1. Introduction

The pollen culture constant temperature shaker is a key equipment in plant biotechnology. It creates a stable and uniform liquid suspension culture environment for isolated pollen by precisely controlling temperature, rotation speed, and light, simulating and optimizing its physiological conditions, thereby inducing it to change its normal gametophyte development pathway, shift towards sporophyte development, and ultimately produce haploid plants or callus tissue.

2、 Common configurations:

• temperature：25°C - 28°C(Core interval), temperature control accuracy needs to reach ±0.5°COr higher, to meet the requirements of heat shock pretreatment (such as33°C）Waiting for complex programs.

• Speed:50 - 120 rpm(Low speed zone), the operation needs to be extremely smooth, ensuring insulation and mixing without damaging fragile pollen cells.

• Light intensity:Usually in dark conditions (equipped with a light shield or placed in a dark room), some studies require light/For dark periods, a controllable lighting system needs to be configured.

3、 Plan Details

Typical scenario:

This program is widely used in genetic breeding and physiological research of important economic crops such as tobacco, rice, chili, rapeseed, and wheat. Its core purpose is to dissociate microspores/Pollen cultivation efficiently produces homozygous double haploids with genetic background（Doubled Haploid, DH）Plants can significantly shorten the breeding period, accelerate the selection of homozygous lines, and study gene function.

Configuration parameters and principles:

The success of pollen cultivation highly depends on the accuracy and stability of the physical and chemical environment provided by the constant temperature shaker. The core principle is to guide pollen from gametophyte development pathway to embryogenesis pathway through precise temperature control, gentle oscillation, and controllable lighting.

• temperature25°C - 28°C(Core interval)

Temperature is one of the most critical parameters, and the dedifferentiation of pollen and embryo initiation are extremely sensitive to temperature fluctuations. Therefore, the equipment must be able to25°Cto28°CMaintain long-term stability within the core range, and the fluctuation range is usually required to be less than ±0.5°CIn addition, heat shock commonly used in chili cultivation, such as33°CThe stress pretreatment, such as processing for several hours, also requires the accuracy of the equipment's heating rate and temperature switching.

• Speed:50 - 120 rpm(Low speed zone)

The control of rotational speed is directly related to the survival rate of cells. Due to pollen being fragile cells lacking cell wall protection, excessive rotational speed can generate destructive shear forces. Therefore, the shaker must be able to50-120 rpmRunning smoothly and uniformly within the low-speed range, this gentle oscillation can ensure uniform exchange of nutrients and gases in the culture medium, effectively prevent cell aggregation, and avoid cell rupture caused by severe shaking.

• Light intensity: usually under dark conditions

Light management is usually chosen in the early stages of cultivation under dark conditions, as darkness has been proven to be more favorable for embryo initiation of microspores. Therefore, it is common practice to choose a shaker with a fully shaded design or place it in a dark room. For certain studies that require investigating the effects of photoperiod on embryonic development, it is necessary to equip the shaker with a programmable lighting system to achieve precise light dark cycle control.

Finally, a sterile environment is the guarantee for the success of the experiment. The rocking bed chamber is commonly used304or316Stainless steel material, due to its corrosion resistance, smooth surface, and easy cleaning and disinfection, can effectively prevent microbial contamination and ensure the growth of pollen in a pure environment.

4、 Source of Literature

• Touraev, A., Vicente, O., & Heberle-Bors, E. (1997). Initiation of microspore embryogenesis by stress. Trends in Plant Science, 2(8), 297-302.

  This is a foundational review that systematically explains that the embryogenesis of microspores in various plants, including tobacco, is induced by stress (such as temperature stress), and discusses in detail the relevant liquid suspension culture methods, clarifying the core position of constant temperature shaker in this technological system.

• Gui, Y., et al. (2018). Improved anther culture efficiency of rice by abiotic stress. Plant Cell, Tissue and Organ Culture (PCTOC), 135(2), 269-279.

  This research paper provides the latest practical case of rice pollen cultivation. In order to optimize the cultivation efficiency, the liquid cultivation system was clearly used in the article, and the cultivation parameters (such as speed and temperature) carried out on a constant temperature shaker were specifically described in the materials and methods section, reflecting the practical application of this equipment in modern crop breeding technology.

• Kim, M., et al. (2013). Production of doubled haploids through anther culture in Capsicum annuum L. Korean Journal of Horticultural Science & Technology, 31(3), 350-357.

  This literature represents a typical example of applying pollen cultivation techniques in difficult crops such as chili peppers. The methodology section of the article clearly states that "anthers are placed on a rotating shaker in liquid medium80 rpmThe rotational speed, in25°CCultivation under dark conditions "provides specific equipment usage parameters and protocol support for haploid breeding of horticultural crops such as chili peppers.