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
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

H. vulgare

(n=7)

H. vulgare

(n=7)
Production of Barley Haploids through
Chromosome Elimination and Embryo Rescue
Possible mechanisms for chromosome elimination:

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

Spindle or centriole abnormalities - spindle formation or centriole

(n=7
attachment of H. bulbosum 4. Production of Haploids In Vitro through
Anther and Microspore Culture

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

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
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

Cold Treatme
Micro
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.

% Anthers

Producing Embryos
 Cold Pretreat

Culture medium
Anther culture - essential micro- and macronutrients, sucrose and

Cold treatment im
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

frequency of sym
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.

formation, control

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