CN116463276B - Method for separating and culturing sugarcane suspension single cells and method for regenerating plants - Google Patents
Method for separating and culturing sugarcane suspension single cells and method for regenerating plants Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology, and provides a method for separating and culturing sugarcane suspension single cells and a method for regenerating plants. The method for separating and culturing the sugarcane suspension single cells comprises the induction and the subculture of the callus; obtaining embryogenic suspension cells and suspension single cells; the method for regenerating plants comprises the steps of suspending and oscillating embryogenic suspension single cells in an MS culture medium for subculture to obtain loose suspension cell particles; transferring loose suspension cell particles to a basic differentiation culture medium for differentiation culture; transferring the irregular compact cell mass to a basic MS rooting culture medium for illumination culture; the invention establishes a sugarcane suspension single cell culture and plant regeneration system for the first time, and provides a foundation for cultivating new sugarcane varieties.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for separating and culturing sugarcane suspension single cells and a method for regenerating plants.
Background
Sugarcane is taken as an important cash crop in China, is always a main source of sugar, and the sugarcane sugar industry is an important pillar of the sugar industry in China. However, the sugar cane variety is single in the production of the sucrose, and the bred new variety can not meet the production requirements of the sugarcane, so that the healthy and stable development of the sucrose industry and the further improvement of the competitiveness are seriously influenced. However, sugarcane is a warm-loving crop, is mainly asexually propagated, and sugarcane cultivated in most areas such as Guangxi, yunnan and the like cannot form complete spikes due to temperature and sunlight condition limitations, pollen spores in the spikes have almost no fertility, so that innovation of germplasm resources of the sugarcane and fine variety breeding work have not been progressed in breakthrough. The development of modern biotechnology and the continuous maturation of sugarcane somatic cell hybridization technology provide new ideas and methods for the development of sugarcane breeding.
Cell suspension culture (Cell suspension culture) is a key element in tissue culture studies. Cell suspension culture refers to the process of obtaining callus by using isolated materials (such as root tips, stem tips, leaves and the like) of plants, then carrying out secondary culture to further form undifferentiated, active and loose callus or other easily dispersible tissues, and then placing the callus or other easily dispersible tissue in a liquid culture medium to carry out vibration dispersion suspension culture, thereby obtaining free single cells or small cell clusters which keep good growth state and can be continuously proliferated. These single cells or cell clusters are referred to as suspension cell lines (Suspension cell line) or suspension lines. And compared with a tightly aggregated cell mass, suspended single cells with uniform size and thick cytoplasm have more important significance in breeding. The cells obtained by the culture of the technology have the advantages of rapid cell growth, relatively consistent cell state, good repeatability, easily controlled conditions, uniform cell or cell mass size, good dispersibility, short culture period and the like, and are widely used for research in a plurality of fields such as molecular biology, developmental biology, biochemistry, cytology, genetics, physiology and the like, and are also directly used for screening mutants on the cell level, transferring gene macromolecular substances, producing secondary metabolites, establishing plant clone, cell hybridization, protoplast separation, culture, fusion and the like. Therefore, the plant cell suspension culture technology has been developed from the 80 s of the 20 th century to the present, has become a new hot spot for biotechnology research and development, is a cell in vitro culture technology with huge application potential, and is widely applied to the cultivation of new plant varieties.
In 1953, cell suspension culture was first achieved by Muir et al on tobacco and marigold, and a single cell culture technique was developed. By 1958, steward et al successfully established a carrot cell suspension culture system by using carrot callus as a material, and obtained complete regenerated plants by embryogenesis. From this point, suspension cell culture techniques are widely used for herbaceous plants. Until now, suspension cell lines of rice, corn, wheat, sorghum, rye and other cereal crops have been successfully established, and suspension culture techniques are relatively mature.
The report of Paul, W.J.T. et al in 1992 has established an embryogenic cell suspension line for sugarcane with low experimental success rate and no mention of the acquisition of single cells in suspension from sugarcane. Liao Zhaozhou et al (sugarcane protoplast culture-characteristics of a cell line of a regenerable plant, sugarcane sugar industry 1999) mainly used homogeneous suspension cell mass rather than suspension single cells when isolating protoplasts from sugarcane suspension cell lines as material, nor did they describe the establishment of cell suspension lines in detail, only the morphology of sugarcane calli and suspension culture were observed. Moreover, the current report about suspension culture of sugarcane cells has been long in many years, and in more than ten and twenty years, except for Hong Cui, 5 kinds of sugarcane with different genotypes are selected as test materials, and the suspension culture is performed by inducing embryogenic callus by young leaves of the sugarcane, little research is related to the field, and she does not mention to establish a perfect sugarcane cell suspension system and a single cell suspension culture method. The sugarcane suspension cell culture system established in the 90 th century of the 20 th century is probably not applicable to the current sugarcane varieties any more. And the research on single-cell suspension culture of sugarcane is less.
Many factors such as genotype of the material, material-taking part, callus induction, rotation speed of the shaking table, hormone level of the culture medium and the like are closely related to whether a fast and stably-proliferating cell suspension system can be established. The calli and suspension cell lines induced by plants of the same species and different genotypes have great differences in indexes such as color, cell growth amount and the like. Therefore, the establishment of a sugarcane suspension single cell line has great difficulty at present.
Disclosure of Invention
The invention aims at: aiming at the problems, the invention provides a method for separating and culturing the suspended single cells of the sugarcane and a method for regenerating plants, which firstly establishes a system for culturing the suspended single cells of the sugarcane and regenerating the plants, and provides a foundation for culturing new varieties of the sugarcane.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the method for separating and culturing the sugarcane suspension single cells comprises the following steps:
(1) Inducing and subculturing the callus: selecting tail sheaths at the beginning of sugarcane elongation as materials for inducing callus, removing outer leaf sheaths, sterilizing, cutting outer leaf sheaths in a sterile environment, taking the innermost layers of pale yellow young leaves 4-6cm away from a growing point, cutting into tissue blocks, and culturing in a callus induction medium to obtain loose callus; then carrying out secondary multiplication culture on loose callus in MS liquid culture medium to obtain secondary callus; the callus induction medium comprises the following components: MS+2, 4-D0.5 mg/L+6-BA0.5 mg/L+30 g/L sucrose+6 g/L agar, and adjusting pH to 5.5-6.0;
(2) Acquisition of embryogenic suspension cells: inoculating the subculture callus into a suspension culture medium for liquid suspension culture, adding suspension culture solution into the suspension culture medium, performing dark culture in a shaking table, performing subculture once every 7 days, continuously performing subculture for 6-8 times, standing the suspension culture for a period of time at the end of each subculture period, removing the upper layer culture solution, and adding fresh suspension culture solution; obtaining embryogenic suspension cells after the subculture is finished; the suspension culture solution comprises the following components: MS+2, 4-D2.0 mg/L+30 g/L sucrose, and adjusting pH to 5.5-6.0;
(3) Acquisition of suspended single cells: shaking the embryogenic suspension cells uniformly, standing for a period of time, filtering cells in the middle layer and the culture solution by using a 200-mesh cell sieve, and obtaining the suspension single cells in the filtrate.
In the present invention, preferably, the sugar cane variety selected is neo-table sugar No. 22.
In the present invention, preferably, the culturing in the callus induction medium in the step (1) is a dark culturing for 21 days at 25.+ -. 1 ℃.
In the present invention, it is preferable that the number of times of the subculture for proliferation in the step (1) is 2, the time interval between the subcultures is 30 days, the culture condition is dark culture, and the culture temperature is 25.+ -. 1 ℃.
In the present invention, preferably, the time taken for the standing for a period of time is 2 to 3 minutes.
In the present invention, preferably, the shaking rotation speed of the shaking culture in the step (2) is 120rpm.
The invention also provides a method for regenerating sugarcane suspension single cells into plants, which comprises the following steps:
(1) Suspending, oscillating and subculturing embryogenic suspension single cells in an MS liquid culture medium to obtain loose suspension cell particles;
(2) Taking out the loose suspended cell particles, cleaning the loose suspended cell particles with sterile water, and transferring the loose suspended cell particles to a basic differentiation culture medium for differentiation culture, wherein the basic differentiation culture medium comprises the following components: MS+6-BA 2 mg/L+KT0.5 mg/L+30 g/L sucrose+6 g/L agar, and adjusting pH to 5.5-6.0; obtaining macroscopic irregular compact cell clusters in a basic differentiation medium;
(3) And (3) clamping the whole irregular compact cell mass in a sterile environment, transferring the cell mass onto a basic MS rooting culture medium for light culture at 25+/-1 ℃, wherein the basic MS rooting culture medium is as follows: 0.5MS+NAA3mg/L+6-BA0.5mg/L+30 g/L of sucrose+6 g/L of agar, and adjusting pH to 5.5-6.0.
In the above method for regenerating plants, preferably, the number of times of the subculture in the step (1) is 4 to 5, and the subculture period is 7d at 25.+ -. 1 ℃.
In the above method for regenerating plants, it is preferable that the time for the differentiation culture in the step (2) is 14d, and the culture is performed in a dark environment at 25.+ -. 1 ℃.
In the above method for regenerating plants, preferably, the light culture in the step (3) has a light intensity of 1800lx and a light time of 16 hours/d, and the culture time is 20 days or more.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. The invention successfully establishes a single-cell suspension system of sugarcane. By selecting a new sugar No. 22 variety, selecting a tail sheath at the early stage of sugarcane elongation as a material for inducing callus, and adopting a proper induction culture system, a secondary culture system, culture conditions and culture time and a proper culture medium hormone, the callus is established to be a embryogenic cell suspension single cell line which can be rapidly and stably proliferated. The embryogenic suspension single cells are cultured in an MS liquid suspension medium, and homogeneous embryogenic cell masses with vigorous growth, clear cytoplasm and low browning degree can be obtained in the exponential phase of a growth curve. In the 2,4-D concentration of 2.0mg/mL, the growth amount of the sugarcane suspension cells shows an S-shaped curve growth trend; after 9d, the cell rapidly grows into the logarithmic phase, and the duration is 9-15d; by 15-17d, the cell growth enters a resting stage, the proliferation amount is stable and reaches the maximum value, which is 2.4X10 5/mL; therefore, the embryogenic suspension single cell line with high yield and strong activity is obtained.
2. The invention successfully utilizes the single-cell suspension system of the sugarcane to obtain regenerated plants, and when the suspended cell particles are subjected to differentiation culture, the browning rate is 17.78 percent, the differentiation number is 12.33, and the cell mass diameter is 1.52cm through the optimized differentiation culture medium and culture conditions; when rooting culture is carried out on compact cell clusters, the number of purple cell clusters reaches 28.67 after 14d culture and the number of green bud point cells reaches 27.67 after 28d culture through a preferable rooting culture medium and culture conditions, and the green seedling rate reaches 81.11 percent.
Drawings
FIG. 1 is a primary suspension cell line and its cellular composition formed after 6-8 times of young leaf callus subculture;
FIG. 2 shows the state of suspended cells after filtration by different mesh cell sieves;
FIG. 3 is a cytological and histological morphology of sugarcane embryogenic single cell suspension system plants at various stages of regeneration;
FIG. 4 is a compact cell mass state;
FIG. 5 is a differentiation process state of compact cell mass;
FIG. 6 is a dynamic growth curve of sugarcane suspension single cells.
Detailed Description
The present invention will be further described with reference to the following examples in order to more clearly illustrate the present invention.
Study materials: the test material is sugarcane (Saccharum officinarum), and the planting variety is neo-sugar No. 22 (ROC 22) by adopting conventional planting from a sugarcane experiment base of university of Guangxi.
EXAMPLE 1 isolation and cultivation of sugarcane suspension Single cells
The method for separating and culturing the sugarcane suspension single cells comprises the following steps:
(1) Inducing and subculturing the callus: selecting tail sheaths at the initial stage of sugarcane elongation as materials for inducing callus on sunny days, stripping off outer leaf sheaths, sterilizing with 75% ethanol for 30s, washing with sterile water for 3 times, cutting off outer leaf sheaths in a sterile environment, taking the innermost layers of pale yellow young leaves with the thickness of about 0.5cm to about 0.5cm and the thickness of 4 cm to 6cm from a growing point, clamping up the young leaves with forceps, and placing the young leaves in a callus induction culture medium for dark culture for 21 days at the culture temperature of 25+/-1 ℃ to obtain loose callus; selecting a beige, loose and high-activity callus, and performing secondary multiplication culture on the loose callus in an MS liquid culture medium, wherein the secondary multiplication culture is carried out for 2 times, the secondary interval time is 30 days, the culture condition is dark culture, and the culture temperature is 25+/-1 ℃ to obtain the secondary callus; the composition of the callus induction medium was: MS+2, 4-D0.5 mg/L+6-BA0.5 mg/L+30 g/L sucrose+6 g/L agar, pH5.5-6.0, in this example to pH5.8;
(2) Acquisition of embryogenic suspension cells: selecting subculture callus which grows vigorously, has bright yellow color and loose texture, slightly crushing the subculture callus by forceps, transferring the subculture callus into a suspension culture medium for liquid suspension culture, and adding suspension culture solution into the suspension culture medium; the container is a 250mL triangular flask, the liquid filling amount is 80-100mL, and the container is placed in a shaking table with the rotating speed of 120rpm for dark culture, and the culture temperature is 25 ℃; repeating the steps for 6-8 times every 7 days, repeating the steps for 7 times in the embodiment, standing the suspension culture for 2-3 minutes at the end of each repeating period, removing the upper layer culture solution, and adding fresh suspension culture solution; obtaining embryogenic suspension cells with high concentration and high density after the subculture is finished; the suspension culture solution comprises the following components: MS+2, 4-D2.0 mg/L+30 g/L sucrose, pH5.5-6.0, pH5.8 in this example;
(3) Acquisition of suspended single cells: shaking the embryogenic suspension cells uniformly, standing for 2-3 minutes, filtering cells in the middle layer and culture solution by using a 200-mesh cell sieve, and obtaining suspension single cells in filtrate.
In the step (2), the outer layer part of the young leaf callus can be quickly fused into an MS liquid culture medium in the process of subculture, and after 6-8 times of subculture, the internal tissue is gradually disintegrated into suspension cells in an oscillation environment. Microscopic examination revealed that the cultures in the initial suspension cell line at this time consisted mainly of cell clusters consisting of round-sphere-shaped embryogenic cells and elongated/irregularly-shaped non-embryogenic cells, and that the cell clusters consisted of a generally large number of embryogenic cells and a small number of non-embryogenic cells (FIG. 1A), and that the single cells dissociated in the medium were initially predominantly irregularly non-embryogenic cells (tube/rod-shaped) and gradually developed into embryogenic cells (round-sphere-shaped) with the extension of the subculture cycle (FIG. 1B).
The present study found that suspension cell lines that underwent 6-8 times of subculture had tended to stabilize, and were the best period for single cell suspension line culture and subsequent study. The embryogenic single cell suspension system is established by first removing the non-embryogenic cell mass to obtain embryogenic cells. By utilizing the difference of embryogenic cells and non-embryogenic cells in indexes such as cell diameter, length, specific gravity and the like, the test firstly utilizes the characteristics that embryogenic cell clusters have high specific gravity and are easy to precipitate and tube/rod-shaped cells have high specific gravity and float, cultures in a triangular flask are uniformly shaken before being filtered by using a cell sieve, then the cultures are kept stand for 30s-3min, most non-embryogenic cells are removed by adopting a method of removing upper cells, cells and culture solutions in middle layers are temporarily reserved, and the culture solutions are filtered by using cell sieves with different meshes. At the same time, large cell clusters precipitated in the lower layer are also removed.
The study found that there was a significant difference in morphology and size (p < 0.05) in cells collected after filtration through different mesh cell sieves, see FIG. 2. The cell mass obtained after filtration using a 60 mesh cell sieve generally consisted of 80-120 embryogenic cells and non-embryogenic cells, and the average area of the cell mass was 0.045mm 2, and when observed under a microscope, the cell mass was thick, light transmittance was not strong, and the brown part of the cells observed in the visual field was large (FIG. 2A). Meanwhile, large cell clusters and massive callus in the aging stage are collected by a 60-mesh cell sieve, and the browning of cells is serious. The cell mass obtained after filtration using an 80 mesh cell sieve generally consisted of 50-80 embryogenic cells and non-embryogenic cells, and the average area of the cell mass was 0.038mm 2, which also stepped into the growth arrest phase, and was large in cell density, thick in cell mass, and weak in light transmittance, and the brown portion of the cells observed under the microscope was large (fig. 2B). The cell mass obtained after filtration by using a 100-mesh cell sieve generally consists of 20-50 embryogenic cells and non-embryogenic cells, the average area of the cell mass is 0.014mm 2, the cells at the moment are generally in a stage of vigorous growth, the cell density is not large, the light transmittance under a microscope is strong, and the browning condition is light (figure 2C). The cells obtained after filtration through a 200 mesh sieve were mostly single cells, the average cell mass area was 0.0008mm 2, but since the cells were still in the initial cell suspension stage at this time, and the 200 mesh filtrate was mainly obtained from the medium layer culture, the single cells obtained after the filtrate were mainly tube/rod-like non-embryogenic single cells (FIG. 2D), and the single cell viability was about 58%.
The method also comprises the steps of obtaining suspended single cells successfully by adopting an enzymolysis method, shaking the embryogenic suspended cells uniformly, sucking out 5mL of suspended culture by using a rubber head dropper, placing the suspension culture in a10 cm centrifuge tube, centrifuging to remove supernatant, adding 5mL of pectase basic enzyme solution for enzymolysis, centrifuging to remove enzymolysis solution after the enzymolysis is finished, repeatedly washing three times by using an MS basic culture medium as a washing solution, and centrifuging to remove the washing solution to obtain the purified single cells. However, the suspension single cells obtained by this method are temporarily incapable of forming regenerated plants. The possible reasons are that the pectase has serious damage to cells when removing the connection between cells, and the enzymolysis process has long operation time, wherein the complex operation process can cause further damage to cells, so that the cells lose normal separation capability.
EXAMPLE 2 sugarcane suspension single cell regeneration plants
The sugarcane suspension single cells are from the suspension single cells obtained in the embodiment 1, and the method for regenerating the sugarcane suspension single cells into plants comprises the following steps:
(1) Suspension single cell culture: the suspension single cells obtained by filtering in example 1 are cultured by using a liquid culture medium of MS+2, 4-D2.0 mg/L+30 g/L sucrose and with pH value of 5.8, the rotation speed of a shaking table is 120rpm, the dark culture is carried out at 25+/-1 ℃ for 21 days, and homogeneous embryogenic cell masses with vigorous growth, clear cytoplasm and low browning degree can be obtained in the exponential phase of a growth curve, so that the homogeneous embryogenic cell masses are obtained, and are shown in figure 3A. The dynamic growth curves of different 2,4-D concentrations are shown in FIG. 6, in the 2,4-D concentration of 2.0mg/mL, the growth amount of the sugarcane suspension cells is in the growth trend of an S-shaped curve, and the cells are in the growth lag phase at 0-7D, so that the growth amount is small, and even the hidden trend is the trend of decline; after 9d, the cell rapidly grows into the logarithmic phase, and the duration is 9-15d; by 15-17d, the cell growth enters into a stationary phase, and the proliferation amount is stable and reaches the maximum value of 2.4X10 5/mL. After 19d the cells grew into senescence and the cell density gradually decreased.
(2) Suspending single cells obtained in the example 1 in an MS liquid culture medium, carrying out suspension oscillation subculture, wherein the rotation speed of a shaking table is 120rpm, subculturing every 7d, carrying out dark culture at 25+/-1 ℃, and carrying out macroscopic irregular loose type suspended cell particles in a culture system after 4-5 times of subculture, wherein the visible irregular loose type suspended cell particles are shown in figures 3B-1 and 3B-2; at the moment, loose cell particles are light yellow in color, soft in texture, high in moisture content, loose in cell-cell connection and easy to damage by applying external force.
(3) Taking out loose suspended cell particles from a triangular flask, washing the loose suspended cell particles with sterile water for 3 times, transferring the suspended cell particles to a basic differentiation culture medium by forceps for differentiation culture, wherein the differentiation culture is carried out in a dark environment at 25+/-1 ℃ for 14d, and the basic differentiation culture medium comprises the following components: MS+6-BA 2 mg/L+KT0.5 mg/L+sucrose 30 g/L+agar 6g/L, pH5.5-6.0, in this example to pH5.8; obtaining macroscopic irregular compact cell clusters in the basic differentiation medium, see fig. 3C-1 and 3C-2; the cell mass is yellowish white, the length of the cell mass is about 2cm, the width is about 1.5cm, and the cells are closely connected.
(4) Slightly clamping the irregular compact cell mass by forceps in a sterile environment, transferring the irregular compact cell mass onto a rooting culture medium for light culture at 25+/-1 ℃, wherein the light time is 16h/d, the light intensity is 1800lx, the culture time is more than 20 days, and the rooting culture medium is: 0.5MS+NAA3mg/L+6-BA0.5mg/L+sucrose 30 g/L+agar 6g/L, and the pH is adjusted to 5.5-6.0, and the pH is adjusted to 5.8 in this example. After 10D, purple spots (FIGS. 3D-1 and 3D-2, the boxes are shown as purple spots) were grown on the cell mass, and the cell mass was taken out with forceps and then dissociated and stained to observe the vessel (FIGS. 3D-3, the boxes are shown as vessels). However, differentiation of the catheter was not observed after dissociation staining of the loose cell mass (FIG. 3B-1) and the compact cell mass (FIG. 3C-2). After the cell mass is full of purple punctate substances, the purple color gradually fades after the cell mass is continuously cultured in rooting medium at 25 ℃ under illumination for 14 days, the cell mass is yellow-white, and green buds grow (fig. 3E, line frame shows green buds). After about 14d, seedlings were generated at the bud points (fig. 3F).
Effect of hormone on sugarcane compact cell mass differentiation culture
In step (3) of example 2, the levels of both 6-BA and KT were designed orthogonally to obtain higher differentiation rates of compact cell mass, with 6-BA concentration levels of 0.5,1.0,1.5,2.0mg/L; KT concentration levels were 0.5,1.0,1.5,2.0mg/L, and the test was repeated for 3 flasks, each flask being inoculated with 5 loose particles of suspension cells. After 14d of culture, irregular compact cell clusters which are visible to naked eyes grow in the MS differentiation medium, and indexes such as browning rate, differentiation number and diameter range of the cell clusters are counted respectively. The statistical results are shown in Table1 below.
TABLE 1 Effect of hormone combinations on sugarcane compact cell mass differentiation
Note that: * Indicating significant difference (P < 0.05), indicating very significant difference (P < 0.01).
From the above table, it is clear that the combination of 6-BA and KT at different concentrations has a significant effect on browning, differentiation and cell mass diameter during the growth of sugarcane compact cell mass.
Further multiplex comparisons were performed using the Duncan new complex polar method to obtain Table 2 and Table 3 below.
TABLE 26 influence of BA concentration on sugarcane compact cell mass differentiation
Note that: multiple comparisons were performed using the Duncan new complex polar difference method, with significant differences in lower case expression (P < 0.05) for the same column, as follows.
As can be seen from table 2, the browning rate and differentiation number of the compact cell mass were significantly different between level 1 and level 4 among the influence factors of 6-BA at different concentrations; cell mass diameters were significantly different between level 1 and 3, and between level 2 and 3, and between level 4. Within the experimental range, when the KT concentration was unchanged, the compact cell mass browning rate decreased with an increase in the 6-BA concentration, and the differentiation number and the cell mass diameter increased with an increase in the 6-BA concentration.
TABLE 3 Effect of KT concentration on sugarcane compact cell mass differentiation
As can be seen from table 3, when the concentration of 6-BA is unchanged, the browning rate, differentiation number and cell mass diameter of the compact cell mass tended to rise and then fall with the rise of the KT concentration, and there was no significant difference in the browning rate and differentiation number of the compact cell mass between the four levels of the KT concentration, indicating that KT hormone was not a major factor affecting the browning rate and differentiation number of the compact cell mass. The compact cell mass has the lowest browning rate, the highest differentiation number and the highest cell mass diameter which are respectively shown at a 6-BA concentration level 4 and a KT concentration level 1and a KT concentration level 4, and when the hormone concentration is 6-BA2.00mg/L+KT0.50mg/L, the browning rate is the lowest, the differentiation number is the highest, the cell mass diameter is the largest, and 17.78%, 12.33 and 1.52cm respectively (the compact cell mass with normal growth is shown in FIG. 4A; and the compact cell mass with browning is shown in FIG. 4B).
In conclusion, adding 6-BA2.00mg/L+KT0.50mg/L into the differentiation medium is the optimal hormone concentration for the growth and differentiation of the sugarcane compact cell mass.
(II) influence of rooting medium hormone ratio on differentiation of compact cell clusters into seedlings
In step (4) of example 2, orthogonal test design was performed on the MS medium macroelement and hormone combination to obtain higher green seedling rate of the regenerated seedlings, wherein the MS macroelement adopts 0.1MS, 0.5MS, 1.0MS, 1.5MS; the concentration of the hormone combination NAA+6-BA is 2.5+0mg/L,2.5+0.5mg/L,3+0mg/L and 3+0.5mg/L respectively, and the basic MS rooting culture medium is as follows: MS+sucrose 30 g/L+agar 6g/L, pH5.8, 6 flasks were repeated for each test, each flask being inoculated with 5 compact cell clusters. And respectively counting the differentiation number of the purple compact cell mass after 14d, the number of green bud point cells after 28d, the browning number and differentiation number of the cell mass, the albino number, the green seedling rate of the regenerated seedlings and the like. The statistical results are shown in Table 4 below.
TABLE 4MS macroelement and hormone combination interactions to differentiate compact cell clusters into shoots
Note that: * Indicating significant difference (P < 0.05), indicating very significant difference (P < 0.01).
The analysis results in table 4 show that the macroelements of different MS culture mediums interact with different hormone combinations, which has a significant effect on the number of purple cell masses generated after the compact cell masses are cultured in the MS rooting culture medium for 14d and the albino seedling number during seedling formation, and has no significant effect on the number of green bud point cells formed when the compact cell masses are cultured in the MS rooting culture medium for 28d, the browning number and the green seedling rate.
Further multiplex comparisons were performed using the Duncan new complex polar method to obtain Table 5 and Table 6 below.
TABLE 5 influence of MS macroelements on compact cell mass differentiation into shoots
Note that: multiple comparisons were performed using the Duncan new complex polar difference method, with significant differences in lower case expression (P < 0.05) for the same column, as follows.
TABLE 6 influence of hormone combinations on differentiation of compact cell clusters into shoots
As can be seen from tables 5 and 6, the ratio of macroelements in the MS medium has a significant effect on the browning number and the green seeding rate of the compact cell mass differentiated into seeding, the purple cell mass that can be formed after 14d of culture has a significant difference between levels 1,2, 1,3, 2,4, 3,4 of the macroelements of MS, the green sprouting point cell mass that can be formed after 28d of culture has no significant difference between levels 1 and 4, and the albino seeding number has no significant difference between levels 1 and 4. The number of purple cell clusters which can be formed after 14d of culture and the number of green bud cell clusters which can be formed after 28d of culture are obviously different among levels 1,2, 1, 4,2 and 3 of hormone combination, the number of brown spots is obviously different among levels 1,2, 1,3, 1 and 4, the number of albino spots is obviously different among levels 1,2, 1, 4,2 and 3, and the rate of green spots is obviously different among levels 1,2, 1, 4,2 and 3. When the compact cell mass was differentiated into seedlings in the rooting medium, the highest values of the number of purple cell masses after 14d culture, the number of green bud point cells after 28d culture and the green seedling rate appeared in the major elements (1/2 MS major elements) of 0.5 proportion of MS, which were 25.33, 25.75 and 64.72%, respectively. For hormone combinations, the highest values of purple cell mass number after 14d culture, green bud point cell number after 28d culture, green shoot rate were found at NAA 3mg/L+6-BA 0.5mg/L, respectively 12.17, 14.75, 45.56% (FIG. 5A is compact cell mass full of purple material; FIG. 5B is compact cell mass brown and albino shoot in MS rooting medium; FIG. 5C is brown cell mass upon shoot formation).
In summary, the optimal culture condition for differentiating compact cell clusters into seedlings in MS rooting medium is that the most suitable culture condition for 0.5MS macroelements is +NAA3mg/L+6-BA0.5mg/L+30 g/L of sucrose+6 g/L of agar, and the green seedling rate at this time is 81.11%.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.
Claims (9)
1. A method for regenerating a plant from sugarcane suspension single cells, comprising the steps of:
S1, separating and culturing sugarcane suspension single cells:
S1.1, induction and subculture of callus: selecting tail sheaths at the beginning of sugarcane elongation as materials for inducing callus, removing outer leaf sheaths, sterilizing, cutting outer leaf sheaths in a sterile environment, taking the innermost layers of pale yellow young leaves 4-6cm away from a growing point, cutting into tissue blocks, and culturing in a callus induction medium to obtain loose callus; then carrying out secondary multiplication culture on loose callus in MS liquid culture medium to obtain secondary callus; the callus induction medium comprises the following components: MS+2, 4-D0.5 mg/L+6-BA 0.5 mg/L+30 g/L sucrose+6 g/L agar, pH5.5-6.0;
s1.2 acquisition of embryogenic suspension cells: inoculating the subculture callus into a suspension culture medium for liquid suspension culture, adding suspension culture solution into the suspension culture medium, performing dark culture in a shaking table, performing subculture once every 7 days, continuously performing subculture for 6-8 times, standing the suspension culture for a period of time at the end of each subculture period, removing the upper layer culture solution, and adding fresh suspension culture solution; obtaining embryogenic suspension cells after the subculture is finished; the suspension culture solution comprises the following components: MS+2, 4-D2.0 mg/L+30 g/L sucrose, pH5.5-6.0;
s1.3 acquisition of suspended single cells: shaking the embryogenic suspension cells uniformly, standing for a period of time, filtering cells in the middle layer and the culture solution by using a 200-mesh cell sieve, and obtaining suspension single cells in filtrate;
S2, suspending, oscillating and subculturing embryogenic suspension single cells in an MS liquid culture medium to obtain loose suspension cell particles;
S3, taking out the loose type suspension cell particles, cleaning the loose type suspension cell particles with sterile water, and transferring the loose type suspension cell particles to a basic differentiation culture medium for differentiation culture, wherein the basic differentiation culture medium comprises the following components: MS+6-BA 2 mg/L+KT0.5 mg/L+sucrose 30 g/L+agar 6 g/L, and adjusting pH to 5.5-6.0; obtaining macroscopic irregular compact cell clusters in a basic differentiation medium;
S4, clamping the whole irregular compact cell mass in a sterile environment, transferring to a basic MS rooting culture medium for light culture at 25+/-1 ℃, wherein the basic MS rooting culture medium is as follows: 1/2MS macroelement+NAA 3 mg/L+6-BA 0.5 mg/L+sucrose 30 g/L+agar 6g/L, and pH is adjusted to 5.5-6.0.
2. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the selected sugarcane variety is Xintai sugar No. 22.
3. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: culturing in the callus induction culture medium in the step S1.1 for 21 days at 25+/-1 ℃.
4. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the number of times of the secondary proliferation culture in the step S1.1 is 2, the time interval between the secondary culture is 30 days, the culture condition is dark culture, and the culture temperature is 25+/-1 ℃.
5. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the time for standing for a period of time is 2-3 minutes.
6. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the shaking table rotational speed of the shaking culture in step S1.2 was 120 rpm.
7. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the times of the secondary culture in the step S2 are 4-5 times, the temperature is 25+/-1 ℃, the secondary culture is dark, and the secondary period is 7d.
8. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the time of the differentiation culture in the step S3 is 14d, and the culture is carried out under a dark environment at 25+/-1 ℃.
9. The method for regenerating a plant from sugarcane suspension single cells according to claim 1, wherein: the illumination culture in the step S4 has the illumination time of 16h/d, the illumination intensity of 1800lx and the culture time of more than 20 days.
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