1. Chapter 3 Plant Organ and Tissue Culture

2. Plant Tissue Culture Bilingual Teaching courseware
Plant organ culture is a technique to carry out culture in vitro of the whole or part of a certain organ or organ primordia in a plant.

3. Plant Tissue Culture Bilingual Teaching courseware
The plant organs cultured in vitro are divided into vegetative organs (roots, stems, leaves) and reproductive organs (fruits, seeds, flower).

4. Plant Tissue Culture Bilingual Teaching courseware
There are many studies on the content of embryo and anther culture, which will be introduced respectively as a chapter.

5. Plant Tissue Culture Bilingual Teaching courseware
Tissue culture refers to the technique of plant tissue (meristem, epidermis, parenchyma) and callus.
分生组织
表皮组织
愈伤组织
薄璧组织

6. Section 1 Basic procedure of plant organ and tissue culture
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Section 1 Basic procedure of plant organ and tissue culture

7. 1、obtainment of sterile explants
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1、obtainment of sterile explants
（1）Sterilization of shoot-tip, stem segment and leaf
Explants are generally washed with tap water and then are absorbed water with absorbent paper.

8. Plant Tissue Culture Bilingual Teaching courseware

9. Plant Tissue Culture Bilingual Teaching courseware
Explants with more hairs are flushed with tap water after being soaked in detergent.

10. Plant Tissue Culture Bilingual Teaching courseware
Firstly sterilized in 70% alcohol for 30-60s and washed with sterile distilled water, and then sterilized in 0.1% HgCl2 for min and washed with sterile distilled water once more.

11. （2）Sterilization of root, tuber, bulb
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（2）Sterilization of root, tuber, bulb
Carefully cleaned, cleaned the rough and uneven surface and the gap with a soft brush and remove the injury.

12. Plant Tissue Culture Bilingual Teaching courseware
Increasing the sterilization time or the concentration of the sterilizing agent.

13. （3）Sterilization of flower bud
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（3）Sterilization of flower bud
In flower bud, anther is wrapped in tepals and is sterile, we can directly carry out sterilization of flower bud.

14. （4）Sterilization of fruits and seeds
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（4）Sterilization of fruits and seeds
Generally speaking, fruits and seeds generally have hairs or wax, need to add a few drops of Tween-80 in the sterilization agent in order to increase the sterilization effect.

15. 2、The establishment of primary culture
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2、The establishment of primary culture
（1）Sterile environment
（2）Standard operation
（3）Suitable condition

16. 3、Morphogenesis and plant regeneration
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3、Morphogenesis and plant regeneration
Morphogenesis of the material cultured in vitro is realized through two pathways, that is, organogenesis pathway and somatic embryogenesis pathway .

17. In organogenesis pathway:
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In organogenesis pathway:
Bud or bud primordium originats from the outer cells in the tissue cultured in vitro, that is, external origin.
Root primordium occurs in the inner cells, that is, internal origin.
There is no connection between bud and root, showing a one-way polarity.

18. Plant Tissue Culture Bilingual Teaching courseware

19. Plant Tissue Culture Bilingual Teaching courseware

20. Plant Tissue Culture Bilingual Teaching courseware
In somatic embryogenesis pathway, most somatic embryos originate from single cells, and formed embryoid is double polarity.

21. Plant Tissue Culture Bilingual Teaching courseware
In the early stage of embryoid, stem end and root end are differentiated at its opposite ends, whose vascular tissue is not associated with that of the parent plant or explant and is expressed as an independent Y type.

22. Plant Tissue Culture Bilingual Teaching courseware
Differentiated cells are composed of different tissues respectively. In general cells of the same form and function form the same tissue.
Tissues related to function form an organ, and further develop into a whole plant.

23. 4、Observation and record of culture product
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4、Observation and record of culture product
（1）Callus
Texture: dense and loose type;

24. Plant Tissue Culture Bilingual Teaching courseware

25. Color: green, light green, yellow, purple, etc.;
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Color: green, light green, yellow, purple, etc.;

26. Plant Tissue Culture Bilingual Teaching courseware

27. Plant Tissue Culture Bilingual Teaching courseware
Callus induction rate = (explant number of callus induction / explant total number) × 100%

28. Plant Tissue Culture Bilingual Teaching courseware
（2）Embryoid
Embryoid induction rate = (explant number of embryoid induction/ explant total number) × 100%

29. Plant Tissue Culture Bilingual Teaching courseware
（3）Bud
Clustered shoots （ multiple shoots）：Germinate from the terminal or axillary bud；

30. Plant Tissue Culture Bilingual Teaching courseware
Adventitious bud：generated from callus or generated directly without callus。

31. Plant Tissue Culture Bilingual Teaching courseware
Bud differentiation rate = (explant number of bud formation/ explant total number) × 100%

32. Plant Tissue Culture Bilingual Teaching courseware
（4）Root
There are no the formation of roots in the tissue culture, and only if they are transferred to the appropriate culture medium, they can only be induced.

33. Rooting rate = (bud number of rooting / total bud number) × 100%
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Rooting rate = (bud number of rooting / total bud number) × 100%

34. Plant Tissue Culture Bilingual Teaching courseware
Roots can also be directly induced by callus，but it is difficult for roots to regenerate buds.

35. Section 2 Plant vegetative organ culture
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Section 2 Plant vegetative organ culture

36. 1、Plant root segment culture
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1、Plant root segment culture
Plant root segment culture refers to the technique of culturing in vitro using root segments of plants as explants.

37. （1）Establishment of root clones ①Direct proliferation of root：
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（1）Establishment of root clones
①Direct proliferation of root：
Low inorganic ions culture media (white or 1 / 2MS medium)；
25-27 ℃ dark culture；
Forming taproot and lateral root.

38. There are three culture methods： ☆solid culture； ☆ liquid culture；
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There are three culture methods：
☆solid culture；
☆ liquid culture；
☆ solid-liquid double layer culture.
——(the upper end of morphology is inserted into solid culture medium, the lower end of morphology is immersed in liquid culture medium.)

39. ②Indirect proliferation of root
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②Indirect proliferation of root
Callus induction：Callus is induced from root.
Plant regeneration：Shoots or roots are alone regenerated from callus (or shoots and roots at the same time ). Shoots with no roots are induced to regenerate roots and then form a complete plant.

40. ③Root formation process
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③Root formation process
The occurrence of root in vitro is carried out in an adventitious root method.
Adventitious root formation is divided into two stages:
☆ The formation of root primordium: Which is associated with auxin.
☆ Elongation and growth of root primordium: Which can be achieved without exogenous hormones.

41. （2）Factors affecting occurrence and growth of root in vitro
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（2）Factors affecting occurrence and growth of root in vitro
①Basic culture medium
Generally MS (or 1/2MS or 1/3MS)、White、B5 medium；
②Growth regulators
The appropriate concentration of NAA；

42. Woody plant is harder than herbaceous plant；
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③Plant materials
Woody plant is harder than herbaceous plant；
The adult tree is harder than the young tree ；
Macrophanerophytes is more difficult than bush.

43. Solid-liquid culture； ⑤Light and temperature Dark culture, 16-25℃； ⑥pH
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④Culture methods
Solid-liquid culture；
⑤Light and temperature
Dark culture, 16-25℃；
⑥pH
Generally

44. Plant Tissue Culture Bilingual Teaching courseware

45. 2、Plant stem segment culture
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2、Plant stem segment culture
Plant stem segment culture refers to technique of culturing in vitro of stem segment with more than one normal bud or adventitious bud (including the cut-off of the young stem of tubers and corms and bulbs.

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47. Plant Tissue Culture Bilingual Teaching courseware

48. Plant Tissue Culture Bilingual Teaching courseware

49. Plant Tissue Culture Bilingual Teaching courseware

50. Plant Tissue Culture Bilingual Teaching courseware

51. Plant Tissue Culture Bilingual Teaching courseware
The main objective of the stem segment culture is rapid propagation in vitro.
Its second purpose is to study division potential and totipotency of stem cells and its ability to induce cell variation and to obtain mutants.

52. Plant Tissue Culture Bilingual Teaching courseware
Stem segment culture is the main way of plant rapid propagation in vitro.
Because of its advantages of easy success, small variation, uniform properties and fast propagation.

53. Plant Tissue Culture Bilingual Teaching courseware
Stem segment culture is the most common method in plant tissue culture, which is widely used in ferns, woody plants and herbaceous plants.

54. Plant Tissue Culture Bilingual Teaching courseware
Single bud, clustered (multiple) bud, complete plant and callus can be obtained through culturing in vitro of stem with buds, while stem without buds usually form callus.
Axillary bud

55. Plant Tissue Culture Bilingual Teaching courseware
Proliferation method of forming single bud and clustered (multiple) bud of stem with a bud is suitable for rapid propagation in vitro of plant.

56. 3、Plant leaf culture technique of culturing in vitro of leaf.
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3、Plant leaf culture
☆Plant leaf culture refers to
technique of culturing in vitro of leaf.
☆ Leaf organs including leaf primordia, petiole, vagina, blade, mesophyll and cotyledon.

57. Plant Tissue Culture Bilingual Teaching courseware
The special use of plant leaf culture is to study the process of leaf morphogenesis and its photosynthesis, chlorophyll formation, genetic transformation, etc..

58. Plant Tissue Culture Bilingual Teaching courseware
Plantlet regenerated from leaf organs culture in vitro has been successed in many plant.
The most is ferns, the second is dicotyledonous, the least is monocotyledonous.
羊齿植物
Ferns
Di-
Mono-

59. Plant Tissue Culture Bilingual Teaching courseware
callus

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61. Plant Tissue Culture Bilingual Teaching courseware

62. Plant Tissue Culture Bilingual Teaching courseware

63. Plant Tissue Culture Bilingual Teaching courseware
Leaf callus

64. But Bryophyllum pinnatum (Lam.) Oken (落地生根) is an exception.
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Note: stem segment culture does not necessarily go through callus stage, but leaf culture generally undergo.
But Bryophyllum pinnatum (Lam.) Oken (落地生根) is an exception.

65. Morphogenesis Plant Tissue Culture Bilingual Teaching courseware
Potential development
Morphogenesis

66. Plant Tissue Culture Bilingual Teaching courseware
红掌，原名：花烛。
Araceae，天南星科
It is a perennial evergreen herbs.
多年生常绿草本植物

67. Callus induced from leaf of monocotyledon
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Callus induced from leaf of monocotyledon

68. Section 3 Plant reproductive organ culture
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Section 3 Plant reproductive organ culture

69. 1、Culture of plant floral organs
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1、Culture of plant floral organs
Plant floral organ culture refers to the technique of culturing in vitro of the whole flower or flower part (including receptacle, pedicel, petal, filament, ovary, anther and ovule etc.).

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Petal culture process

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74. Plant Tissue Culture Bilingual Teaching courseware
In the fourth chapter, the embryo culture and anther culture will carry out a detailed introduction. ☆
anther ☆
embryo ☆
embryo

75. 2、Plant immature fruit culture
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2、Plant immature fruit culture
Plant immature fruit culture refers to the technique of culturing in vitro of plant immature fruits at different developmental stages.

76. Plant Tissue Culture Bilingual Teaching courseware
Its special purpose is to study fruit development, seed formation and development, and other aspects.

77. Plant Tissue Culture Bilingual Teaching courseware
3、Plant seed culture
Definition: Plant seed culture refers to the technique of culturing in vitro of immature seed and mature seeds after fertilization.

78. Plant Tissue Culture Bilingual Teaching courseware
Special purpose: to break seed dormancy, shorten life cycle; save distant hybrids, improve the germination rate of hybrid etc..

79. Culture requirements:
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Culture requirements:
for the germination of mature seeds, only need culture medium with simple components and no growth regulators.

80. Plant Tissue Culture Bilingual Teaching courseware
For the germination of immature seeds, some nutrients and growth regulators need to be added.

81. Plant Tissue Culture Bilingual Teaching courseware
If it needs to form callus, the corresponding growth regulators and nutrients need to be added.
Also need to damage it.

82. Sucrose concentration in normal culture was 3%.
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The requirement for seed culture on sucrose concentration is lower, generally 1%-3%.
Sucrose concentration in normal culture was 3%.

83. Section 4 Plant tissue culture
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Section 4 Plant tissue culture

84. 1、 Plant meristem culture meristem: Shoot-tip and root-tip
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1、 Plant meristem culture
meristem: Shoot-tip and root-tip
Shoot-tip culture is widely used in plant regeneration and virus-free.

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91. Plant Tissue Culture Bilingual Teaching courseware

92. Small to 10-100µm, large to dozens of millimeters.
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Shoot-tip culture is the culture of promeristem and derived meristem at the top of the plant.
Small to µm, large to dozens of millimeters.
The survival rate of shoot tip culture was positively correlated with the shoot tip size.

93. Plant Tissue Culture Bilingual Teaching courseware

94. Stereo microscope Anatomical needle
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Stereo microscope
Anatomical needle

95. Plant Tissue Culture Bilingual Teaching courseware
Shoot-tip dissection

96. When rapid propagation, a few millimeters of shoot-tip is cultured.
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When rapid propagation, a few millimeters of shoot-tip is cultured.
When virus-free, shoot-tip whose size is less than 1mm is cultured.

97. Possible development direction of shoot-tip culture： ①Bud germination
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Possible development direction of shoot-tip culture：
①Bud germination
②Produce embryoid
③Produce adventitious organs
④Callus formation: avoid using 2,4-D

98. Shoot tip culture may appear the following response：
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Shoot tip culture may appear the following response：
①Growth is too slow
②Growth is too fast and callus increased
③Normal growth

99. Plant Tissue Culture Bilingual Teaching courseware
2、 Plant callus culture
Callus culture is the technique of induction and culture of parenchyma cells with disorderly growth generated from plant explants.
Parenchyma cells
薄璧细胞

100. Calli is a source of suspension cell culture and protoplast culture .
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Calli is a source of suspension cell culture and protoplast culture .

101. The texture of callus was loose and dense.
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The texture of callus was loose and dense.
Dense callus could be converted to loose callus with high concentration of auxin.
The loose callus can be converted to dense callus with high concentration of cytokinin (CTK).

102. Calli color: colorless, green, yellow, red, purple (anthocyanin花青素)
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Calli color: colorless, green, yellow, red, purple (anthocyanin花青素)
Calli need subculture, generally culture in the dark or weak light, the temperature is 25℃.

103. 3、 Plant thin layer tissue culture
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3、 Plant thin layer tissue culture
It is a technique of culturing in vitro of plant thin layer tissue.
Thin layer tissue: 3-6 layer cell.
Continuous illumination, the temperature is ℃.

104. Plant Tissue Culture Bilingual Teaching courseware

105. See you next week！