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EXHIBIT C

        (a)  Colchicine.

                (i)  Synonyms.  Colchicine may also be found referred to as the following:

                (ii)  Structure.  Colchicine has a structure shown in the following diagram.  COLCHICINE CRYSTALLINE;

C22H25NO6
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                        (iii)  Chromosome numbers

(A)  Polyploidity

    Polyploid plants have chromosome numbers of 3x or higher.

    Polyploidy is common in plants. About 1/3 to 1/2 of all angiosperms are polyploid.

    Polyploids include:

    Polyploids may be:

  • naturally induced - possible causes:
    • fusion of unreduced gametes: chromosome number is not reduced during meiosis, so gametes have 2n chromosomes
      • (2n + n), unreduced female gamete & reduced male gamete
      • (n + 2n), reduced female gamete & unreduced male gamete
      • (2n + 2n), unreduced female gamete & unreduced male gamete
    • spontaneous chromosome doubling - this is probably rare
  • artificially induced by:
    • environmental shock
    • chemicals that disrupt chromosome division - e.g., colchicine disrupts the spindle

    Compared to their diploid parents, artificially induced autoploids usually have

  • more vegetative growth (gigas features: larger cells, thicker leaves, bigger flowers, larger plants)
  • less seed production   

    The best candidates for artificial autopolyploidy are:

  • crops that are harvested for their vegetative parts (forages, root crops, vegetables, flowers)
  • diploids with low chromosome numbers
  • cross-pollinated species (greater recombination. . . more chance of finding a balanced polyploid genotype)
Basic chromosome number (x)
= number of chromosomes in a genome.
Haploid chromosome number (n)
= number of chromosomes in a gamete

In a diploid plant species

  • x = n
  • each somatic cell has 2n = 2x chromosomes
  • each gamete has n = x chromosomes

(B)  Euploid plants

have exact multiples of the basic chromosome number (eg. 1x, 2x, 3x....)

Euploids include

  • monoploids (x)
  • diploids (2x)
  • triploids (3x)
  • tetraploids (4x)
  • pentaploids (5x)
  • hexaploids (6x)
  • septaploids (7x)
  • octoploids (8x)

Euploids with an uneven number of genomes (triploids, pentaploids, etc.) are usually sterile because their chromosomes can not pair regularly in meiosis.

(C)  Aneuploid plants

Aneuploidy results from the addition or deletion of specific chromosomes.

Aneuploids include:

  • nullisomics (2n-2)
  • monosomics (2n-1)
  • trisomics (2n +1)
  • tetrasomics (2n+2)

Aneuploidy can be caused by cytological accidents

  • non-disjunction during meiosis (unequal sets of chromosomes go to opposite poles)
  • unpaired chromosomes may fail to reach a pole during meiosis

Result: unbalanced gametes, with extra or missing chromosomes

(I)  Aneuploids are more common in polyploid species than in diploid species.  Polyploids can tolerate the loss or addition of a chromosome, because they have more copies of each chromosome.

(1)  Aneuploids are more common in clonally propagated species than in seed-propagated species.  

Seed formation requires normal meiosis and the fusion of functional gametes.

In aneuploids

  • chromosomes may not be able to pair normally during meiosis
  • gametes with missing or extra chromosomes may not be functional

(II)  Uses of Aneuploids

(1)  to identify which chromosome a gene is on.

(2)  to transfer specific chromosomes into cultivars or other species ("chromosome engineering")

(D)  Haploidy in plants

Haploid
= a plant in which the somatic cells contain n chromomes (ie. the number that is normally found in the gametes)

Doubling of the chromosome number from n to 2n gives a doubled haploid.

A haploid of a diploid is . . . monoploid.

  • A haploid of a polyploid has more than 1 set of chromosomes. It is called a polyhaploid. (Example: a haploid of a tetraploid has 2 sets of chromosomes. It is called a dihaploid)

(I)  Procedures for producing haploid plants

(1) Identification of naturally occurring haploids

Spontaneous haploid plants occur at a low frequency in many plants.

Example: In corn (maize), haploid plants occur at a rate of 1 haploid per 1000 diploids. They develop by parthenogenesis (development of an unfertilized egg into an embryo).

Problem: How can we distinguish the haploids from the diploids?
Solution: Marker genes (eg. a dominant gene, P, purple plant colour)

To derive haploids from an F1 plant (green).

  • Pollinate the F1 plant (pp) with pollen from a homozygous purple plant (PP).
  • All diploid progeny will be purple (Pp).
  • All haploid progeny will be green (p).

Expected ratio: 999 purple diploids:1 green haploid.

(2)  Interspecific hybridization & chromosome elimination

After an interspecific cross, the chromosomes of one parent species may be eliminated, leaving only 1 haploid set from the other parent.

Example: the bulbosum method in barley

Pollen is collected from plants of Hordeum bulbosum, a wild relative of cultivated barley (Hordeum vulgare).

The H. bulbosum pollen is brushed onto emasculated barley florets.

A hybrid zygote forms, but during the first few cell divisions, the H. bulbosum chromosomes are eliminated. The seeds that develop contain haploid embryos with one set of H. vulgare chromosomes.

The haploid embryos must be "rescued" and transferred to tissue culture to obtain haploid plantlets.

The haploid plants can be treated with colchicine to obtain doubled haploids

(3)  Anther, pollen & microspore culture

In tissue culture, haploid microspore cells may develop into haploid plants.

Cultured anthers may give rise directly to embryoids & then haploid plantlets, or callus may develop first.

(4)  Chromosome doubling

The chromosomes of haploids may double spontaneously or can be doubled with colchicine to obtain doubled haploids.

(5)  Uses of haploids & doubled haploids

The main use of haploidy is for the rapid production of completely homozygous doubled haploid lines (even in self-incompatible species).

Because haploids are hemizygous (only one allele per locus) they are useful for

  • mutation studies (recessive mutants can be observed immediately)
  • selecting against undesirable recessive alleles

Genetic segregation is less complex in polyhaploids than in polyploids, so breeders of autopolyploid crops sometimes select among polyhaploids, then double the chromosome number.

It may be possible to cross polyhaploids with related diploid species.  (McGill University Plant Breeding 367-535B).