# Development and Application of an In Vitro Pollen Viability Assay for Comparative Safety Assessment of Transgenic Alfalfa (Medicago sativa L.)

## TL;DR

This study developed a method to assess pollen viability in transgenic alfalfa to evaluate environmental safety and found no significant difference compared to non-transgenic plants.

## Contribution

A novel in vitro pollen viability assay for transgenic alfalfa under varied environmental conditions was developed and validated.

## Key findings

- No significant difference in pollen viability was found between transgenic line SA6-8 and non-transgenic parent ZM-1.
- The in vitro method was optimized with parameters at 1/4/8 h and 20/30/40 °C for assessing pollen viability.
- The approach provides insights for biosafety protocols and planting strategies for transgenic alfalfa.

## Abstract

Alfalfa (Medicago sativa L.) is a vital global forage crop. Transgenic technology promises enhanced yield and quality, but requires rigorous environmental risk assessment, particularly regarding pollen-mediated gene flow, for which standardized protocols are lacking. Based on an optimized in vitro culture medium, this study developed a method to assess alfalfa pollen viability. Using a single-factor experimental design, key assessment parameters were established at 1/4/8 h and 20/30/40 °C. A comparative analysis revealed no significant difference (p > 0.05) in pollen viability between the transgenic line SA6-8 and its non-transgenic parent “ZM-1” within this evaluation system. This result indicates that the genetic modification did not impact the pollen viability of SA6-8. By establishing this in vitro germination-based pollen viability assessment system and comparatively analyzing pollen viability between transgenic alfalfa and its non-transgenic parent under diverse environmental conditions, our approach provides crucial insights for optimizing transgenic alfalfa planting strategies and strengthening biosafety review protocols.