Haploid and double haploid Production and their roles in crop improvement by shahnul pathan

1. RAJMATAVIJYA RAJE SINDHIYA KRISHI VISHWA VI
DHYALAY Department of Genetics & plant breeding
Presented by: SHAHNUL
(22131608 )
Msc Ag.Final year
R.A.K. College of Agriculture Sehore (m.p)
Credit Seminar
Topic –
Submitted To -
Dr. MD Yasin
Dr. Ashok Saxena
Dr. Lekharam
Dr. A.N. Bhanu
Dr. Abha shrivastava
Haploid and double haploid Producti
on and their roles in crop improveme
nt

2. Haploids are plants (sporophytes) that contain a gametic chromosome number (n). They can or
iginate spontaneously in nature or as a result of various induction techniques. Spontaneous de
velopment of haploid plants has been known since 1922
when Blakeslee first described this phenomenon in Datura stramonium (Blakeslee et al., 1922)
This was subsequently followed by similar reports in tobacco (Nicotiana tabacum), wheat (Tritic
um aestivum) and several other species (Forster et al., 2007)
The potential of haploidy for plant breeding arose in 1964 with the achievement of haploid em
bryo formation from in vitro culture of Datura anthers (Guha and Maheshwari, 1964, 1966
Haploid
Doubled haploid
A doubled haploid (DH) is a genotype formed when haploid cells undergo chro
mosome doubling. Artificial production of doubled haploids is important in pla
nt breeding.

3. Production of haploids and doubled haploids
• Haploids produced from diploid species (2n=2x), known as monoploids, contain only one set of
chromosomes in the sporophytic phase (2n=x).
• They are smaller and exhibit a lower plant vigor compared to donor plants and are sterile due to
the inability of their chromosomes to pair during meiosis. In order to propagate them through se
ed and to include them in breeding programs, their fertility has to be restored with spontaneous
or induced chromosome doubling.
• The obtained DHs are homozygous at all loci and can represent a new variety (self-pollinated cr
ops) or parental inbred line for the production of hybrid varieties (cross-pollinated crops). In fac
t, cross pollinated species often express a high degree of inbreedi For these species, the inducti
on process per se can serve not only as a fast method for the production of homozygous lines b
ut also as a selection toolng depression the elimination of genotypes expressing strong inbreed
ing depression. Selection can be expected for traits caused by recessive deleterious genes that
are associated with vegetative growth. Traits associated with flower fertility might not be related
and should be eliminated by recurrent selection among DH lines.
• The production of pure lines using doubled haploids has several advantages over conventional
methods. Using DH production systems, homozygosity is achieved in one generation, eliminatin
g the need for several generations of self-pollination.
• The induction of DH lines in dioecious plants, in which sex is determined by a regulating gene,
has an additional advantage.

4. Origin of Haploids
• Haploids arise spontaneously, and can be experimentally induced. In na ture, t
hey arise through the development of egg cells without fertilization (par cheng
enesis) or through preudogamy. There are many animals where haploidy is co
mmon and is involved in sex determination. For example, in insects of the ord
er Hymenoptera (honey bees, wasps etc.), unfertilized eggs (haploid) develop i
nto males, while fertilized eggs (diploids) develop into females: Spontaneous a
nd induced haploidy has been reported in several animals such as frog, mouse
, chicken and newt. Several plant species, such as, flax, cotton, rape, coconut, t
omato, pearl millet, wheat, barley, are known to produce haploids spontaneou
sly. In cinkorn wheat (Triticum monococcum) Smith reported the occurrence o
f haploid plants at a rate of 1/1000. The rate increased upto 20/1000 a intersp
ecific and upto 200/1000 when combined with delayed pol-lination.

5. Some genotypes show very high of haploid production. The first report of induc
ed haploidy in plants was that by Blakeslee et al. in 1922, through cold treatmen
t of young buds of Datura stramonium. Later, haploids were induced in several o
ther plant species through different techniques. In general, there are three main
approaches for the production of haploids:
(1) par- thenogenesis and apogamy,
(2) somatic reduction and chromosome elimination, and
(3) anther, pollen and ovule culture.

6. Parthenogenesis and apogamy
• The term parthenogenesis is used for the development of embryo from ovum/egg cel
l without fertilization, whereas the term apogamy is used for the development of an e
mbryo from a vegetative cell of the embryo sac without fertilization. The haploids aris
ing from the maternal cells in the embryo sac are called "gynogenetic haploids", wher
eas, those arising from the male (sperm) nucleus in the embryo sac are called "andro
genetic haploids".
• The various methods used to produce parthenogenetic haploids are as follows.
• (i) Temperature treatments
• (ii) Irradiation
• (i) Chemical treatments
• (iv) Delayed pollination
• (v) Wide hybridization
• (vi) Alien cytoplasm
• (vii) Inducing genes
• (viii) Haploid initiator genes
• (ix) Semigamy
• (x) Selection of twin seedlings

7. Temperature treatments
• Parthenogenetic haploids may be produced by high or low temperature treat
ments. In Datura stramonium, Blakeslee and coworkers obtained haploids usin
g cold treatment of young buds. Randolph ob- tained haploids in corn (Zea ma
ys) through heat treatment just after pollination. Pollination with heat-treated
pollen has also been employed for producing haploids.
Chemical treatments:
Certain chemical agents have been used for in- duction of haploidy in plants. The chemical toluidine blue,
which inactivates the sperm nuclei, could induce haploidy in some plant species such as, Vinca, tomato, mai
ze and Populus. Nitrous oxide was found to stimulate embryogenesis
in the cells of embryo sac resulting in haploid production in Capsicum annum

8. Alien cytoplasm
• This method of haploid production was reported by Kihara and Tsunewaki in
wheat. Haploids are produced when the nuclei of strain Salmon of bread wh
eat (T. aestivum) are placed into the cytoplasm of certain species of Aegilops,
c.g., Ae. caudata. Alongwith haploids, diploid see- dlings were also observed
(n/2n twins). The 2n seedlings were produced from fertilization of the synerg
id cells by the male gamete, while the egg cell was stimulated to develop par
thenogenetically into haploid embryo.
• Certain genes are known to induce haploidy in plants. The gene indeterminate gam
etophyte' (ig) induces haploids of both female (gynogenetic) and male (androgeneti
c) origin in maize (Zea mays). There is an irregular development of the megagameto
phyte in plants homozygous for this gene (ig ig); their embryo sacs contain 1-5 egg
cells and 1-7 polar nuclei. This gene leads to the production of 3 per cent haploids
among which the gynogenetic and androgenetic haploids occur in the ratio of abou
t 2: 1. Combination of the ig gene with certain marker genes, such as, " (colourless
scutellum and green plants) and R (purple pigmented kernel crown, scutellum, plu
mule and see- dlings) enables the easy identification of androgenetic and gynogene
tic haploids. For example, in the cross ig ig r5QX Ig Ig Ri Ri 6, the haploids showing
colourless scutellum and green seedlings will be gynogenetic, whereas in the cross
ig ig RR XIg lg, the haploids with colourless scutellum and seedlings will be androg
enetic.
Inducing genes

9. Haploid initiator genes
• A gene for haploid induction was reported by Hagberg in barley; this gene is
called "haploid initiator gene" (hap). When plants homozygous for the hap g
ene are used in crosses as females or are selfed. they produce 15-40 per cent
haploid progeny. However, when the hap plants are used as male parent, no
haploid progeny are produced. The heterozygous F plants (Hap hap) obtaine
d from the cross hap hap QX Hap Hap & produceper cent haploid progeny. In
the F generation, the genotypes of diploid Hap Hap, Hap hap and hap hap in
the ratio 1:2:1; plants with Hap Hap genotype produce no haploid progeny, t
hose having Hap hap genotype plants are produce 3-6 per cent haploids, whi
le hap hap plants produce 15-40 per cent haploids (Fig. 17-2). In contrast, 50
% of the haploid F2 plants will possess hap allcle, while the rest 50% will hav
e the Hap allele; the haploids possessing Hap allele are of value in breeding p
rogrammes:

10. Haploid production in barley
• through chromosome elimination from interspecific hybrid. 8 and V represent the chromosomes of H. bulbosu
m and H. vulgare, respectively. production of Datura innoxia haploids through anther culture. They noted the
appearance of numerous embryoids inside the cultured anthers; subsequently these embryoids developed int
o haploid plantlets, Haploid production through anther culture has been successfully extended to a large num
ber of plant genera and species, c.g., barley, rice, Brassica and tobacoo. The pollen present within anthers may
either directly develop into embryoids or it may form a callus from which plantlets may differentiate.

11. The main factors affecting haploid induction and subsequent
regeneration of embryos are
1. genotype of the donor plants
2. physiological condition of donor plants (i. e. growth at
lower temperature and high illumination)
3. developmental stage of gametes, microspores and ovule
4. Pre-treatment (i. e. cold treatment of inflorescences prior
to culture, hot treatment of cultured microspores)
5. composition of the culture medium (including culture on
“starvation” medium low with carbohydrates and/or
macro elements followed by transfer to normal
regeneration medium specific to the species)
6. physical factors during tissue culture (light, temperature).

12. Haploid techniques
1. Induction of maternal haploids
(a) In situ induction of maternal haploids
(b) Wide hybridization
(c) irradiated pollen
2. In vitro induction of maternal haploids
gynogenes
3. Induction of paternal haploids - androg
enesis

13. Induction of maternal haploids
(a) In situ induction of maternal haploids
In situ induction of maternal haploids can be initiated by pollinat
ion with pollen of the same species (e.g., maize), pollination with
irradiated pollen, pollination with pollen of a wild relative (e.g., b
arley, potato) or unrelated species (e.g., wheat). Pollination can
be followed by fertilization of the egg cell and development of a
hybrid embryo, in which paternal chromosome elimination occu
rs in early embryogenesis or fertilization of the egg cell does not
occur, and the development of the haploid embryo is triggered b
y pollination of polar nuclei and the development of endosperm.

15. (c) irradiated pollen
• Pollination with irradiated pollen is another
possibility for inducing the formation of
maternal haploids using intra-specific
pollination. Embryo development is
stimulated by pollen germination on the
stigma and growth of the pollen tube within
the style, although irradiated pollen is unable
to fertilize the egg cell. It has been used
successfully in several species

16. In vitro induction of maternal haploids
- gynogenesis
 In vitro induction of maternal haploids, so-called gynogenesis, is another
pathway to the production of haploid embryos exclusively from a female
gametophyte. It can be achieved with the in vitro culture of various un-po
llinated flower parts, such as ovules, placenta attached ovules, ovaries or
whole flower buds. Although gynogenetic regenerants show higher genet
ic stability and a lower rate of albino plants compared to androgenetic on
es, gynogenesis is used mainly in plants in which other induction techniq
ues, such as androgenesis and the pollination methods above described,
have failed. Gynogenic induction using un-pollinated flower parts has be
en successful in several species, such as onion, sugar beet, cucumber, s
quash, gerbera, sunflower, wheat, barley etc. (for a detailed list and proto
cols overview, see Bohanec, 2009 and Chen et al., 2011) but its applicatio
n in breeding is mainly restricted to onion and sugar beet.

17. Fig. 1. Production of onion haploid plants with in vitro gynogenesis.
(A) In vitro culture of un-pollinated flower buds on BDS medium supplemented with 500 mg/l
myo-inositol, 200 mg/l proline, 2 mg/l BAP, 2 mg/l 2,4-D, 100 g/l sucrose and 7 g/l agar;
(B) germination of haploid embryos after 60 to 180 days in culture;
(C) elongation of haploid plantlets and
(D) acclimatization of haploid plants in the greenhouse.

18. Induction of paternal haploids
- androgenesis
• Androgenesis is the process of induction and regeneration of haploids and double haploids originating
from male gametic cells. Due to its high effectiveness and applicability in numerous plant species, it has
outstanding potential for plant breeding and commercial exploitation of DH. It is well established for
plant breeding, genetic studies and/or induced mutations of many plant species, including barley,
wheat, maize, rice, triticale, rye, tobacco, rapeseed, other plants from Brassica and other genera (for
protocols, see Maluszynski et al., 2003
• Androgenesis can be induced with in vitro culture of immature anthers, a technically simple method
consisting of surface sterilization of pre-treated flower buds and subsequent excision of anthers under
aseptic conditions.
• The anthers are inoculated and cultured in vitro on solid, semi-solid or liquid mediums or two-phase
systems (liquid medium overlaying an agar-solidified medium). Anther culture was the first discovered
haploid inducing technique of which efficiency was sufficient for plant breeding purposes (Maluszynski
et al., 2003). It is still widely used, although isolated microspore culture is an improved alternative.
During isolation of microspores, the anther wall tissues are removed, thus preventing interference of
maternal sporophytic tissue during pollen embryogenesis and regeneration from somatic tissue.
Moreover, basic research of haploid embryogenesis can be performed directly at the cellular,
physiological, biochemical and molecular levels

19. Fig. 2. Microspore culture of cabbage:
(A) first divisions of microspores in NLN medium,
(B) regenerated embryos,
(C) embryos at desiccation treatment needed for regrowth,
(D) selfing of DH lines

20.  Following regeneration, haploid plants obtained from either anther or ovule culture may grow n
ormally under in vitro conditions or can even be acclimatized to form vital mature plants. Such
plants often express reduced vigor but in some crops such as onion, even haploid plants might
grow vigorously. At the flowering stage, haploid plants form inflorescences with evident malfor
mations. Due to the absence of one set of homologous chromosomes, meiosis cannot occur, so
there is no seed set. Duplication of the chromosome complement is therefore necessary
 Various methods have been applied over several decades and are still in development. The mos
t frequently used application is treatment with anti-microtubule drugs, such as colchicine (or
iginally extracted from autumn crocus Colchicum autumnale), which inhibits microtubule polym
erization by binding to tubulin. Although colchicine is highly toxic, used at a millimolar concentr
ation and known to be more efficient in animal than in plant tissues, it is still the most widely us
ed doubling agent. Other options are oryzalin, amiprophosmethyl (APM), trifluralin and pronami
de, all of which are used as herbicides and are effective in micromolar concentrations. Anti-micr
otubule drugs might be applied at various stages of androgenesis, such as being incorporated i
nto microspore pretreatment media. Colchicine application on anther culture medium, for insta
nce, showed a significant increase in embryo formation and green plant regeneration in wheat (I
slam, 2010). More often, duplication treatments are applied after regeneration at either embryo,
shoot or plantlet level. Similarly, treatments of gynogenically derived embryos with colchine ha
ve also been found to be appropriate (Jakše et al., 2003). The treatment of plants at later develo
pmental stages has the advantage that only already tested haploid regenerants are treated eithe
r in vitro (for instance at the shoot culture stage) or in vivo following acclimatization.
Chromosome doubling

21. 1. The induction and regeneration of haploids followed by spontaneous or
induced doubling of chromosomes are widely used techniques in adva
nced breeding programs of several agricultural species
2. They have been successfully used for commercial cultivar production o
f species such as asparagus, barley, Brassica juncea, eggplant, melon,
pepper, rapeseed, rice, tobacco, triticale, wheat and more than 290 varie
ties have already been release
3. Another feature that should be considered is the breeding strategy. Wit
hin the breeding process, DH lines can be induced as soon as from F1
generation (note that gametes on F1 plants represent the F2 generation)
4. The role of DH in the breeding process largely depends on the plant mo
de of reproduction. In self-pollinated species, they can represent final c
ultivars or they can be used as parental lines in hybrid production or te
st-crosses of cross-pollinated species. The basic breeding scheme in s
elf-pollinated species starts with crossings of desired genotypes, leadin
g to hybrids containing chromosome sets of both parents. During game
te formation, recombinations enable new gene combinations, which are
fixed in the process of doubled haploid production. Doubled haploids t
hus represent recombinant products of parental genomes in a complete
ly homozygous state.
Applications of doubled haploids in plant breeding

22.  DHcanbepropagatedastruebreedinglines,facilitatinglarge-scaletestingofagronomicperformanceoverth
eyears.Duetocompletehomozygosity,theefficiencyofselectionforbothqualitativeandquantitativecharacte
rsisincreasedsincerecessiveallelesarefixedinonegenerationanddirectlyexpressed.Additionally,doubled
haploidscanbeusedinarecurrentselectionschemeinwhichsuperiordoubledhaploidsofonecyclerepresent
parentsforhybridizationforthenextcycle.Severalcyclesofcrossing,doubledhaploidproductionandselectio
nareperformedandgradualimprovementoflinesisexpectedduetothealternationofrecombinationandsele
ction.
 Similarly as with self-pollinated species, the use of doubled haploids in cross-pollinated species im
proves selection efficiency and can be used at any or each cycle of recurrent selection. Cross-pollin
ated species are known to possess numerous deleterious recessive alleles that are not expressed i
n heterozygous states. They are gradually fixed during selfpollination, causing inbreeding depressi
on and difficulties in producing homozygous lines during conventional breeding. Doubled haploid t
echnology helps to overcome these problems through the rapid fixation of genes in one generatio
n and early elimination of deleterious alleles from populations. The recovered recombination prod
ucts thus represent more viable combinations of genes. Their complete homozygosity enables true
breeding and stable field performance over generations of progeny, although the complete lack of
heterozygosity and heterogeneity in varieties is thought to be more vulnerable to environmental c
hanges and altered cropping systems.

23.  Mutation breeding is another area of plant improvement for w
hich doubled haploid techniques can help to accelerate the p
rocess. Homozygosity of regenerants and true breeding prop
agation enables the fixation of mutations in the first generatio
n after mutagenic treatment. All mutated traits are immediatel
y expressed, allowing screening for both recessive and domi
nant mutants in the first generation without the need for self-
pollination. The first option is, that mutagenic treatment is ap
plied to dormant seeds that, on germination and flowering, pr
oduce M1 gametes, which are used as donor material for hapl
oid culture.
 Refrence-: Jana Murovec and Borut Bohanec (2012). Haploids
and Doubled Haploids in Plant Breeding, Plant Breeding, Dr. I
brokhim Abdurakhmonov (Ed.)