1. Faculty of Science, School of Sciences, Natabua Campus Lautoka
BIO706: Embryology
Lecture 34: Production of Double Haploids through Anther culture

2. Production of double haploids through anther culture

3. Introduction
Haploids - defined as the sporophyte plants that contain a gametic chromosome number (n).
Monohaploid: e.g. Rose n = x = 7
Polyhaploid: e.g. French Marigold n = 2x = 24

4. Doubled Haploid (DH) lines – What are they?
• Haploid: an individuals with the gametic chromosome number (n) in its somatic cells.
• A Doubled Haploid: is a genotype formed when haploid cells (n), i.e. egg or sperm cell undergo chromosome doubling (2n).
• The resulting individual is completely homozygous.

5. Doubled haploids allow breeders to stabilize desired traits in a single year, reducing the time required for new variety development by up to seven years.
The process of selection can be performed at a very young stage itself.
The efficiency of the doubled haploid method can overcome competitive interactions between plants and environment which is a major problem in conventional plant breeding.

6. History
The history of DHs began with the observation of natural sporophytic haploid in Datura stramonium L. reported by Bergner in 1921.
This was followed by similar discoveries in other plant species e.g. Nicotiana tabacum and Triticum compactum
First report on haploid production was published by Blakeslee et al. (1922) in Datura stramonium.
Guha and Maheshwari (1964, 1966) developed an in vitro anther culture technique for the production of haploid Datura innoxia plants.
Haploids were reported in many other species, i.e. barley (Kasha and Kao, 1970), tobacco (Burk et al., 1979), rice, maize, brassica.

7. At present DHs production via anther culture has been reported in more than 250 plant species belonging to 100 genera and 40 families. (V.K.Misra et al., 2014)
The first in vitro induced haploid plants of female tissues origin was achieved by San Noeum (1976) in barley.
Commercial varieties developed through DH protocols - reported for many crops and more than 290 varieties have already been released (Ferrie and Caswell, 2011).
Compared to agronomic species, there has been very little work on DH in ornamental species despite the tremendous potential benefits.

8. Methods to Induce DHs In vivo occurrence of haploid plants
Spontaneous haploids
Hybridization
a) Intraspecific hybridization
b) Wide hybridization
Parthenogenesis

9. In vitro Methods
Haploids from male gametes
a) Anther culture
b) Pollen/Microspore culture
Haploids from female gametes
a) Ovary slice culture
b) Ovule culture

10. ANDROGENESIS
Androgenesis is defined as the process of embryo development from the male gametophytes (i.e. microspores or anthers), with the subsequent regeneration of haploid and doubled haploid plants from these cells.

12. Half anther culture in Anthurium
PCTO, Budi Winarto et.al., 2012

13. Pollen in hanging drops Isolated microspore culture
Microspore culture – Zantedeschia aethiopica
Medium
Microspores
Filter paper
Anthers
80 pollen grains/drop
Pollen in hanging drops
Isolated microspore culture

14. Stages of Anther Culture

15. Stages of Anther Culture
Chromosome analysis

16. GYNOGENESIS
Gynogenesis is the culture of unfertilized female gametophytes (i.e. ovules, ovaries).
This method is used when plants do not respond to androgenic methods, there is a problem with regenerating albino plants from anther culture, or the donor plants are male sterile
Gynogenesis is usually less efficient than androgenesis.
Used in plant families that do not respond to androgenesis
- Liliaceae
- Compositae

17. Gynogenesis – Spathiphyllum wallisii

18. Gynogenesis – Spathiphyllum wallisii

19. Factors affecting haploid induction
Donor plant genotype
Physiological condition of donor plants & Pre-treatment
Culture medium composition
Physical factors during tissue culture (light, temperature)
Organic addenda/sugar/sugar alcohol
Developmental stage of gametes, microspores and ovules.
Other miscellaneous factors.

20. Factors affecting haploid induction

21. Stage and alternatives of microspores

22. In vitro generated haploids in ornamental crops

23. In vitro generated haploids in ornamental crops

24. Identification of Haploids
Morphological observation
Differences of leaf morphology A) Haploid, B) Diploid and C) Triploid in Anthurium andreanum cv. ‘Tropical’

25. One year old seedlings of Dendrocalamus latiflorus a, C & d Anther regenerated plants of 3x, 6x and 12x b) Seedling sample 6x

26. Identification of Haploids cont.
Chloroplast number in stomatal guard cells
Anthurium
D) Haploid cell with 19, E) Diploid cell with 30, F) Triploid cell with 52 chloroplasts

27. Chrysanthemum stomata cells
Haploid, Tetraploid (4x) and Pentaploid (5x)

28. Identification of Haploids cont.
Chromosomal count & Flow cytometry
Determination of ploidy level of regenerated plants of Popular by flow cytometry (a-c) and chromosome counting (d-f).
a and d plant displaying a haploid histogram (2n=x=19)
b and e plant displaying a diploid histogram (2n=2x=38)
c and f plant displaying a triploid histogram (2n=3x=57)

29. Genome doubling methods.
Spontaneous Chromosome doubling Artificial Chromosome doubling

30. Spontaneous Chromosome doubling

31. Anti-microtubule drugs
Colchicine
Oryzalin
Amiprophosmethyl (APM)
Trifluralin
Pronamide

32. Stages of application of anti mitotic in DH production
Anther treatment
Microspore treatment
Haploid embryo treatment
Young haploid seedling treatment
Young haploid Root tip treatment

34. Double haploids in Crop Improvement

35. Rapid technique homozygous plants can be achieved in one generation
(inbred lines)
Production of homozygous lines of the cross pollinating species and hybrids are highly desirable
(to avoid inbreeding depression)
The conventional method of inbreeding is impractical for self-incompatible, male sterile and tree species.
Protocols already developed for Albizza lebbek, Azadirachta indica, Cassia siamea, Ceratonia siliqua, Hevea brasiliensis, Peltophorum pterocarpum, Populus sp.

36. Shortens the breeding cycle
Normally, in a hybridization programme evaluation of lines is possible only after 4-5 years of pedigree breeding and it takes another 4-5 years to release a new variety.
By anther culture of F1 hybrids the various genotypes of gametes can be fixed and evaluated in the first generation

37. New plant breeding techniques (NPBT)
Homozygous parental lines of a selected heterozygous plant are reproduced.
The genes involved in the meiotic recombination process are silenced through transgenesis.
Consequently, nonrecombined haploid lines are obtained from the heterozygous plant and their chromosomes are doubled through the double-haploid technique.
The doubled haploids obtained are screened to find a pair that, would reconstitute the original heterozygous plants.
Only nontransgenic plants are selected, thus the offspring of the selected parental lines would not carry any additional genomic charge.

38. Other advantages of Double Haploids (DH)
DHs can be represent as new variety (self-pollinated crops)
Induction of mutations
DH lines are also valuable tools in marker-trait association studies, molecular marker-assisted or genomic selection – based breeding and functional genomics
Useful in cytogenetic research
Genetic engineering
Useful in development of chromosome substitution lines.
QTL mapping, genetic and physical mapping

39. Conclusion
Doubled haploids (DH) technology is highly desirable can be implemented in all environments.
Rapid production of homozygous lines.
Speed up the breeding by fixing of the elite inbreds.
Shortened breeding cycle and cost saving.
Elimination of unfavorable genes and enrichment of favorable genes.
Improvement of germplasm lines.
Early release of commercial hybrids and resource saving.
Combination with other tools including precision phenotyping, bioinformatics, genomic selection and modeling can improve the efficiency of the breeding process.

40. Thank you! Questions are welcome