CN106538382B - Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants - Google Patents
Method for establishing efficient eremochloa ophiuroides regeneration system by taking young ears as explants Download PDFInfo
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Abstract
The invention relates to a method for establishing a high-efficiency regeneration system of eremochloa ophiuroides by taking young spikes as explants. The method provided by the invention has the advantages of simple explant disinfection, high callus induction rate and easy implementation, and the method has short callus induction period, high adventitious bud differentiation rate of the obtained callus, easy test-tube plantlet rooting and suitability for modern biotechnology operations such as eremochloa ophiuroides germplasm innovation, genetic transformation and the like.
Description
Technical Field
The invention relates to a method for establishing a high-efficiency regeneration system of eremochloa ophiuroides in-vitro young ear in a way, belonging to the field of plant biotechnology research.
Background
Radix Rhodiolae [ Pseudocentipede ], [ alpha ], [ beta ], [ alpha ], [ betaEremochloa ophiuroides(Munro.) Hack ] is a perennial C4 herbaceous plant of the genus Centipede of the subfamily Gratelou of the Gramineae family, and is the only good warm-season turf plant of this genus that can be used as turf grass. In addition to a small amount of eremochloa ophiuroides in southeast Asia, eremochloa ophiuroides are mainly distributed in the Yangtze river basin and south areas of China, and the central south of China is the natural distribution center of eremochloa ophiudes, which is the best lawn grass recognized at home and abroad and originated from China, so the Chinese lawn grass (Chinese lawn grass) is reputed (Hanna et al 1995). This grass is distinguished by poor resistance to barrenness, low pest damage and low maintenance levels compared to other turf grass and is therefore also known as "lazy grass". Frugal coalThe grass can be widely used for the construction and planting of courtyard lawns, rest lawns and slope protection lawns, is particularly suitable for water and soil conservation and large-area landscape construction (Hanson et al 1969; Hanna 1995), and is one of the three warm season type lawns in the world. However, the eremochloa ophiuroides are sensitive to salt, cold-resistant and poor in drought resistance compared with other lawn grass, and the inherent defects are always bottlenecks which restrict the wide application of the eremochloa ophiuroides; in addition, because eremochloa ophiuroides are originally single and relatively small in distribution range, the genetic basis is narrow. Therefore, the innovation and genetic improvement of eremochloa ophiuroides germplasm resources are fundamental ways to overcome the inherent defects and are also the premise and key for effectively utilizing the resources.
Germplasm innovation based on somatic mutation technology and application of agrobacterium-mediated genetic transformation technology to lawn grass are in the future. However, the low regeneration efficiency restricts the process in breeding eremochloa ophiuroides. It is therefore of great importance to explore high frequency regeneration pathways. The selection of explants is the first step in the establishment of a regeneration system. Although any tissue and organ of a plant body can be used as an explant for establishing a regeneration system theoretically, even used as an explant for gene transformation, the dedifferentiation and redifferentiation capability, the potential tendency of cell totipotency and the competence degree of the explants are greatly different, and even different parts of the same tissue and organ are obviously different. In addition, the search for an optimal regeneration medium is also crucial, and the type, concentration and ratio of hormones play a critical role in the growth and differentiation of the explant.
To date, research on tissue culture and regeneration systems of eremochloa ophiuroides has been carried out for some time, but the research progress is not great. Mature seeds are used as explants in 2004 by the university college of Zhanjiang and university of Hunan agriculture, loose callus is obtained through induced culture for about 30 days, and then yellow compact nodular embryogenic callus is obtained through successive subculture for 60 days. In 2008, the university scholars of south China agricultural uses centipede grass mature seeds as explants, soft and water-like callus is observed to appear after 2-3 weeks of induction and culture, a certain amount of embryogenic callus is obtained after 5 weeks of continuous culture, and the whole callus induction period is as long as 7-8 weeks. A plant research institute of Chinese academy of sciences of Jiangsu province, 2008, 6 months discloses a patent of a lateral bud induction callus and plant regeneration method of eremochloa ophiuroides, wherein a stem section with a lateral bud is adopted as an explant material to induce a callus through 4 weeks (28 days), and the green seedling differentiation rate of the callus at the later stage is lower. In the prior art, the callus is induced by the seeds or lateral buds and then is differentiated into plants, the whole process lasts for a long time, most of the time is more than 3 months, the needed time, manpower and material resources are more, and the callus induction rate and the increment coefficient thereof are low. Therefore, the invention aims to establish a rapid and efficient regeneration system of eremochloa ophiuroides by selecting explants and hormones, thereby providing possibility for creating somatic cell variants and transferring excellent genes, and aiming to realize innovation of eremochloa ophiuroides germplasm resources, improvement of drought resistance, cold resistance and salt tolerance of eremochloa ophiuroides and the like.
Disclosure of Invention
Technical problem
The technical task and the technical problem to be solved by the invention are to overcome the defects of long induction period, low induction efficiency, poor differentiation capability of the callus obtained by induction, being not suitable for micropropagation scale, large-scale screening of somatic mutants, high-efficiency agrobacterium-mediated genetic transformation and the like of the callus in the existing centipede grass in-vitro culture technology. The invention takes the young ear of eremochloa ophiuroides at the beginning of flowering period as an explant material, and granular callus can be obtained in a short time by regulating and controlling exogenous hormone, so that a high-efficiency regeneration technology system suitable for large-scale micropropagation, germplasm innovation and genetic transformation is established.
Technical scheme
The invention mainly provides a method for establishing a high-efficiency regeneration system of eremochloa ophiuroides by taking young ears as explants, the provided method has the advantages of simple explant disinfection, high callus induction rate and easy implementation, the callus induction period is short, the obtained callus has high adventitious bud differentiation rate, and test-tube plantlets are easy to root. The technical scheme is as follows:
1) material
Collecting fresh young spikes (inflorescence) without extracting leaf sheaths at the beginning of the flower season of the eremochloa ophiuroides as explant material;
2) culture medium
The basic culture medium is an MS culture medium consisting of major elements, trace elements and vitamins, and 30.0g/L of sucrose and 8.0g/L of agar are added;
callus induction medium MS 1: 2.0-4.0 mg/L of 2,4-D and 0.1mg/L of BAP are respectively added into the MS minimal medium;
subculture medium MS 2: adding 1.5mg/L of 2,4-D and 0.1mg/L of BAP into a minimal medium;
green shoot differentiation medium MS 3: 2.0mg/L KT and 0.1mg/L NAA are added into a basic culture medium;
test-tube plantlet rooting medium MS 4: adding 0.6mg/L NAA and 0.2mg/L KT into a basic culture medium;
all media were adjusted to pH 5.8 with potassium hydroxide and hydrochloric acid before autoclaving;
3) method of operation
Explant treatment: selecting fresh, tender and robust young ear (inflorescence) without leaf sheath extraction in the initial flowering stage of eremochloa ophiuroides, peeling off outer-layer leaf sheath, soaking in 70-75% ethanol for 35-45s on a super-clean workbench, washing with sterile water for 2-3 times, and then washing with 10% H2O2Sterilizing for 8-10min, washing with sterile water for 3-5 times, and drying on sterile filter paper;
callus induction: cutting the bract of the young ear with sterile knife, clipping with sterile forceps, cutting into 0.5-1.0cm long segments, inoculating into callus induction culture medium MS1, performing callus induction culture for about 1 week;
subculturing the callus: selecting yellow, compact and granular callus at 3 weeks of callus induction culture, transferring to callus subculture medium MS2, performing proliferation subculture, and performing secondary subculture at 3-4 weeks of culture;
green seedling differentiation and test-tube seedling rooting: transferring the callus after 2 successive subcultures to a green seedling differentiation culture medium MS3, performing adventitious bud differentiation and test-tube seedling growth culture, and replacing the culture medium every 2 weeks. Transferring the regenerated plant to a test-tube plantlet rooting culture medium MS4 after 4 weeks, and carrying out rooting culture for 2 weeks;
seedling hardening and transplanting: when the root of the regenerated plant grows to 2.0 cm and the regenerated seedling grows to 5.0-6.0 cm, opening the cap of the tissue culture seedling bottle and hardening the seedling. Taking out the regenerated seedlings after hardening, washing off root culture medium, transplanting the regenerated seedlings into a seedling culture medium, placing the regenerated seedlings in a greenhouse for 2 weeks, and transplanting regenerated plants into a pot or a field of the greenhouse;
the temperature of a culture room is controlled to be 25 +/-1 ℃ in the whole in vitro culture process, callus induction and proliferation subculture are carried out in the dark or scattered light, green seedling differentiation, growth and test-tube seedling rooting culture are carried out under the conditions of 12h illumination and 12h dark alternately and 3000Lux illumination.
Has the advantages that:
compared with the prior art, the invention has the following advantages and positive effects:
1. the invention adopts the young ear (inflorescence) at the beginning of flowering phase as the explant, has simple disinfection operation, is easy to implement, does not need to use toxic reagents such as mercuric chloride and the like, and is safe and environment-friendly;
2. the method provided by the invention greatly improves the callus induction rate, has short time for generating callus, and obviously improves the induction efficiency compared with the existing eremochloa ophiuroides callus induction method;
3. most of the callus obtained by the method provided by the invention is yellow, compact and granular, has high differentiation rate and is easy to root, so the efficiency of obtaining embryogenic callus and establishing a regeneration system is greatly improved;
the invention is suitable for modern biotechnology operations such as germplasm innovation and genetic transformation of eremochloa ophiuroides, and is also suitable for establishment of other lawn grass regeneration systems and subsequent theoretical and application researches on aspects such as germplasm innovation, character improvement and the like.
Drawings
FIG. 1 shows young and fresh ears without the leaf sheaths removed;
FIG. 2 freshly inoculated young ear explants;
FIG. 3 initial induced callus;
FIG. 4 subcultured calli;
FIG. 5 callus differentiated test-tube plantlets;
FIG. 6 rooting of test tube plantlets.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
(1) Experimental Material
Taking a fresh young ear (inflorescence) without extracting leaf sheath from eremochloa ophiuroides seed source E092-1 in the initial flowering stage as an explant material (as shown in figure 1);
(2) culture medium
The basic culture medium is an MS culture medium consisting of macroelements, microelements and vitamins, and 30.0g/L of sucrose (Sigma) and 8.0g/L of agar (Japan) are added;
callus induction medium MS 1: adding 2.0mg/L of 2,4-D (Sigma D-7299) or 4.0 mg/L of 2,4-D (Sigma D-7299) or 6.0 mg/L of 2,4-D (Sigma D-7299) and 0.1mg/L of BAP (Sigma B3408) into MS minimal medium;
subculture medium MS 2: adding 1.5mg/L of 2,4-D (Sigma D-7299) and 0.1mg/L of BAP (Sigma B3408) into a basic culture medium;
green shoot differentiation medium MS 3: 2.0mg/L KT (Sigma K-3378), 0.1mg/L NAA (Sigma N0640), or 2.0mg/L BAP (Sigma B3408), 0.1mg/L NAA (Sigma N0640), or 2.0mg/L CPPU (Sigma C2791), 0.1mg/L NAA (Sigma N0640) was added to the minimal medium;
test-tube plantlet rooting medium MS 4: adding 0.6mg/L NAA (Sigma N0640) and 0.2mg/L KT into a basic culture medium;
all media were adjusted to pH 5.8 with potassium hydroxide and hydrochloric acid before autoclaving;
(3) method of operation
Explant treatment: selecting fresh, tender and robust young ear (inflorescence) of eremochloa ophiuroides E092-1 at the initial flowering stage without leaf sheath, peeling off outer leaf sheath, soaking in 70-75% ethanol for 35-45s on a super clean bench, washing with sterile water for 2-3 times, and washing with 10% H solution2O2Sterilizing for 8-10min, washing with sterile water for 3-5 timesFinally, placing the mixture on sterile filter paper to control the moisture;
callus induction: the young ear with surface sterilization is firstly cut into bracts by a sterile knife, then the young ear is taken out by a sterilized forceps, is cut into small segments with the length of 0.5-1.0cm, is inoculated on a callus induction culture medium MS1 (shown in figure 2) containing different hormones, is cultured under the conditions of 25 +/-1 ℃ and 12 hours of scattered light/12 hours of darkness, and then the callus appears at the 7 th day (shown in figure 3). After 21d of culture, the induction rate of the callus was counted (as shown in Table 1). As can be seen from Table 1, the formation of callus was induced at different hormone concentrations, but the induction rate (healing rate) was greatly different at different gradients. Wherein when the concentration of 2,4-D is 4.0 mg/L, BAP (6-benzylpurine) is 0.1mg/L, the induction rate of the callus is the highest and reaches 76.7 percent. Therefore MS + 4.0 mg/L2, 4-D + 0.1mg/L BAP + 30.0g/L sucrose + 8.0g/L agar, pH =5.8 is the optimal medium for callus induction;
TABLE 1 Induction of callus tissue by different hormone combinations and ratios
Subculturing the callus: selecting yellow, compact and granular callus at the 21d callus induction culture, transferring to callus subculture medium MS2, performing proliferation subculture (as shown in figure 4), wherein the culture room temperature is 25 + -1 deg.C, and the illumination condition is 12 hr scattered light/12 hr dark, and performing secondary subculture at the 21d callus induction culture;
green seedling differentiation: transferring the callus after 2 successive subcultures to a green seedling differentiation medium MS3 containing different hormones, performing differentiation of adventitious buds and growth culture of test-tube seedlings, controlling the temperature of a culture room at 25 +/-1 ℃ in the culture process, alternately treating the illumination for 12h and the darkness for 12h under the condition of 3000Lux light intensity, and replacing the culture medium once at 14d (shown in Table 2). Table 2 shows that the eremochloa ophiuroides callus can induce adventitious buds on differentiation culture media with different hormones, but the adventitious bud differentiation rate and test-tube plantlet growth show significant difference under different hormone proportions. The hormone combination of KT + NAA is selected, the adventitious bud induction and the test-tube plantlet growth are both optimal, namely all callus tissues are successfully induced and differentiated to form adventitious buds, and the test-tube plantlet growth is prolonged to 2.0 cm at the 14 th day (as shown in figure 5). Therefore, the optimal culture medium for green seedling differentiation is MS + 2.0mg/L KT + 0.1mg/L NAA + 30.0g/L sucrose + 8.0g/L agar, and the pH is = 5.8;
TABLE 2 induced differentiation of adventitious buds of callus with different hormone combinations
Rooting the test-tube plantlets: transferring the regenerated plant on the optimal green seedling differentiation medium to a test-tube seedling rooting medium MS4 after 4 weeks, maintaining the culture condition consistent with green seedling differentiation, and performing rooting culture for 14d (shown in figure 6);
seedling hardening and transplanting: when the root of the regenerated plant grows to 2.0 cm and the regenerated seedling grows to 5.0-6.0 cm, opening the cap of the tissue culture seedling bottle and hardening the seedling. And taking out the regenerated seedlings after hardening, washing off the root culture medium, transplanting the regenerated seedlings into a seedling culture medium, placing the regenerated seedlings in a greenhouse for culturing for 14 days, and transplanting the test tube plants into a pot of the greenhouse.
Various other modifications and changes may be made by those skilled in the art based on the above teachings and design considerations, and all such modifications and changes are intended to fall within the scope of the claims.
Claims (5)
1. A method for establishing a high-efficiency regeneration system of eremochloa ophiuroides by taking young spikes as explants is characterized by comprising the following steps:
(1) selecting fresh young ear inflorescences without extracting leaf sheaths in the beginning flowering phase of eremochloa ophiuroides, peeling the outermost leaf sheaths, then sterilizing in a super clean bench, and peeling bracts outside the young ears to obtain sterile young ears;
(2) inoculating the aseptic young ear explant on a callus induction culture medium MS1, and performing induction culture to obtain a callus;
(3) peeling the callus from the explant, transferring the callus to a subculture medium MS2, and performing proliferation subculture;
(4) transferring the callus after subculture to a green seedling differentiation medium MS3 for culture, and performing differentiation induction of adventitious buds to obtain test-tube plantlets;
(5) transferring the differentiated test-tube plantlets to a rooting culture medium MS4, and culturing until the test-tube plantlets are rooted;
(6) hardening the rooted regeneration plant;
(7) after hardening off, taking out the regenerated seedlings, washing off the root culture medium, transplanting the regenerated seedlings into a seedling culture medium, and placing the regenerated seedlings in a greenhouse for culture;
the explant used in the step (2) is cut into sterile young ears with the length of 0.5-1.0cm by using sterile scissors and is cut off; callus induction medium MS1 included: MS culture medium, 2-4mg/L of 2,4-D, 0.1mg/L of BAP, 30.0g/L of sucrose, 8.0g/L of agar, and adjusting the pH value of the culture medium to 5.8 before autoclaving; the induction culture time of the explant in a callus induction culture medium MS1 is 2-3 weeks, and the temperature of a culture room is kept at 25 +/-1 ℃ in the process of in vitro culture and is carried out in the dark or under scattered light;
culturing the callus in the step (4) in a green seedling differentiation culture medium MS3 for 3-4 weeks; the green seedling differentiation medium MS3 includes: 2.0mg/L KT, 0.1mg/L NAA, 30.0g/L sucrose and 8.0g/L agar are added into an MS culture medium, and the pH value of the culture medium is 5.8; in the process of adventitious bud induction differentiation and test-tube plantlet growth, the temperature of the culture room is kept at 25 +/-1 ℃, 12h of illumination and 12h of darkness are alternately treated, and the culture medium is replaced every 10-14d under the condition of 3000Lux illumination.
2. The method of claim 1, wherein the method comprises the steps of: the method for sterilizing young ear without leaving leaf sheath in step (1) comprises sterilizing bract surface wrapping young ear with 70-75% ethanol for 35-45s, washing with sterile water for 2-3 times, and adding 10% H2O2Sterilizing for 8-10min, washing with sterile water for 3-5 times, and placing on sterile filter paperAnd (5) drying.
3. The method of claim 1, wherein the method comprises the steps of: the subculture medium MS2 in the step (3) comprises: adding 1.5mg/L of 2,4-D, 0.1mg/L of BAP, 30.0g/L of sucrose and 8.0g/L of agar into MS culture medium, wherein the pH value of the culture medium is 5.8; the proliferation culture time is 3-4 weeks, and the subculture is carried out for 2 times continuously; the subculture is carried out at a culture room temperature of 25. + -. 1 ℃ in the dark or under scattered light conditions.
4. The method of claim 1, wherein the method comprises the steps of: the rooting medium MS4 in the step (5) comprises: adding 0.6mg/L NAA, 0.2mg/L KT, 30.0g/L sucrose and 8.0g/L agar into MS culture medium, wherein the pH of the culture medium is 5.8, the culture conditions of the culture chamber are completely consistent with those of the green seedling differentiation period, and the time from culture to rooting is 2 weeks.
5. The induction method for establishing a high-efficiency regeneration body of eremochloa ophiuroides by using young ears as explants according to claim 1, which is characterized in that: and (6) opening a tissue culture seedling bottle cover when the roots of the plants to be regenerated grow to about 2cm and the regenerated seedlings grow to 5-6cm, and hardening the seedlings.
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| CN107996400B (en) * | 2017-11-30 | 2021-04-16 | 广西壮族自治区中国科学院广西植物研究所 | Method for inducing adventitious buds of salvia miltiorrhiza by taking anthurium andraeanum inflorescence as explant |
| CN108849528B (en) * | 2018-08-24 | 2020-01-03 | 江苏省中国科学院植物研究所 | Method for obtaining eremochloa ophiuroides mutant |
| CN111826381B (en) * | 2020-04-13 | 2021-09-17 | 江苏省中国科学院植物研究所 | Centipede grass root promoting gene EoSINAT5, plant expression vector and application thereof |
| CN117327712A (en) * | 2023-10-24 | 2024-01-02 | 江苏省中国科学院植物研究所 | Method for regulating and controlling root growth of eremochloa ophiuroides through transferring EoBBR gene |
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| CN101011035A (en) * | 2007-01-25 | 2007-08-08 | 新昌县白云草业研究所 | Seashore paspalum young spike isolated culture strain-reproducing technique |
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