Haploids and doubled haploids: Their generation and application in plant breeding

Authors: Vijay Sharma1, Kumara Swamy R.V1., S.G. Khandagale1 and Sunil Kumar Paswan2
1Maharana Pratap University of Agriculture & Technology, Udaipur- 313001
2C.S.A. University of Agriculture & Technology, Kanpur- 208002


Abstract

The attaining homozygous line in the normal breeding was crucial in order to develop hybrids. The best method to shortcut breeding technology is induction of haploid lines. Haploid plants are intensively utilized for investigation and improvement of many agricultural crops. Haploids are unique plants and can provide researchers with genetic information not possible with normal diploid individuals. The methods of generation and some advantages of using haploids in plant breeding are discussed here.

What are haploids?

Haploids are plants that contain a gametic chromosome number (n). They can originate spontaneously in nature or as a result of various induction techniques.

Spontaneous haploid plants are reported in several crop species, like tomato, potato, soybean, maize, barley, wheat, rice, rye etc. The recent Biotechnological techniques provide tools for induction of the haploid plants with in limited time period. It also enables the breeding methods in the fruit crops.

Classification of Haploids

In general, haploids are classified into three types.

1. Maternal haploid: These haploids contain only nuclear material and cytoplasm from the maternal parent. They result either from the elimination of the chromosomes provided by the paternal parent during embryo development.

2. In vitro androgenic haploid: These haploids obtained through the anther or microspore culture and contain both the cytoplasm and nucleus of the developing microsporocyte. The microspore developed haploids better and accurate.

3. In vivo androgenic haploid: These haploids develop from an egg cell or any other cell of the embryo sac by having the chromosomes of the maternal parent being lost during embryogenesis. Such haploids contain the cytoplasm of maternal plant and only the chromosomes of the paternal parent.

What are double haploid?

A plant or line obtained by doubling the chromosome number of a haploid plant or individual. Double haploid technique is a valuable method for genetic cartography of complex traits.

Why we need double haploids (DH)?

• Development of homozygous lines.
• Fixation of heterosis.
• Mutational studies and easy to induce mutation.
• Production of biotic and abiotic stress resistant plants.
• Cytogenetical research.
• Induction of genetic variability at haploid level.
• Double haploids in genome mapping.
• Evolutionary studies.

Advantages in the utilization of haploids

• Haploid plants can be utilized for the accelerated development of homozygous lines and pure cultivars. For this purpose it is essential to double the chromosome number after a haploid individual is generated.
• Haploids can also be utilized for the selection of genotypes that contain favorable genes. Since haploids possess only a single dose of their respective genomes. This signi?cantly facilitates the search and selection of favorable genes and the development of superior breeding genotypes.
• Natural selection on haploids can be utilized as a genetic ?lter to identify or remove harmful mutant genes.

Generation of haploids and doubled haploids

1. Chromosome elimination
It was discovered that haploid plants of Hordeum vulgare could be obtained on a large scale following the hybridization of Hordeum vulgare with Hordeum bulbosum (Kasha and Kao, 1970). When H. vulgare and H. bulbosum are crossed, a normal double fertilization event occurs. However, during seed development, chromosomes of H. bulbosum are eliminated in both the embryo and endosperm. At approximately 10 days post-fertilization, most dividing cells in the embryo are haploid. Colchicine, a mitotic inhibitor is applied to the haploid seedlings generating fertile spikelet/seed sectors with double the chromosome number. Haploids with these fertile sectors generate seed that have a normal diploid chromosome number.

Chromosome elimination is an alternative method for producing haploids commonly utilized in wheat. In this approach, pollen from either H. bulbosum or maize pollen is applied to the silks of an emasculated wheat spike. The application of maize pollen has proven to be the most successful approach by providing the highest frequency of haploids.

2. In vivo androgenesis
Kermicle (1969) reported on the possibility of obtaining androgenic haploids in maize. He found that pollination of plants containing the homozygous gene ig1 (indeterminate gametophyte 1) results in the development of 1-3% of seed with an androgenic haploid embryo.

3. In vitro androgenesis
In vitro androgenesis refers to the culturing of the male gamete either in the form of an anther or as isolated microspores onto an appropriate culture media. For most crop species appropriate in vitro androgenesis culture media has been developed.

4. Induction of maternal haploids in maize
In maize, maternal haploids can occur spontaneously. Their rate is usually about one haploid per one or two thousands of normal diploid plants. However, the low frequency of natural haploid generation prevents an effcient use of this approach in breeding programs.

An alternative approach to obtain and investigate maternal haploids in maize was reported following the discovery of a line called "Stock 6" (Coe, 1959). In this report, it was observed that pollen of Stock 6 induced the generation of haploidy. The discovery of a maize pollen source that contains a haploid-inducing factor, simplified and facilitated the ability to obtain haploids from a wide array of different genotypes. Stock 6 has since been utilized for the development of many new haploid-inducing lines that possess dominant marker genes for isolation of haploids. Typically, dominant marker genes conferring anthocyanin production are utilized. Such genes cause the development or a deep red or purple pigment in the seeds, seedlings and/or plants. One such marker is called R1-nj (R-navajo). This gene expresses anthocyanin in both the aleurone layer of the crown of the seed as well as the embryo. Following pollination of a breeding material by a haploid-inducing line that possesses marker gene R1-nj, seeds that develop with pigment in the aleurone layer but no pigment in the embryo will provide haploid plants . Seeds with pigment in both the aleurone and the embryo are hybrid.

5. Generating doubled haploids
Doubling the chromosome number in haploids is often conducted through the use of colchicine or other mitotic inhibitors, such as nitrous oxide gas and some herbicides. Following treatment of the apical meristem by a mitotic inhibitor, chromosome numbers are doubled in small sectors of the haploid plant. Normally the doubled sectors produce seeds. These seeds are doubled haploids, pure line cultivars.

Application of haploids and doubled haploids in plant breeding

• Development of homozygous inbred line and pure cultivars. At present, more than 200 varieties have been developed by utilizing a doubled haploid approach (Thomas et al., 2003).
• Provide a possibility of screening breeding material for the presence of advantageous genes.
• Giving an immediate product of stable recombinants from species crosses or fixation of heterotic combination.
• No masking effects because of high homogeneity.
• High efficiency in stacking specific targeted genes in homozygous line.
• Increased performance per se due to selection pressure in the haploid phase or during first generation of DHs.
• Simplified logistics for seed exchange between main and off season programmes since each line is fixed and can be represented by a single plant.
• Development of substitution and addition lines.

Utilization of haploids and doubled haploids can simplify the identification of genotypes that can provide a significant improvement in a variety of agronomic traits. In addition, haploids and doubled haploids can accelerate the generation of homozygous lines and pure cultivars. Therefore, haploid inducement technologies have a bright future in plant breeding.

Achievement

Crop

Haploid production route

Varieties

Country

Rice

Anther culture

Tanfeng 1, Tan Fong 1, Hua Yu 1, Hua 03, Xin Xiu, Xhongua 8, Ta Be 78, Guan 18

China

Anther culture

Dama

Hungry

Anther culture

Parag 401 (ACR 401)

India

Anther culture

CR Dhan 801 (CRAC 2224-1041; IET 18720)

CRRI,India

Anther culture

Patei and Moccoi

Argentina

Wheat

Anther culture

Hua Pei 1, Lung Hua 1, Jinghua 1, Yunhua 1, Yunhua 2

China

Anther culture

Kharoba

Morocco

Anther culture

Florin

France

Wheat x Maize

Glosa, Faur F, Liter, Miranda

Romani

Tobacco

Anther culture

Tan Yu 1, Tan Yu 2, Tan Yu 3,

China

Anther culture

F211

Japan

Barley

H. bulbosum

Mingo, Gwylan

Canada



References:

1. Acquaah, J. (2012). Principles of Plant Genetics and Breeding. John Wiley & Sons, Ltd, publication, pp. 115-119.

2. Coe, E.H. (1959). A line of maize with high haploid frequency. Amer. Nat., 93:381-382.

3. Kasha, K.J. and Kao, K.N. (1970). High frequency haploid production in barley (Hordeum vulgare L.). Nature, 225:874-876.

4. Kermicle, J.L. (1969). Androgenesis conditioned by a mutation in maize. Science, 116:1422-1424.

5. Thomas, B., W.T., Forster, B.P. and Gertsson, B. (2003). Doubled haploids in breeding, in Doubled haploid production in crop plants (eds M. Maluszynski, K. Kasha, B. P. Forster and I. Szarejko) Klewer Academic Publishers, pp. 337-350.


About Author / Additional Info:
I am currently pursuing Doctoral degree in Genetics & Plant Breeding from MPUAT, Udaipur.