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Terminology and Background of Haploids Advantages and Limitations- 2 Haploid-Production Systems- 3 A- Chromosome Elimination (Bulbosum (Method (B- Androgenesis (Anther Culture (C- Genogenesis (Ovary Culture D- Haploid Initiation Gene
Terminology and. 2 Background
Haploid: an individuals with the gametic chromosome number in its somatic cells Doubled Haploid: an individual with the doubled chromosome number of the haploid Monoploid - haploid derived from a diploid, x is one genomic complement (n=x) as Barley (n=x=7). Polyhaploid - haploid from a polyploid (n> x) , wheat n = 3x = 21; durum n = 2x = 14 as
1st aabb 2nd
crosses made F1
3rd F2 1AABB 2AABb 1AAbb 2AaBB 4AaBb 2Aabb 6.25% 1aabb 1aaBB 2aaBb
Population 1st AABB x aabb
Haploid production Cochicine treatment Doubled Haploid aaBB aabb 3rd (25%) AABB AAbb
Head Row Test
First : Homozygous lines can be obtained in the .shortest possible time
By extracting haploids from F1 hybrids of diploid barley and then doubling the chromosome numbers , this saves at least 3 to 4 generations of self-pollination, which are normally required when producing uniform lines for conventional breeding of self-pollinated species.
Reduction of 3-5 years for cultivar release
Second: selection is more reliable and effective at the DH than at the . diploid level
100% homozygosity of doubled haploid Using DH lines , we can evaluate quantitative characters knowing that all genes have been fixed and that desirable alleles will not be lost due to segregation in later generations.
The additive genetic variance of DH lines is always greater than the diploid lines ; therefore, the response to selection should be correspondingly greater in
populations of DH lines than in diploid populations.
Third: the homozygosity of DH lines
is reached without the selection .pressure of a field environment
can be repeated anytime can be used in different Lab can be used by different researchers
Because of this, one could set up facilities at one location to produce new lines for evaluation in any part of the world.
Fourth: DH lines can be used for quantitative genetic studies if they are a random sample of gametes . from F1 hybrids
A. Detection of Gene Interaction B. Estimation of Genetic variances C. Detection of Linkage D. Estimation of the Number of Genes E. location of polygenes
Fifth: haploids have a value in mutation breeding:
(1) Both recessive and dominant mutations will be expressed. (2) Selection on the basis of gamete frequency as haploid plants dose not require large population compared with diploid. (3) Any mutations associated with deleterious effects should be selected against during haploid production.
Sixth, the population size required to obtain a desired genotyoe from haploids is samller than that from an F2 population
Limited numbers of DH lines could be produced, therefore, the number of crosses one can utilize per year is limited. Both the bulbosum method and anther culture techniques require controlledenvironment growth facilities and skilled technical personnel.
Barley haploids can arise spontaneously or by chemical induction. Because the frequency of such spontaneous haploids is very low and unreliable, they have little value for breeding. Based on the derivations of haploids, haploidproduction systems for breeding barley can be classified into four categories : chromosome elimination (bulbosum method), androgenesis (anther culture), genogenesis (ovary culture), and haploid initiation gene.
A- Chromosome Elimination (Bulbosum Method) and Embryo Rescue
The phenomenon of chromosome elimination was discovered in interspecific cross between (Hordeum vulgare x H. bulbosum ).
For long time , barley breeders have shown interest in crossing Hordeum vulgare x H. bulbosum in the hope of transferring desirable characters from the wild species to cultivated barley. A few hybrids had been obtained from this cross but the success rate was low .
Hordeum vulgare bulbosum
Kasha and Kao (1970) pointed out that a high frequency of haploids could be obtained from this cross using various lines of diploid .Hordeum vulgare as female parent
In addition to the H. vulgare x H. bulbosum cross, chromosome elimination and haploid plants have been obtained from many other interspecific and intergeneric crosses ( barley x maize , barley x Italian ryegrass ). However, the frequencies are too low to be of interest as a barley breeding systems.
Day 0 – emasculation Day 2 - pollination with H. bulbosum pollen Day 3 (to 5) - 40% of the embryonic cells are haploid, endosperm abortion occurs, GA3 treatment enhances retention of florets Day 11 - 94% of the embryonic cells are haploid Day 14 (to 16) - embryos are dissected and cultured in the dark at 18 to 22 C, embryos develop in vitro Day 22 (to 28) - embryos are transferred to light for seedling development
Day 50 – plants
Double fertilization dose occur and chromosome elimination proceeds gradually in both the embryo and endosperm . By 8-10 days post fertilization, most dividing cells in the embryo are .haploid
The preferential loss of bulbosum chromosomes has been demonstrated cytologically and by gene markers.
Chromosome elimination process in embryos derived from H. vulgare x H. bulbosum
Days after pollination
No. of Embryo Scored
No. of cells containing Chromosome 14 13 12 11 10 9 8 7
Mean Chromosome number
Kanto Nijo 25 Yoshikei 15 Kanto Nijo 25 Yoshikei 15 Kanto Nijo 25 Yoshikei 15 Kanto Nijo 25 Yoshikei 15 Kanto Nijo 25
15 15 13 13 10 10 10 10 8 8
117 124 15 20 54 55 56 51 35 33
0 1 0 3
24 13 4 11
2 0 1 0
16 9 20 15
2 15 1 12
176 160 79 77 54 55 56 51 35 33
12.5 13.0 9.6 10.5 7.0 7.0 7.0 7.0 7.0 7.0
3 10 26 0 8 20
Up to 50-60 % of the embryos cultured have given rise to mature haploid plants. More than 90 % of these mature haploid of H.vulgare and the remainder are diploid hybrids that can be readily distinguished on the basis of their decumbent growth habit and other bulbosumlike .characteristics Thus, chromosome checks are not required to
identify haploids. Using this method , one technician can produce 50-100 haploids/week, or under ideal conditions and with specific genotypes, up to 300 haploids/week .
Androgenesis (Anther (Culture
Androgenesis – haploid plant derived from male gamete, most common method in vitro Anther and microspore (pollen) culture - haploid plants are derived from microspores (pollen) cultured individually or in anthers
In barley, Clapham (1971) first reported the induction of callus from cultured anthers , and in (1973) the production of haploid plants
Subsequently, significant progress has been made in haploid production . If reliable and productive anther culture techniques became available, this system could have greater potential than the bulbosum method for use in barley breeding.
The callus can arise by one of the following three :pathways
Division of the vegetative cell while the generative cell is non functional Division of the microspores in which a generative cell is not formed Division involving both the vegetative cell and the generative cell
3 to 5°C
3 to 5°C Microspore Similar nuclei
Factors affecting the development of haploid plants in vitro
Anther stage - most responsive cells for haploid embryo formation are those between the tetrad stage of microsporogenesis to just past the first pollen mitosis. Donor plant or anther pretreatment – enhances haploid embryo formation Actively growing plants and the first set of flowers are most responsive Cold pretreatment of anthers - either pre- or post-culture treatment (3 to 5 oC for 2 to 4 days), symmetric rather than asymmetric division of the microspore nuclei or division of the vegetative nucleus
Although these results are most encouraging, the following areas require :further research
the strong genotypic response to culture condition the relatively high frequencies of albino plants the variation in chromosome numbers of plants derived from callus establishing the stability of microsporederived plants in subsequent generation
Genogenesis (Ovary (Culture
All plants obtained have been green and haploid, but the frequencies were very low , ranging from 0.2 to 1.4% of the ovaries culture . There has been an influence of genotype , and perhaps as in the case of parthenogenesis , some stimulus will be required to increase haploid frequencies . At present , haploids from ovary culture are not interest for breeding barley .
Parthenogenesis - from unfertilized egg
Apogamy - from other cells of the megagametophyte
Parthenogenesis and Apogamy
Haploid Initiation Gene
Hagberg and Hagberg (1980 ) discovered this mutant gene accidentally in a cytological study. The haploid Initiation Gene ( hap ) appears to be a single gene that controls the abortion or the survival of abnormal embryos and endosperms, and especially favors the formation and survival of haploids.
The original mutant line produced 11-14% haploids in its progeny when a plant homozygous for the hap gene was used as the female parent in crosses with other cultivars
8% of the progeny were maternal haploids: in contrast, their reciprocal crosses produced no .haploids
Thus the hap locus acted through the maternal tissue, either to
-prevent fertilization of the egg cell
nucleus -stimulate the egg cell nucleus to divide prematurely, - possibly to allow the haploid or unbalanced embryos to develop normally
The F2 progeny of the crosses between
Homozygous mutant parent x Normal parent hap/hap x +/+ or its reciprocal crosses included between 2 and 3% haploids Indicting that the hap/hap gene was partially dominant.
The haploid plants, when their chromosome numbers were doubled , gave ratio of 34 hap/hap : 14 +/+ , a significant deviation from the expected 1:1 . ratio Suggesting that the maternal
Suggesting that the maternal genotype had an important influence on the formation and survival of haploid embryos . The F3 progeny lines often had 3040% viable haploid plants.
The Limitations of this method
The viability and the vigor of haploids differed from cross to cross . Many hap/hap seed did not germinate because of embryo abortion. The mechanism of this haploidproduct system and the reason for embryo abortion are not clear .
The spontaneous doubling rate of barley haploids ranges from 1 to 3% . this rate is too low for barley breeders and geneticists to achieve an efficient and rapid transformation of sterile , haploid materials into fertile homozygous .lines
It doesn’t affect chromosome replication, but does prolong the time for mitosis
Consequently, artificial means have to be used to doubling chromosome number is spindle inhibition by colchicines.
Normal versus colchicine Mitosis
Barley haploids can be treated with colchicines at various growth stages. Three-leaf stage Two tiller stage Three tiller stage
powder (1%), 20 ml dimethylsulphoxide (DMSO), and 10 drop of Tween 20 per liter • Post - treatment after vernalization – take more time but may save more haploid plants – vernalize green plants right after regeneration – transfer haploid plants to vermiculite after 6-8 weeks – treat them with colchicine
Before Colchcinie Treatment
After Colchicine Treatment
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