Double haploid production

1. PBG-502 - Assignment
Prepared by:-
Kalpataru Nanda
1st yr M.Sc.(Ag)
Adm. No. - 03PBG/16
Submitted to:-
Dr. T .K . Mishra
Professor
(Dept. of Plant Breeding & Genetics)

4. “Dihaploid": -The chromosomal constitution of cells formed
by haploidization of polyploids.
Dihaploids resulting from halving the chromosome copy
number of tetraploids are especially useful in selective
breeding of crop plants.

5. Introduction
Conventional methods employed by plant breeders for homozygous
lines production are cumbersome, time-consuming, labourious and
rather inefficient. Sometimes it may take years to produce a pure
line. The introduction of in- vitro techniques, especially anther
culture for the induction of androgenesis has accelerated the
production of haploids for plant breeding programs. Positive results
have been obtained especially with rice, wheat, potato, barley,
maize, asparagus, sunflower, brassica, tobacco, etc. Among these,
rice and wheat are the best examples in which a number of improved
varieties have been released. In wheat, the breeding cycle can be
shortened by three or four generations when the pollen haploid
breeding method is used instead of conventional cross-breeding. The
release of the wheat varieties Jinghua 1 and Florin is a typical
example of what can be achieved with other crops. In the present
review, emphasis is also given to haploid induction through
unfertilized ovule/ovary culture(gynogenesis).

6. In-vitro haploid and doubled haploid plant
production via unfertilized ovule culture
The process of haploid regeneration through unpollinated
female gametophytes is called as gynogenesis.
Ist described in barley by San Noeum (1976)
Haploids of 21 angiosperm species have been obtained by
gynogenesis, in most cases using explant at uninucleate to
mature embryo sac stages
Most Successful in onion , sweet potato ,maize ,cucumber,
wheat

7. PROTOCOL
1.Genotype selection :-The donor genotype is thought to
play a decisive role in unfertilized ovary/ovule culture.
 Gynogenesis efficiency in plants is highly dependent on the
variety used, the growth condition of the plants, and the
quality of the donor material.
 Inbred lines and hybrid F1 tend to have higher rates of
embryo production and plant regeneration compared with
open pollinated populations.
 The percentage of gynogenic ovules ranged from 0% to
48.8%, depending on genotype.

8. 2.Stage of ovule development:- The development
stage of the ovules has a profound influence on
gynogenesis in-vitro .
 In many crop species, such as barely, sugar beet,
maize, and sunflower, several authors found that
optimal gynogenesis was obtain with nearly
matured embryo sacs .
 The most responsive ovaries (ovules) had nearly
mature or fully mature embryo sacs .
 In general, ovaries with yellow stigma were the
best explants for direct shoot regeneration.
 It was observed that only ovules collected on the
day of, or one day before, anthesis were responsive
to gynogenesis.

9. 3.Pretreatment:- Stress treatments are the most
common factor affecting embryogenesis, with
cold/heat shock and starvation treatment being
commonly used.
 Certain physical treatments (e.g. low/high
temperature, dark period or starvation medium)
applied to donor plants in in- vitro culture may have a
strong influence on embryo induction, and are also
important to switch from gametophytic pathway to
sporophytic development.
Pretreatment can be applied at different levels of
explants, such as intact flowers, isolated ovules, or
inflorescence.

10. Duration of pretreatment are different, and the
regeneration efficiencies vary as well.
Ovules exposed to 32 0C for 4 days produced the
greatest number of gynogenic ovules, followed by
ovules exposed to 40C for 4 days, and produced better
embryogenic response (28% and 22%, respectively).
 Dark incubation favors gynogenesis and minimizes
somatic callusing.

11. 4. Culture medium:- Culture media formulation has
also contributed to the progress of gynogenic
methods. The most often modified components are:
(1) source of organic nitrogen,
(2) Carbohydrates and
(3) growth regulators.
During induction, ovaries require low levels of
growth regulators and to be kept in the dark or light,
while for regeneration they are transferred to
medium with higher growth regulator concentration
and incubated in light. During cultivation of female
gametophytes, the principle of empirical selection of
nutrient media, vitamins, amino acid, and growth
regulators is the predominant approach.

12.  Sucrose is usually used at concentration of 2–3%.
Several amino acids and vitamins stimulate
gynogenesis.
Auxins are widely used for induction of gynogenesis,
and their optimum concentrations have been
reported to vary considerably from species to species.
 Specific ratios of NAA and BA supported
caulogenesis.
 Thidiazuron (TDZ) is another widely used and active
growth regulator in induction and regeneration media
for improving gynogenic response.
 Addition of polyamine (spermidine and putrescine)
in induction and regeneration media resulted in
improved gynogenic embryos and haploid plantlets .

14. Fig. 1. Production of onion haploid plants
with in vitro gynogenesis. (A) In vitro
culture of un-pollinated flower buds on
BDS medium (Dunstan and Short, 1977)
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.

15. Isolated microspore culture techniques for haploid
and doubled haploid plant production
An isolated microspore culture provides an excellent system
for the study of microspore induction and embryogenesis and
can produce doubled haploid (DH) plants, which are used to
accelerate plant-breeding programs . Microspore culture is
defined as isolating the microspores from the anther prior to
culture, whereas anther culture involves culturing the whole
anther.
 Lichter (1982) mechanically isolated microspores
from Brassica buds prior to culturing them, which launched
the field of isolated microspore culture research.
Major research has been done in cereals & brasssica sps.

16. Protocol
1. Growing donor plants:- A prerequisite for successful
and consistent microspore culture response is healthy,
pest-free donor plants.
 Donor plants can be grown in the field, the greenhouse, or
in environmentally controlled growth chambers , Seeds
are planted with adequate spacing to allow for vigorous
growth, watered regularly, and screened and treated to
minimize disease and insect infestations .
 The temperature at which the donor plants are grown
plays a critical role in microspore culture response. The
cold temperature stress of the donor plants results in a
higher frequency of microspore embryogenesis, and while
embryos can still be obtained from greenhouse grown
plants, the response is decreased.

17. 2.Harvesting floral organs:- The developmental stage
of the microspores used for culture is crucial for success.
 Buds or tillers are typically harvested when the
microspores are at the uni-nucleate to early binucleate stage.
Acetocarmine stain is most commonly used for determining
the developmental stage of the microspore .
 For many species the plant material is collected and used
immediately, while for most cereals, tillers are selected,
placed in nutrient solution, media, water, or inducer
chemicals and kept for up to several weeks prior to
microspore isolation.
Most temperature pretreatments are at 4–10°C, but short
heat shock conditions of 33°C for 48–72 h can also be used.

18. 3.surface sterilization:-The tillers and buds are surface
sterilized prior to microspore isolation to eliminate bacterial or
fungal contaminants. The most common surface sterilization
protocols involve briefly (1–2 min) immersing the plant material
in ethanol (70%), followed by immersion in sodium hypochlorite
(6% or less) with a drop of tween for several minutes (up to
15 min), followed by several washes with sterile distilled water.
4. Isolating microspores:- There are two main techniques
used to isolate microspores for culture.
The first method involves mechanically crushing the
surface-sterilized buds to release the microspores from the
anthers, using a mortar and pestle or a blender. The resulting
slurry is then passed through a filter, or a series of filters, so
that the somatic anther wall and bud tissue is separated from
the microspores. The microspores are subsequently collected
by centrifugation.

19. Maltose density gradients and Sucrose gradients have been
used to separate the developmental stages of the
microspores to give a more uniform and debris-free sample.
The second method is shed microspore culture, wherein
anthers are extracted from the buds, placed in a liquid
medium, and microspores are allowed to dehisce.
The removal of somatic tissue (debris) from the microspore
preparation is critical because its presence may negatively
affect the microspores through the release of phenolic
compounds, and in some cases, it may produce diploid calli,
embryos, and plants, complicating the search for haploid or
DH embryos and plants.

20. 5.Culture and induction of microspores:-
Isolated microspores must be provided with suitable,
nutrient-rich medium and appropriate culture
conditions. Macro and micro-nutrients, vitamins and a
carbohydrate source must be provided. Antibiotics,
such as cefotaxime can also be added to the medium
if contamination is a problem.
Stress is considered to be the inducer of
embryogenesis in microspores: without stress,
microspores follow their normal gametophytic
pathway to form pollen grains . Stress can be applied
through pretreatment of the buds, tillers or isolated
microspores, and culture media.

21. The widely used stresses were cold or heat shock,
sugar starvation, and colchicine treatment .
while γ-irradiation, ethanol stress, hypertonic shock,
centrifugal treatment, reduced atmospheric pressure,
feminizing agents, and abscisic acid were considered
neglected stresses.
Novel stresses included high medium pH,
carrageenan oligosaccharides, heavy metal stress,
inducer chemicals, and 2,4-D pretreatment.
Pyramiding stress agents may trigger the switch to
sporophytic development of microspores in
recalcitrant species.

22. 6.Regeneration of embryos:- Microspores can take a
direct or an indirect route to develop into an embryo.
The indirect route involves a number of irregular,
asynchronous divisions which results in a callus, the callus
undergoes organogenesis, and subsequently haploid embryos
are formed.
 The direct and preferred route is similar to zygotic embryo
development, wherein the embryos develop directly and
proceed through the globular, heart-shaped, torpedo, and
cotyledonary stages. Direct embryogenesis is primarily
observed in the Brassica and Solanaceae species.
 For both routes, once cotyledonary embryos have
developed, they can be removed from the culture media for
regeneration into plants. Microspore-derived embryos are
usually plated onto a solid media in the light for conversion of
the embryos into plants.

23. Doubling of chromosomes, if required
The ploidy level of regenerated plants can be determined
through chromosome counts or using flow cytometry .For
some species, there is a high percentage of spontaneous
chromosome doubling, which results in homozygous DH
plants. If spontaneous doubling does not occur or occurs at a
low frequency, the haploid plants will need to be treated with
a doubling agent in order to produce fertile, homozygous, DH
plants.

26. 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

32. References:-
Google docs & google scholarly articles