The article "Molecular breeding of water lily: engineering cold stress tolerance into tropical water lily" provides an in-depth examination of the molecular breeding of water lilies, specifically focusing on enhancing the cold tolerance of tropical water lilies. The research primarily employs the pollen tube pathway to introduce a gene encoding choline oxidase (CodA) into tropical water lilies, using a cold-inducible promoter Rd29A. Here's a detailed summary of the study's key aspects:

1. Principle:

- Tropical water lilies are limited in their cultivation range due to their inability to withstand winter cold. - Genetic engineering techniques are used to introduce the CodA gene, capable of producing glycine betaine, into tropical water lilies to improve their cold tolerance. Glycine betaine is a common osmoprotectant in plants that helps them resist adverse conditions.

2. Steps of Genetic Transformation:

Fig. 1. a Flowering on the first day. b Pistil. c Sepals (abaxial and adaxial view). d Petals (abaxial and adaxial view). e Stamens (abaxial and adaxial view). f Stigmatic secretions in a centrifuge tube.

- Collection of pollen and floral organs from tropical water lilies.

- Introduction of a plasmid containing the CodA gene and the Rd29A promoter into the water lilies via the pollen tube pathway.

- Screening of transformed seeds and subsequent cultivation.

- Winter cold tolerance testing of the transformed plants to observe their survival rates.

- Transgene test. To confirm if the gene was successfully introduced into water lily using PCR, southern blot, and qRT-PCR.

Fig. 2. a The plasmid used for transformation. b Pollinated flower with a paper bag. c Collected stamen and pollen. d Collecting pollen with a brush. e Pour the mixture onto the stigma.

3. Considerations:

- The transformation efficiency is relatively low, requiring a substantial amount of experimental material.

- Transformed plants need to undergo multiple rounds of molecular testing, such as PCR and Southern blot, to ensure correct integration and expression of the exogenous gene.

- The physiological characteristics of transformed plants should be analyzed in detail to assess improvements in cold tolerance.

4. Results:

- Transformed plants with enhanced cold tolerance were successfully obtained.

- These plants successfully overwintered in the Hangzhou area (at 30.3N latitude), demonstrating better cold tolerance compared to non-transformed plants.

- Under cold stress, transformed plants showed lower electrolyte leakage rates and malondialdehyde contents, as well as higher glycine betaine contents and antioxidant enzyme activities.

5. Academic Value:

- The study offers a new method for the molecular breeding of water lilies, potentially expanding their cultivation range.

- The findings are significant not only for water lily breeding but also for functional genomics research.

This article showcases the potential of molecular breeding techniques in improving the cold tolerance of plants, providing new insights for the genetic improvement of aquatic plants like water lilies.