Principles of Plant Genetics and Breeding

178 CHAPTER 10
promote rapid pollen tube growth or extend the
period over which the pistil remains viable.
4 Modify ploidy level. A diploid species may be converted
to a tetraploid to be crossed to another species.
For example, narrow leaf trefoil (Lotus tenuis, 2n = 12)
was successfully crossed with broadleaf bird’s foot
trefoil (L. corniculatus, 2n = 24).
5 Use mixed pollen. Mixing pollen from a compatible
species with pollen from an incompatible parent makes
it possible to avoid the unfavorable interaction associated
with cross-incompatibility.
6 Remove stigma. In potato, wide crosses were
accomplished by removing the stigma before pollination
and by substituting it with a small block of agar
fortified with sugar and gelatin.
7 Grafting. Grafting the female parent to the male
species has been reported to promote pollen tube
growth and subsequent fertilization.
8 Protoplast fusion. A protoplast is all the cellular
component of a cell excluding the cell wall.
Protoplasts may be isolated by either mechanical or
enzymatic procedures. Mechanical isolation involves
slicing or chopping of the plant tissue to allow the
protoplast to slip out through a cut in the cell wall.
This method yields low numbers of protoplasts. The
preferred method is the use of hydrolytic enzymes
to degrade the cell wall. A combination of three
enzymes – cellulase, hemicellulase, and pectinase – is
used in the hydrolysis. The tissue used should be
from a source that would provide stable and metabolically
active protoplasts. This calls for monitoring
plant nutrition, humidity, day length, and other
growth factors. Often, protoplasts are extracted from
leaf mesophyll or plants grown in cell culture. The
isolated protoplast is then purified, usually by the
method of flotation. This method entails first centrifuging
the mixture from hydrolysis at about 50×
the force of gravity, and then resuspending the protoplasts
in a high concentration of fructose. Clean,
intact protoplasts float and can be retrieved by pipetting.
Protoplasts can also be used to create hybrids
in vitro (as opposed to crossing mature plants in
conventional plant breeding).
Overcoming the problem of inadequate hybrid
seed development
Abnormal embryo or endosperm development following
a wide cross may be overcome by using proper
parent selection and reciprocal crossing as previously
described. In addition, the technique of embryo rescue
is an effective and common technique. The embryo is
aseptically extracted and nurtured into a full plant under
tissue culture conditions (see Chapter 11).
Overcoming lack of hybrid vigor
Hybrids may lack the vigor to grow properly to flower
and produce seed. Techniques such as proper parent
selection, reciprocal crossing, and grafting the hybrid
onto one of the parents may help.
Overcoming hybrid sterility
Sterility in hybrids often stems from meiotic complications
due to lack of appropriate pairing partners. Sterility
may be overcome by doubling the chromosomes of the
hybrid to create pairing mates for all chromosomes, and
hence producing viable gametes.
Bridge crosses
Bridge crossing is a technique of indirectly crossing
two parents that differ in ploidy levels through a transitional
or intermediate cross (Figure 10.2). For example
Italian ryegrass
(Lolium multiflorum)
2n = 2x = 14
Italian ryegrass
2n = 2x = 14
×
Tall fescue
(Festuca arundinacea)
2n = 6x = 42
Not possible
Meadowgrass
×
(Fescue pratensis)
2n = 2x = 14
Hybrid
(sterile; diploid)
Chromosome
doubling
Bridge hybrid
(fertile; unstable)
2n = 4x = 28
Figure 10.2 An example of a bridge cross. In order to
hybridize Italian ryegrass and tall fescue, the breeder may
first make an intermediary cross with meadowgrass,
followed by chromosome doubling.
×
Hybrid
(select 6x)
F. arundinacea
2n = 6x = 42
×
BC 1
F. arundinacea
×
BC 2
BC n
F. arundinacea
Select 6x F. arundinacea with
ryegrass desired traits