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 (n>x), 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
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
Vegetative Generative
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 Datura 0
20 40 60 80 100
5°C
3°C
C
C
% Anthers Producing Embryos Tobacco
Days in Culture
5°C for 72 h
Control 0 0
3 7 12
5 10
% Pollen w/identical nuclei
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.
