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

Plant Cell, Tissue and Organ Culture HORT 515

Haploids In Vitro

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 (nx), prefix indicates genome complement number, e.g. tobacco is a dihaploid

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

Agricultural applications for haploids - Rapid generation of homozygous genotypes after chromosome doubling

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

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

Monoploid Production of Barley (H. vulgare)

Barley Monoploid Production

H. vulgare(n=7)

H. vulgare

H. bulbosum(n=7)

H. bulbosum

X

Chromosome Elimination

Embryo Culture and Haploid Plant Production

Hybrid Zygote

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

VegetativeGenerative

Similar Nuclei

Germination

Haploid Proembryo

Haploid Embryo

First Mitosis

Microspore Tetrad

Microspore Mother Cell

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

Similar nuclei

3 to 5°C

Microspore

Embryo

3 to 5°C

Generative

Vegetative

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

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 Datura0

20

40

60

80

100

5°C

3°C

C

C

% A

nth

ers

Pro

du

cin

g E

mb

ryo

s Tobacco

Days in Culture

5°C for 72 h

Control

00

3 7 12

5

10

% P

olle

nw

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l nu

clei

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.