All experiments were performed using the tomato cultivar VFN8 (kindly provided by the Petoseed Co., Woodland, CA). The seeds were sterilized and germinated in Magenta GA-7 boxes containing MS agar (Murashige and Skoog 1962) at 24C with a 16 hr/8 hr light/dark cycle. The MS medium contained 2% sucrose and an FeCl3/EDTA concentration of 0.2 uM. Seedlings were harvested after 14 days. The root system of each seedling was detached at the base of the hypocotyl and transferred to a 125 ml Erlenmeyer flask containing 50 ml of MSSV media (Fillatti et al. 1987) and incubated at a temperature of 24C and light fluence of 50 uEinsteins/m2/sec with rotary shaking at 110 rpm. Two root systems were cultured per flask. After treatment, the roots were submerged in FAA solution (50% ethanol, 5% glacial acetic acid, 10% formalin) for several days to permit direct visualization of lateral root primordia.
The ability of different auxins to stimulate lateral root formation was tested by incubating detached root systems in liquid culture for 96 hours in the presence of 0, 1, 3, 5, or 10 uM auxin. Dose-response curves for NAA, IAA and IBA are shown in Figure 1. Lateral root initiation was stimulated to the greatest extent by NAA, followed by IAA and IBA. Data were collected only from basal roots; however, the primary root and longer laterals exhibited similar increases in lateral root initiation. The response of a given root was not always uniform, with some roots containing segments with a high concentration of laterals adjacent to segments with relatively few laterals. NAA was more effective at inducing lateral root initials than IAA or IBA over the concentration range tested, exhibiting an approximately 10-fold increase over the control at 5 uM. The time course of new lateral root initiation during auxin treatment was determined by periodically harvesting seedling root systems growing in liquid culture with 1.6 uM NAA (Figure 2). At the earliest point tested, 13 hours, the first new initials were just becoming visible, and by 36 hr approximately 50% of the laterals that would ultimately arise had been initiated.
The effect of coincident exposure of the roots to cytokinin and auxin was tested by growing roots in liquid culture with 1.6 uM NAA and various concentrations of the cytokinin benzyladenine (BA). The results are shown in Figure 3. Lateral root formation was reduced by BA at all of the concentrations tested, with total suppression of the NAA-induced LRP formation at concentrations of 1.5 uM and above. We conclude that treatment of excised tomato roots with auxin results in a significant increase in the frequency of lateral root initiation, an effect that can be completely suppressed by simultaneous treatment with cytokinin.
Fillatti JJ, Kiser J, Rose R & Comai L (1987) Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio/Technology 5: 726-730Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497
Figure 1. Concentration dependence of lateral root induction by auxin. Detached root systems from 14 day old VFN8 seedlings were incubated in MSSV with the indicated concentrations of NAA, IBA and IAA. Roots were harvested after 96 hr. Vertical bars indicate standard error.
Figure 2. Time course of lateral root induction. Seedling root systems were incubated in MSSV medium with (closed circles) or without (open circles) 1.6 uM NAA for the times indicated. Vertical bars represent standard error.
Figure 3. Effect of cytokinin on induction of lateral roots by NAA. (a) Closed circles: tomato seedling roots incubated in liquid culture for 96 hours with 1.6 uM NAA and various concentrations of the cytokinin benzyladenine. Open circle: control roots without exogenous hormone. (b) Roots treated with 1.6 uM NAA and 1.5 uM BA for 96 hr.