# Defining the Optimal Microspore Developmental Window for Efficient Anther-Derived Somatic Embryogenesis in Rubber Tree (Hevea brasiliensis)

## TL;DR

This study identifies the best time to collect male flower buds from rubber trees to maximize the production of embryos in lab cultures, improving breeding efficiency.

## Contribution

The research defines a precise developmental window for anther culture and confirms the somatic origin of embryogenic tissues in rubber trees.

## Key findings

- Anthers with uninucleate microspores show the highest callus and embryo formation rates.
- The optimal stage corresponds to buds 1.42–1.57 mm in diameter with green to yellowish-green coloration.
- Embryogenic tissues originate from anther wall cells, not microspores.

## Abstract

Anther-derived somatic embryogenesis is a valuable approach in rubber tree (Hevea brasiliensis) breeding; however, its effectiveness is highly influenced by the developmental stage of the microspores. The present investigation focused on male flower buds of the cultivar Reyan 73397 at successive developmental stages to examine the relationship between visible bud characteristics and internal microspore development, assess how microspore developmental stage affects callus induction and somatic embryo formation, and identify the stage with the greatest embryogenic potential. Cytological observations distinguished six well-defined phases of microspore development, spanning from microspore mother cells to fully mature pollen grains, each reliably linked to particular bud diameters, coloration, and anther morphology. Anthers corresponding to each developmental phase were cultured in vitro, and their ability to initiate callus and produce somatic embryos was systematically evaluated. Anthers containing uninucleate microspores exhibited the highest rates of both callus formation and somatic embryogenesis, with the early-uninucleate stage showing the strongest response. This stage consistently matched flower buds measuring 1.42–1.57 mm in transverse diameter and displaying a green to yellowish-green appearance. In contrast, anthers collected at the microspore mother cell and tetrad stages did not produce embryogenic responses. Histological evidence has indicated that both callus and somatic embryos originate from diploid somatic tissues of the anther wall, particularly connective parenchyma cells, rather than from microspores themselves. Based on these findings, a rapid, non-destructive selection method integrating bud diameter, bud color, and sieve-based size separation was developed to identify responsive explants efficiently. Overall, this study defines the optimal developmental window for anther culture in rubber trees, verifies the somatic origin of embryogenic tissues, and provides a practical morphological and cytological basis for improving anther culture efficiency in rubber tree breeding programs.