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Plant Cell, Tissue and Organ Culture

HORT 515
Haploids In Vitro

1. Terminology and Background

2. Processes Leading to Production of Haploid Plants

3. Production of Haploids through Chromosome Elimination and


Embryo Rescue

4. Production of Haploids In Vitro through Anther and


Microspore Culture
1. Terminology and Background

Haploid - gametic number of chromosomes, n which may not be


equivalent to x

Monoploid - haploid derived from a diploid, x is one genomic


complement

Polyhaploid - haploid from a polyploid (n> x), prefix indicates


genome complement number, e.g. tobacco is a dihaploid
Agricultural applications for haploids - Rapid generation of
homozygous genotypes after chromosome doubling

Reduce time for variety development, e.g. 10 to 6 years or less

Homozygous recombinant line can be developed in one generation


instead of after numerous backcross generations

Selection for recessive traits in recombinant lines is more efficient


since these are not masked by the effects of dominant alleles
2. Processes Leading to Production of Haploid
Plants

Androgenesis – haploid plant derived from male gamete, most


common method in vitro

Parthenogenesis - from unfertilized egg

Apogamy - from other cells of the mega-gametophyte, example

Chromosome elimination - chromosome elimination in somatic cells,


most common method used with plant breeding
Parthenogenesis and Apogamy
Androgenesis – haploid plant derived from male gamete, most
common method in vitro

Parthenogenesis - from unfertilized egg

Apogamy - from other cells of the mega-gametophyte

Chromosome elimination - chromosome elimination in


somatic cells, most common method used with plant
breeding
3. Production Haploids through Chromosome Elimination
and Embryo Rescue

Production of haploids by chromosome elimination - There are


numerous examples, primarily achieved by wide crosses and embryo
culture

The barley example - Achieved by an interspecific cross between barley


(Hordeum vulgare, 2n = 2x = 14, VV, female) x H. bulbosum (2n = 2x =
14, BB, male), see examples
Monoploid Production of Barley (H. vulgare)

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
Cross (VV x BB)
Progeny: V VV VB VBB
n= (7) (14) (14) (21)
1517 0 26 0
Barley Monoploid Production

X
H. vulgare H. bulbosum
(n=7) (n=7)

Hybrid Zygote

H. vulgare Chromosome Elimination H. bulbosum

Embryo Culture and Haploid Plant Production


Production of Barley Haploids through Chromosome
Elimination and Embryo Rescue

Possible mechanisms for chromosome elimination:

Asynchrony of mitotic cycle times - H. bulbosum cell cycle is much


longer

Spindle or centriole abnormalities - spindle formation or centriole


attachment of H. bulbosum chromosomes is defective in the H.
vulgare nucleus
4. Production of Haploids In Vitro through Anther and
Microspore Culture

Definition, History and Background

Anther and microspore (pollen) culture - haploid plants are derived


from microspores (pollen) cultured individually or in anthers

History:
Tulecke (1953) - haploid callus (but no plants) derived Ginkgo biloba

Guha and Maheshwari (1964) - haploid plants derived from cultured


Datura anthers

Nitsch, C (1974) - haploid plants derived from cultured tobacco


microspores

Background – micro-sporogenesis and micro-gametogenesis leading


to pollen development, example
Microsporogenesis/microgametogenesis leading to
haploid embryo formation

Haploid embryo formation based on continued divisions of


the vegetative or generative cells - embryos are derived from
continued proliferation of either of these cells rather than pollen
formation

Haploid embryo formation based on symmetric division of


the microspore - rather than asymmetric division that leads to
pollen formation, most common path to haploidy, example
Microspore Mother Cell

Microspore Tetrad

First Mitosis
Similar Nuclei

Vegetative
Generative

Germination

Haploid Proembryo

Haploid Embryo
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, example

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
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, example

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, examples
Cold Treatment (3 to 5°C) Enhances Symmetric Division of
Microspores or Division of VegetativeNuclei

3 to 5°C

Vegetative Microspore

Similar nuclei

Generative

3 to 5°C

Embryo
Cold Pretreatment of Anthers Enhances the Embryogenic
Response

Cold treatment imposed prior to the first pollen mitosis increases the
frequency of symmetric divisions of the microspore leading to embryo
formation, control – room temperature.

Tobacco
Producing Embryos

100 3°C

w/identical nuclei
80 5°C for 72 h
% Anthers

5°C

% Pollen
60 C 10
5 Control
40
C
20 0
0 3 7 12
0 Days in Culture
Tobacco Datura
Culture medium

Anther culture - essential micro- and macronutrients, sucrose


and vitamins; bicellular pollen types require 2 to 4% and
tricellular types 6 to 12% sucrose

Hormone dependency as follows:

Hormone independent group - embryos directly from the


microspores w/o callus, predominantly bi-cellular pollen types,
e.g. tobacco
Hormone dependent group - bi- or tri-cellular pollen types and
plants are regenerated through a callus intermediary, typically
requires auxin and, in some instances cytokinin, e.g. grasses.

Microspore/pollen culture – bi-cellular pollen types only -


basal components + glutamine, serine and elevated levels
of i-inositol, example
Bajaj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y.
Yamada (eds.), Handbook of Plant Cell Culture. Volume 1. Techniques
for Propagation and Breeding. MacMillan, New York. p. 228-287.

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