1. Chapter 7 Plant Protoplast Culture and Cell Fusion

2. Plant protoplast culture and cell fusion are the key techniques of plant cell engineering.

3. Protoplast is the exposed and viable protoplasm without cell wall.
Except cell wall, protoplast has all the characteristics of living cells.

4. Section 1 Plant protoplast isolation

5. 1、Protoplast isolation
（1）Structure and chemical composition of plant cell wall
Cell wall is divided into：
①Primary wall：Cellulose（纤维素）, hemicellulose （半纤维素） and pectin （果胶）。
②Secondary wall： Cellulose（纤维素） and hemicellulose （半纤维素）。

7. （2）Degradation enzymes of cell wall and their degradation mechanism
① Cellulase;
② Hemicellulase;
③ Pectinase.

8. Cellulose: %:
Hemicellulase: %
Pectinase: It generally contain harmful enzymes, such as protease (蛋白酶) and lipase (脂肪酶), which should be centrifuged before use and shorten the enzymolysis time.

9. （3）Material source
①Material source: leaf is often used as material, but embryogenic callus or embryogenic suspension cell line is the most ideal.

10. ②Pretreatment：
Objective: to improve the yield and activity of protoplast.

11. Methods:
Low temperature treatment: 4℃, in dark for 1-2h;
ISO-osmotic solution (等渗溶液) treatment: several hours, increase the yield and activity. Especially in plants containing polyphenols (多酚), such as apple and pear.

12. （4）Isolation method
①Mechanical isolation method
Cells are pretreated in hypertonic sugar solution (高渗糖溶液).
After cells occurred slight plasmolysis (质壁分离) and protoplast is contracted to be spherical, cell wall is cut and the intact protoplast is released from the wound.

13. Shortcomings：
The number of obtained intact protoplasts is few, and this method can not be used for cells with a low degree of vacuolation (液泡化).
Therefore, the mechanical isolation method has not been widely applied.

14. Advantage：
The method can avoid damage effect of enzyme preparation (酶制剂) on protoplast.

15. ②Enzymolysis isolation method
It is widely used and the most effective method for protoplast isolation.
It is divided into two-step method and one-step method.

16. Two-step method:
Materials are firstly treated with pectinase (果胶酶), single cell is released, and then single cell is treated with cellulase (纤维素酶) and hemicellulase (半纤维素酶) and protoplast is isolated.

17. Advantage:
Obtained protoplasts are consistent and the quality is good. But due to the operation complexity, two-step method has gradually been eliminated (淘汰).

18. One-step method:
Materials are treated with mixed enzyme solution prepared by cellulase (纤维素酶) , hemicellulase (半纤维素酶) and pectinase (果胶酶) , protoplast is obtained by one step.
The method is simple and easy to operate. At present, the method is widely applied.

19. ——★ Sampling:
Calli or suspension culture cells or tissue and organ of sterile plantlets are widely used because of their strong redifferentiation ability.
If plant leaves in natural environment are used, young leaves of healthy and strong plants need to be selected.
After routine disinfection and pretreatment, the material is ready for use.

21. ——★ Enzyme liquid preparation:
According to the experimental material, the reasonable combination of enzyme liquid is determined.
We should pay attention to the type of enzyme preparation, the ratio of enzyme preparation and the pH of the enzyme liquid.

24. After enzyme liquid is centrifuged, it is filtered and sterilized with 0.45um Microporous filter membrane (微孔滤膜) and packed （分装 ） and frozen at - 20 ℃ to conserve.

25. In order to improve the stability of protoplast membrane, CaCl2·2H2O and dextran (葡聚糖) potassium sulfate (硫酸钾) are added to enzyme liquid.

26. ——★ Enzyme liquid treatment:
The plant material is placed in enzyme solution (if it is young leaves, its lower epidermis should be torn off) and treated with vacuum pump （真空泵）for 5 min in order to promote the enzyme liquid penetration.

28. After vacuum pumping, the plant material is placed in a reciprocating oscillating shaker (往复振荡式摇床), treated at 26℃ for 2-8 h by enzymolysis.

29. Mannitol (甘露醇) is generally used as osmotic pressure regulator.

31. Shortcoming：
Enzyme preparations generally contain nucleic acid (核酸) enzymes, protease (蛋白酶), phenols (酚类) and other substances, which will affect the vitality of protoplasts.

32. Advantage：
It can obtain a large number of protoplasts.
It is almost suitable for organs, tissues and cells of all plants.

33. 2、Protoplast purification
（1）Sedimentation （沉降） method
Using specific gravity (比重) principle, low speed centrifuge makes protoplasts sedimentate in the bottom.

34. Advantages and disadvantages：
①The purification and collection are convenient and the protoplasts are rarely lost.
②Protoplast purity is not high.
③At present, it is the most widely used method.

35. （2）Floating (漂浮) method
The purification method of protoplasts floating on the surface of liquid is made by 150g centrifuge using a high osmotic solution (e.g. 20% sucrose), whose density is larger than the protoplast density.

36. ① Advantage
The purity of obtained protoplast is high.

37. ② Shortcoming
The yield of protoplasts is low.

42. （3）Discontinuous gradient (梯度) centrifugation method
Also known as interface method (界面法).
Using two different density of solution to form discontinuous gradient, centrifuge at 150g, the protoplasts and damaged cells are in different liquid phase (液相), thus purify the protoplast.

45. ① Advantage
The protoplast size is uniform and the purity is high.

46. ② Shortcoming
The yield of protoplasts is low, and the operation is complicated.

48. 3、Protoplast activity determination测定
（1）Morphological observation method
—— Visual method目测法:
If the color of the protoplast is bright, its shape is complete, and it is rich in cytoplasm, thus the protoplast is active.

49. ——Osmotic pressure change method
Put the protoplast in hypertonic (高渗的) or hypotonic (低渗的) solution and observe the situation of expansion and contraction to determine its vitality.

50. If the volume of protoplast can change with the osmotic pressure of the solution, it is the activated protoplast.

51. （2）FDA method
FDA:荧光素二乙酸fluorescein diacetate
FDA itself does not have the fluorescence荧光, and can freely pass through the cell membrane of dead and living cells.

52. FDA mother liquid preparation:
FDA isn’t soluble in water, it is soluble in acetone丙酮.
2mg FDA is firstly dissolved in 1 ml acetone in order to prepare mother liquor, and then conserved at 4℃ refrigerator, whose conservation time should not be too long.

53. When being used, 0.1ml mother liquor is added in 10ml newly prepared mol/L mannitol 甘露醇 solution, the final concentration of FDA is 0.02%.

54. Staining observation:
Take a drop of 0.02%FDA solution and a drop of protoplast suspension and mix fully on glass slides载玻片, which is stained at 25 ℃ for 5-10 min and observed with fluorescence microscope.
荧光显微镜

55. However, FDA in living cells can be cleaved by esterase酯酶, which can release fluorescein (F) of FDA.

56. Fluorescein (F) is not free to pass through the cell membrane and accumulate in the intact 完整的living cells.

57. So activated cells have fluorescence荧光, dead cells have no fluorescence.
Live protoplasts: yellow green fluorescence

58. This method is convenient and reliable, and is the most common method.

59. （3）Phenosafranine酚藏花红staining method
Phenosafranine is a kind of basic dye碱性染料, which is soluble in water, appears red, and has yellow fluorescence.

60. The living protoplasts can be absorbed phenosafranine and appear red, and the protoplasts without activity are colorless because they have no absorption ability.

61. 4、Factors affecting the number and activity of protoplasts
（1）Tested materials
Callus, suspension cell line, or plant explants are generally chosen for the test materials.

62. If calli or suspension cell lines are selected, the material in the phase of vigorous division after subculture is selected.
Callus also need to pay attention to the selection of light yellow, granular material.

63. If the explants are selected, we must pay attention to the age, growth and development status of the plant, and the maturity of the tissue and organs of the explants.

64. We should choose the young tissue in the robust健壮的plants.
The protoplasts of this kind of materials have high yield, strong vitality and high plating rate after culture.

65. It is best to use sterile plantlets, which can exempt 免去material disinfection procedures, avoid the damage of the disinfection liquid on the materials, and greatly enhance the vitality of protoplasts.

66. （2）Type, combination and enzymolysis time of enzymes
Enzyme quality: need high purity;
Combination: combination is determined according to cell wall characteristics and enzyme activity;

67. Enzyme concentration: don't be too high;
Enzymolysis time: don't be too long, not more than 8h.

68. （3）Osmotic pressure stabilizer
Osmotic pressure stabilizer is mainly organic solutions consisting of sugar alcohols or soluble sugars (such as mannitol甘露醇, sucrose, etc.), is currently used.
Mannitol is mostly used.

69. （4） Plasma membrane stabilizer
To prevent the destruction of plasma membrane, increase the number of intact protoplasts to promote cell wall regeneration.
Dextran potassium sulfate葡聚糖硫酸钾, and calcium chloride氯化钙 are commonly used.

70. Calcium chloride: calcium can be bound to membrane protein, increase the calcium content of the membrane, and thereby can increase the stability of the plasma membrane.

71. Potassium dextran sulfate: inhibit the activity of some enzymes such as RNAase, which is helpful to the stability of plasma membrane.

72. （5） pH
In general, it is ;
If the material is plant tissue and organ, pH buffering agent缓冲剂 should be added to enzyme liquid in order to maintain its pH.

73. Generally 0. 05-0. 1 mol/L phosphate磷酸盐or 3. 0-5
Generally mol/L phosphate磷酸盐or mmol/L MES 【2-(N-吗啡啉）乙磺酸】 is added.

74. Section 2 Plant protoplast culture

75. 1、Protoplast culture method
（1）Plate culture method
2-fold agar is added into the culture medium, and then mixed with the same amount of protoplast suspension (suitable density).

76. Advantages:
The uniform distribution of protoplasts is favorable to its division and is easy to obtain single cell lines and observe the growth and development of single protoplast on localization.

77. Disadvantages:
The protoplasts are easily heated and easily broken;
The protoplasts are always in high osmotic pressure, and its growth and development is slow, its plate rate is also low.

78. （2） Thin layer liquid culture method
The thickness of culture medium is about 2-3mm.

80. Every other week每隔一周, the original liquid medium is replaced by fresh liquid medium using a graduated pipette刻度吸管.
When protoplasts form a large cell mass, it is transferred onto solid culture medium.

81. Advantages:
It is easy to operate, and can reduce the damage of heating on protoplasts, and also can reduce the osmotic pressure in time and supply the fresh culture medium, its cell plate rate is high.

82. Disadvantages:
The protoplast is easy to precipitate沉淀, the distribution is not uniform, formed clusters of cells gather together, it is difficult to select single cell clones.

83. （3） Solid-liquid combined culture method
The liquid medium of the same composition is added onto the solid medium containing protoplasts.

84. When the cells start to divide, the original liquid medium can be replaced by the fresh liquid medium regularly each week.
This method is the best in all the methods.

85. Advantages:
The distribution of protoplasts is uniform, it is advantageous to divide and easy to obtain single cell lines.
Moreover, because it can remove the harmful substances which inhibit the division, its cell plating rate is high.

86. Disadvantages:
Protoplasts are easy to break by heating.

88. 2、Factors affecting protoplast culture
①Protoplast viability:
Obtaining large numbers of viable protoplasts is the primary condition for the success of culture.

89. The callus or suspension cell line with strong division must be selected.

90. The enzyme concentration is not too high, the enzymolysis (酶解) time is not too long.

91. ②Initiation density of protoplast
——Suitable density is 5×103~1×104/mL;
——When cells are cultured by nursing, the culture density can reduce.

92. ③Osmotic stabilizer
In general, mol/L mannitol (甘露醇) is used.

93. ④Medium composition培养基成分
——Basic medium:
MS, B5 etc..
——Carbon source:
glucose, but some plants need sucrose, the others require a variety of carbon sources.

94. ——Concentration of inorganic salt and nitrogen氮 forms:
Don't be too high, some plants need ammonium nitrogen (铵态氮), some plants need nitrate nitrogen (硝态氮).

95. —— The concentration ratio of plant growth regulators:
Auxin: NAA, IAA, 2,4-D;
Cytokinins (CTKs): KT, ZT, 6-BA.

96. ⑤Culture conditions
—— Light:
Its initial stage does not need light, after the formation of callus, it need light.
—— Temperature:
26±1 ℃.

97. ⑥Plant material and genotype
—— In general, woody plants are more difficult than annual-biennial herbaceous (一、二年生草本) plants.

98. —— In woody plants, plants with high polyphenol (多酚) content are more difficult, such as apples and pears.

99. 3、Protoplast regeneration
Protoplast regeneration process refers to the process of separated and purified protoplasts very fast recover cell wall and regenerate cells;

100. And then continue to divide to form calli;

101. Finally differentiate and develop into the whole plant from organs or embryoid through appropriate culture methods under good culture conditions.

102. ①Cell wall regeneration
Under the suitable conditions, the protoplasts are cultured for 1-2 days, the protoplasts begin to lose its spherical 球形appearance, and begin to form a new cell wall.

103. The presence of the cell wall is directly related to the cell division, and the protoplasts whose cell wall can not be regenerated can’t undertake normal cell division.

104. —— Factors affecting cell wall regeneration
★Plant genotype
★ Donor cell differentiation status

105. ★ Medium composition:
More than 0.3M sucrose or more than 0.5M sorbitol can inhibit the formation of cell wall.
Cell wall regeneration in some plants needs be added 2,4-D, etc..

106. —— Identification of regenerative cell wall
★Fluorescence荧光staining and other identification methods :
They are the most commonly used and convenient and effective methods.

107. ★ Commonly used fluorescein荧光素is calcafluor white卡氏白(CFW).
Specific process:
※ ※ ※ Preparation of the staining solution:
CFW dissolved in M mannitol甘露醇.

108. ※ ※ ※ Staining:
CFW solution be mixed with protoplast suspension 悬浮液. After being stained for 1min, protoplasts are microscopic examined using more than 410 nm filter滤光片.

109. If there is the presence of cell wall, we can see blue light (420 nm).

110. If the protoplast contains chlorophyll, we can see the red fluorescence荧光, and the red light can remove using the filter 滤光片.

111. ②Cell division and the formation of callus or embryoid
—— The division of regenerative cells and the formation of callus or embryoid

112. ※ ※ ※ Cell division:
2-3d, the cell division occurs very rapidly.
The first cell division can happen in 2-7 d.
After the cells begin to divide, the osmotic pressure of the medium must be reduced timely.

113. ※ ※ ※Callus or embryoid form
Continuous division may form callus or embryoid.
Does not need to change the components of the culture medium.

114. Just 只要the osmotic pressure is reduced降低, the fresh medium is added, and the light intensity is increased.

115. —— Factors influencing the initiation of cell division
※ ※ ※ Genotype:
Division rate of Solanaceae茄科 is high.
Division rate of Gramineae禾本科is low.

116. ※ ※ ※ The developmental state of the donor供体 material
※ ※ ※ Protoplast activity

117. ※ ※ ※ Medium composition:
Concentration ratio of growth regulators, osmotic pressure, etc..

118. ③Plant regeneration
Two pathways:
—— ★ ★ ★
Callus induction→organ regeneration (bud) → plant regeneration
Most is this pathway.

119. —— ★ ★ ★
Direct formation of embryoids→regeneration plants
This pathway is the most ideal.

120. Section 3 Plant cell fusion

121. Plant cell fusion is the technique developed in the 1970‘s and matured at the end of the 1980's.

122. 1、Protoplast fusion
A method of fusing protoplasts by a physical or chemical method to obtain a descendant后代of two parents with all or part of the genetic material.
also known as somatic cell hybridization体细胞杂交.

124. ①Fusion principle
The existence of the difficulties:
The cell membrane surface has a stable hydrophobic groups疏水性基团, which have membrane potential电位.

125. Because of its electrostatic静电repulsive force排斥力so that the protoplast can not be adsorbed together.

126. ——Fusion principle of chemical method
★ ★ PEG (polyethylene glycol聚乙二醇) and other molecules with anion阴离子form common electrostatic bonding静电键with protoplast surface anion under Ca2+ connection , so as to promote the adhesion and binding among protoplasts.

127. ★ ★Under the treatment of high Ca2+ ---high pH, Ca2+ and PEG molecules combined with the plasma membrane are eluted洗脱, resulting in charge balance disorders and re-distribution.

128. Charge balance disorders and re-distribution make some positive charges of protoplasts connect with negative charges of other protoplasts.

129. Protoplasts happen adsorption polymerization吸附聚合, finally protoplasts fuse together.

130. ——Fusion principle of physical method
★ ★High frequency AC (alternating current,交流) voltage is applied to the two parallel平行electrodes电极of the fusion slot融合槽 and produce electrophoretic电泳effect.

131. ★ ★ The protoplasts in the fusion slot dipolarization偶极化 and arranged with a beaded-like along the direction of the electric field电场.

132. ★ ★ The instantaneous瞬间的 high voltage DC (direct-current,直流) pulse is applied, adhesive and adjacent protoplast membrane partially happen reversible可逆性instantaneous perforation瞬间穿孔.
Then the protoplast membrane connect and close, finally they fuse as a whole.

133. 2、Fusion types and methods
——Symmetric fusion对称融合
※ ※ Nucleus and nucleus fusion;
Cytoplasm and cytoplasm fusion.
※ ※ Homologous fusion同源融合;
Heterologous fusion异源融合.

134. ※ ※ ※ Fusion body ：
Homokaryon同核体(Homologous );
Heterokaryon（Heterologous ）.

135. ※ ※ It can be used to realize the recombination of parents' good traits, but it can not eliminate the bad characters, and it can not be solely used the nuclear or cytoplasmic genes of single parent.

136. ——Asymmetric fusion不对称融合
※ ※ Nucleus inactivation失活(cytoplast胞质体) of a parent, cytoplasmic genome基因组inactivation (karyoplast核质体) of another parent.

137. ※ ※ Methods of nucleus inactivation:
Physical methods: X-ray, gamma (γ) ray, ultraviolet irradiation紫外线照射.
Chemical method: iodine acetic acid碘乙酸.

138. Note: Radiation辐射 treatment has mutagenic诱变effect, but chemical substances have some damage on the protoplast, so its dose 剂量is not too high.

139. 微原生质体融合 ——Microprotoplast fusion
※ ※ Also known as micronucleus technique微核技术;

140. ※ ※ The micronucleus微核is formed after the micronucleation微核化treatment of cell, coated with biofilms被膜, and contains one or several chromosomes;

141. ※ ※ As a donor, the micronucleus fuses with protoplast of the recipient受体, thus achieve the transference of partial genome基因组.

142. —— Chemical fusion ②Method of protoplast fusion
Definition定义: The chemical fusion agent is used, which can promote the protoplast approach each other and adhesively fuse.

143. Main types:
High calcium钙 and high pH fusion method
PEG fusion method

144. PEG fusion method In 1974, Gao Guonan et al. created this technique;
The operation is simple, the fusion effect is good, and it does not need expensive instrument and equipment, therefore it is widely used.

145. Mechanism of induced诱导fusion
※ ※ PEG has the function of molecular bridge;
Protoplast ⊙ ⊙ (hydrogen bonded) ⊙ ⊙ PEG ⊙ ⊙ (hydrogen bond) ⊙ ⊙ protoplast

146. ※ ※Breaking charge balance
After PEG treatment and then PEG is eluted using high Ca2+ and high pH solutions instead of other solutions;

147. Elution using high Ca2+ and high pH solutions may make positively charged groups of a protoplast attach to negatively charged groups of another protoplast, which may result in the protoplast fusion and greatly improve the fusion rate.

149. High calcium钙 and high pH fusion method
Keller and Melchers (1973) first found high pH and high calcium can induce fusion.

150. Mechanism：
※ ※ The calcium concentration determines the stability and plasticity可塑性of the cell membrane, which influences the binding of the protoplast membrane;

151. ※ ※ High pH can change the surface charge of the plasma membrane, which is conducive to cell fusion;

152. The concentration of calcium ion and pH range范围varies depending on the plant species.
For example: Tobacco protoplast fusion need pH10.5, 0.05 mM CaCl2.

154. ——Physical fusion method
Definition定义: Mechanical methods such as electrical stimulation, centrifugation, vibration and so on are used, which can promote the fusion of protoplast.

155. Mainly include electric fusion method and ultrasonic fusion method

156. Electric fusion method
※ ※ Basic principle:
Under the induced condition of DC (direct current直流)electric pulse, the charge on the surface of protoplasts membrane and its oxidation-reduction potential氧化还原电位will change;

157. This change will make heterogeneous异种protoplasts bond and happen membrane Instantaneous瞬间rupture破裂;

158. Then the plasma membranes begin to connect until closing into integral完整的membrane and forming fusion body.

159. Advantages:
No chemical residue残毒;
Good repeatability.
Disadvantages:
Expensive equipment.

161. 3、Culture and development of the fusion body
①Types of fusion products
Heterokaryon异核体：
Heterologous fusion ；
Polykaryon多核体：
Containing different proportion of parents；

162. Homokaryon同核体：
Homologous fusion；
Heteroplasmon异胞质体：
Different cytoplasmic sources, Most of them are the fusion bodies derived from来源于 one sub-protoplast with no nucleus and the other protoplast with a nucleus.

163. ②Developmental process of the fusion body
There are 3 main processes:
※ ※ Cell wall regeneration
※ ※ Nuclear fusion
※ ※ Cell proliferation

164. 4、Somatic hybrid杂种 selection
Why to choose:
Because the products after protoplast fusion treatment include:
homokaryons同核体;
heterokaryon异核体;
mixed groups of parental protoplasts with no fusion;
some effective methods must be taken to selected out the heterokaryons异核体 and true hybrid plantlets.

165. ※ ※According to the selection period, it can be divided into:
The selection of hybrid杂种 cells;
The selection of hybrid plants.

166. ①Selection of hybrid cells
※ ※Complementary screening method
互补筛选法
Being selected using the complementary effects of parental cells on the physiological or genetic characteristics.

167. On the selective medium,
Only the hybrid cells with complementary function can grow and develop;
But the non hybrid cells with no complementary function can not grow and develop.

168. According to the different types of complementary, they can be divided into:
★ ★ Hormone autotrophic complementary (growth complementary) selection
Any parent:
The medium needs to be added to the hormone;

169. Hybrid cells:
Due to the complementary, their can produce endogenous hormones, they can also grow on the medium which does not need to add hormones.

170. ★ ★Albino complementary selection
白化互补选择
One parent can differentiate a albino mutant;
The other parent only form small cell clusters.

171. Hybrid cells: Albino complementary白化互补, can form green callus and green plantlets.

172. ★ ★ Nutritional deficiency complementary selection
营养缺陷型互补选择
One parent:
NR-(硝酸还原酶缺失突变体), the mutation site is CNX;
The other parent:
NR-, the mutation site is NIA.

173. Hybrid cells:
Due to complementation, can form NR+ (具有正常的硝酸还原酶) and can grow on the culture medium with NO3- as sole nitrogen source.

174. ★ ★ Resistant mutant complementary selection
抗性突变体互补选择
Parent 1:
Only form some small cell masses on the defined medium, but is not inhibited by actinomycin D放线菌素-D;

175. Parent 2:
Can differentiate into plantlets on the defined medium, but is inhibited by actinomycin D;

176. Hybrid cells:
Can differentiate into plantlets, and is not subject to the inhibition of actinomycin D.

177. ★ ★Gene complementation selection
基因互补选择
Parent 1:
Chloroplast叶绿体 deletion缺失mutant突变体, which is controlled by recessive隐性gene a;

178. Parent 2:
Chloroplast deletion mutant, which is controlled by recessive gene b;

179. Hybrid cells:
Normal chloroplast.

180. ※ ※Mechanical screening method
机械筛选法
Complementary selection method互补选择法requires a variety of mutants突变体, so the application is limited, mechanical screening method is not subject to this restriction限制.

181. ★ ★Natural color marker separation
天然颜色标记分离
Protoplasts respectively have different colors (such as white and green);
But hybrid cells have two kinds of color at the same time, are a mixture of two colors.

182. ★ ★Fluorescein labeling separation
荧光素标记分离
Protoplast of two parents are imported different fluorescent dyes with no toxicity;

183. But among the hybrid cells fused from two parents, the heterokaryon异核体and homokaryon同核体can be separated according to the existence of two types of fluorescence.

184. ★ ★Fluorescein labeling separation
荧光活性自动细胞分类器分类融合体
荧光活性自动细胞分类器(Fluorescence Activated Cell Sorter, FACS), 也称为流式细胞计，Flow Cytometry, FCM.

185. Both of parents are labeled with different fluorescent agents, after they fuse, heterokaryon异核体 contains two kinds of fluorescent labeling;

186. When the mixed cell population passes through the cell classifier分类器, the fluorescence characteristics are identified by electronic scanning and classified into three groups: Parent 1, Parent 2, and the fusion body.

187. ②Selection of hybrid plants
Morphological identification;
Cytological identification;
Biochemical identification;
Molecular biological identification.

188. 5、Cytoplasmic engineering细胞质工程
also known as cell reconstruction细胞拆合工程;

189. Separating the cytoplasm from the nucleus using physical or chemical methods;
And then carrying out the re-combination of nucleus and cytoplasm among different cells;
Finally reconstructing a new cell.

190. ①Characteristics of the organelle细胞器 genome基因组
※ ※Nuclear genome:
The largest, is divided into:
Photosynthetic genes;
Storage protein genes;
Sugar metabolism genes;

191. Nitrogen metabolism genes;
Developmental regulation genes;
Stimulate response genes;
Membrane protein genes;
etc..

192. ※ ※Chloroplast叶绿体genome:
Semi-autonomous半自主性, is divided into:
Genetic system genes;
Photosynthetic system genes;
Biosynthetic genes.

193. ※ ※ Mitochondrial线粒体genome:
Semi-autonomous;
Cytoplasmic male sterility细胞质雄性不育is determined by the mitochondrial genome.

194. ②Organelle 细胞器separation
Cell organelles such as nucleus and mitochondria线粒体etc. are isolated by differential centrifugation差速离心, rate zonal centrifugation速度区带离心 and isopyknic centrifugation等密度梯度离心.

195. ③Organelle transplantation
Organelle transplantation mainly includes:
Chloroplast uptake;
Mitochondrial uptake;
Nucleus transferring.

196. ④Uptake of microorganisms by protoplast
Intake of yeast cells;
Uptake of nitrogen fixing固氮 bacteria and blue-green algae藻.

197. But there is still a lack of sufficient evidence for the normal survival and reproduction繁殖 of these ingested摄取 microbes微生物 in the host 宿主cell.