Chapter 3 Protoplast Culture & Somatic Hybridization Wenwu Guo

Part 2 Somatic hybridization （体细胞杂交） Definition Fusion and culture Devel. process & hybrid verification Application

1: Definition of SH SH, i.e. protoplast fusion, a process of PPs from two different parents were induced to fuse and then regenerate into plantlets, also named as SH (A + B) Several different expressions: 体细胞杂交：Somatic hybridization 细胞融合：Cell fusion 原生质体融合：Protoplast fusion 无性杂交：asexual hybridization 超性杂交：Parasexual hybridization 超性融合：Parasexual fusion 细胞操作：Cell manipulation 细胞工程: Cell engineering

Comparison of SH with Sexual crossing Items Somatic fusion Sexual cross Time 时间 No season limit Only bloom season (花期限制，母本花期) Phylogeny 亲缘关系 Can circumvent graft/sexual incompatibility 克服嫁接/有性不亲和性 Can not Results 结果 nuclear, cytoplasmic genomes recombined Ploidy level increaed Double fertilization, no recombination and ploidy level unchanged

Possible Fusion Results of Two Genetically Different Protoplasts = chloroplast = mitochondria Fusion = nucleus heterokaryon cybrid hybrid hybrid cybrid

Terminology-1 对称杂种 (symmetric hybrid)（双亲给杂种贡献的遗传物质均为100%） 非对称杂种(asymmetric hybrid)（双亲给杂种贡献的遗传物质不等（相对值） 胞质杂种（cytoplasmic hybrid, Cybrid）：细胞质重组，细胞核未重组 对称融合（symmetric fusion） 也称标准融合（Standard fusion） 非对称融合（asymmetric fusion）：融合前对一方进行处理，使其染色体丢失一部分

Terminology-2 供-受体融合：Donor-recipient fusion 体配融合（gameto-somatic fusion）（性细胞与体细胞融合） 亚原生质体-原生质体融合： Subprotoplast-protoplast fusion 小原生质体：Miniprotoplast 微原生质体：Microprotoplast 胞质体：Cytoplast 胞质体-原生质体融合: Cytoplast-protoplast fusion

Technical developmental history 1970s, establish and optimize of PP technique 1972, tobacco interspecific somatic hybrid 1975, high Ca-high pH PEG fusion; electro-fusion Numerous reports, some were not realistic Early 1980s, model plants to staple/economic crops, symmetric to asymmetric, more realistic Late 1980s, many reports on perennial woody plants 1990s to date, a conventional & safe ‘biotech’ for plant breeding in citrus, rapeseed, wheat etc.

Potato + Tomato = Pomato ?

Factors affecting fusion 2: protoplast fusion Fusion methods Fusion process Factors affecting fusion

Fusion methods Polyethylene glycol （PEG，聚乙二醇） PEG-mediated fusion Electro-fusion Polyethylene glycol （PEG，聚乙二醇） Electrofusion, Electrically induced fusion

1. PEG-mediated fusion  Characteristics: 1) Merits include: low input, high frequency of hetero-karyons, no species limit; 2) Defects include: tedious fusion procedure, toxicity to cells  Working mechanism: PEG分子具有轻微的负极性，可与具有正极性基团的水、蛋白质和碳水化合物等形成H键，从而在原生质体之间形成分子桥，从而使原生质体发生粘连进而促进原生质体融合；PEG能增加类脂膜的流动性，也使原生质体的核、细胞器发生融合成为可能

Several stock solutions to prepare Fusion sol: CaCl2 2H2O 8-10 mmol KH2PO4 0.7 mmol Mannitol 0.5-1.0 mol pH 5.6 Inducing sol: Fusion sol + PEG 20-45％ Diluting sol: sol A (g/100 ml) pH 6.0 sol B (g/100 ml) pH 10.5 Glucose 7.21 Glycin 0.375 CaCl2 2H2O 0.79 NaOH 0.169

PEG induced fusion procedure Fusion sol P1 P2 Mix, keep intact 1 min Screening P1 P2 Add PEG Culture Fusion Add diluting sol Mix sols A & B Add medium Dilution Wash Note: Mix solutions A & B (9:1) to make working solution

2．Electrofusion, Electrically induced fusion Three advantages compared with PEG fusion No toxicity to protoplasts Higher fusion frequency Simpler manipulation Electro-fusion: A high frequency AC field is applied between 2 electrodes immersed in the suspension of protoplasts- this induces charges on the protoplasts and causes them to arrange themselves in lines between the electrodes to form pearl-chain. They are then subject to a high voltage discharge which causes their membranes to fuse where they are in contact.

Electro-fusion instrument in NKL, HZAU

The process of electrically induced fusion

Fusion parameters Alternate current voltage (交流电压) Time duration of AC (交变电场处理时间) Direct current voltage (直流高频电压) Pulse width (脉冲宽度) No. of Pulses (脉冲次数)

Fusion process Membrane fusion (膜融合) Cytoplasm fusion (细胞质融合) Nucleus fusion (核融合)

Factors affecting PP fusion   PP quality (intact, viability etc) Fusion method (PEG or electro-fusion) Fusion parameters (电融合参数、溶液浓度等)