15 Oct 2020

The Techniques of Haploid Production

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

Haploid plants have been a useful resource for genetic studies in identifying recessive mutations and plant breeding programs. Haploid production reduces the time required to produce the improved cultivar of a specific plant. Haploid plants are produced from haploid culture.

What is Haploid culture?

Haploid culture is an in vitro technique used to produce haploid (cells have half the number of chromosomes) plants.

Blackslee et al. (1922) first reported the natural occurrence of the haploid condition in Datura plants, due to parthenogenesis (embryo development from an unfertilized egg). The process of parthenogenesis is the only natural cause that leads to the spontaneous production of haploids.

Until 1960, haploid production was achieved by hybridizing two species of the plants or pollen irradiation. Then, Guha and Maheswari (1964) introduced the first haploid embryos and plantlets by culturing the excised anthers of Datura innoxia in lab conditions. The study was then performed on the Nicotiana tabacum, which is considered a model species for the anther culture experiments.

After the first report from Guha and Maheswari, several scientists worked in the direction of in vitro production of haploids. Now, much progress has been made in producing the haploids of several economically important plants, including wheat, rice, maize, etc. So far, the haploids have been produced from anther culture of more than 170 species, and in approximately 30 species, it is reported from the in vitro female gametophyte culture.

This article presents you a brief on the different in vivo (inside the living organism or cell) and in vitro (in lab condition, outside the living organisms) techniques to produce haploid cultures.

What are the Techniques for Haploid Production?

Haploids can be produced in vivo (within the living organism) or in vitro (outside of a living organism). The techniques of haploid production in both conditions are given below:

In vivo techniques

1. Androgenesis

This is a spontaneous process of in vivo development of a male derived haploid embryo from a fertilized egg in the absence of, or after the elimination of, the female nucleus. Now, advances in research have generated several approaches to perform the procedure under in vitro conditions.

2. Gynogenesis

This is a process of development of an embryo from unfertilized eggs or ovaries. In vivo, it occurs when the zygote formation/fertilization event does not occur but the cell division of the egg is initiated. This condition forms haploid seeds of completely maternal origin.

3. Distant hybridization

This is defined as a cross between species of the same or two different genera. So, it’s a selective elimination of chromosomes of one parent for haploid production. Distant hybridization is of two types:

• Interspecific hybridization: Hybridization between the individuals of two distinct species of the same genera.
• Intergeneric hybridization: Hybridization between individuals of two different species belonging to two different genera.

How Haploids are Produced from Distant Hybridization?

Several hypotheses have been proposed to justify the event, some of them include:

• The timing of the cell cycle between parents is different.
• Degradation of chromosomes by a host-specific enzyme.
• The proteins attached to chromosomes and required for the process are not synthesized together.

4. Irradiation effects

Ionizing (x-ray and gamma-ray) and non-ionizing radiation are used to treat pollens employed in the pollination of normal plants. The process leads to the destruction of the germination of pollens but doesn’t affect the capability of the egg cell. This induces parthenogenetic embryo development in plants.

The first study of radiation-induced haploid production was reported in Triticum monococcum and later experimented in several commercial crops. The process is affected by several factors, including genotype, physiological status of parents, source, and the dose of irradiation.

5. Chemical treatments

Chemicals like colchicine, nitrous oxide, and maleic hydrazide are used to produce haploids. However, some mitotic inhibitors have proved to be very helpful in the production of double-haploids (the number of chromosomes is doubled). These include: colchicine, oryzalin, amiprophosmethyl, trifluralin, and pronamide.

In vitro methods of haploid production

1. Androgenesis

The production of haploids through anther or pollen culture is called androgenesis and to-date, it has been reported in 135 species. The principle involved in the process is to halt the development of pollen cells into a gamete and induce it in a suitable environment to develop into a haploid plant.

The two types of androgenesis include:

• Direct androgenesis: the formation of an embryo directly from pollen or microscope without callus.
• Indirect androgenesis: the formation of an embryo with an intermediary callus stage.

The development of haploids through androgenesis depends on several factors that include the genotype of donor plants, stage of microspore or pollen, physiological status of donor plants, and pretreatments of anthers.

2. Gynogenesis

The process of development of haploids through ovary or ovule culture is known as gynogenesis. It was first reported in Hordeum vulgare (Barley) by San Norm in 1976, and later the technique was used for haploid production in wheat, maize tobacco, sunflower, sugarbeet, and other economically important plants.

This approach is used where androgenesis is not effective in producing haploids. To-date, haploids have been produced in about 19 species (distributed to 10 families) using the approach of gynogenesis. This method has a two-way approach, which includes direct embryogenesis and indirect embryogenesis that goes through callus formation followed by plant regeneration on another medium.

These methods have been very useful in quantitative trait analysis, mutational studies, identification of recessive traits, and hybrid production with particular traits. These methods have shortened the time required for cultivar development. Future research may provide more insights into the factors critical to haploid production and expand into the production of some other species of the plants.

References

1. Seguí-Simarro, J. M. (2010). Androgenesis Revisited. The Botanical Review, 76(3), 377–404. DOI:10.1007/s12229-010-9056-6
2. Watts, A., Kumar, V., Raipuria, R. K., & Bhattacharya, R. C. (2018). In Vivo Haploid Production in Crop Plants: Methods and Challenges. Plant Molecular Biology Reporter. DOI:10.1007/s11105-018-1132-9.
3. Sestili, S., & Ficcadenti, N. (1996). Irradiated pollen for haploid production. Current Plant Science and Biotechnology in Agriculture, 263–274. DOI:10.1007/978-94-017-1860-8_15.
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