CN114258858B

CN114258858B - Method for inducing embryogenic callus of sugar beet

Info

Publication number: CN114258858B
Application number: CN202111561835.0A
Authority: CN
Inventors: 谭文勃; 兴旺; 刘大丽; 李思琪
Original assignee: Heilongjiang University
Current assignee: Heilongjiang University
Priority date: 2021-12-20
Filing date: 2021-12-20
Publication date: 2023-04-28
Anticipated expiration: 2041-12-20
Also published as: CN114258858A

Abstract

A method for inducing embryogenic callus of sugar beet, belonging to the technical field of callus induction. In order to obtain embryogenic callus of sugar beet and establish a regeneration system, sugar beet resources 92005-1 are selected, aseptic seedlings are obtained after seed treatment and germination, aseptic seedling growing point explants are collected, and the explants are inoculated into a callus culture medium containing 0.75 mg/L6-BA and 0.1mg/L NAA, and embryogenic callus is obtained after 7-8 weeks of culture. The method for inducing callus by utilizing sugar beet resources 92005-1 provides a cytological theoretical basis for establishing a sugar beet plant regeneration system, and lays a foundation for better utilizing genetic engineering means to provide new varieties with high quality and high yield.

Description

Method for inducing embryogenic callus of sugar beet

Technical Field

The invention relates to a method for inducing embryogenic callus of sugar beet, belonging to the technical field of callus induction.

Technical Field

Beet is one of the important sugar crops in the world, is also the second largest sugar crop in China, is planted in a large area in northeast, northwest and North China, and is an important economic crop and sugar crop in the north China. Different types of sugar beets also have different values, sugar beet being a cultivated beet population of the genus beta that has the greatest relationship with human production and life. The method is mainly used for producing sucrose and is a raw material of sugar factories. Many byproducts are produced after sugar beet sugar production, so that the beet sugar production has high economic value and wide application. For example, molasses may be fermented or chemically produced into products such as methanol, ethanol, butanol, glycerol, monosodium glutamate, acetone, etc. The filter mud after sugar production contains rich calcium and other nutrients, and can be used as fertilizer and for improving acid soil. The sugar-making waste silk (grain meal) is an excellent feed for livestock, and the waste silk processed by beet sugar factories in China is exported in large quantity, so that japan and other countries are in the market. Sugar beet is derived from the hybridization between leaf beet and fodder beet with larger storage root during evolution, and is formed by long-term artificial selection. The breeding of new varieties of sugar beet is mainly aimed at improving the sucrose content in roots and improving the quality of extractables. However, due to the short cultivation and domestication time of beet, narrow genetic background, poor resources and high incompatibility of selfing, the genotype of a good single plant is easy to lose, and a lot of difficulties are brought to the propagation and preservation of breeding materials of good varieties. With the rapid development of molecular biology and plant transgenic technology, the current use of genetic engineering technology to improve crop quality and cultivate new varieties is one of the hot spots of crop breeding research, while the genetic engineering technology needs to rely on the support of tissue culture technology, and the plasmid with target genes is guided into embryogenic callus by inducing the embryogenic callus, and the complete plant is formed by differentiating the embryogenic callus by utilizing the redifferentiation capability of the embryogenic callus, so as to establish a complete regeneration system, thus forming transgenic plants. Therefore, the culture of the embryonic callus of the beet and the establishment of a regeneration system thereof provide a new way for beet genetic engineering and new and good variety cultivation.

Callus can be divided into two main categories based on histological observations, appearance characteristics, and its reproducibility, regeneration pattern, etc: embryogenic and non-embryogenic calli. The embryogenic callus is firm in texture, milky or yellow in color, and has spherical particles on the surface, which grow slowly; from the cytology perspective, embryogenic callus is composed of cells with equal diameters, the cells are smaller, the protoplasm is thick, no vacuoles exist, starch grains are often rich, the nucleus is large, the division activity is strong, the content is rich, the embryogenic callus has typical characteristics of torpedo embryos and heart-shaped embryos, the non-embryogenic callus with poor differentiation capability is opposite, the tissue structure is loose, the cells are relatively large, a large vacuole exists in the embryogenic callus, almost no organelles exist, and the embryogenic callus is in a water immersion brown state. In order to obtain callus with higher differentiation capacity, embryogenic callus should be selected for induction. However, at present, the domestic study on sugar beet callus is that a set of beet tissue culture and plant regeneration system is established by taking beet variety "Ganyang No. 7" petiole as explant through the steps of inducing callus, callus to differentiate adventitious buds, bud rooting, transplanting and the like ([ 1] Wu Guojiang, liu Hailong, li Zhijiang ]. The construction of beet tissue culture and plant regeneration system [ J ]. Chinese sugar materials, 2018,40 (06): 14-18.DOI: 10.13570/j.cnki.scc.2018.06.005.). However, it is only a complete set of researches on the establishment of beet regeneration system, and no intensive cyto-logical observation of callus is performed. Foreign aspects we cannot determine the type of resource they use by just the name of the breed they provide. Thus, there is a need to find a method for inducing embryogenic callus of sugar beet.

Disclosure of Invention

The invention provides a method for inducing sugar beet embryogenic callus for obtaining sugar beet embryogenic callus and establishing a regeneration system thereof, which is characterized by comprising the following steps:

s1, seed treatment and aseptic seedling obtaining: picking full sugar beet seeds, removing hulls, sterilizing, repeatedly flushing with distilled water, inoculating on an MS basic culture medium to germinate the seeds, obtaining sugar beet aseptic seedlings after 18-22 days, and collecting sugar beet aseptic seedling explants;

s2, inducing callus: and adding 0.75 mg/L6-BA and 0.1mg/LNAA into the MS basal medium to serve as an induction medium of the callus, and inoculating the sterile seedling explant of the S1 into the callus medium for 7-8 weeks.

Further defined, the sugar beet variety is sugar beet resource 92005-1.

Further defined, the sterilization in S1 is to sterilize with 75% alcohol for 2min and then with 12% sodium hypochlorite for 5min.

Further defined, the MS basal medium described in S1 and S2 has a composition of 3% sucrose, 0.8% agar, and the balance water.

Further defined, the explant of S1 is taken from a leaf, petiole or growing point of a sterile seedling.

Further defined, the explant of S1 is taken from a sterile seedling growth point.

Further defined, the culture temperature of S2 is 22-24 ℃.

The invention has the beneficial effects that:

according to the invention, sterile seedlings of sugar beet resources 94002-1, 92005-1 and 92011/1-6/1-1 are respectively used as base materials, leaves, petioles and growing points are respectively used as explants, plant cytokinins with different types and concentrations are selected to generate somatic embryos, and as a result, the result shows that the sugar beet resources 92005-1 are treated by seeds and germinated to obtain the sterile seedlings, the growing points are collected and used as the explants, the explants are inoculated into a callus culture medium containing 0.75 mg/L6-BA and 0.1mg/LNAA, embryogenic callus can be obtained after 7-8 weeks of culture, the embryogenic callus can be subjected to re-differentiation induction to obtain primordia, and the primordia can proliferate or develop to form early somatic embryos, so that the primordia further develop to mature somatic embryos, and a complete plant is obtained.

According to the invention, the induced callus is subjected to paraffin section preparation, callus of different types, appearance forms and resources is observed, embryogenic callus and non-embryogenic callus with good regeneration capability are distinguished in cell forms, the relationship between the regeneration capability and the cell forms of the sugar beet callus is researched and analyzed, a cytologic theoretical basis is provided for establishing a sugar beet plant regeneration system, and a foundation is laid for better utilization of genetic engineering means to provide new varieties with high quality and high yield. In addition, the re-differentiation of beet callus is always a bottleneck for restricting the establishment of a beet regeneration system, and the change rule of the beet callus can be analyzed from the aspects of tissue structure and cytomorphology, so that the problem can be more intuitively found, and proper measures are taken for promoting the differentiation of the callus aiming at the structural change of each stage.

Description of the drawings:

FIG. 1 is a graph of calli induced by different concentrations of hormone, wherein A in FIG. 1 is the condition of calli growth of sugar beet resource 94002-1 leaf on MS+0.75 mg/L6-BA; b in FIG. 1 is the callus growth condition of sugar beet resource 92005-1 petiole on MS+0.75mg/L6-BA; c in FIG. 1 is the callus production of sugar beet resource 92005-1 petiole on MS+0.25 mg/L6-BA; d in FIG. 1 is the callus growth of sugar beet resources 92005-1 on MS+0.75 mg/L6-BA+0.1 mg/LNAA.

FIG. 2 is a diagram showing the difference between embryogenic and non-embryogenic cells of beet, wherein A in FIG. 2 is the case of callus of sugar beet resource 92005-1 growth point on MS+0.75 mg/L6-BA+0.1 mg/LNAA, embryogenic cells and non-embryogenic cells are distributed in the callus (white arrow represents embryogenic cells of the second type, blue arrow represents embryogenic cells of the first type; yellow arrow represents non-embryogenic cells of the first type, black arrow represents non-embryogenic cells of the second type), B in FIG. 2 is the case of callus of red beet resource 357 petiole on MS+0.5 mg/L6-BA, and the callus is non-embryogenic cells (white arrow represents non-embryogenic cells of the first type, black arrow represents non-embryogenic cells of the second type);

FIG. 3 is a diagram showing the morphological observation of calli of sugar beet resources 92005-1 growth point on MS+0.75 mg/L6-BA+0.1 mg/LNAA;

FIG. 4 is a histological observation of beet Somatic embryogenesis, wherein A in FIG. 4 is a paraffin section of callus at MS+0.75 mg/L6-BA+0.1 mg/L NAA at the point of growth of 92005-1 sugar beet, se, somatic embryo (embryoid); FIG. 4B is a paraffin section of callus at the growth point of 92005-1 sugar beet at MS+0.75 mg/L6-BA+0.1 mg/LNAA, me, multicellular embryos, multicellular proembryoid Z, ma, material embryo, mature embryo.

The specific embodiment is as follows:

the MS basal medium in the following specific embodiment has the composition of 3% of sucrose, 0.8% of agar and the balance of water.

Sugar beet resources 94002-1, 92005-1 and 92011/1-6/1-1 of sugar beet resources are all derived from the national beet germplasm metaphase library as described in the following embodiments.

The invention adopts the traditional paraffin slicing method to manufacture paraffin slices of the obtained callus, and specifically comprises the steps of fixing, dyeing, dehydrating, transparentizing, waxing, embedding, slicing, pasting, spreading, dewaxing and sealing for observing the material. The method mainly comprises the following steps: fixing each tissue material with FAA fixing solution, and storing in 70% alcohol at 4deg.C; the fixed tissue material is soaked in the stain solution of the hematoxylin for dyeing for 7-15 days. After dyeing, washing the material with running water, and observing the dyeing degree by using a microscope in the washing process; gradually removing water in the tissue by alcohol with different concentration gradients after washing is finished (generally, dehydrating by 70% alcohol, 85% alcohol and 95% alcohol and then dehydrating by 100% alcohol for 2 times), wherein each stage is separated by 1-2 h; then 1 time of 1/2 dimethylbenzene and 1/2 pure alcohol mixed solution is carried out; transparent in pure xylene twice for 2h each time, and finally waxing, embedding and slicing, wherein the slice thickness is 8 μm.

Example 1: induction of sugar beet resource 92005-1 callus

(1) Seed treatment and aseptic seedling obtaining

Selecting plump sugar beet resources 92005-1, grinding the seeds with a mortar to remove the shells, sterilizing with 75% alcohol for 2min, sterilizing with 12% sodium hypochlorite for 5min, repeatedly washing with distilled water for 4-5 times, inoculating on an MS basic culture medium to germinate, obtaining sugar beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the sugar beet aseptic seedlings for callus induction.

(2) Induction of callus

MS basal medium was added with (1) 0.25 mg/L6-BA; (2) 0.5 mg/L6-BA; (3) 0.75 mg/L6-BA; (4) 0.25 mg/L6-BA+0.1 mg/LNAA; (5) 0.5 mg/L6-BA+0.1 mg/LNAA; (6) and (2) taking 0.75 mg/L6-BA+0.1 mg/LNAA as a callus induction culture medium, respectively inducing the sugar beet explants obtained in the step (1), and counting callus induction rate after culturing for 8 weeks at 24 ℃, wherein the callus induction rate is= (number of formed callus explants/number of inoculated explants) ×100%.

Example 2: induction of sugar beet resource 94002-1 callus

(1) Seed treatment and aseptic seedling obtaining

Selecting full sugar beet resources 94002-1, grinding the seeds with a mortar to remove the shells, sterilizing with 75% alcohol for 2min, sterilizing with 12% sodium hypochlorite for 5min, repeatedly washing with distilled water for 4-5 times, inoculating on an MS basic culture medium to germinate, obtaining sugar beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the sugar beet aseptic seedlings for callus induction.

(2) Induction of callus

Example 3: induction of sugar beet resources 92011/1-6/1-1 callus

(1) Seed treatment and aseptic seedling obtaining

Selecting plump sugar beet resources 92011/1-6/1-1 seeds, grinding the seeds with a mortar to remove the shells, sterilizing with 75% alcohol for 2min, sterilizing with 12% sodium hypochlorite for 5min, repeatedly washing with distilled water for 4-5 times, inoculating on an MS basic culture medium to germinate, obtaining sugar beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the sugar beet aseptic seedlings for callus induction.

(2) Induction of callus

The calli obtained in examples 1-3 were evaluated:

(1) The callus induction rate, color and texture of each treatment method in examples 1 to 3 were counted, and the results are shown in Table 1 and FIG. 1, and the treatment method not shown in the table is a treatment method for induction failure.

TABLE 1 Effect of different induction methods on sugar beet callus induction

As can be seen from the results in Table 1, the growth point explants of sugar beet resources 92005-1 were induced to a higher rate of 50% on MS medium containing 0.75 mg/L6-BA+0.1 mg/LNAA or 0.25 mg/L6-BA, and the obtained calli were better in color, whereas the calli or explants of sugar beet 92011/1-6/1-1 were not induced to die gradually, probably due to the hormone concentration of the medium and the resource variety of beet.

(2) Observing the difference between beet embryo cells and non-embryo cells and the formation process

It was observed by paraffin section that only the growing point explant of sugar beet resource 92005-1 was cultured on MS medium containing 0.75 mg/L6-BA+0.1 mg/LNAA to obtain callus as embryogenic callus containing four different cell types (as A in FIG. 2), the first embryogenic cell type, the cytoplasm was dense, the nucleus was located in the middle, the starch grain content was more, and the cell type was located at the edge of the differentiation center. The second type of embryogenic cells, which are dense in cytoplasm, are easily stained, have a nucleus at the edge and a small starch granule content, generally near the differentiation center, presumably have completed primary differentiation. The first type of non-embryogenic cells, with a central large vacuole, has a low starch granule content, contains almost no organelles, and has a nucleus centrally located. The second type of non-embryogenic cells are distributed on the periphery of the first type of non-embryogenic cells, and only contain a few cytoplasm, and no other organelles for differentiation exist in the second type of non-embryogenic cells to form vacuoles. B in FIG. 2 is the case of calli of the leaf stalk of red beet resource 357 on MS+0.5 mg/L6-BA, which calli are non-embryogenic cells, and the calli were compared.

(3) Observing the cytomorphology of beet callus under the treatment of different plant growth regulator substances from different sources

FIG. 3 is a diagram showing the morphology of calli grown on MS+0.75 mg/L6-BA+0.1 mg/LNAA at the growth point of sugar beet 92005-1, which shows that calli contain first and second embryogenic cells, the cell volume is smaller (about 15-25 μm in diameter), the cell arrangement and shape are more regular and the cytoplasm is rich, the cytoplasm is stained deep, the cell nucleus is larger, and the capacity of vigorous cell division is provided.

(4) Somatic development process and origin mode of sugar beet cell embryo

As shown by the observation of sections, embryoid bodies formed by gradually differentiating embryoid cells which are supposed to be of internal origin by se in FIG. 4A gradually differentiate to form other embryoid structures in the later stage, and then develop to form complete plants. The observation of three cell primordia together in FIG. 4B is presumed to convey nutrients for the formation of mature embryos. There are also differences in the way somatic embryos originate from different plant varieties and different pathways. From the cytological observation of sugar beet callus, the somatic embryo has two origins, namely external origin and internal origin, but the appearance time and the appearance number are different. These differences may be related to the embryogenesis pathway and induction rate of sugar beet somatic cells, for which specific reasons and related molecular mechanisms need to be further explored. Regardless of the manner in which the somatic embryos originate, their ultimate goal is to develop into embryoid bodies to further obtain regenerated organs.

Claims

1. A method of inducing embryogenic callus of sugar beet comprising the steps of:

s1, seed treatment and aseptic seedling obtaining: picking full sugar beet seeds, removing hulls, sterilizing, repeatedly flushing with distilled water, inoculating on an MS basic culture medium to germinate the seeds, obtaining sugar beet aseptic seedlings after 18-22 days, and collecting sugar beet aseptic seedling explants; the variety of the sugar beet is sugar beet resource 92005-1; the explants are taken from the growing point of the aseptic seedlings;

s2, inducing callus: and adding 0.75 mg/L6-BA and 0.1mg/L NAA into the MS basal medium to serve as an induction medium of the callus, and inoculating the sterile seedling explant of the S1 into the callus medium for 7-8 weeks.

2. The method of claim 1, wherein the sterilizing of S1 is performed with 75% alcohol for 2min and with 12% sodium hypochlorite for 5min.

3. The method of claim 2, wherein the MS basal medium of S1 and S2 comprises 3% sucrose, 0.8% agar, and the balance water.

4. A method according to claim 3, wherein the cultivation temperature of S2 is 22-24 ℃.