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Induction of double haploids

The in vivo induction of double haploid requires a specific genotype which is called “inducer.” The first inducers introduced in literature are tock 6 (Coe 1959) and WS14 (Lashermes and Beckert 1988) and the synthetic “KEMS (Shatskaya et al. 1994). In the past decade, scientists at the University of Hohenheim in Germany have developed two inducing additional lines: the line “RWS”, a descendant of a cross “KEMSxWS14″ and the “UH400″ directly Synthetic “KEMS”.
The purpose of a cross with the inducer is the production of haploid plants. Female flowers that were pollinated with pollen of the inductor produce ear seeds with embryos that contain only the haploid maternal genome. If these seeds are sown the resulting plants will be sterile because they can not form regular gametes.

For an efficient production of haploids, the rates of induction have to be high. The rate of induction is defined as the number of seeds with haploid embryo divided by all seeds investigated. These rates vary from 2% to 10% depending on the inducer used.
To identify seeds with haploid embryo requires a marker phenotype. For example the lines RWS and UH400 pose a red marker in the embryo and the stem. Additionally the inductor has to produce enough pollen and has to have acceptable agronomic characteristics to facilitate maintenance. The donor plants which are crossed with the inducer have to be a type of heterozygous germplasm.

The mechanism of inducing double haploids
Until now the mechanism of induction of haploidie in vivo is not clarified completely. It is generally believed that only one of the two sperm nuclei functiones properly.
If the endosperm is fertilized, it produces seeds with haploid embryos (maternal genome only) and a triploid endosperm. This is the desired product in the double haploid technique. If the embryo is fertilized, it produces seed with a diploid embryo (maternal and paternal genome) and a diploid endosperm. With this no viable seed is produced because the triploid endosperm is responsible to nurture the embryo.

Selection of the haploid seeds
The key to successful commercial application of the double haploid technique is a system for efficient selection of the haploid seeds. The selection is based on distinguishing between haploid embryos and diploid embryos. Visual markers associated with a color, morphological and biochemical markers can be used for identification.
The marker for identification have to possess the following characteristics:
1. easy, cheap and early detection
2. sure to distinguish
3. independent of the environment and the genetic wealth of the donor
4. dominant expression (e.g. R1-nj (Nanda and Chase 1966, Neuffer et andalusia. 1997).
For example the R1-nj gene belongs to a family of genes that regulate the expression of anthocyanin pigment. The R1-nj allele produces a phenotype with scutelum and purple aleurone grains F1 (Li et al. 2001). These two features can be used as marker in the embryo and the endosperm.

Duplicating the genome
The haploid seeds are sterile and can not be autofecundar to develop homozygous lines. Sometimes some of the seeds have a diploid genome resulting from a spontaneous doubling of the genome. The rates of spontaneous duplication vary depending on the genotype of 1 to 10% (Beckert 1994, Chase 1969, Deimling et al. 1997).
The remaining haploid seeds need to be treated to induce its dihaploidización. For this colchicine, an alkaloid derived from Colchicum autumnal can be used to double the number of chromosomes of the cell. Colchicine prevents cellular mitosis by affecting the functionality of tubulin, the protein of microtubules. As a result, the chromosomes cells are duplicated but not divided. Typically, the treatment with colchicine is not complete.” For example, sometimes only the number of chromosomes in the ear is doubled and sometimes only the female inflorescence.

Multiplication of double haploid plants
The plants developing from seeds treated with Colchicine are called the D0 generation. These plants should be fertile and thus able to produce the next generation (D1) which will lead to a DH line which in theory is 100% homozygous.
Depending on the intensity of the expression of color in the embryo associated with the phenotypic marker, it is sometimes not possible to distinguish uhaploid embryo seeds from diploid seeds. These plants can be distinguished from the rest of haploid plants by their phenotype. The characteristics of these plants are:
1) force and high altitude
2) Stem color purple (presence of anthocyanin inherited inducer)
3) godson
4) Ear large, highly branched and with a lot of pollen
By contrast, the D0 haploid plants are very weak and produce little pollen. The vulnerability of these plants can be mitigated with special measures.

Beckert M (1994) Advantages and disadvantages of the use of in vitro/in situ produced DH maize plants. pp. 201-213. In Bajaj YPS (ed.) Biotechnology in Agriculture and Forestry. Maize, Vol. 25. Springer Verlag, Berlin, Heidelberg

Chase SS (1969) Monoploids and monoploid-derivatives in maize (Zea mays L.). The Botanical Reviews 35:117-167

Coe EH (1959) A line of maize with high haploid frequency. The American Naturalist 93:381-382

Deimling S, Röber FK, Geiger HH (1997) Methodik und Genetik der in-vivo-Haploideninduktion bei Mais. Vortr. Pflanzenzüchtung 38:203-224

Gayen P, Mandan JK, Kumar R, Sarkar KR (1994) Chromosome doubling in haploids through colchicine. Maize Genet Coop Newslett68:65

Lashermes P, Beckert M (1988) Genetic control of maternal haploidy in maize (Zea maize L.) and selection of haploid inducing lines. Theoretical and Applied Genetics 76:404-410

Li Y, Bernot JP, Illingworth C, Lison W, Bernot KM, Egglestone WB, Fogle KJ, DiPaola JE, Kermicle J, Alleman M (2001) Gene conversion within regulatory sequences generates maize r alleles with altered gene expression. Genetics 159:1727-1740

Nanda DK, Chase SS (1966) An embryo marker for detecting monoploids of maize (Zea maize L.). Crop Science 6:213-215

Neuffer MG, Coe EH, Wessler SR (1997) Mutants of maize. Cold Spring Harbor Laboratory Press, New York

Röber FK, Gordillo GA, Geiger HH (2005) In vivo haploid induction in maize – performance of new inducers and significance of doubled haploid lines in hybrid breeding. Maydica 50:275-283

Shatskaya OA, Zabirova ER, Shcherbak VS, Chumak MV (1994) Mass induction of maternal haploids. Maize Genetics Cooperation Newsletter 68:51

Wan Y, Petolino JF, Widholm JM (1989) Efficient production of doubled-haploid plants through colchicine treatment of anther-derived maize callus. Theor. Appl. Genet. 77: 889-892

March 4th, 2009
Topic: Crop Science, Plant breeding Tags: None

One Response to “Induction of double haploids”

  1. Line development and hybrid evaluation (example maize) | Says:

    [...] of this genome in the population of gametes. Induction of haploid lines can be achieved by an inducer or by anthere culture or intraspecific crosses (such as maize x [...]

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