1. Chapter 8 Plant Somaclonal Variation

2. Section 1 Concept and selection of plant somatic clone variation

3. 1、Concept
Larkin and Scowcroft (1981) first proposed that the plant generated from any form of cell culture, which is called somatic cell clone (somaclones体细胞无性系 ).

4. In the development stage, the phenomenon which leads to the genetic changes in the regenerated plants is called somaclonal variation体细胞无性系变异.

9. 2、Somaclonal variation is mainly related to two aspects:
（1）Morphological characteristics, growth habit, resistance variation and so on.
As an important source of genetic variation in crop breeding, somaclonal variation can be an important source of plant cell engineering breeding.

10. （2）Chromosome variation
①Chromosome number variation

12. ②Chromosome structural variation
For example:
Chromosome deficiency缺失、chromosome exchange互换、chromosome translocation易位and chromosome inversion倒位etc.

14. Advantages and disadvantages of somaclonal variation:
Suitable for all kinds of tissue culture plants;
Continuously and rapidly provide source of variation
Eliminate one or a few defects 缺陷of fine varieties;
To provide a new variant 变异株系.

15. Disadvantages:
After being subcultured for many times, plant regeneration ability is weakened;
Variation is not stable and will change after crossing杂交 or selfing自交;
Variation is not predictable, and discomfort variation 不适变异often occurs.

16. （1）Selection of materials Under the condition of culturing in vitro :
3、In vitro screening 筛选 of somaclonal variation
（1）Selection of materials
Under the condition of culturing in vitro :
★Callus→Small cell mass or single cell suspension culture →Mutant selection
Free individual protoplast

17. （2）Selection of mutant cells
★ ①Types of cultured cells
Mutant can be produced by protoplasts or suspension culture cells.

18. ★ ②Selection method of mutant cells
——Direct selection method
Selecting a medium containing a specific substance, only the mutant cells can grow on this medium, but the non mutant cells can not grow, so that directly screen mutant.

19. Specific substances include biological toxins毒素, herbicides除草剂, high concentrations of heavy metal ions重金属离子and salts盐类, etc..

21. ——Indirect selection method
Indirect selection is a screening method for selecting the parameters related to the mutation.

22. Dorffling et al., using hydroxyproline (羟脯氨酸, HYP) as the selective agent, obtained somaclonal variants resistant to HYP, its cold resistance is stronger than the donor 供体 parent, and it also has rather high genetic stability.

23. ★ ③Genetic basis and stability identification of mutant traits
——Mutant cells or tissue identification
Mutant cells or tissue subcultured for several times on normal culture medium is used for identification.
If it appears the selected target traits, it can be identified as mutant cells or tissues.

24. ——Mutant plant identification
The dentification of regenerated plants；
The identification of selfed自交 seeds or progeny plants of regenerated plants.

25. Advantages of in vitro screening of somaclonal variation
Effectively shorten the screening period, save manpower, material resources and financial resources.

26. Problems of in vitro screening of somaclonal variation
Not all of somaclonal variation can be stably inherited;
These variations are not the first to appear, some of which have occurred in the traditional breeding materials, although the frequency is low;
Somaclonal variation can cause more than one character variation, but the variation results are difficult to predict.

27. Section 2 Factors affecting plant somaclonal variation

28. 1、Explant
①Generally speaking, the higher degree of specificity culture tissues have or the older culture tissues are, the greater the odds of producing a mutation are.

29. ②When meristems分生组织 or young explants are cultured (such as apical buds顶芽 or axillary buds腋芽), variation rarely occurs.

30. 2、 Species and genotypes
①Genotype: Variation frequency difference among genotypes is large.
②Ploidy level: the plants which are polyploids or have more chromosome number, have higher variation frequency than diploid二倍体 and haploid单倍体.

31. 3、 Culture medium
（1）Certain components of basic culture medium can make the ploidy倍性 of culture materials change.
For example: MS, 1/4MS, and 1/2MS can make the ploidy change.

32. （2）Plant growth regulator
①2,4-D:
It can increase the frequency of variation .
②Cytokinins (CTKs):
The high concentration of CTKs can improve the variation frequency of polyploids.
③combination of auxin and CTK:
Different proportions can change the proportion of different ploidy cells.

33. 4、 Subculture time
Somaclonal variation caused by subculture times is common.
Generally speaking, the longer the subculture time is, the more the subculture times are, the higher the probability of cell variation is.

34. Section 3 Mechanism of plant somaclonal variation

35. 1、Pre-existing mutation expression
The variation of some somatic clones is due to the expression of pre-existing variation in the cells of the explant.

36. In general, unless a single cell or protoplast is cultured, otherwise, if the multicellular explant 多细胞外植体composed of different cell types is cultured, and the phenotype of the regenerated plants is not uniform.

37. There are two forms of pre-existing variation:
① The cells, which is derived from different tissues and their chromosome ploidy is different, is also called extracellular explants, such as the phloem韧皮部cells, parenchyma薄壁组织cells and xylem木质部cells.

38. The differentiation and growth of these cells in different tissues is not synchronized同步的.

39. When the external 外在的conditions change (such as under different growth regulator conditions), the development of these cells will change direction, resulting in variation.

40. ②Chimeras嵌合体
Harmann (1983) pointed out:
The existence of chimeras is mainly due to the different genetic background of the tissue, or the mutant变异体cells in the meristem, so the chimeras in tissue culture can produce a high frequency of mutants.

41. 2、Chromosome number variation
The main causes of producing aneuploid非整倍体in the tissue culture have:
①The emergence of multi-spindle多级纺锤体;
②Chromosome non-disjunction不分离 or chromosome lagging滞后;
③Nuclear fragmentation核裂.

42. In a divided cell, if there are three or multi spindle, chromosome unequal segregation分离will inevitably必然happen, which will result in the variation of chromosome number.

43. If chromosome non-disjunction不分离 or chromosome lagging滞后phenomenon appears in the anaphase分裂后期of cell division;
When the cell enters the stage of telophase分裂末期, non-disjunction chromosomes will be still in a daughter cell;

44. Forming a hyperploid超倍体cell whose chromosome number more than parent亲本 cell, and the other cell has become hypoploid亚倍体 cell.
Lagging chromosomes may be lost and may also enter a cell.

45. In addition, nuclear fragmentation核裂 often led to the emergence of dikaryon双核.
The higher nuclear fragmentation frequency the material has, the higher emergence frequency the polyploid多倍体, hypopolyploid亚多倍体, and aneuploid非整倍体 cell have.

46. 3、Point mutation
Also known as single base substitution单碱基替换, which refers to a mutation that occurs by a single base change.

47. Point mutation can be divided into two types: Transitions转换and transversions颠换.

48. Transitions转换:
Substitution between purine嘌呤and purine, or substitution between pyrimidine嘧啶and pyrimidine嘧啶.

49. Transversions颠换:
Substitution between purine and pyrimidine.

50. 4、Chromosome exchange of somatic cells and exchange of sister chromosome
Unequal crossing over不等交换 refers to the exchange between the section区段of two chromosomes in synapsis联会.

51. The result causes that one chromosome has repeat segment and the other one has a missing segment.

52. However, when homologous chromosomes in synapsis联会 can not be accurate pairing, exchange will occur in a non corresponding position.
As a result, one chromosome will miss a part, while the other has a small part.

53. Because repeated and redundant genes can be mutated again into new genes, unequal exchange is believed to be one of the sources of new genes in the evolutionary process.

54. 5、DNA replication and deficiency缺失
DNA replicates itself, which can increase the number of copies and cause a single base change or chromosomal number change.

55. 6、The activation of transposable elements (TE)转座因子
Transposable elements can duplicate复制a copy拷贝through self-replication and inserted into a new position (target) of the genome基因组, and make the target gene inactivate失活.

56. On the other hand, transposable elements转座因子also have very important evolutionary significance, which can cause gene rearrangement directly or indirectly.

57. Direct effects include:
Replication unit fusion induced fusion caused by transposition;
Gene deletion缺失;
Gene inversion倒位or translocation易位;

58. Indirect effects:
Indirect effect refers to the same transposable elements转座因子, which insert in the different chromosomes or different positions on the same chromosome, can allow recombination to occur as micro homologous regions微型同源区域, and result in gene deletion缺失, amplification扩增, inversion倒位and translocation易位.

59. Although in 1951 McClintock early found transposable elements in maize and called it “ controlling element控制成分”. but because of the people were in the shackles束缚 of traditional ideas of orderly gene on the chromosome and difficult to accept “jumping genes” this new concept, so until 1967 Shapiro found transposable element转座因子 in E.coli .

61. 7、DNA methylation甲基化
Some scholars put forward a hypothesis that most of the mutations in tissue culture may be directly or indirectly related to the change of DNA methylation status.

62. When DNA is highly methylated甲基化, the activity of the gene is inhibited, and the activity of the gene can be improved when the degree of methylation is decreased.

63. Methylation甲基化changes may cause:
The structure change of chromatin染色质;
Delayed replication延迟复制of heterochromatin异染色质;
Chromosome breakage断裂;
Gene expression changes.

64. 8、Changes in cytoplasmic DNA
The variation of mitochondrial线粒体genome基因组was significantly higher than that of the chloroplast叶绿体genome.
The most classic example is the cytoplasmic细胞质 DNA control of cytoplasmic male sterility细胞质雄性不育(CMS).

65. 9、Epigenetic variation外遗传变异
Epigenetic variation, also known as developmental variation, refers to changes in gene expression caused by external influences, which can lead to phenotypic表型variation.

66. Common epigenetic外遗传variation is rejuvenization复幼phenomenon in tissue culture;
Namely under the environment of culture in vitro, in order to adapt to the environment, the explants derived from an adult plant change step by step in the direction of young.

67. Therefore,
Tissue cultures培养物can be any kind of development status from adult to young;
Regeneration plantlets vary with the developmental stages of the cultures and show different developmental states, which may be still maintained or disappear after a period of time;

68. Another example of epigenetic variation is the adaptive effect of tissues or cells (or domestication驯化), namely losing the heterotrophism异养(or demand) of auxin生长素, cytokinin细胞分裂素 or vitamin and become autrophism自养.

69. Other epigenetic外遗传variation, including strong growth advantage after transplanting移栽, which may be related to the young state reversal幼态性逆转or virus-free脱毒.

70. Transient短暂的stuntedness矮化may also belong to epigenetic variation, which may related to growth regulating substances remaining in vivo体内 in the tissue culture.

71. Transient dwarf plants will resume normal growth habit in the field or greenhouse after one or two seasons.

72. Section 4 Application of plant somaclonal variation in breeding

73. 1、Improving crop varieties品种, increasing germplasm种质 resources

74. 2、Strengthening the gradual permeation 渐渗of exogenous 外源 genes into cultivated species

75. When somatic hybrids杂种, monosomic alien addition lines 单体异附加系and alien substitution lines 异代换系 and other materials are used for tissue culture, they can occur genetic exchange, which can increase the gradual permeation of exogenous genes into cultivated species.