CN114258859B - Method for inducing red beet embryogenic callus - Google Patents

Abstract

A method for inducing red beet embryogenic callus, which belongs to the technical field of callus induction. In order to obtain embryogenic callus of red beet and establish a regeneration system, red beet resource 357 is selected, aseptic seedlings are obtained after seed treatment and germination, aseptic seedling explants are collected, and the embryogenic callus is obtained after inoculation in a callus culture medium containing 0.25 mg/L6-BA for 7-8 weeks. The method for inducing red beet callus provides a cytological theoretical basis for establishing a beet plant regeneration system, and lays a foundation for better utilizing genetic engineering means to provide high-quality and high-yield new varieties.

Description

Method for inducing red beet embryogenic callus

Technical Field

The invention relates to a method for inducing red beet embryogenic callus, 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 beet have different values, wherein the red beet has rich nutrition, is rich in natural red vitamin B12 and iron, has the function of enriching blood, and the nicotinic acid which is rich in the red beet can participate in the synthesis of hemoglobin, so that the content of the hemoglobin is increased, and the oxygen carrying capacity is further improved. In addition, the byproducts of red beet processing can also be used for producing chemical products such as alcohol, methanol, butanol and the like. Meanwhile, the beet red pigment which is an important secondary metabolite in the red beet has detoxification and antioxidation activities and has stronger medical and health care values.

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, there are many factors that affect embryogenic callus formation, and thus, there is a need for a method of inducing embryogenic callus in red beet.

Disclosure of Invention

The invention provides a method for inducing red beet embryogenic callus, which comprises the following steps of:

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

s2, inducing callus: and (3) adding 0.25 mg/L6-BA 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.

Preferably, the red beet aseptic seedlings are obtained after 20 days.

Further defined, the variety of red beets is red beet resource 357.

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 explants of S1 are taken from leaves, petioles and growth points of sterile seedlings.

Preferably, the explant of S1 is taken from the petiole of a sterile seedling.

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

The invention has the beneficial effects that:

the invention takes aseptic seedlings of red beet resource Y7, red beet resource S14 and red beet resource 357 as basic materials, respectively takes leaves, petioles and growing points of the aseptic seedlings as explants, and selects plant cytokinins with different types and concentrations to generate somatic embryos, and as a result, the invention discovers that the petioles of the aseptic seedlings are obtained after the red beet resource 357 is treated and germinated by seeds, and the embryogenic callus can be obtained after the petioles are inoculated in a callus culture medium containing 0.25 mg/L6-BA and cultured for 7 to 8 weeks.

The induced callus is subjected to paraffin section making, callus of different types, appearance forms and resources is observed, embryogenic callus and non-embryogenic callus with good regeneration capacity are distinguished in cell forms, the relationship between the regeneration capacity and the cell forms of the beet callus is researched and analyzed, a cytological theoretical basis is provided for establishing a 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 is 20d 357 red beet petiole calli growth; b is callus of S14 red beet leaf on MS+0.25mg/L6-BA+0.1mg/L NAA; c is callus of Y7 red beet petiole on MS+0.75mg/L6-BA+0.1mg/L NAA; d is the callus of Y7 red beet growing point on MS+0.25mg/L6-BA+0.1mg/L NAA; e is 357 red beet petiole callus on MS+0.25mg/L6-BA; f is 357 red beet growing point callus on MS+0.75mg/L6-BA; g is 357 red beet petiole callus on MS+0.25 mg/L6-BA+0.1 mg/L NAA; calli of red beet petiole with H of 357 on MS+0.5mg/L6-BA;

FIG. 2 is a graph showing the difference between embryogenic and non-embryogenic cells of beet, wherein A is the distribution graph of embryogenic and non-embryogenic cells of callus of leaf stalk of red beet resource 357 on 0.25 mg/L6-BA (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 is the callus of leaf of red beet resource S14 on MS+0.5 mg/L6-BA+0.1 mg/L NAA, the callus being 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 the cells of calli of sugar beet treated with different plant growth regulator substances, wherein A, a:357 red beet petioles callus on MS+0.25 mg/L6-BA+0.1 mg/L NAA; b, B: s14 red beet leaf callus on MS+0.5 mg/L6-BA+0.1 mg/L NAA; c, C:357 red beet petiole callus on ms+0.25 mg/L6-BA; d, D:357 red beet petiole callus on ms+0.5 mg/L6-BA; e, E:357 red beet growing points callus on MS+0.75mg/L6-BA; f, F: s14 red beet leaf callus on MS+0.5 mg/L6-BA+0.1 mg/L NAA;

FIG. 4 is a diagram showing external morphology and cytology observation of callus differentiation induced by 20d beet petiole, wherein A in FIG. 4 is 357 red beet petiole growth state in MS+0.25 mg/L6-BA callus induction medium 20 d; b in FIG. 4 is 357 red beet petiole in MS+0.25 mg/L6-BA callus induction medium 20d cytomorphology map, me, multicellular embryos, multicellular proembryoid; v, vessel elements, catheter molecules;

FIG. 5 is a histological observation of somatic embryogenesis of beet, wherein, A in FIG. 5 is 357 red beet petiole in MS+0.25 mg/L6-BA callus paraffin sections, and embryogenic cells begin to disperse; b is 357 red beet petiole callus paraffin section on MS+0.25mg/L6-BA, two cell embryo; c is 357 red beet petiole callus paraffin section on MS+0.25mg/L6-BA, three cell embryo; d is dc, dyad cell, two cell embryos being generated by division; t, tetracyte embryo; tri, tri cell three-cell embryos; e is 357 red beet petiole callus paraffin sections on MS+0.25 mg/L6-BA, exogenous embryogenic cells, me, multicellular embryos, multicellular primordial embryogenic mass; f is callus paraffin section of s14 red beet leaf on MS+0.5 mg/L6-BA+0.1 mg/L NAA, exogenous embryogenic cell, me, multicellular embryos, multicellular proembryogenic mass.

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.

The red beet 357, red beet Y7 and red beet S14 described in the following embodiments are all derived from the national beet germplasm metaphase library.

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 callus from red beet resource 357

(1) Seed treatment and aseptic seedling obtaining

Selecting full red beet resource 357 seeds, grinding the seeds with a mortar to remove the shells, sterilizing the seeds with 75% alcohol for 2min, sterilizing the seeds with 12% sodium hypochlorite for 5min, repeatedly washing the seeds with distilled water for 4-5 times, inoculating the seeds on an MS basic culture medium to germinate the seeds, obtaining red beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the red beet aseptic seedlings for inducing callus.

(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/L NAA; (5) 0.5 mg/L6-BA+0.1 mg/L NAA; (6) and (2) taking 0.75 mg/L6-BA+0.1 mg/L NAA as a callus induction culture medium, respectively inducing the red beet explants obtained in the step (1), and counting callus induction rate after eight weeks, wherein the callus induction rate is= (the number of formed callus explants/the number of inoculated explants) multiplied by 100%.

Example 2: induction of red beet resource Y7 callus

(1) Seed treatment and aseptic seedling obtaining

Selecting full red beet resource Y7 seeds, grinding the seeds with a mortar to remove the shells, sterilizing the seeds with 75% alcohol for 2min, sterilizing the seeds with 12% sodium hypochlorite for 5min, repeatedly washing the seeds with distilled water for 4-5 times, inoculating the seeds on an MS basic culture medium to germinate the seeds, obtaining red beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the red beet aseptic seedlings for inducing callus.

(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/L NAA; (5) 0.5 mg/L6-BA+0.1 mg/L NAA; (6) and (2) taking 0.75 mg/L6-BA+0.1 mg/L NAA as a callus induction culture medium, respectively inducing the red beet explants obtained in the step (1), and counting callus induction rate after eight weeks, wherein the callus induction rate is= (the number of formed callus explants/the number of inoculated explants) multiplied by 100%.

Example 3: induction of red beet S14 callus

(1) Seed treatment and aseptic seedling obtaining

Selecting full red beet S14 seeds, grinding the seeds with a mortar to remove the outer shell, sterilizing the seeds with 75% alcohol for 2min, sterilizing the seeds with 12% sodium hypochlorite for 5min, repeatedly washing the seeds with distilled water for 4-5 times, inoculating the seeds on an MS basic culture medium to germinate the seeds, obtaining red beet aseptic seedlings after about 20d, and respectively collecting leaf stems, leaves and growing points of the red beet aseptic seedlings for inducing callus.

(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/L NAA; (5) 0.5 mg/L6-BA+0.1 mg/L NAA; (6) and (2) taking 0.75 mg/L6-BA+0.1 mg/L NAA as a callus induction culture medium, respectively inducing the red beet explants obtained in the step (1), and counting callus induction rate after eight weeks, wherein the callus induction rate is= (the number of formed callus explants/the number of inoculated explants) multiplied by 100%.

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 red beet callus induction

The sterile seedling petiole explant of red beet 357 is inoculated on callus induction medium (MS+0.25 mg/L6-BA) for 10-14 d, and after petiole expansion starts, 20-30 d, compact white callus is formed at the cut of petiole (as A in figure 1).

As shown in the test results shown in Table 1, the petiole has the best effect as an explant for inducing callus, the induction rate is over 50%, the induction rate of leaves and growing points is lower than 50%, and the efficiency of inducing callus is low. Wherein MS+0.25mg/L6-BA is used as an induction medium, the induction rate obtained by inducing the leaf stalks of the red beet 357 is highest and reaches 100%, and the leaf explant of the red beet S14, the leaf stalk explant of the red beet Y7 and the growth point explant can induce callus, but the induction rate is not high when the explant of the red beet 357 is selected for induction. Therefore, the present study uses leaf stalks of red beet 357 as the optimal explants for inducing callus, and MS+0.25mg/L6-BA as the optimal medium for inducing callus.

(2) Observing the difference between the embryonic cells and the non-embryonic cells of the red beet and the formation process

The red beet resource 357, the leaf stalk, was observed to contain four different cell morphologies (as a in fig. 2 a) in the calli obtained by culturing on MS medium containing 0.25 mg/L6-BA, the first embryogenic cell type, the cytoplasm was dense, the nucleus was in the middle, the starch granule content was more, and the edge at 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 leaf of red beet resource S14 on MS+0.5 mg/L6-BA+0.1 mg/L NAA, the calli being non-embryogenic cells.

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

The cytomorphology of the beet calli treated by different plant growth regulating substances is shown in figure 3, and the difference of colors and textures of the beet calli treated by different plant growth regulating substances can be seen from the figure. A, d, e, f in FIG. 3 contains first and second non-embryogenic cells, respectively, which have large cell volumes (about 30-80 μm in diameter), irregular shapes, pale cytoplasm, shallow staining, and not abundant organelles, and often contain only one large vacuole. B and c in FIG. 3 contain first and second embryogenic cells, which have smaller cell volumes (about 15-25 μm in diameter), more regular cell arrangement and shape, abundant cytoplasm, deep cytoplasmic staining, larger cell nuclei, and vigorous cell division ability.

In addition, we observed that 357 red beet petioles began to expand and white dense nodular tissue appeared at the petiole wound 10-20 d in the 20d growth state on MS+0.25 mg/L6-BA callus induction medium (see A in FIG. 4). We performed paraffin section production to find that in the occurrence area of white nodular tissue, the connection part of the white nodular tissue and the petiole has a conduit structure (see B in fig. 4), and the direction is diversified, so as to provide nutrient and moisture absorption and transportation for somatic embryo and multicellular primordial embryo mass. It can be seen that the outer cells divide vigorously, are closely and regularly arranged, are deeply stained, are embryogenic cells, and initially form multicellular primordia (me).

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

The cells gradually spread out and the cell wall thickens at the beginning of the embryogenic cell division (a in fig. 5), and these individual embryogenic cells begin to divide in a mostly unequal manner, forming a large basal cell and a small apical cell (B in fig. 5), with little basal cell division and the apical cell continuing to divide into multiple cells (C in fig. 5), forming a large number of three-and four-cell embryos (D in fig. 5). These somatic embryos gradually divide to form somatic primordia (F in FIG. 5) and then gradually differentiate to form embryoid bodies of other shapes.

In the embryogenic callus group of beet, it was observed that the embryogenic pattern of beet somatic cells was both of external and internal origin. Wherein E, F in FIG. 5 are all embryogenic cells of exogenesis which form multicellular primordia (me) and which, upon slicing, were found to be initially divided by cells of embryogenic callus, thereby producing multicellular primordia. The multicellular primordia are closely arranged, small in shape, dense in cytoplasm, large in nucleus and capable of vigorous division, and then the multicellular primordia which are continuously divided are continuously increased to gradually form early somatic embryos. Although the red beet resource 357 has a different way of origin of the cell embryo, it will develop into embryoid bodies and thus further into regenerated organs.

Claims (4)

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

s1, seed treatment and aseptic seedling obtaining: selecting full red beet seeds, removing shells, sterilizing, repeatedly flushing with distilled water, inoculating on an MS basic culture medium to germinate the seeds, obtaining red beet aseptic seedlings after 18-22 days, and collecting red beet aseptic seedling explants; the variety of the red beet is red beet resource 357; the explant is taken from the petiole of a sterile seedling;

s2, inducing callus: and (3) adding 0.25 mg/L6-BA 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 ℃.

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