Validation of CRISPR construct activity and gene function in melon via a hairy root transformation system

Area of Science:

• Plant biotechnology
• Molecular biology
• Agricultural science

Background:

• Melon (Cucumis melo L.) is a globally cultivated fruit crop with significant nutritional value.
• Efficient gene editing protocols are crucial for accelerating functional gene analysis in melon.
• Hairy root induction offers a rapid system for plant genetic studies.

Purpose of the Study:

• To develop an efficient protocol for inducing and transforming hairy roots in melon.
• To establish a system for rapid validation of CRISPR/Cas9 gene editing constructs in planta.
• To analyze the function of melon genes, such as CmRHL1, in root development.

Main Methods:

• Melon cotyledons were used as explants for inducing hairy roots using Agrobacterium rhizogenes K599.
• CRISPR/Cas9 constructs were transformed into induced hairy roots to assess gene editing efficiency.
• The effect of sgRNA expression cassette number on gene editing efficiency was evaluated.
• The function of CmRHL1 in root hair development was investigated through gene editing.

Main Results:

• A high efficiency of hairy root induction (68.61%) and transformation was achieved within a month.
• The system allowed for rapid assessment of CRISPR/Cas9 target site activity in planta.
• Gene editing efficiency was positively correlated with the number of sgRNA expression cassettes.
• Mutation of CmRHL1 significantly inhibited melon root hair development.

Conclusions:

• The established hairy root editing method provides a complementary tool for rapid CRISPR/Cas9 construct validation in melon.
• This system facilitates efficient gene function characterization during root development.
• The protocol accelerates functional genomics research in melon, complementing heritable genome editing approaches.