The second nursery practice applying the commercial rooting powder Seradix 2 (3 g kg-1 indole-3-butyric acid [IBA]) negatively affected the survival and subsequent rooting of cuttings of clones 1 and 2. Subsequent studies examined: 1) the effect of the method of IBA application (powder vs. liquid);. The results showed that regardless of the method of application, IBA significantly reduced the percentage survival and rooting in cuttings of clones 1 and 2. In addition, the survival and rooting of cuttings was seasonally variable, with particularly low rooting in winter (e.g. for clone 1, 32 %) compared to summer (eg for clone 1, 83%).
The application of exogenous IBA may have disrupted the endogenous PGR balance of the cuttings, resulting in inhibition of survival and rooting. Essentially, both cuttings and in vitro shoots showed adverse survival and rooting responses when exposed to excessive IBA concentrations. Means ± standard errors within columns followed by lowercase letters are significantly different between types of cuttings within each clone; capital letters indicate significant differences between clones (p < 0.05) (n = 100).
Means ± standard errors across rows followed by different letters indicate significant differences between cuttings and in vitro shoots with respect to percent survival and roots (p < 0.05).
LIST OF ABBREVIATIONS
INTRODUCTION
Belonging to the family Myrtaceae, Eucalyptus is a commercially important genus with a wide variety of uses in the timber, pulp and pharmaceutical industries (Turnbull 1999; Moyo et al. 2011). Eucalyptus plantations were introduced into South Africa in the late nineteenth century to meet the growing timber needs that exceeded the availability of native forests (Wise et al. 2011). However, plantations established from seeds are characterized by a large variation in growth, shape and vigor, characteristics that are undesirable for commercial forestry (Ahuja 1993; Eldridge et al. 1994).
Once superior genotypes have been produced, it is essential that efficient breeding strategies exist to exploit the genetic gain (Watt et al. 1995; Wilson 1998a; Naidu and Jones 2009). The role of auxins as a key determinant in the process of adventitious root formation is well established (Zaerr and Mapes 1982; Blakesley 1994; Blythe et al. 2007). The discovery that exogenously supplied auxin could potentially induce root formation in cuttings greatly improved plant reproductive activities (Blythe et al. 2007; Pop et al. 2011 and references therein).
If it is desirable that Eucalyptus genotypes have difficulty rooting as cuttings, in vitro techniques can be applied with greater success (Mokotedi et al.
LITERATURE REVIEW
- The history and importance of Eucalyptus
- Vegetative propagation of Eucalyptus via cuttings
- Parent Plants
- Selection of cuttings
- The role of auxins in adventitious root formation
- Environmental factors
- In vitro propagation of Eucalyptus .1 Routes of regeneration
- Direct Organogenesis
Many eucalypt species within subgeneric groups have the capacity for natural hybridization (Griffin et al. 1988). Propagation of eucalypts by rooted cuttings (macrocuttings) reached commercial scale in the 1980s (de Assis et al. 2004). These trees are then felled and the re-sprouted scions are used as rooted cuttings (macrocuttings) (Eldridge et al. 1994).
Plant development is characterized by three phases consisting of a juvenile, transitional and mature (mature) phase (Hartmann et al. 1997). De Almeida et al. 2007) showed that by increasing the concentration of IBA applied to E. The endogenous levels of auxins change with the different phases of root formation (Pop et al. 2011).
Mature embryos are then transferred to PGR-free media for further germination (George et al. 2008). Further, shoots subjected to GA3 during the elongation phase subsequently failed to initiate roots (Brondani et al. 2012a). 27 Acclimatization of plants from in vitro (heterotrophic conditions) to an external environment (autotrophic) is the decisive, final stage in the micropropagation process (Hartmann et al. 1997).
MATERIALS AND METHODS
- Plant Material
- Standard nursery practices
- Cutting types
- Mode of application of Seradix a) Powder Substrate
They were made into mini cuttings (3.5 – 4 cm long) (hereafter referred to as cuttings) consisting of an apical meristem and a pair of leaves reduced to 1/3 of its original surface area to minimize water loss through transpiration (Figure 2). Cuttings were placed in a solution of Nufilm® (Miller Chemical and Fertilizer Corporation, Pennsylvania, USA) (active ingredient, poly-1-p-menthene (875 g l-1)) before planting. Nufilm® is an adhesion extender designed to extend the life of foliar insecticides and fungicides applied to cuttings, even in the presence of overlying dew.
All results were recorded five weeks after cuttings were initially set and evaluated parameters for each mini-alley included survival, root formation and callus formation. Cuttings planted in Unigro 98® trays were placed in polyethylene root tunnels (Figure 3) with the air temperature regulated between 28 – 38 °C by thermostatically controlled fans. 30 Figure 3 Eucalyptus cuttings placed in Unigro 98® trays and placed in a polyethylene tunnel with thermostatically controlled fans and an automatic overhead misting system, with a) and without b) misting.
31 maintained as close to 100% as possible with an automated overhead misting system set to release a continuous mist for 10 seconds every 4 minutes. There were no artificial light sources in the rooting tunnel, and the light intensity in winter ranged from ~ 300 μmol s-2 m-1 to. As this study focused on root survival and induction, all results were recorded after five weeks in the root tunnel.
Standard nursery practice involved applying the commercial rooting powder, Seradix 2 (Bayer Crop Science, Leverkusen, Germany) (active ingredient, 3 g kg-1 IBA) to the basal end of eucalyptus cuttings immediately before placement in the growth medium. For the initial study, cuttings (n = 100) from two Eucalyptus grandis x Eucalyptus nitens clones (clones 1 and 2) were dipped in Seradix 2 to determine percent survival and rooting according to standard nursery practice. Shoots harvested from parent plants (clones 1 and 2) biweekly were used to make 'soft' cuttings (n = 100) (standard nursery practice).
This study consisted of three treatments: a control (without application of Seradix or talcum powder), cuttings dipped in talcum powder and pieces dipped in Seradix 2. For the 10 sec IBA treatment, the base of each cut was kept in the IBA solution (in a depth of 0.5 cm) for.
HARD
An IBA solution, 3 g l-1 was made by first dissolving IBA in ethanol and then distilled water.
SOFT HARD
SOFT
- Different Seradix concentrations and time of application
- In vitro studies
- Decontamination and culture establishment
- Elongation and Rooting
For 5 h IBA treatment, the prepared IBA solution was mixed with pure vermiculite and placed in containers. This was done to provide a support medium thereby ensuring that only the base of cuttings of clones 1 and 2 were exposed to the IBA solution for the required 5 h period before being planted out in the growth medium. To obtain a lower concentration of Seradix than was commercially available, Seradix 1 was mixed with talc in equal amounts to obtain Seradix 0.5 (0.5 g kg-1 IBA).
Cuttings were supplied with different concentrations of Seradix either at initial establishment (T0 week) or two weeks after establishment of cuttings (T2 week). The basal part of the cutting was immersed in clean water to allow Seradix to adhere to the cutting which was then replanted in the growth medium. In both studies, there were five treatments consisting of five replicates with 20 cuts in each (n = 100).
Shoots were taken from the parent plants of clones 1, 2 and 3 and placed in a solution of 1 g l-1 methyl N-(1-butylcarbamoyl-2-benzimidazole) carbamate (Benlate, Volcano Agroscience (Pty) Ltd, South Africa), 1 g l-1 boric acid, 0.5 ml l-1 chlorothalonil (Bravo 500, Volcano Agroscience (Pty) Ltd, South Africa) and a drop of Tween® -20 placed on a shaker for 30 minutes and then transferred to laminar flow. After the shoots were rinsed with sterile distilled water, they were immersed in 0.2 g l-1 HgCl2 and a drop of Tween® -20 for two minutes. The shoots were then rinsed three times in sterile, distilled water and then treated for another two minutes with 10 g l-1 calcium hypochlorite and a drop of Tween® -20.
After three rinses with distilled water, each stem was divided into 2 – 3 cm long explants with a pair of cut leaves and then placed in bud induction medium. After decontamination, each explant was placed in 10 ml bud induction medium in a 50 ml culture tube for two weeks. Shoots were grown in 100 ml culture bottles with 20 ml of medium for a period of 3 – 4 weeks.
For the root studies, shoots ≥ 1.5 cm elongated on E1 and E2 were placed on 10 ml of root medium in a 50 ml culture tube. The concentrations of IBA (0, 0.1 and 1.0 mg l-1 IBA) were chosen to typify the range of Seradix concentrations applied to cuttings (i.e. no IBA, low and high IBA concentrations).
- RESULTS
- Macropropagation of three Eucalyptus grandis x E. nitens clones .1 Survival and rooting under standard nursery practices
- Comparison of survival and rooting responses of the two vegetative propagation methods
- DISCUSSION
- Effects of types of cuttings and season on survival and rooting
- The relationship between the survival and rooting of cuttings and auxin supply
- Shoot survival and rooting responses in vitro
- Comparison of cuttings and in vitro survival and rooting responses
- The relationship between callus formation and rooting
- Concluding remarks
- REFERENCES
Although Clone 1 cuttings achieved almost the same percentages of survival and rooting in the control and talcum powder treatments, the percentage of cuttings that developed callus (45% and 28%, respectively) varied significantly. The highest percent survival for Clone 1 cuttings (74%) was achieved in the absence of Seradix. Although Clone 3 shoots demonstrated similar survival (90% and 100%, respectively) in the control and 0.1 mg l-1 IBA treatments, the percentage of shoots that rooted (0% and 40%, respectively) and developed callus (0% and 100%, respectively) were significantly different (Table 15).
In clone 1, in the absence of IBA, only significant differences were observed between shoots elongated at E1 (Table 14) and E2 (Table 15) in terms of percent callus formation (Table 16). There were no significant differences in any of the parameters tested in clone 2 shoots in the absence of IBA. There were no significant differences in any measured parameter between shoots elongated in different media and then exposed to 1.0 mg l-1 IBA at the rooting stage.
Cuttings and in vitro shoots of clone 1 showed significant differences in percent survival and rooting in the absence of IBA (Table 17). In the absence of IBA, the percentage survival of both cuttings and shoots in vitro was high (> 95%). However, cuttings rooted best in the absence of IBA, while in vitro shoots did not root.
61 As with clone 1, cuttings from clones 2 and 3 rooted best in the absence of IBA, while in vitro shoots failed to root (Table 17). Under the moderate IBA treatment, in vitro shoots from clones 2 and 3 had a significantly higher survival rate than those from cuttings. The highest percentage of survival and rooting for clone 1 cuttings was achieved in the absence of Seradix (95% and 83% respectively) (Table 10).
In the absence of IBA, cuttings and in vitro shoots of all clones had a high survival rate (>80%). However, although the cuttings rooted successfully, the in vitro shoots failed to root due to the absence of IBA. In this study, callus formation in cuttings and in vitro shoots of clones 1, 2, and 3 varied significantly across studies.
Cuttings rooted best in the absence of IBA, while in vitro rhizogenesis required 0.1 mg l-1 IBA.
