Principles of Plant Genetics and Breeding

(a) (b)
(c)
Figure 1 (a) Germinated seed (× 50), (b) callusing seeds
(× 50), and (c) multiple shoot-forming callus (× 50).
POLYPLOIDY IN PLANT BREEDING 223
Callus formation
Sterile, husked, dry, mature seeds were used as an
explant organ for callus induction in tall wheatgrass
(Figure 1a, b). Although grass hulled seeds could be
sterilized using standard sterilization methods, tall
wheatgrass hulled seeds were surface-sterilized with
10% sodium hypochlorite for 15 minutes and 70%
ethanol for 5 minutes, and rinsed three to four times
in distilled water. The sterilization method may vary
depending upon the physiological state of the seed.
Dry mature seeds are the most suitable for this sterilization
method; alcohol exposure should be reduced
to 1–2 minutes or eliminated from the protocol when
using immature seeds. This is necessary because the
soft, young seeds are easily bruised during hulling, thereby allowing the alcohol to be absorbed into the tissues, with lethal consequences.
Unlike cytokinin, auxin may be used alone to induce a callus, or in combination with cytokinin. Although treatment combinations
of kinetin (KIN) or 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA) were used
to induce healthy callus from the seeds, the greatest amount of friable callus was obtained by using treatment combinations of
equal concentration (2–20 µM) of kinetin or BA and 2,4-dichlorophenoxyacetic acid (2,4-D) (Table 1). Further, the amount of callus
formed increased with the growth regulator concentration. It was discovered that 2,4-D could also be successfully used alone
to induce friable callus, although in smaller amounts. Callus formation may occur prior to, or after, seed germination, but often
both occurred simultaneously. It was also observed that “organogenic” callus formation potential decreased with seed germination
capacity. Seeds that had lost their germination capacity could also form callus; however, such callus growth was only temporary,
and it could not form either friable callus or organogenic structures. This may suggest the existence of a correlation between
“totipotency” of seed cells (cotyledonary or embryo) and zygotic embryo viability. When Murashige and Skoog (MS) (1962) and
Gresshoff and Doy (GD) (1974) nutrient media were evaluated, the MS medium caused the greater rate of callusing of seeds (86%
and 88%) over 4–6 weeks of culturing (Table 1). This rate could be improved to 100% by using freshly harvested seeds and/or
including about 2% wood charcoal. The effect of darkness was later evaluated and found not to be a major factor for callus formation.
Cultures grew well when cultured at 26–28°C under an 8-hour photoperiod. The morphogenetic growth responses were
enhanced when the nutrient media were supplemented with 3% sucrose and 500 mg/l magnesium chloride. The pH of the media
was adjusted to 5.7 with potassium hydroxide prior to autoclaving. All attempts to induce callus in leaf and root explants were
unsuccessful. However, under unspecified conditions callus formed sporadically from the tips of non-severed shoots and roots.
When such callus was collected and subcultured on fresh media, it formed normal shoots.
Table 1 Organogenic responses of tall wheatgrass tissues to the combinations of 20 µM 6-benzylaminopurine (BA)
or kinetin to 20 µM auxins on Murashige and Skoog (MS) or Gresshoff and Doy (GD) media.
% Shoot-forming Shoot average
% Seed callusing callus per callus
Cytokinin GD MS GD MS GD MS Auxin
Kinetin 86 88 82 90 8.2 9.2 2,4-D
14 15 10 13 4.4 6.1 NAA
3 4 1 3 2.2 3.2 IAA
BA 79 86 78 84 6.2 8.3 2,4-D
11 10 5 6 3.0 6.2 NAA
4 5 2 3 2.1 2.4 IAA
Standard error 1.57 1.57 1.64 1.64 0.42 0.42
least squares mean
Coefficient of variation (CV): 30.70%, seed callusing; CV: 25.86%, shoot-forming callus; CV: 18.33%, average shoots per callus.
2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; NAA, α-naphthalene acetic acid.