Use of multinodal explants for micropropagation of Leucadendron ‘ Safari Sunset ’

Leucadendron ‘Safari Sunset’ is one of the most valuable Proteaceae cultivar, due to its economic importance as cutflower. In vitro tissue culture of this cultivar and others may offer a massive cloned plant production that permit to satisfy the market demand. Multinodal explants of Leucadendron ‘Safari Sunset’ were established in vitro on a half strength Murashige and Skoog medium supplemented with 2.2 μM benzylaminopurine (BAP). Various antioxidant treatments were used to reduce the presence of phenolic compounds in culture. No important differences in growth and multiplication between these treatments were detected during in vitro establishment and multiplication, but explants cultured on a medium with 1 g L of polyvinylpyrrolidone (PVP) developed a higher number of buds in the establishment phase. Proliferation of axillary shoots was achieved with 0.88 μM BAP, being slightly higher on a medium with 2.2 μM BAP although the buds were deformed, showing abnormal leaves. The use of phytagel in this phase increased proliferation rate and shoot growth. Low percentages rooting were obtained when microcuttings were cultured on the same basal medium supplemented with several indole-3-butyric acid (IBA) concentrations. The best rooting percentage (37.5%) was obtained with 44.1 μM IBA. This work shows that micropropagation of L. ‘Safari Sunset’ is possible with multinodal explants, although is necessary to optimize the protocol for commercial applications. Additional key words: antioxidant treatment, in vitro tissue culture, ornamental plants, phenolic compounds, Proteaceae.


Introduction
'Safari Sunset' (L.salignum × L. laureolum), is an ornamental plant widely cultivated for cut flower.It is the most cultivated Leucadendron on a worldwide basis.First bred in New Zealand, it expanded rapidly to other zones.It is present in all the protea producing regions: New Zealand, Israel, Australia, Hawaii, California, Chile, Equador, South Africa, Zimbabwe, Azores and Madeira among others.Presently, millions of floral stems are sold at flower auctions in Holland, being extensively used by florists for floral arrangements (baskets, centrepieces, etc.) and bouquets.
It has dark green leaves and its terminal bracts present a luminous red during summer, darkening with the course of the season to adopt a dark wine-red colour.Owing to its fast growth and early entrance in production it has become a highly profitable cultivar.Its propagation by stem cuttings is easy, but it is frequently difficult to satisfy the demand for plants using conventional methods of vegetative propagation.Micropropagation could be a valid alternative to solve this problem, as well as to provide the indispensable plant health guarantee for vegetative material exchange at an international level.The need to establish protocols for the micropropagation of different types of proteas has been supported by the IPA (International Protea Association) at the International Protea Conference held in 2006 in San Diego, California.
Although diverse authors have studied the micropropagation of some proteas (Hu and Wang, 1983;Ben-Jaacov and Jacobs, 1986;Seelye et al., 1986;Rugge et al., 1989;Kunisaki, 1989Kunisaki, , 1990;;Bunn and Dixon, 1992a,b;Watad et al., 1992a,b;Pérez-Francés et al., 1995;Rugge, 1995;Dias Ferreira et al., 2003;Wu and du Toit, 2004), results have not been totally satisfactory, as at present there is still no firm that specialises in marketing in vitro produced protea plants.A previous report by Pérez-Francés et al. (1995) showed the effect of some factors on in vitro multiplication of this cultivar.In this study, the use of benzylaminopurine (BAP) improved bud proliferation rates.The addition of naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) or gibberellic acid (GA 3 ) to the proliferation medium containing BAP did not significantly affect the formation of new buds per explant or their length.The presence of activated charcoal in the medium clearly inhibited bud proliferation and an MS medium (Murashige and Skoog, 1962) at half strength produced the best response.Dias Ferreira et al. (2003) worked with the same cultivar, obtaining the best results on a modified MS medium containing ascorbic acid and a combination of BAP and GA 3 when shoot tips were used for establishment.They obtained only 20% elongated plants with explants from the tender parts of the shoots.
In the present paper, we report a method for micropropagating Leucadendron 'Safari Sunset' from multinodal (2-3 node) explants.These are a good option for this technique, since they provide 2-3 buds per explant.

Establishment
Explants were removed from actively growing terminal shoots of plants growing in a greenhouse and used to initiate in vitro culture.Shoots 20 cm in length were dipped into a 2 g L -1 Benlate solution (50% Benomyl) for 1 hour.The clean materials were given a quick (5 s) rinse in 70% ethanol, followed by washing in distilled water.These were then surface-sterilized in a 5% sodium hypochlorite solution with one drop L -1 of Tween 80 (surfactant) under vacuum for 15 min, followed by three washes in sterilized distilled water.Before planting segments in the culture medium, they were dipped into different antioxidant solutions to diminish excessive oxidation.
Multinodal segments (3-5 cm length with 2-3 nodes) were placed in basal MS medium (Murashige and Skoog, 1962) containing half-strength macronutrient salts (1/2 MS), 3% sucrose, 7 g L -1 agar, and 100 mg L -1 myo-inositol.The 1/2 MS medium was supplemented with plant growth regulators and the antioxidant substances and adjusted to pH 5.7 before autoclaving.Antioxidant treatments were used in the establishment phase to reduce explant oxidation: a combination of citric (1,500 mg L -1 ) and ascorbic acid (100 mg L -1 ) (Pérez-Francés et al., 1995) or polyvinylpyrrolidone (PVP) (1 g L -1 ), employed in different ways: T1 (ascorbic and citric acid solution shaken for 30 min before culture), T2 (T1 followed by darkness for three days after culture), T3 (T1 then subculture every three days during the first week of culture), T4 (ascorbic and citric acid included in the medium), P1 (PVP solution shaken as in T1), P2 (P1, then treated as in T2), P3 (P1 then subcultured as in T3), P4 (as T4 but with PVP), O (as T2 without antioxidants), SF (subcultured as in T3 without antioxidants).The cultures were maintained for 8 weeks during which they were transferred once after 4 weeks of incubation.Twenty millilitres of medium was dispensed into each (20 × 150 mm) glass tube and autoclaved at 103.5 kPa and 121°C for 20 min.Explants were divided in two groups (Fig. 1).Leaves of group A explants were cut off leaving 1-2 mm petioles, whereas leaves of explants B were totally eliminated.During establishment, explants were inserted into the basal medium supplemented with 2.2 µM BAP.Cultures were maintained at 25 ± 2°C, in a culture room illuminated with cool white fluorescent tubes (110 µmol m -2 s -1 ) on shelves during the 16 h photoperiod.Within 2 weeks, axillary buds sprouted and 2 weeks later, buds were excised and subcultured in a multiplication medium.
At least 24 tubes were raised for each treatment and the experiment was repeated two times.
At least 24 tubes were raised for each treatment and all the experiments were repeated three times.Number of bud-breaks were recorded at weekly intervals.
Results were analysed statistically by analysis of variance, using the SPSS statistical software package (version 17.0).Standard error of the mean was calculated and is indicated by ± sign.

Establishment
When multinodal segments from green shoots were used, 66.7% of explants sprouted buds on a medium with PVP and BAP 2.2 µM, producing approximately two buds per explant.Table 1 shows the mean values of buds developed 4 weeks after culture for each treatment.Explants A developed a higher number of buds after all treatments assayed, and a mean of 25 buds sprouted when PVP was included in the medium (P4), in comparison with the 7 buds developed by explants B during the same treatment.The highest number of buds (10) produced by explants B was obtained when explants remained Table 1.Buds mean developed four weeks after culture with each antioxidant treatment in each explant type C (control, without antioxidant treatment), T1 (ascorbic and citric acid solution shaken for 30 min before culture), T2 (T1 followed by darkness for three days after culture), T4 (ascorbic and citric acid included in the medium), P1 (PVP solution shaken as in T1), P4 (as T4 but with PVP), O (as T2 without antioxidants), SF (subcultured every three days during the f irst week of culture without antioxidants).Explants A (leaves were cut off leaving 1-2 mm petioles), Explants B (leaves were totally eliminated) All antioxidant treatments used were effective for explant oxidation and necrosis control, but no important differences between them were detected.Nevertheless, some observations suggested that explants smaller than 30 mm in length presented greater browning symptoms, but statistical data are insufficient to verify this.

Multiplication
The buds with greatest development during the establishment phase were isolated from the explants and subcultured on a multiplication medium consisting of 1/2 MS medium and two different BAP concentrations (2.2 µM and 0.88 µM).Multiplication rates obtained were low in both cases and no significant differences were identified between them (Fig. 2a).However, shoot cultures growth with BAP 2.2 µM showed stunted growth with small and abnormal leaves.Moreover these buds presented more symptoms of necrosis than buds obtained on a 1/2 MS medium supplemented with 0.88 µM BAP, which showed normal development (Fig. 3a-b).
Though the multiplication rates did not alter for the different antioxidant treatments used (Table 2), they did depend on the subculture number, showing a significantly decline in the third subculture (Table 2).With the use of phytagel as a gelling agent, multiplication rates continued to be low (Fig. 2b), and no significant differences were observed in the multiplication rates in comparison with the buds multiplied in a medium gelled with agar.However, buds obtained with phytagel showed stronger growth in both BAP concentrations without hyperhydricity symptoms and presented less symptoms of browning (Fig. 3c-d).

Rooting
When microshoots (1.5-3 cm length) were cultivated in a 1/2 MS rooting medium containing NAA or IBA, after 4 weeks in the establishment phase and 16 weeks in a multiplication phase (4 subcultures), IBA gave better results than NAA (Table 3).
Root induction was preceded by callusing at the base of the shoot on IBA concentrations higher than 34.3 µM.On a 1/2 MS medium with 49 µM IBA, which proved to be the best, 37.5% of shoots formed roots.Roots appeared after 4 weeks and the maximum response was observed after 7 weeks, reaching between 25-40 mm in length (Fig. 4).
Roots obtained with IBA concentrations above 49 µM showed very little growth (1-2.5 mm in length).No rooting was seen when microshoots were dipped in an IBA solution and then cultured on a 1/2 MS medium without growth regulators.The addition of activated charcoal to the medium did not improve the rooting percentage or root length.

Discussion
Use of multinodal explants provided more than one bud per explant and reduced browning.Dias Ferreira et al. (2003) studied three types of 'Safari Sunset' explants: shoot tips (5-10 mm), explants from the green part of the shoots and explants from the remaining lignified shoots.They achieved a 65% bud elongation from shoot tips but only 20% from explants from the green part of the shoots, in an MS medium including a combination of growth regulators.They did not obtain positive results from the oldest explants.Similar results were found by Rugge et al. (1989), who reached a higher percentage of sprouted buds using multinodal green explants than with semi-lignified and lignified explants.
Our results were better when the explant leaves were cut off leaving 1-2 mm petioles, than with explants without petioles.This could be due to excessive phenols production in the zone that produced damage on the axillary meristems.In previous studies with members of the Proteaceae, the utilization of explants with and without leaf remnants gave rise to differences.In many of them, authors used explants with 1-5 mm of leaf (Ben-Jaacob and Jacobs, 1986;Watad et al., 1992b;Rugge, 1995), though others used explants without remains of leaf (Seelye et al., 1986;Kunisaki, 1989), nevertheless only Pérez-Francés et al. (2001b) compared the development of axillary buds in these two types of explants with Leucadendron discolor, with an inferior response in explants with 1-3 mm of petiole than with the other type.
The need for a study on the action of antioxidant treatments on the in vitro culture of this plant arises from the Proteaceae being rich in phenolic compounds (Malan, 1992;Watad et al., 1992b;Rugge, 1995   and du Toit, 2004).After the dissection of explants, these compounds may be oxidized by different enzymes (polyphenoloxidases and tyrosinases) which are synthesized and/or liberated due to injury during excision and sterilization of tissues (Torres, 1989).Oxidation products are known to inhibit the enzymatic activity, browning the tissues and darkening the medium.Darkening can be reduced by limiting the production of phenolic compounds, modifying the redox potential, inactivating the phenolases, and reducing the activity of the phenolase and/or the availability of substrate (Hu and Wang, 1983).
In several reports concerning Proteaceae, the use of an antioxidant solution is necessary in some phases of culture: citric and/or ascorbic acid (Pérez-Francés et al., 1995;Dias Ferreira et al., 2003;Wu and du Toit, 2004), PVP (Pérez-Francés et al., 2001a,b), or BAP applied to the entire mother plant at 200 mg L -1 .This latter significantly reduced browning and promoted bud sprouting in vitro (Rugge, 1995;Pérez-Francés et al., 2001b).It is also possible to apply an etiolation treatment to a new actively growing shoot on the mother plant (Watad et al., 1992b).In our present study, we evaluate the effect of different antioxidant treatments on explant oxidation, using PVP and an ascorbic and citric acids solution combined with darkness and frequent subculture.The PVP absorbs the phenolic substances so they cannot be oxidized by the polyphenoloxidases, whereas the citric and ascorbic acid prevent oxidation of the phenolic compounds.Explant incubation in darkness in the initial period and the frequent transfer of explants into fresh medium were the other treatments assayed.No differences in tissue oxidation were observed between the different treatments.Results during the multiplication phase were similar to those reported by Pérez-Francés et al. (1995); nevertheless Dias Ferreira et al. (2003) obtained higher multiplication rates, although they used a combination of different growth regulators.Buds obtained in a medium with 2.2 µM BAP browned rapidly due to their poor development.There are cases in which the effect of the BAP at certain concentrations inhibits bud elongation, causing browning.To diminish this, the BAP concentration can be reduced in this phase, or be combined with an auxin or gibberellin to eliminate the inhibitory effect of BAP on bud growth (Orellana Pérez, 1998).In our work, using a lower BAP concentration was sufficient to promote bud development and reduce the risk of browning.
Multiplication rates obtained were slightly higher but not significantly different when phytagel was used as agar substitute, moreover these buds were longer.Similar results in 'Safari Sunset' using a gelling agent were obtained by Pérez-Francés et al. (1995).Any differences observed may be a result of the higher purity of phytagel and unidentified impurities in the agar (Sharma and Ramamurthy, 2000), which could contribute to inhibiting shoot growth (Hu and Wang, 1983).There are some reports of phytagel addition improving the multiplication phase in different plants (Saadat and Hennerty, 2002;Thimmappaiah and Sadhana, 2002).
The major problem in our study was the low rooting percentage obtained.Rooting in vitro may be reduced due to a long period in culture (Norton and Norton, 1986), moreover, the presence of a cytokinin in the medium during the establishment and multiplication phases (Torres, 1989;Peddaboina et al., 2006) can also negatively affect rooting.This can be increased using high cytokinin concentrations (Figueiredo et al., 2001).In our case, presence of BAP during the establishment and multiplication phases could have reduced the rooting rates.
In conclusion, the present work shows that micropropagation of Leucadendron 'Safari Sunset' is possible with multinodal explants, although for commercial applications it will be necessary to optimize the protocol and study the factors that affect rooting in this cultivar after a relatively long period in culture.

Figure 2 .Figure 3 .
Figure 2. Effect of a) different BAP concentrations and b) gellificant agent on the multiplication rate of Leucadendron 'Safari Sunset'.Means and standard deviations for independent variables and ANOVA tests.The same letters indicate no significant differences at P = 0.05%.

Table 2 .
Effect of different treatments and subcultures on the multiplication rates of L. 'Safari Sunset'.Multiplication rates means obtained by Duncan's test a Same letters within a column indicate no significantly differences at P = 0.05%.C: control.P1: PVP solution for 30 min on sharking before culture.O: darkness during three days after culture.