(1) Liquid shallow culture: The purified protoplasts are suspended in liquid culture medium. A small amount is taken to form a very thin layer at the bottom of the culture dish, and the dish is sealed for culture.

Advantages: Simple operation and easy to add fresh culture medium.

Disadvantages: Protoplasts are prone to adhesion, making it difficult to observe at a fixed point.

(2) Solid culture: also called agarose plate method or embedding culture method. After the protoplasts are suspended in liquid culture medium, they are mixed with a coagulant (mainly agar or low-melting-point agarose) in a certain proportion to form a thin layer at the bottom of the culture dish. After solidification, the dish is sealed and cultured.

Advantages: easy to observe, helpful to reduce the impact of harmful releases

(3) Solid-liquid double-layer culture method: first spread a layer of solid culture medium at the bottom of the culture dish, and wait for it to solidify.

After solidification, liquid shallow culture is carried out on it.

Advantages: The nutrients in the solid culture medium can be absorbed and utilized by the protoplasts in the liquid layer, and the toxic substances produced by the protoplasts can be absorbed by the solid culture medium

What are the main applications of protoplast fusion (somatic cell hybridization)?

(1) Breaking through reproductive barriers, overcoming sexual hybrid incompatibility, and creating new heterokaryotic somatic hybrids

(2) Transfer excellent agronomic traits and create new germplasm materials

(3) Selectively transfer cytoplasmic genes and genomes to create cytoplasmic hybrids and study cytoplasmic-nuclear interactions and somatic-cytoplasmic gene inheritance

What are the methods of protoplast fusion?

(1) Spontaneous fusion: During the enzymatic degradation of the cell wall, some adjacent protoplasts fuse with each other to form homokaryons. Protoplasts from actively dividing cells have a higher frequency of spontaneous fusion.

(2) Chemical fusion: NaNO3 method, high pH-high calcium method, polyethylene glycol (PEG) induction method, polymer compound method

(3) Electric field induction method (cell electrofusion, mainly two-dimensional electrophoresis): protoplast contact - plasma membrane fusion - spherization - nuclear fusion

Advantages: Avoid potential toxicity of chemicals

Fusion rate: related to factors such as protoplast source, volume, chain length, pulse duration and voltage.

Laser induction method

Cell fusion based on microfluidic chips

(6) High-throughput cell fusion chip

(7) Other cell fusion technologies: such as ion beam cell fusion

technology

What is the possible mechanism of PEG-induced protoplast fusion?

Briefly describe the characteristics of several main protoplast fusion methods

(1) Symmetrical fusion: This is a fusion method in which the parental protoplasts are not treated before fusion. It combines all the characteristics of the parents. While introducing favorable traits, it also inevitably introduces some unfavorable traits.

(2) Asymmetric fusion: A method in which the nucleus or cytoplasm of a parent is inactivated by physical or chemical methods before fusion is performed.

What are the main products of protoplast fusion and what are their functions?

(1) Symmetrical hybrid: refers to a hybrid that has all the nuclear genetic material of both parents.

(2) Asymmetric hybrids: Asymmetric hybrids are hybrids in which the nuclear genetic material of one or both parents is lost.

(3) Cytoplasmic hybrid: A type of asymmetric hybrid in which the nuclear material of one fusion partner is completely lost and the cytoplasmic genetic material of both partners is present.

Heteroplasmic hybrid: A type of cytoplasmic hybrid. This refers to a hybrid in which the nucleus comes from one species and the cytoplasm comes from another.

What are the methods for screening and identifying somatic cell hybrids, and which one is the most effective?

(1) Morphological identification: Hybrids are often morphologically intermediate between their parents, including plant shape, leaf shape, and flower organs.

(2) Cytological identification

Chromosome observation: The number of chromosomes is the sum of the parents, or one or more chromosomes less; cell fusion can create non-intact chromosomes.

ploid, substitution lines, chromosome segment replacement lines and other materials

Flow cytometry ploidy analysis: The ploidy of hybrid cells can be roughly analyzed using a cell ploidy detector, which is generally

In the parental control analysis, the DNA content of the hybrid measured was basically the sum of the parents.

(3) Molecular marker identification: the most effective detection method, which can detect small reorganizations of genetic material

(4) Fluorescence in situ hybridization (FISH) identification: Identify the recombination of heterologous chromosomes and locate the recombinant fragments on the chromosomes.

(5) Protein electrophoresis analysis: To determine true or false hybrids from the gene expression products, parents should generally be used as controls, and multiple proteins can be analyzed.

The most effective is: molecular marker identification

What are the characteristics of Class II restriction endonucleases?

(1) Able to recognize specific DNA recognition sites, which are usually double-stranded palindromic structures.

(2) The double-stranded DNA molecules formed after cutting can complement each other and reconnect.

(3) The cutting and modification functions are performed by two different enzymes.

(4) ATP and SAM (S-adenosylmethionine) are not required as active cofactors.

2. Compare the characteristics of plasmid vectors, λ phage vectors and cosmid vectors.

Plasmid vector: (1) autonomous replication (2) binding of plasmid DNA

λ phage vector: (1) λ phage vector is a bacterial virus that can be packaged in vitro and can efficiently infect Escherichia coli. (2) The genome is a double-stranded DNA molecule with a length of about 50 kb. (3) The genomic DNA is linear, with 12 bases of complementary single strands at the 5' end (sticky ends, cos sites). (4) Lysogenic state: integrated into the Escherichia coli chromosome and then replicated. (5) Lytic state: after entering the host cell, the complementary single strands at both ends form a circular DNA molecule through base pairing, and replicate with the host cell. After a growth cycle of 40 to 45 minutes, about 100 infectious phage particles are released, which lyse the Escherichia coli to complete self-packaging.

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