Principles of Plant Genetics and Breeding

224 CHAPTER 13
Microcloning of tall wheatgrass for breeding
Within 6 weeks of culturing sterile, hulled seeds of tall wheatgrass on MS or GD media to which kinetin or BA and 2,4-D, NAA, or
IAA were added, adventitious shoot development could be observed from the callus (Figure 1c), irrespective of whether the callus
originated from germinated or non-germinated seeds. Generally, shoot development was preceded by callus greening, which
was a sign of meristem development. Such a physical manifestation has precedents in the first report on micropropagation of tall
wheatgrass (Kindiger 2002) and reports on other crops such as cassava (Matand et al. 2004) and peanut (Matand et al. 1994). It
has been previously reported that such plants, or other indirectly formed shoots, could result from a mono- or multicellular
organogenetic process (George 1993). In this report, light microscopy observations suggested that most, if not all, tall wheatgrass
shoots that were observed resulted from an aggregation of several adjacent callus cells. Single and multiple adventitious shoots
were observed on both MS and GD semisolid media (Figure 1c).
As in the case of callus formation, treatments containing 2,4-D in combination with either kinetin or BA induced a significantly
greater amount of shoots (9.2 or 8.3 shoots per callus on MS; and 8.2 or 6.2 shoots per callus on GD) and percentage of shoot formation
(90% or 84% on MS; and 82% or 78% on GD) than those induced with either IAA or NAA combined with similar levels of
cytokinin (Table 1). Overall, kinetin outperformed BA for morphogenetic responses; however, morphogenetic responses
observed on the MS basal medium were generally comparable to those observed on the GD basal medium.
One of the main contributions of this report is that it delineates, for the first time, a one-step method of microcloning for the
improvement of tall wheatgrass. In the first report on tissue culture of tall wheatgrass, Kindiger (2002) depicted a three-treatment
microcloning approach corresponding to the three phases of callus, shoot, and root induction, which is a common practice in tissue
culture of other crop species such as cassava as described by Matand et al. (2004). Callus was induced from seeds in the dark,
on MS medium containing basal salts, 3% sucrose, 0.5 g/l casein, 8 g/l phytagel, and 0.005 g/l 2,4-D and benzylaminopurine
(BAP). Then, callus was transferred to the shoot-inducing medium containing an increased concentration of sugar (5%), a
reduced concentration of phytagel (5 g/l), a growth regulator combination treatment with a lower concentration of BAP (0.001
g/l) and a similar concentration of NAA (0.005 g/l) without 2,4-D and casein. Lastly, shoots were rooted on half-strength MS
medium containing decreased concentrations of sucrose (1.5%) and 2,4-D (0.75 mg/l), an addition of IAA (3.75 mg/l) and kinetin
(1.075 mg/l), and an increased concentration of phytagel (10 g/l) without casein. The present report expounds on a related but
one-step microcloning approach in which all the three morphogenetic phases including callus, shoot, and root formation were
controlled under a single treatment. Not only could it reduce a long culturing period, but it could also eliminate the cost of
increased concentrations of, and/or additional, chemicals.
However, when producing in vitro plants through indirect adventitious shoot formation, it should be borne in mind that there is
a potential for indefinite rejuvenation of shooting callus. Maintaining such a property requires that the callus be subcultured in
relatively mid-sized amounts. Frequent break-offs of callus may enhance the wounding effect that normally stimulates callus
formation in plant tissues in nature. This induces plant wound healing that is underlain by intense cell division. After the callus
has formed shoots, it is necessary to frequently break them off about every 2–3 weeks to stimulate rapid shoot growth. Further, it
was observed that a cluster of young shoots would grow uniformly, with new shoot formation at their base, as long as none of the
shootlets significantly outgrew the others. Based upon our laboratory experience, it was surmised that any more rapidly growing
shoot might exert an inhibitory effect on its immediate surrounding shootlets, similar to the apical dominance. This was further
corroborated by the finding that the removal of the tallest shootlet of the cluster resulted in a rapid elongation of all the surrounding
shootlets and an initiation of new shoot development on the same callus.
Shoot rooting
Generally, in vitro-induced shoots of appropriate height, according to the desires of the researcher, may be rooted in a variety of
ways: (i) shoots may be transferred to fresh basal media; (ii) shoots may be transferred to fresh media with different growth regulator
treatments, primarily auxin alone, or with a trace amount of cytokinin; and (iii) shoots may be repeatedly transferred onto fresh
shoot-inducing media. When root-inducing treatment media are different from shoot-inducing treatment media, it is recommended
that the concentrations of growth regulators be judiciously decreased. As is the experience of other scientists working
under other experimental conditions, it is strongly recommended that root-inducing media be devoid of cytokinins but have a low
concentration of a single auxin. However, because each species may respond differently to similar treatments, it is recommended
that researchers apply their own best judgment based upon their knowledge of the crop.
In vitro adventitious shoots that were formed in tall wheatgrass were initially rooted in media devoid of growth regulators.
However, it was further observed that shoot-inducing media could also easily induce roots by extending the incubation of shoots
eligible for rooting on shoot-inducing media by 1 or 2 weeks. Following the standard practice, in vitro-induced shoots are systematically
acclimatized to the environmental conditions prior to their transfer into the greenhouse. During the course of our
investigation with tall wheatgrass, it was observed that an in vitro rooting system could eventually supply nutrients to support
plants in the greenhouse, when the acclimatization step was omitted. Thus, all in vitro-formed rooted plants were thereafter successfully
transferred into the greenhouse directly from their test tubes without incurring any loss. Similar observations were also
recently made on cassava (Matand et al. 2004). Plants regenerated in vitro developed normally in the greenhouse as well as in the
field (Figure 2).