CN112189562B - Method for inducing callus to regenerate plant by bamboo joint - Google Patents
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Abstract
The invention provides a method for regenerating plants by induction of callus through bamboo joints, and belongs to the technical field of plant tissue culture. The method comprises the steps of cutting a sterilized test-tube seedling into bamboo joints with buds, wherein the bamboo joints with the lengths of 0.1-8 cm are used as explants, and inoculating the bamboo joints to an MS culture medium containing 1-3 mg/L2,4-D for induction culture to obtain callus; inoculating the callus onto an enrichment culture medium for enrichment culture, and inoculating the obtained compact-particle callus onto an MS culture medium containing 0.5-2 mg/L6-BA, 2-4 mg/LKT, 1mg/LZT and 0.5-1 mg/LTDZ for differentiation culture to obtain a regenerated seedling. According to the invention, plant growth regulating substances are screened, bamboo joint size and position, culture period and the like are optimized and screened, a callus induction and differentiation system of bamboo joints is promoted, a high-efficiency regeneration system is established, and technical support is provided for establishing a floral-leaf vector bamboo transgenic technical system and directionally improving and culturing new bamboo species.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for regenerating plants by bamboo joint induced callus.
Background
The bamboos have special flowering habits, long flowering period and difficult prediction, and the bamboos usually die after flowering and have low fruiting rate (forest tree swallows and the like, 2008), so that the bamboos are difficult to breed and breed new varieties through a sexual propagation approach; the asexual propagation causes high afforestation cost due to factors such as difficult long-distance transportation, low transplanting survival rate, limited rooting capacity and seasonal dependence. The tissue culture has the advantages of high propagation speed, high propagation coefficient, low cost and the like, is not limited by seasons, and is widely applied to in-vitro rapid propagation of plants (Shenchu, 2018; Tan hong super and the like, 2017). The genetic breeding improvement of the plants can be realized by a genetic transformation technology, but the establishment of a stable and efficient regeneration system is a prerequisite for the success of the genetic transformation technology. The current reports on genetic transformation are limited to Dendrocalamus latiflorus (YES et al, 2017), Dendrocalamus malabaricus (Dendrocalamus asper) (geffy, 2018) and pleione bulbocodioides (dendorocalamus farinosus) (dawn et al, 2012), mainly because of the difficulty in establishing a bamboo regeneration system. In 1968, Alexander and the like successfully obtain a complete plant by taking mature seed embryos of bamboos as explants by utilizing an in vitro culture technology for the first time. Then, the tissue culture technology of bamboo is gradually developed, and at present, nearly 80 bamboo species obtain regeneration plants (tangram, etc., 2016). Metha et al (1982) first reported the use of somatic embryos of India bamboo (Bambudinaceae) to establish their regeneration system, and then studied various explants to establish their regeneration system, including seed-explanted bamboo (Dendrocalamus striatus) (RAO et al, 1985), Mexico bamboo (Otatea acuminata) (Woods et al, 1992), Dendrocalamus latiflorus (Sinocola latifolia) (Yeh et al, 1987), Dendrocalamus domestica (Dendrocalamus hatonii Nees Arn. exMunro) (Zhang, 2010), Bambusa vulgaris (Bambu multiple) (Yuan2009), and Dendrocalamus malabaricus (Dendrocalamus nigra) 2018; dendrocalamus latiflorus (d. latiflorus) with anther as explant (Tsay et al.1990); examples of the explant include those of Bambusaoldhami (Yeh et al 1986), Bambusa multiplex (Yuanhing et al 2009), and Phyllostachys bambusae (Bambusa beecheyana) (Yeh et al 1986) in which young inflorescence is used as an explant; dendrocalamus hamiltonii Neeset arn. ex Munro (Zhang Ning et al, 2016), green bamboo (Bambusa oldhami), green bamboo (Sasa pygmaea), Phoenix bamboo (Bambusa multiplex), and Arhat bamboo (phyllostachys aureus) with buds as explants (Huang et al, 1988). However, due to the specific flowering characteristics of bamboo, seeds and anthers are difficult to obtain, and the materials cannot be obtained at any time. The bamboo joint intermediate meristem has sufficient material and can be obtained at any time, and the explant selection has great advantages.
The reports on the bamboo callus regeneration system are still few, only a few sympodial bamboos and only one scattered bamboo species phyllostachys pubescens are concentrated on the callus regeneration system of the mixed bamboo species at present, the main difficulties are concentrated on the selection of a few explants capable of regeneration, the induction rate of effective callus is low, the differentiation of callus is difficult, the bamboo species regeneration frequency is very low and cannot be compared with other gramineous plants or model plants, and the research process of gene function verification is greatly limited.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for inducing callus to regenerate plants by using bamboo joints, wherein the callus is induced by using bamboo joints as explants and matching with a unique culture medium formula, so as to obtain the regenerated plants with high induction rate and high differentiation rate.
The invention provides a method for inducing callus to regenerate plants by bamboo joints, which comprises the following steps:
1) cutting the bud-bearing bamboo joints of the disinfected test-tube seedlings as explants, and inoculating the explants to a callus induction culture medium for induction culture to obtain callus;
the callus induction culture medium contains 1-3 mg.L-12,4-D MS medium;
the length of the bamboo joint with the buds is 0.1-8 cm;
2) inoculating the callus onto a proliferation culture medium for proliferation culture to obtain compact-particle callus; the proliferation medium contains 0.5-1 mg.L-12,4-D MS medium;
3) inoculating the callus with compact particles to a differentiation culture medium for differentiation culture to obtain a regenerated seedling;
the differentiation medium contains 0.5-2 mg.L-16-BA、2～4mg·L-1KT、0.5～1mg·L-1ZT、0.5～1mg·L-1MS culture medium of TDZ.
Preferably, the length of the bamboo joint with the bud is 1-8 cm.
Preferably, the bamboo joint with buds comprises a base bamboo joint with buds and the length of the base bamboo joint is 5-8 cm.
Preferably, the callus is grown for 14 to 21 days and then subjected to propagation culture.
Preferably, after the propagation culture, subculture is further performed; the number of subcultures is 1-2.
Preferably, the test-tube plantlet comprises mixed-growth type bamboo species.
Preferably, the symbiotic bamboo species comprises a floral leaf arrowroot (Pseudosasa japonica f.
Preferably, the temperature of the induction culture or the proliferation culture is 23-27 ℃; the light condition of the induction culture or the proliferation culture is dark culture; the induction culture or proliferation culture is carried out in a dry and sterile environment.
Preferably, the light-dark period of the differentiation culture is 16h/8 h; the illumination intensity is 2000-2500 Lux;
the temperature of the differentiation culture is 23-27 ℃;
the differentiation culture is carried out in a sterile culture room.
The invention provides a method for inducing callus to regenerate plants by bamboo joints, which comprises the following steps:
1) cutting the bud-bearing bamboo joints of the disinfected test-tube seedlings as explants, and inoculating the explants to a callus induction culture medium for induction culture to obtain callus; the callus induction culture medium contains 1-3 mg.L-12,4-D MS medium; the length of the bamboo joint with the buds is 0.1-8 cm; 2) inoculating the callus onto a proliferation culture medium for proliferation culture to obtain compact-particle callus; 3) inoculating the callus with compact particles to a differentiation culture medium for differentiation culture to obtain a regenerated seedling; the differentiation medium contains 0.5-2 mg.L-16-BA、2～4mg·L-1KT、1mg·L-1ZT、0.5～1mg·L-1MS culture medium of TDZ. According to the invention, through researching the influence of different plant growth regulators on callus induction, the treatment only by adding 2,4-D is more suitable for induction of callus by bamboo joints, and the concentration of 2,4-D is 1-3 mg.L-1The method has high callus induction rate and is obviously different from other concentration treatment. Proliferating and culturing callus to obtain great amount of callus material for differential culture, and screening callus differential culture medium with 0.5-2 mg/L content-16-BA、2～4mg·L- 1KT、1mg·L-1ZT、0.5～1mg·L-1After being cultured on the MS culture medium of TDZ, the callus has good differentiation condition, the callus is in faint yellow, the buds grow into clusters or single buds, the buds are stronger, cluster buds can grow, the differentiation rate is higher, and the callus is obviously different from other treatment groups. Therefore, by optimally screening plant growth regulating substance combinations, bamboo joint lengths, culture periods and the like, induction and differentiation systems of bamboo joint callus are promoted, a high-efficiency regeneration system is established, and technical support is provided for further establishing a floral leaf cornus transgenic technical system and directionally improving and culturing new bamboo species.
Furthermore, the invention specifically limits the position of the bud bamboo joint of the explant. The experiment of the invention shows that the positions of different bamboo joints have obvious influence on the induction rate of the callus. Compared with the top bud bamboo joint and the middle bud bamboo joint, the induction callus rate and the compact callus rate of the base bud bamboo joint are both the highest and have obvious difference.
Furthermore, the invention specifically limits the specific length of the bud bamboo joint of the explant. The experiment of the invention shows that the lengths of different bamboo joints have obvious influence on the induction rate of the callus. Compared with the bud bamboo joints with the length of 1-3 cm and the bud bamboo joints with the length of less than 1cm, the induction callus rate and the compact callus rate of the bud bamboo joints with the length of more than 5cm are both the highest and are obviously different.
Further, the invention specifically limits the culture time of the callus during propagation culture. Experiments show that when the generation time of the callus is 14 d-21 d, the growth condition of the callus is good, the generation of compact granular callus is facilitated, the generation time is too long (28d) or too short (7d), the multiplication times are low, the shape of the callus is not good, the influence is visible, and different generation times influence the growth condition of the callus.
Further, the present invention specifically limits the number of subcultures. Experiments show that the subculture frequency of the callus also has an influence on the proliferation of the callus. When the callus is subcultured for 0 time, the multiplication factor is 1.31, which is obviously lower than that of other treatments, and the callus at the time is mostly milky white and is not suitable for differentiation. When the subculture frequency is 2 times, the multiplication times are the highest and reach 4.42, which is obviously higher than other treatments, and the callus at the time is mostly dense granular faint yellow callus, has strong differentiation capability, and is most suitable for callus differentiation tests.
Drawings
FIG. 1 is a graph of the effect of 2,4-D on callus induction;
FIG. 2 is the effect of 6-BA on callus induction;
FIG. 3 is the effect of NAA on callus induction;
FIG. 4 shows the browned callus morphology;
FIG. 5 shows the root callus morphology;
FIG. 6 shows the effect of bamboo joints with different lengths and positions on callus induction;
FIG. 7 is a morphological diagram showing the gradual appearance of green spots in callus after differentiation culture;
FIG. 8 is a diagram showing the morphology of a differentiated seedling gradually growing after differentiation culture;
FIG. 9 is a morphological diagram showing the growth of differentiated seedlings in clusters.
Detailed Description
The invention provides a method for inducing callus to regenerate plants by bamboo joints, which comprises the following steps:
1) cutting the bud-bearing bamboo joints of the disinfected test-tube seedlings as explants, and inoculating the explants to a callus induction culture medium for induction culture to obtain callus;
the callus induction culture medium contains 1-3 mg.L-12,4-D MS medium;
the length of the bamboo joint with the buds is 0.1-8 cm;
2) inoculating the callus onto a proliferation culture medium for proliferation culture to obtain compact-particle callus; the proliferation medium contains 0.5-1 mg.L-12,4-D MS medium;
3) inoculating the callus with compact particles to a differentiation culture medium for differentiation culture to obtain a regenerated seedling;
the differentiation medium contains 0.5-2 mg.L-16-BA、2～4mg·L-1KT、0.5～1mg·L-1ZT、0.5～1mg·L-1MS culture medium of TDZ.
The invention cuts the bud bamboo joints of the disinfected test-tube seedlings as explants, and inoculates the explants to callus induction culture medium for induction culture to obtain callus.
The present invention is applicable to all bamboo species, especially to the mixed growing type bamboo species, and in order to specifically illustrate the tissue culture method and the effect obtained by the method, the embodiment of the present invention is illustrated by using the floral leaf sago bambusoides (pseudosasa japonica f. akebonouji) as an example, but it should not be construed as limiting the scope of the present invention.
The method of sterilization according to the present invention is not particularly limited, and a method of sterilizing explants known in the art may be used. The length of the bamboo joint with the buds is 0.1-8 cm, preferably 0.1-1 cm, 1-3 cm or 5-8 cm, and most preferably 0.1-1 cm. The bamboo joints with buds can be divided into bamboo joints with buds at the top ends, bamboo joints with buds at the middle parts and bamboo joints with buds at the base parts according to positions. The bamboo joint with buds at the top is a bamboo joint which is cut from the top of the test-tube plantlet with a certain length. The bamboo joint with the bud at the base part is a bamboo joint with a certain length which is cut and extended upwards from the stem part connected with the root. The bamboo joint with the buds is preferably a bamboo joint with the base part and the length of 5-8 cm, and the bamboo joint with the buds is cultured as an explant, and the induced callus rate and the compact callus rate are both 93.33% and 36.67%.
In the present invention, the callus induction medium preferably contains 1 mg.L-1MS culture medium of 2, 4-D. When the concentration of 2,4-D is 1 mg.L-1In the process, the callus induction rate is 55%, and the compact granular callus induction rate is 26.67%, so that the method has obvious difference with other treatments.
In the present invention, the temperature of the induction culture or the propagation culture is preferably 23 to 27 ℃, and more preferably 25 ℃. The light condition of the induction culture or the proliferation culture is preferably dark culture; the induction culture or proliferation culture is carried out in a dry and sterile environment.
After obtaining the callus, inoculating the callus to a proliferation culture medium for proliferation culture to obtain compact-particle callus; the proliferation medium contains 0.5-1 mg.L-1MS culture medium of 2, 4-D.
In the invention, the callus is preferably grown for 14-21 d and then is subjected to propagation culture, and more preferably, the callus has the highest reproductive rate of 2.48 in 21d production and 1.95 times of 7d treatment.
In the present invention, after the propagation culture, it is preferable to further include performing a subculture; the number of subcultures is 1-2.
In the invention, the light-dark period of the differentiation culture is preferably 16h/8 h; the illumination intensity is preferably 2000-2500 Lux, and more preferably 2000 Lux; the temperature of the differentiation culture is preferably 23-27 ℃, and more preferably 25 ℃. The differentiation culture is carried out in a sterile culture room.
In the invention, the differentiation medium contains 0.5-2 mg.L-16-BA、2～4mg·L-1KT、1mg·L- 1ZT、0.5～1mg·L-1TDZ in MS medium, most preferably 0.5 mg.L-16-BA、2mg·L-1KT、1mg·L-1ZT、0.5mg·L-1MS culture medium of TDZ. The most preferable differentiation culture medium has the highest number of green spots, the highest callus differentiation rate can reach 50.00%, and 0.5 mg.L-16-BA、2mg·L-1KT、1mg·L-1ZT、0.5mg·L-1The MS culture medium of TDZ shows green spots at first, and the callus differentiation rate reaches 43.33%.
The method provided by the invention is a method for inducing callus and differentiating cluster buds by combining a specific bamboo species type and an explant type with a unique culture medium formula so as to obtain a regenerated plant with high induction rate, high differentiation rate, simple formula and low cost. Firstly, typical underground stem mixed-growing type bamboo species flower and leaf cornflower is taken as a representative, most researches on regeneration of bamboo callus at present are concentrated in research and exploration of sympodial bamboos and scattered bamboo mao bamboos, the regeneration capability of mixed-growing bamboos is weak, and the researches on the regeneration of mixed-growing bamboo callus are rare; secondly, in the invention, the explant for inducing the callus adopts the bamboo joint intermediate meristem of the test-tube plantlet, the induction rate of the base callus (H3) reaches 93.33 percent, which is very difficult to achieve efficiency on bamboo and even woody plants, mainly because the intermediate meristem part with higher activity is found in research, and the higher induction rate is obtained by proper culture medium induction; finally, after the callus is formed, adding the callus into a culture medium with four hormone combinations, and stimulating the differentiation of the callus from different angles to directly obtain strong sprouts.
The following examples are provided to illustrate the method for regenerating plants by callus induced by bamboo joints, but they should not be construed as limiting the scope of the present invention.
Example 1
Callus induction of floral leaf arrowroot
Bamboo joints with buds, which are about 0.5cm in length, are cut from sterilized container seedlings of the mosaic-bamboo plants and taken as explants, MS culture medium is taken as basic culture medium, the influence of different concentrations and combinations of cytokinins 6-BA, KT and TDZ and auxin 2,4-D, NAA and IBA on callus induction under different concentrations (0.05, 0.5 and 2mg/L) is researched, and the total treatment is 61. The callus induction culture is carried out in the dark in a dry sterile culture room, and the culture temperature is controlled within the range of 25 +/-2 ℃.
The influence of different plant growth regulators on callus induction is researched by a 61-hormone proportioning scheme, callus is induced from bamboo joints after 5 days, and the statistic callus induction rate after 20 days is shown in table 1. The induction rate of the callus of the treatment 7-9 is more than or equal to 30 percent, when only 2,4-D is added, the induction rate of the callus is higher than that of other treatments, and when the 2,4-D is combined with other hormones, the induction rate of the callus is lower, which indicates that the treatment only adding the 2,4-D is more suitable for induction of the callus of the bamboo joint.
TABLE 1 Effect of different plant growth regulators on callus induction and bud induction of container seedling bamboo joints
Example 2
Study of the Effect of different concentrations of plant growth regulators on callus induction
Cutting bamboo joints of sterile mosaic-leaf-vector bamboo with length of about 0.5cm as explants, inoculating to the explants containing 2,4-D (0, 0.3, 0.5, 1, 3, 5 mg. L) with different concentrations-1) The callus of the bamboo node of the pleione bambusicola is induced and processed in the MS culture medium; inoculating bamboo joint in 6-BA (0, 0.5, 1, 2 mg. L) of different concentrations-1)、NAA(0、0.5、1、2mg·L-1) Containing 1 mg.L-12,4-D in MS culture medium to induce and treat the callus of the bamboo node of the pleiones mossambica. Each treating 15 bamboo jointsThe cells were cultured in a plastic culture dish having a diameter of 90mm for induction, and the experiment was repeated 3 times.
Effect of different concentrations of 2,4-D on callus induction
The induction culture lasts for about 5 days, the bamboo joint of the flower-leaf cornflower expands to form a light yellow callus, the two ends of the bamboo joint gradually begin to brown, and the diameter of the callus can reach about 3mm after about 3 weeks of induction. As shown in fig. 1. When 2,4-D is not added, the induction rate of the callus is only 8.33 percent, and the induction rate is lower. When the concentration of 2,4-D is (0-3) mg.L-1Within the range, the higher the 2,4-D concentration, the higher the callus and compact callus induction rate. When the concentration of 2,4-D is 3 mg.L-1Then, the callus induction rate reaches 56.67%, and the compact callus induction rate is 16.67%. When the concentration of 2,4-D is 1 mg.L-1In the process, the callus induction rate is 55 percent, and the compact granular callus induction rate is 26.67 percent, so that the difference with other treatment is obvious; when the concentration of 2,4-D is 5 mg.L-1The induction rate of the callus and the compact callus is 36.67 percent and 5.77 percent, and the concentration of the 2,4-D is 1 mg.L-1The treatment difference is obvious, which indicates that the high concentration of 2,4-D can inhibit the induction of callus, and the microscopic observation shows that some callus has vitrification and browning. Thus, when the concentration of 2,4-D is 1 mg.L-1Is suitable for inducing callus of Phyllostachys sieboldii.
Effect of BA on callus proliferation and differentiation
The results are shown in FIG. 2. Under the condition of adding 6-BA, the inductivity of the callus and the compact granular callus shows a gradually reduced trend along with the increase of the concentration of the 6-BA, when the concentration of the 6-BA is 0, the inductivity of the callus and the compact granular callus respectively reaches 46.67 percent and 25 percent, and the inductivity is obviously higher than that of other treatments with the addition of the 6-BA. It was observed that the callus induced by the addition of 6-BA was easy to brown and root, and not conducive to the subsequent proliferation and differentiation (FIG. 4). Therefore, 6-BA is not suitable for the induction of the floral leaf sagittaria officinalis callus, so 6-BA is not added into the induction culture medium.
Effect of NAA on callus proliferation and differentiation
As shown in fig. 3. Followed byIncreasing NAA concentration and gradually decreasing callus and dense granular callus inductivity, adding 0.5 mg.L-1、1mg·L-1、2mg·L-1When NAA is used, the callus induction rate is not different, and the compact granular callus induction rate is obviously lower than that when NAA is not added. It was observed that callus induced by NAA-containing medium was susceptible to rooting during proliferation and differentiation experiments, which was not favorable for callus proliferation and differentiation (FIG. 5). Therefore, no NAA was added during callus induction in this experiment.
Example 3
Research on influence of different length stem sources and positions thereof on induction of bamboo joint callus
Respectively select the length of>5cm、1～3cm、<Three single sterile flower leaf sagittaria bud of 1cm length>Cutting a single seedling of 5cm into three parts, wherein each section is arranged in the middle, and the three parts are arranged according to the positions of the sections in a single branch: the top part is 1-2 cm long, the number is H1, the middle part is 2cm long, the number is H2, and the number of the base part is H3; and (3) cutting a single seedling with the length of 1-3 cm into three parts: the top (the length is 0.3-1 cm), and the number is M1; the length of the middle part (0.3-1 cm) is numbered as M2; a base part (length is 0.3-1) cm, and the number is M3;<the 1cm single seedling is not cut, but only one section is numbered as L. Adding the above numbered materials into a container with 1 mg.L-12,4-D in MS culture medium, culturing in dry sterile culture room in dark at 25 + -2 deg.C. The callus induction was observed and recorded every day for the first 3d, and then every 2 d. Each treatment of 15 bamboo joints, repeated 4 times.
Differential analysis results of explants of different positions and lengths on callus induction
Selecting three stalks with different lengths as research objects, taking bamboo joints as explants, and putting the bamboo joints containing 2,4-D with the concentration of 1 mg.L-1And the basic culture medium is MS culture medium. Observation shows that after 1d, the bamboo joint begins to expand, the stem with the length of more than 5cm induces callus at first, and the length of the stem is 1-3 cThe stem of m induces the callus at last, and the length is<The time for inducing the callus from 1cm of the stem is between the two. After 3d, the two ends of the bamboo joint begin to brown, and the condition growth of the callus H is generally better than that of the callus M. After 3 weeks, the maximum diameter of the callus reached 3 mm. The callus induction rate and the compact callus induction rate are shown in FIG. 6, and the average compact callus induction rate is as follows: l is>M ≈ H. In conclusion, the length of the stem most suitable for callus induction of the Maoshuazi bamboo is L, and then H.
When a 1cm base material of the bamboo joint material with the stem length of more than 5cm is used as an explant, the induction callus rate and the compact callus rate of (H3) are both highest and are 93.33% and 36.67%, wherein the induction rates of the base bamboo joint (M3) with the stem length of 1-3 cm and the base bud bamboo joint (L) with the stem length of <1cm reach 86.67% and 91.67%, and no significant difference exists between H3 and M3. The compact callus induction rate H3 is more than M3 and L, but the three have no significant difference. The compact callus induction rate H2 is the lowest, is only 11.67%, has no significant difference with the compact callus induction rate (23.33%) of M1, and has significant difference with other treatments. In conclusion, the position of the stem which is most suitable for callus induction of the mosaic-saghyz bamboo is the bamboo joint with buds at the base.
Example 4
Selection of callus differentiation and proliferation time
The calli prepared in example 3 were inoculated into growth media for growth culture after 7d, 14d, 21d, and 28d had been generated, respectively. After 25d, the callus proliferation rate is counted, wherein the callus proliferation rate is the weight of the proliferated callus/the weight of the callus before proliferation.
As is clear from Table 2, the callus formation time had a large effect on the growth, and the difference was significant among the treatments 7d, 14d, 21d and 28d, with the highest fold increase of 21d being 2.48, which is 1.95 fold of the 7d treatment. When the generation time is 28d, the browning of the callus is obvious. When the generation time is 14d and 21d, the callus growth condition is good, and compact granular callus is generated.
TABLE 2 influence of callus formation time on proliferation
Example 5
Selection of callus proliferation frequency
The callus prepared in example 3 was cultured for 25 days with 0 proliferation times, 1 proliferation culture and 2 proliferation cultures, respectively, and then the growth of the callus was observed.
The results show that the number of subcultures of the callus also has an effect on the proliferation of the callus. As shown in Table 3, the number of subcultures of callus was 1.31, which is significantly lower than that of other treatments, and the callus was milky white in color and was not suitable for differentiation. When the subculture frequency is 2 times, the multiplication times are the highest and reach 4.42, which is obviously higher than other treatments, and the callus at the time is mostly dense granular faint yellow callus, has strong differentiation capability, and is most suitable for callus differentiation tests.
TABLE 3 influence of callus subculture frequency on proliferation
Example 6
Differentiation of callus with different hormone combinations
MS is used as a basic culture medium, and 6-BA (0.5, 1, 2) mg.L with different concentrations is designed-1、KT(1、2、4mg·L-1)、ZT(0.5、1、2mg·L-1)、TDZ(0.5、1、2mg·L-1) And (3) performing a four-factor three-level orthogonal test, namely selecting compact callus with consistent growth vigor and inoculating the compact callus to a differentiation culture medium for 9 treatments in total. And observing and counting the green point callus rate, the green seedling rate and the growth vigor after 20 days, treating 10 calli each time, and repeating for 3 times. The differential culture of different treatments is placed in a sterile culture room with a photoperiod of 16h/8h and a temperature of 25 +/-2 ℃.
Screening of callus differentiation medium formula
Selecting compact granular faint yellow callus with the same growth state and the diameter of about 5-7 mm, and inoculating the faint yellow callus to differentiationThe results are shown in Table 4. After 3d of culture, the green spots appear on the surface of the treated 9 callus at the beginning, and after 7d, the green spots appear successively (figure 7), and after 25d of culture, the green spot rate is basically stable and can differentiate and emerge successively (figure 8). After further culture for 25 days, the differentiated seedlings grew into clusters (FIG. 9). As shown in Table 4, 0.5 mg. multidot.L was added to the MS medium-16-BA、2mg·L-1KT、1mg·L-1ZT and 1 mg. L-1The green spot rate of treatment 2 in TDZ can reach 50.00% at most, and the differentiation rate of callus of treatment 9 with green spots appearing at first reaches 43.33%, which is not different from treatment 2. The difference from other treatments is obvious. The differentiation conditions of the treatment 2 and the treatment 9 are good, the callus presents light yellow, buds grow into clusters or single buds, the buds are strong and can grow into cluster buds, but the callus of the treatment 9 is observed to have a slight vitrification phenomenon. Therefore, the most preferred differentiation medium for this assay was MS +0.5 mg.L-16-BA+2mg·L-1KT+1mg·L-1ZT+1mg·L-1TDZ。
TABLE 4 Effect of different phytohormone combinations on callus differentiation
Example 7
Method for inducing callus regeneration plant by bamboo joint of cornus floribunda
Cutting sterilized test-tube plantlet of cornus floribunda into 5cm long bamboo joints with buds as explants, inoculating to callus induction culture medium containing 1 mg.L for induction culture-12,4-D MS medium; the callus induction culture is carried out in a dry sterile culture room in the dark at the culture temperature of 25 +/-2 ℃. The callus induction was observed and recorded every day for the first 3d, and then every 2 d. Each treatment of 15 bamboo joints, repeated 4 times.
Forming the obtained callus for 21d, inoculating to proliferation medium containing 1 mg.L for proliferation culture-12,4-D, inoculating the obtained compact-grained callus to a proliferation culture medium again for subculture to obtain a large amount of compact-grained callus.
Inoculating the obtained large amount of compact callus to differentiation culture medium containing 0.5 mg/L, inoculating 10 pieces of callus to each culture medium, placing in sterile culture chamber with photoperiod of 16h/8h and temperature of 25 + -2 deg.C-16-BA、2mg·L-1KT、1mg·L-1ZT、0.5mg·L-1And (3) observing and counting the green point callus rate, the green seedling rate and the growth vigor of the green point in the TDZ MS culture medium after 20 days of culture, and repeating for 3 times to obtain regenerated seedlings.
The statistic result shows that the 2,4-D concentration in the callus culture medium is 1 mg.L-1When the callus induction rate is 55% and the compact granular callus induction rate is 26.67%, the bamboo joint with the bud at the base part and with the length of 5cm is used as an explant, and the proliferation culture and the culture are carried out twice after the callus is formed for 14 d. The cultured callus has good growth condition, is milk white or light yellow in color, is mostly compact granular light yellow callus, and has obvious volume proliferation. After differentiation culture, the obtained differentiated bud grows in clusters or single buds, the buds are strong and can grow cluster buds, and the callus differentiation rate is 50%.
Example 8
Method for inducing callus regeneration plant by bamboo joint of cornus floribunda
Cutting sterilized test-tube plantlet of cornus floribunda into bamboo joints with buds at top of 0.5cm, inoculating to callus induction culture medium containing 3 mg.L for induction culture-12,4-D MS medium; the callus induction culture is carried out in a dry sterile culture room in the dark at the culture temperature of 25 +/-2 ℃. The callus induction was observed and recorded every day for the first 3d, and then every 2 d. Each treatment of 15 bamboo joints, repeated 4 times.
Forming the obtained callus for 14d, inoculating to proliferation medium containing 1 mg.L for proliferation culture-12,4-D MS culture medium, and obtaining compact granular callus.
Inoculating the compact callus to differentiation culture medium containing 2mg/L, and inoculating 10 callus to each culture medium, placing in sterile culture chamber with photoperiod of 16h/8h and temperature of 25 + -2 deg.C-16-BA、4mg·L-1KT、1mg·L-1ZT、1mg·L-1And (3) observing and counting the green point callus rate, the green seedling rate and the growth vigor of the green point in the TDZ MS culture medium after 20 days of culture, and repeating for 3 times to obtain regenerated seedlings.
The statistic result shows that the 2,4-D concentration in the callus culture medium is 3 mg.L-1When the callus induction rate is 56.67 percent and the compact granular callus induction rate is 16.67 percent, the bamboo joint with the bud at the base part and the length of 5cm is used as an explant, and the proliferation culture is carried out 14 days after the callus is formed. The cultured callus has good growth condition, is milk white or light yellow in color, is mostly compact granular light yellow callus, and has obvious volume proliferation. After differentiation culture, the callus has slight vitrification, is easy to brown, has clustered or single buds, stronger buds and can differentiate cluster buds, and the differentiation rate of the callus is 43.33%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A method for inducing callus to regenerate plants by bamboo joints is characterized by comprising the following steps:
1) cutting the bud-bearing bamboo joints of the disinfected test-tube seedlings as explants, and inoculating the explants to a callus induction culture medium for induction culture to obtain callus;
the callus induction culture medium is composed of 1-3 mg.L-12,4-D + MS culture medium;
the length of the bamboo joint with the buds is 0.1-8 cm;
2) inoculating the callus onto a proliferation culture medium for proliferation culture to obtain compact-particle callus; the proliferation culture medium is composed of 0.5-1 mg.L-12,4-D + MS culture medium;
3) inoculating the callus with compact particles to a differentiation culture medium for differentiation culture to obtain a regenerated seedling; the differentiation medium is composed of 0.5 mg.L-16-BA+2mg·L-1KT+1mg·L-1ZT+1mg·L-1TDZ + MS culture medium or 2 mg.L-16-BA+4mg·L-1KT+1mg·L-1ZT+0.5mg·L-1TDZ + MS culture medium composition;
the test-tube plantlet is a mixed-growth type bamboo seed.
2. The method according to claim 1, wherein the length of the bamboo joint with buds is 1-8 cm.
3. The method according to claim 2, wherein the bamboo nodes with buds comprise bamboo nodes with buds at the base, and the length of the bamboo nodes with buds is 5-8 cm.
4. The method according to claim 1, wherein the callus is cultured for 14 to 21 days after the callus is produced.
5. The method according to claim 1, further comprising performing a subculture after the propagation culture; the number of subcultures is 1-2.
6. The method of claim 1, wherein the intergrown bamboo species comprises a floral-leaf sagittaria bambusicola.
7. The method according to any one of claims 1 to 6, wherein the temperature of the induction culture or the proliferation culture is 23 to 27 ℃; the light condition of the induction culture or the proliferation culture is dark culture; the induction culture or proliferation culture is carried out in a dry and sterile environment.
8. The method according to any one of claims 1 to 6, wherein the light-dark cycle of the differentiation culture is 16h/8 h; the illumination intensity is 2000-2500 Lux;
the temperature of the differentiation culture is 23-27 ℃;
the differentiation culture is carried out in a sterile culture room.
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