21 Oct 2020

9 Factors That Affect In Vitro Androgenesis

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Production of haploids are essential for mutagenic studies in labs and in plant breeding programs to produce plants with specific qualitative or quantitative traits. In the previous posts on haploid cultures, we offered a brief on the theory and technique of haploid culture and in vitro androgenesis. In this article, you will learn about nine factors that affect the androgenic response in the anther/pollen cultures.

1. Physiological status of the donor plants

The physiology of the plant is affected by the age and conditions in which it has been grown and this charcateristic of the donor plant affects the in vitro androgenesis. For example, the flowers that bloom early have a better response to culturing than the flowers that bloom at later stages. Similarly, a low frequency of sporophyte formation has been observed in anthers excised at the end of the season.

In some plants (like Brassica rapa), anthers excised at the old and sticky stages result in a higher number of embryos being formed than those excised at the young stage. In some other plants, a stress condition or treatment of plants, with substances like feminizing agents and gametocidal compounds (which interfere with the normal development of the plant), results in enhanced anther response.

2. Stage of pollen development

The androgenic response of anthers depends on the mitotic stage of the plants as well. For example, in plants like Datura innoxia and Nicotiana tabacum, pollens just before or after the mitosis give the best androgenic response.

In some plants, the binucleate stage of the pollens has proven to be non-embryogenic. It also creates toxicity in the cultures that suppress the embryogenic response in other uninucleate grains.

3. Anther walls

The anther wall of pollens consists of the epidermis, endothecium, middle layers, and tapetum. Several studies have shown the effectiveness of anther wall in pollen cultures. The pollen from one cultivar of a plant develops into an embryo even if transferred to the anther cultures of another cultivar.

Some other studies have shown the extracts of the anther in the pollen culture enhance productivity and embryo development. These studies have proved that the nursing nature of anther walls in pollen cultures is an essential factor in androgenic response.

4. Genotype

The genotype of plants is one of the main factors that influences embryo development in microscopic cultures. Some species of plants show a better response to pollen-derived embryo development than others. For example, japonica rice shows a better androgenic response than indica rice.

Hybrids of some plants show a range of responses. Some hybrids show a higher androgenic response, some moderate, and some may be unresponsive. This variability of the microscopic culture responses can be due to the interaction between the genotype of the plant and the environment in which they are raised.

The intercross of poor-responsive lines of some plants (like Solanum tuberosum) may produce lines with better androgenic responses. However, in tetraploid Melandrium, X-chromosomes enhance the androgenic response whereas the presence of even a single Y-chromosome suppresses the response of all three X-chromosomes.

5. Pre-treatment of cultures

Pretreatments of anther/pollen culture result in the enhancement of androgenic response.

• The cold treatment (at 5℃ for 72 hours) of anthers/pollen of the Nicotiana tabacum, before shifting to 25 ℃, enhances the embryo yield. Pretreatment of buds of white burley at 7-9 ℃ is more effective than those treated at 5 ℃. In some plants, species of Brassica and genotypes of wheat show a better androgenic response after giving a high-temperature shock at 30-35℃ for 1-4 days.
• In some species, cold treatment followed by centrifugation results in improved androgenic anthers and rapid and synchronous development of embryos.
• In some plants, like species of Datura and Nicotiana, low dose irradiation of anthers before culturing has been observed to promote callusing and embryogenesis. The mechanism behind the response is the inactivation of nuclei and alteration of auxin and cytokinin in tissues that promote androgenesis by low irradiation.

6. Culture medium

The addition of ethereal in the nutrient culture media or planting the excised anthers on agar-sucrose plates has been observed to enhance the androgenic response in tobacco. The presence of sucrose in different concentrations has been useful in promoting pollen callusing. For example, 6% sucrose is required for potato and wheat, and 12-13 % for Brassica species.

Iron is required in the culture media at the post-inductive stages of anthers culture for proper embryo development. Some studies have shown that a low concentration of nitrogen in the media promotes androgenesis.

7. Culture density

Many studies have shown that the minimum culture density of 3000 pollen/ml of the culture medium is required for embryogenesis but the highest embryo yield has been observed at 10000-40000 pollen/ml of culture medium.

8. Gaseous environment

An elevated concentration of CO2 in the culture vessel has been observed to double the androgenic response. Some other chemicals (inhibitors of ethylene) such as AgNO3 and n-propysallate resulted in the promotion of pollen embryogenesis.

9. Light

Anther cultures do not require light for growth. So, initial incubation of cultures in dark followed by diffuse lights found to induce embryogenesis in anther cultures.

References

1. Haploid production. (1996). Studies in Plant Science, 167–213. DOI:10.1016/s0928-3420(96)80009-7
2. https://www.biologydiscussion.com/genetics/factors-affecting-in-vitro-androgenesis-genetics/71811
3. http://ecoursesonline.iasri.res.in/mod/page/view.p...