the comparative example 6 is an MS basal medium added with 0.28mg/L GA3, 10g/L sucrose and 15g/L agar; the procedure for the in vitro preservation of the remaining germplasm resources was the same as in example 3.
Comparative example 7
Compared with the example 3, the culture medium for the stem tip proliferation and seedling formation in the step (3) of the sugarcane germplasm resource in vitro preservation method of the comparative example 7 is an MS basal medium added with 0.14mg/L TDZ, 10g/L sucrose and 15g/L agar; the procedure for the in vitro preservation of the remaining germplasm resources was the same as in example 3.
Comparative example 8
Compared with the example 3, the method for storing the sugarcane germplasm resource in vitro in the comparative example 8 does not carry out the rhizome basal culture in the step (5), and the steps for storing the rest germplasm resources in vitro are the same as the example 3.
The following table 1 gives the observation results of the in vitro preservation status of the sugarcane germplasm resources of examples 1 to 4 and comparative examples 1 to 8.
TABLE 1
Serial number Peeling offNumber of stem tips/number Leaf emergence rate/%) Number of seedlings/number Percent seedling rate/%) Number of virus strains/(strain) Detoxification rate/% Root number/root Root length/cm
Example 1 550 96 524 95.27 514 98.09 57 6.8
Example 2 550 96 526 95.64 515 97.91 56 6.9
Example 3 550 98 536 97.45 530 98.88 60 7.2
Example 4 550 96 538 97.82 524 97.40 58 5.4
Comparative example 1 550 94 507 92.18 470 92.70 55 6.5
Comparative example 2 550 96 506 92.00 467 92.29 56 6.7
Comparative example 3 550 94 512 93.09 465 90.82 57 6.8
Comparative example 4 550 96 498 90.55 458 91.97 57 6.7
Comparative example 5 550 94 515 93.64 467 90.68 58 6.6
Comparative example 6 550 86 465 84.55 423 90.97 56 6.8
Comparative example 7 550 84 433 78.73 392 90.53 55 6.9
Comparative example 8 550 96 519 94.36 498 95.95 57 6.5
The calculation method of each item of data in table 1 above is: leaf emergence rate (%) = number of stem tips emerging from stem tip ÷ number of stem tips peeled ÷ 100;
seedling rate (%) = number of seedlings formed ÷ number of stem tips peeled × (100); detoxification rate (%) = number of detoxified strains ÷ number of seedlings × (100);
as can be seen from the observation of the root colors of the seedlings of sugarcane stored at low temperatures in examples 1 to 4 and comparative examples 1 to 8, the root colors of the seedlings of sugarcane in examples 1 to 4 and comparative examples 1 to 7 were all green, and the root color of the seedling of sugarcane in comparative example 8 was white; when the seedlings of the sugarcane stored at low temperature in the examples 1 to 4 and the comparative examples 1 to 8 are subjected to seedling hardening, the root and stem base culture is performed, the root and stem base of the seedlings of the sugarcane can be greened, and compared with the seedlings of the sugarcane which are not subjected to the root and stem base culture, the adaptability of young seedlings to the outside can be improved after the root and stem base culture, the greenhouse seedling hardening time is shortened, shading treatment is not needed during seedling hardening, the greenhouse seedling hardening difficulty is reduced, and the planting survival rate of the young seedlings is improved.
When the sugarcane seedlings stored at low temperatures in examples 1 to 4 and comparative examples 1 to 8 were acclimatized and transferred to a seedbed for cultivation for 15 days, it was found that the seedling height of the sugarcane seedlings subjected to root-stock culture was increased by about 12% as compared with the sugarcane seedlings not subjected to root-stock culture, and thus it was found that the growth rate of young seedlings could be accelerated by root-stock culture.
According to the in vitro conservation method of the sugarcane germplasm resources, IBA, IAA and TDZ are added into a culture medium during the culture of the stem tip meristem, and white light illumination and red light illumination are combined, so that the proliferation rate is increased, the pollution rate is reduced, and the quality of non-toxic seedlings for the conservation of the sugarcane germplasm resources is ensured; when the stem tip is propagated and cultured into a seedling, the GA3 and TDZ are added into the culture medium, so that the leaf emergence rate can be improved, and the leaf emergence rate of the meristem of the green stem tip is over 95 percent; because the growth of the leaves causes a large amount of nutrient consumption and is not beneficial to the growth of the root system of the sugarcane seedlings, the sugarcane seedlings need to be subjected to rooting culture, and a proper amount of NAA is added into a rooting culture medium, so that the root system of the sugarcane seedlings can be developed, the number of roots is more than 55, the sugarcane seedlings can be conveniently stored at low temperature, and the in-vitro storage time is prolonged; nicotinic acid is also added into the rooting culture medium, so that the longitudinal growth of the root system of the sugarcane plantlet can be promoted, and the root system of the sugarcane plantlet is lengthened.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims (9)

1. A method for preserving sugarcane germplasm resources in vitro is characterized by comprising the following steps: the method comprises the following steps of obtaining sugarcane germplasm resources, performing detoxification culture, rejuvenation and low-temperature storage by adopting a stem tip meristem, wherein the storage method comprises the following steps:
(1) taking sugarcane germplasm resources for sterilization: cutting the top end of the sugarcane stem tip for 2.5-5 cm to obtain the stem tip; washing the stem tip with disinfectant, washing the stem tip with sterile water, and sucking water on the surface of the stem tip with filter paper to obtain disinfected stem tip;
(2) shoot apical meristem culture: obtaining a stem tip meristem from the stem tip after disinfection treatment, inoculating the obtained stem tip meristem into a stem tip meristem culture medium for culture until the stem tip meristem turns green, and obtaining a green stem tip meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.3-0.5 mg/L IBA, 0.12-0.15 mg/L IAA, 0.08-0.12 mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 8-10 hours, and the white light illumination intensity is 800 LX; the daily red light illumination time is 6-8 hours, and the red light illumination intensity is 1000 LX;
(3) and (3) stem tip proliferation and seedling culture: transferring the green shoot apical meristem in the step (2) to a shoot apical multiplication seedling culture medium for culture to obtain sugarcane seedlings with the height of 2.5-3.5 cm; the stem tip propagation seedling culture medium is an MS basal medium added with 0.25-0.35 mg/L GA3, 0.13-0.15 mg/L TDZ, 10g/L sucrose and 15g/L agar;
(4) rooting culture: transferring the sugarcane plantlets in the step (3) to a rooting culture medium for culture to obtain sugarcane seedlings subjected to rooting culture; the rooting culture medium is 1/2MS basal culture medium added with 0.25-0.35 mg/L NAA and 1.2-1.5 mg/L nicotinic acid;
(5) rhizome basal culture: transferring the sugarcane seedlings subjected to rooting culture in the step (4) into a root and stem basal medium for culture to obtain sugarcane seedlings subjected to basal culture; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 5-8 g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of a bean curd tree;
(6) and (4) low-temperature preservation: transferring the sugarcane seedlings cultured by the base part in the step (5) to a low-temperature storage culture medium for culturing to obtain the sugarcane seedlings stored at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.0-1.2 mg/L6-BA, 0.2-0.3 mg/L NAA, 0.5-0.7 mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 8-10 hours.
2. The in vitro preservation method for the sugarcane germplasm resources according to claim 1, wherein 2.5-5 cm of the top end of the sugarcane stem tip is cut in the step (1) to obtain the stem tip; wiping the outer surface of the stem tip with 70% alcohol, washing the stem tip with Tween with the mass fraction of 0.3% as a disinfectant, washing the stem tip with sterile water, and finally sucking water on the surface of the stem tip with filter paper to obtain the stem tip after disinfection treatment.
3. The method for in vitro preservation of sugarcane germplasm resources according to claim 1, wherein in the step (2), the shoot apical meristem is obtained from the sterilized shoot tip, and the obtained shoot apical meristem is inoculated into a shoot apical meristem culture medium for culture until the shoot apical meristem turns green, so as to obtain a green shoot apical meristem; the stem tip meristem culture medium is an MS basal culture medium added with 0.35-0.5 mg/L IBA, 0.13-0.15 mg/L IAA, 0.09-0.12 mg/L TDZ, 10g/L sucrose and 15g/L agar; the culture conditions of the shoot apical meristem are as follows: the temperature is 25 +/-3 ℃, the daily white light illumination time is 9-10 hours, and the white light illumination intensity is 800 LX; the red light illumination time is 7-8 hours per day, and the red light illumination intensity is 1000 LX.
4. The method for in vitro preservation of sugarcane germplasm resources according to claim 1, wherein in the step (3), the green shoot apical meristem in the step (2) is transferred to a shoot apical multiplication seedling culture medium for culture, and a sugarcane plantlet with the height of 2.5-3.5 cm is obtained through culture; the stem tip proliferation and seedling culture medium is an MS basal medium added with 0.28-0.35 mg/L GA3, 0.14-0.15 mg/L TDZ, 10g/L sucrose and 15g/L agar.
5. The method for in vitro conservation of sugarcane germplasm resources according to claim 1, wherein in the step (4), the sugarcane plantlets in the step (3) are transferred to a rooting medium for culture to obtain rooting-cultured sugarcane plantlets; the rooting culture medium is 1/2MS basal culture medium added with 0.28-0.35 mg/L NAA.
6. The method for in vitro preservation of sugarcane germplasm resources according to claim 1, wherein in the step (5), the sugarcane seedlings subjected to rooting culture in the step (4) are transferred to a rootstock basal medium for culture to obtain basal-culture sugarcane seedlings; the rhizome basal culture medium is 1/2MS basal culture medium added with 1.2 g/L gypsum, 6-8 g/L bean curd leaf juice and 10g/L agar; the bean curd leaf juice is juice obtained by squeezing fresh leaves of bean curd trees.
7. The method for in vitro preservation of sugarcane germplasm resources according to claim 1, wherein in the step (6), the sugarcane seedlings cultured in the basal part in the step (5) are transferred to a low-temperature preservation culture medium for culture, so as to obtain the sugarcane seedlings preserved at low temperature; the low-temperature preservation culture medium is 1/2MS basal culture medium added with 1.1-1.2 mg/L6-BA, 0.25-0.3 mg/L NAA, 0.6-0.7 mg/L sorbitol and 60mg/L penicillin; the storage conditions are as follows: the temperature is 12 +/-3 ℃, and the daily white light illumination time is 9-10 hours.
8. The method for in vitro preservation of sugarcane germplasm resources according to claim 1, wherein the method for restoring and culturing the sugarcane seedlings preserved at low temperature in the step (6) comprises the following steps: 1) putting the sugarcane seedlings stored at low temperature into a greenhouse for hardening the seedlings for 1-3 days; 2) the roots of the sugarcane seedlings are cleaned by clean water and then transplanted to a seedbed.
9. The method for in vitro preservation of sugarcane germplasm resources according to claim 8, wherein the method for restoring and culturing the sugarcane seedlings preserved at low temperature in the step (6) comprises the following steps: 1) putting the sugarcane seedlings stored at low temperature into a greenhouse for hardening the seedlings for 2-3 days; 2) the roots of the sugarcane seedlings are washed clean by clear water and then can be transplanted to a seedbed.
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