Transformation of the Cotton Plastid Genome

Introduction

As one of the most important economic crops in the world, cotton is an excellent source of natural textile fibers. However, to date, plastid transformation has only been efficient in tobacco. Cotton is particularly challenging to manipulate in vitro due to difficulties encountered during plant regeneration through somatic embryogenesis. Scientists have produced genetically modified cotton through nuclear genetic engineering, but this cotton is only suitable for growing in areas without wild relatives to avoid potential outcrossing with related weeds. Furthermore, nuclear-engineered transgenic cotton is not completely immune to attack by insect pests due to low expression of the transgene. The chloroplast genome in cotton has no paternal or biparental inheritance, and its chloroplast transgenic traits are maternally inherited. Therefore, transgenic escape can be avoided by chloroplast genetic engineering.

Fig. 1. Transformation of the cotton plastid genome using the double barrel vector. (Daniell H, et al., 2005)

Our Services

Most chloroplast transformation is achieved by organogenesis with green leaves as explants. Meanwhile, most economically important crops undergo regeneration through somatic embryogenesis when grown in vitro. Lifeasible is committed to the stable and reproducible plastid transformation of cotton from embryogenic callus. Our selection of appropriate regulatory sequences and selectable markers that function in non-green and green plastids and the generation of regenerated chloroplasts in recalcitrant crops through somatic embryo technology can easily assist you in achieving cotton plastid transformation.

Here, our engineers used the selectable markers aph A-6 and npt Ⅱ as chloroplast vectors for cotton plastid transformation by combining them with an efficient regeneration system through somatic embryogenesis. We have developed simple and reliable protocols to provide you with professional cotton plastid genome transformation services.

(1) Construction of cotton chloroplast transformation vector. Designing primers according to the available sequence information of tobacco, and amplifing the flanking regions from cotton genomic DNA to construct marker genes.
(2) Embryonic cotton callus was bombarded with gold-coated particles for chloroplast transformation.
(3) Transgenic cell cultures and plantlets were tested for the stability of site-specific transgene integration using PCR and Southern blotting.
(4) Transfer the confirmed transgenic plants to a growth chamber for flowering and fruiting.

Advantages of Cotton Plastid Transformation Using Somatic Embryogenesis

• A simple and repeatable process.
• Achieving zero pollution of food crops through chloroplast gene transformation and meet the requirements of various regulatory agencies.
• Improving cotton gene transformation efficiency and resistance gene expression.
• Selectable marker genes can detoxify antibiotics regardless of tissue or plastid type.
• Transformation procedures for cotton cell cultures can be optimized to achieve high frequencies of transformation.
• Only one or two chloroplasts are transformed in plant cells, other untransformed plastids are eliminated during selection.

Lifeasible has successfully used somatic embryogenesis technology to transform chloroplasts through plastid genomes in a variety of crops. With our team's extensive experience in somatic embryogenic chloroplast transformation, we are able to provide cotton plastid genome transformation services to customers around the world. If you are interested in our services, please do not hesitate to contact us for more information.

References

1. Daniell H, Kumar S, Dufourmantel N. (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops[J]. TRENDS in Biotechnology. 23(5): 238-245.
2. Kumar S, Dhingra A, Daniell H. (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes[J]. Plant molecular biology. 56(2): 203-216.