Text/Normative KnowledgeEditorial/Normative KnowledgeintroductionThe widespread nuclear instability in plant cells cultured in vitro has been confirmed in a wide range of plant materials - this instability typically manifests as changes in the number and structure of the culture chromosomes and the regenerated plant chromosomes.The author will use cytological techniques to conduct experimental observations on the effectiveness of genetic stability in regenerated plants of wild chives, in order to verify the feasibility and effectiveness of cytological techniques in confirming the genetic stability of regenerated plants

Text/Normative Knowledge

Editorial/Normative Knowledge

introduction

The widespread nuclear instability in plant cells cultured in vitro has been confirmed in a wide range of plant materials - this instability typically manifests as changes in the number and structure of the culture chromosomes and the regenerated plant chromosomes.

The author will use cytological techniques to conduct experimental observations on the effectiveness of genetic stability in regenerated plants of wild chives, in order to verify the feasibility and effectiveness of cytological techniques in confirming the genetic stability of regenerated plants.

Wild chives (Alliaceae) are important wild relatives of garden onions. Diploid (2n=16) and tetraploid (2n=32) cell types are known to exist in wild chives, making them a highly valuable research object for regenerated plants.

In recent years, due to the excessive exploitation of garlic flavored inflorescences and leaves by local residents, and the lack of organized planting, only harvesting and not planting have led to a continuous reduction in the wild genetic resources of economically important species in their natural habitats, making it impossible to achieve sustainable and healthy development of biological resources.

Based on the severe threat of gene extinction, the National plant tissue culture Bank strives to protect this important species and other related types in vitro. Subsequently, we successfully completed cloning and reproduction through in vitro bud proliferation, attempting to study the genetic stability of different generations of regenerated plants using cytological techniques.

Cytological studies on regenerated cells have shown that the majority (> 90%) of cells have normal complement, and mutations occur only in a small number of cells, such as tumor transformation, polyploidy, and structural changes.

Compared with direct regeneration, this structure and numerical distortion have a very high frequency, which is particularly prominent when regenerated through callus tissue.

It must be mentioned that when ex situ conservation is the main goal, in both direct regeneration and callus regeneration cases, all clone proliferation should be regularly monitored and verified for the genomic integrity of the culture material.

In addition to biochemistry, histology and histochemistry, cytogenetic analysis is one of the most informative and reliable technologies to determine whether any changes have taken place in nuclear substances during regeneration and organogenesis.

In cytogenetics, karyotype studies are the most important because they usually provide true information about the structure, number and overall morphology of chromosomes.

On the other hand, meiosis focuses on the study of the details of chromosome pairing behavior and recombination frequency, and pays more attention to the separation mode of chromosomes at the later stage I and II. The pairing details and recombination frequency cannot be directly inferred in the study of mitosis.

Let's learn about the experimental methods, observation process, and result analysis of in vitro cultivation of wild chives.

The standard plan for establishing in vitro regeneration of small plants has been discussed in other articles, and will not be elaborated here. The materials and methods of this experiment will be introduced in detail below.

The experiment conducted nuclide studies on both in vivo (control) and in vitro (regenerated plant) materials, including four consecutive passages labeled as P1, P2, P3, and P.

Subsequently, P (female parent) was established in the field, and the passaged plants were cultured and named P, (F). In order to determine the changes in nuclear morphology, five regenerated cells cultured in vitro were also studied.

Prepare at least five slides from the root tips of each regenerated material, averaging four cell tissues on each slide to ensure that the deviation of the scoring data is within a reasonable range.

In order to conduct meiosis analysis, flower buds of appropriate size were collected from control plants and P4 (F) plants at the same time.

Collect target samples:

Fix the flower buds directly in the above-mentioned fixing solution, add a drop of FeCl3 solution, fix for at least 24 hours, and then store in 70% ethanol at a temperature of 10 degrees Celsius.

Chromosome preparation:

Save slide:Remove the cover glass using liquid nitrogen freezing technology and dehydrate it using a 1:3,1:6,1:9 (glacial acetic acid: 95% ethanol) series. Then, deform it twice with 95% ethanol and pack it in D.P.X. For the analysis of mitosis and meiosis preparation, only cells with good flat shape and countable chromosomes were scored.

3.1 Supplementary Observations on Chromosome Mitosis

Most of the cells analyzed in the root tip cells of the control plant had a chromosome number of 32, but a special single cell isomer with only 30 chromosomes was encountered (2n=30).

In P1, P2, and P3, the proportion of cells with normal chromosome numbers is extremely high.

In P, 96% of cells showed a normal chromosome number of 2n=32, while the remaining 4% of cells were found to be aneuploid or polyploid in 3 and 1 cells, respectively.

In the cases of P2 and P3, the proportion of normal cells was calculated and significantly higher (Table 1). The critical analysis of the three abnormal cells encountered in P2 showed that,One cell developed an adenoma, while the other two cells underwent some structural changes, resulting in significant changes in chromosome morphology.

On the contrary, all abnormal cells (3) in P3 plants were recorded as aneuploidy. About 97% of P4 plants analyzed before field transfer had normal cells, while a small portion (3%) of cells were observed to be abnormal. Among them, 2% were recorded as aneuploidy, and 1% were recorded as structural changes (Table 1)

Growing under wild conditions, cytological analysis of the secondary developing roots showed a higher number of normal cells (97.6%). Appearing as an adenoma in 2 cells (2n=31) and polyploid in 1 cell (> 4-fold).

Another very important finding suggests that micronuclei are the main influencing factor of nuclear instability.Micronucleus chromosomes have never participated in numerical and/or structural changes, and they maintain good integrity in terms of size and overall morphology.

In the control plant, all 27 pollen mother cells (PMCs) had tetravalent, divalent, and univalent mixtures at diploid or metaphase I, ranging from all univalent to the aforementioned combinations (Table 2, Figure 7). There is no polyvalent beyond the tetravalent level.

One characteristic observed in PMC is that compared to divalents and unit prices, the proportion of tetravalent opponents is extremely high. On average, each cell has 5.77IV+2.6211+3.621 (Table 2).

The number of tetrads per cell ranges from 0 to 8 (Table 4). Out of the 27 cells analyzed, up to 8 cells were associated in the form of all tetrads.

In the tetrad, the ring type dominates (average=4.88; range=0-8) and the chain type (0.88; 0-4) (Table 2).

This results in some cells having only circular tetrads, while the chain type does not exist at all. Similarly, in the case of divalent compounds, circular structures are more commonly encountered than rod-shaped (chain like) structures.

On average, each cell has 2.18 rings and 0.44 rods divalent. Although up to 12 ring divalents can be observed in a few cells, only two rod divalents are recorded at most.

Out of 27 cells, 3 showed loss of connectivity and encountered 32 univalent units in all 3 cells.

In the regenerated plants of P4 (F) transferred in the field, the chromosomal associations in the double line/final withering stage and metaphase I stage are very interesting - they show significant changes in the average values of different associations for each cell.

Among the 25 analyzed PMCs, except for one sample with 32 unit prices, all other PMCs showed different proportions of tetravalent, divalent, and unit prices, with an average of 5.40IV, 4.56II, and 1.4I per cell.

A comparison of this material revealed that, like the control plant, no association beyond the tetrad level was observed in all analyzed cells.

The relative differences in the proportion of ring and chain/rod binding in the case of tetravalent and divalent plants follow similar trends observed in the control plants (Table 2).

The incidence of chain types (0.96; 0-2) and cyclic divalents (3.68; 0-9) with a range of 0-8 is higher than that of divalent rods (0.8; 0.5).

Another interesting feature observed in P4 (F) plants is that the number of quartiles per cell ranges from 0 to 8 (Table 4), and compared to 29.6% of the control group, 20% of the analyzed cells have all quartiles.

3.2 Combination analysis of chromosome crossover frequency between different groups

The average chromosome crossover frequency of each PMC in the control group was 28.88, of which 22.70 were terminated with a termination coefficient of 0.79 (Table 3), while the chromosome crossover frequency and the number of terminated chromosome crosses in P, (F) plants slightly increased.

According to the calculation, the average number of crosses per cell is 30.56, of which 25.28 were terminated. In the PMC of the control and regenerated plants of Forsythia suspensa, the termination coefficient is 0.84 (Table 3) (Table 2 continued)

In the control plant, 13 of the 20 cells observed in the later stage I showed normal distribution (16:16) (Table 5). The remaining cells exhibit uneven distribution and/or a series of abnormal phenomena, such as delayed/delayed divalents and delayed segregation of chromosomes.

P. The difference between (F) and the control is that P, (F) have more (75%) cells with normal distribution of chromosomes, and the remaining 25% cells have uneven distribution or some of the above abnormalities (Table 5).

Two cells showed the presence of micronucleus numbered 2 in anaphase II, while some cells showed chromosome hysteresis and/or late segregation in addition to normal distribution.

The control plant has 75.06% of stained pollen, while the P, (F) plants have 73.07% of stained pollen (Table 5).

Chromosome instability is a phenomenon related to the in vitro culture system and its regenerated plants. Many researchers have proved that nuclear instability occurs from time to time through the nuclear studies of root tips, but there are few reports on chromosome stability and pairing behavior during meiosis.

We have conducted research on mitosis and meiosis, with the main purpose of monitoring the genetic stability of plants regenerated through tissue culture, not only during the culture and passage period, but also after their establishment in the field.

For various onion plants in general, especially wild chives, there are few reports on chromosome number, size, morphology, pairing behavior, and segregation patterns. Therefore, our experiment is the first attempt in this direction.

As mentioned earlier, diploid and tetraploid cell types are widespread in nature.

In the root tip cells of control plants, the appearance of polyploid complements (2n=32) in the vast majority (99.2%) confirmed that the materials used in this study were tetraploid cells.

An abnormal cell with 2n=30 is usually not related to the effectiveness of ploidy level or 8 as the basic chromosome number.

Meiosis analysis showed that 29.6% of pmc had 8 tetrads, while Mathur and Tandon claimed 16 tetrads in their academic reports.

It can be seen that in the polyploid group, a small proportion of chromosomes with abnormal numbers naturally exist.

From Table 1, it can be clearly seen that the regenerated cells of each generation (P1, P2, P3, P4), even after establishment in the field, are not affected by the extension of culture time and maintain the original chromosome number (2n=32).

More interestingly, the number of aberrations and karyotypes only occur in a small number of cells.

These observational data indicate that the cultivated explants (stem bases) contain most homogeneous cells that can form adventitious buds and regenerate plants with stable genotypes.

Chromosome abnormalities such as aneuploid, tripolar and micronucleus were observed in the tetraploid cell type of 1~18 months old wild Chinese chive.

On the contrary, this distortion is rarely observed in callus culture of a diploid group a. cepa.

Based on this observation, we can conclude that the ploidy level of the explant material does indeed affect the range and frequency of chromosomal abnormalities.

Although the explant material is tetraploid, the regenerated body maintains the integrity of chromosome number and structure in consecutive generations due to the absence of indirect callus tissue, and even if there are abnormalities, they are limited to a very small proportion of cells.

In polyploid species, especially autotetraploid species, whether naturally occurring or artificially cultivated, the average decrease of tetravalent and the corresponding increase of bivalent frequency are common in successive generations, and have been recorded in many research materials related to plant tissue culture, such as rape, verbena, amaranth, orchid and edamame.

This phenomenon often leads to 'diploid', which in turn helps plants adapt to new physical and physiological environments. Although it is difficult to compare one-on-one, the trends observed in P4 (F) plants are highly similar to this.

The data in Table 2 on the average values of tetravalent and divalent in the control and P4 (F) plants show a significant decrease in the former (from 5.77 to 5.40), and an increase of approximately 57.5% in the divalent situation (from 2.62 in the control to 4.56 in P4 (F)).

From this, it can be clearly demonstrated that P4 (F) plants exhibit diploid phenomenon. Although the degree is relatively mild, compared to the control group, the smaller proportion of unit prices in the regenerated plants seems to indirectly promote the diploid process.

The complete absence of multivalent and trivalent further confirms this, as trivalent and multivalent are the only other possible configurations. The high crossover frequency of P4 (F) plants indicates that there may be more recombination in the genetic composition of the material. However, more chromosome crosses are terminated, resulting in a higher value of the termination coefficient.

The corresponding values for the control plants are 28.88 and 0.79, respectively (Table 2). In the later stages of P and (F) plants, the marginal increase (16:16) in the average distribution of I chromosomes in the percentage of cells may be directly related to the average increase in divalence per cell. Compared to the multivalent configuration, it usually tends to separate in a more orderly manner.

This is mainly due to the simple configuration and suitable orientation of the intermediate I divalent. The pollen coloring rate of regenerated plants (P, (F)) from normal meiosis is low, indicating that the latter is not due to any chromosome factor.

At this point, we can boldly conclude that the participation of genes and physiological factors may have played a crucial role.

summary

The author conducted a study on the genetic stability of regenerated plants of wild chives after 4th generation culture and field establishment using cytological techniques.

Nuclear studies on regenerative cells have shown that current research has found that the majority (> 90%) of cells have normal complement and variation limited to a small number of cells, such as tumor transformation, polyploidy, and structural changes.

The meiosis analysis of regenerated plants showed that there was no difference between them and the control plants in general, and all parameters (correlation, cross frequency, terminal coefficient and pollen coloration) had no significant change compared with the control plants.

The tetravalent range and frequency indicate a significant decrease in regenerated plants, further confirming the effectiveness of cytological techniques in confirming the genetic stability of regenerated plants.

reference

Guo Yangdong, "Research Report on the Propagation of Male Sterile Clones of Chinese Chive by Tissue Culture Method"

Yang He, Application and Development Prospects of Plant cell engineering

Ma Shubin, "Breeding and Application of Male Sterile Lines in Chinese Chives"

Zhang Xiansheng, "Cytological observation of fertile and sterile anther and pollen development in Chinese chives"

Jing Shaoling, Application of cell engineering in Maize Germplasm Improvement

Li Rongqian, "Karyotype Analysis of Different Varieties of Leek"

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