Plant material and culture conditions.

Apical buds and axillary buds (explants) with 1 to 2 cm of stem of E. pulcherrima ‘Prestige™ Red’ were excised during Jan. 2008 from healthy plants grown in the greenhouse under long-day photoperiods. Long days were created during October to Dec. 2007 using night interruption lighting with incandescent bulbs (1.8 μmol·m⁻²·s⁻¹) from 2200 to 0200 hr. The buds were surface-disinfected by placing them in a 1% Tween 20 solution for 5 min followed by 70% ethyl alcohol for 1 min and then 20% bleach (97% sodium hypochlorite) for 15 min (Pickens et al., 2005). Finally, buds were dipped in sterile water three times for 5 min. Basal medium (20 mL) containing MS salts (4.4 g·L⁻¹), myo-inositol (0.1 g·L⁻¹), sucrose (30 g·L⁻¹), and agar (7 g·L⁻¹) (pH 5.7 to 5.8) (Pickens et al., 2005) was poured into 100-mL baby food jars and autoclaved at a temperature of 121 °C at 15 psi for 15 min. Various concentrations of plant growth regulators (PGRs) were incorporated into MS basal medium depending on treatment type. BA was incorporated before autoclaving the liquid medium and IAA (filter-sterilized) was incorporated into the liquid medium after autoclaving. Disinfected buds were placed on the autoclaved medium solidified with agar in aseptic conditions under a laminar flow hood and incubated in a growth chamber with a 16-h photoperiod (125 μmol·m⁻²·s⁻¹ illumination with fluorescent bulbs) at 25 °C day and 22 °C night temperatures. Effects of PGRs on callus induction and rooting were evaluated in two studies.

Callus study.

The callus study was composed of two experiments. For the first experiment, BA was incorporated into the basal medium at concentrations of 0 μM, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM; and for the second experiment, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM of BA were incorporated in combination with 4 μM of IAA. An experimental control with 0 μM of BA and 0 μM of IAA was also incorporated in the second experiment. After 1 month, explants were visually evaluated for percentage of explants having red callus at the base and callus color. Furthermore, the number of micro-buds and micro-shoots was examined. Explants were subcultured four times at monthly intervals onto a new medium with the same ingredients.

Rooting study.

Four-month-old in vitro-grown poinsettias were transferred onto a rooting media consisting of one of the treatments: MS with 28.5 μM IAA (Roy and Jinnah, 2001), half-strength MS salts and vitamins with 28.5 μM IAA, or MS with 4 μM BA. An experimental control with MS and no PGRs was also included. The plants were evaluated for average number of days for root initiation and the number of roots initiated per shoot. The average number of days for root initiation was calculated as the total number of days taken for root initiation (number of days from shoots sticking into the medium through root initiation) divided by the total number of plantlets per treatment. The experiment was ended after 5 months.

Statistical analysis.

Each experiment had five explants per treatment (a vessel with an explant is an experimental unit) and each experiment was repeated once within each subculture cycle. All treatments were arranged in a randomized complete block design and data were analyzed by the General Linear Model using Statistical Analysis System (SAS Version 9.1.2; SAS Institute, 2004). Treatment means were separated by the least significant difference method at α = 0.05.

Explant disinfection, plant material, and growth conditions.

Surface disinfection of explants was difficult as a result of latex secreted at the cut surfaces of explants. At least 15% to 25% of the explants were contaminated per each micropropagation cycle during a preliminary study. The contaminations were greater (40%) without using 1% Tween 20 solution during the disinfection process of explants. Thus, Pickens et al. (2005) disinfection protocol was used to minimize contaminations encountered. Explants (apical and axillary buds) of poinsettia ‘Prestige™ Red’ responded to the PGR combinations (IAA and BA) by developing calluses at the base of the explant before organogenesis. Monthly repeated subculture onto a new medium was necessary to achieve organogenesis and plantlets.

Callus study.

Primarily two types of calluses were identified in poinsettia micropropagation: red callus (Fig. 1A) and white callus (Fig. 1B). With continuous subculturing in a medium with BA (4 to 12 μM), red callus produced more micro-buds, micro-shoots (data not shown), and healthy plants, whereas white callus remained unchanged. White callus is a result of recalcitrant cells, which are unproductive. Red callus is the most important in poinsettia proliferation; it proliferates fast and produces into micro-buds and micro-shoots. The results observed were consistent with other experiments of poinsettia such as De Langhe et al. (1974) and Pickens et al. (2005) in which red callus produced plantlets efficiently. Apart from red and white calluses, gray–green callus was also observed with low BA levels (2 to 4 μM). This was consistent with the study of De Langhe et al. (1974), in which the dominant callus color was red with low IAA:Kn. The higher the ratio, the more grayish green callus produced.

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Poinsettia ‘Prestige Red’ explants (apical buds and axillary buds) producing red and white calluses, micro-buds, and micro-shoots in poinsettia micropropagation. (A) Red callus on a medium with 10 μM 6-benzylaminopurine (BA) (1 month old); (B) white callus on a medium with 4 μM indole-3-acetic acid (IAA) and 10 μM BA (1 month old); (C) micro-buds on a medium with 12 μM BA (2 months old); (D) micro-shoots on a medium with 10 μM BA (3 months old).

Citation: HortScience horts 45, 7; 10.21273/HORTSCI.45.7.1126

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Explants grown in media supplemented with BA produced red callus at the base of the explant, whereas explants in media without BA did not give rise to any callus. There is a significant (α = 0.05) difference between plants that are treated with BA and without BA (Table 1). Therefore, use of BA in poinsettia micropropagation is effective and increased red callus production. BA can be used to generate red callus (Table 1), micro-buds (Fig. 1C), and micro-shoots (Fig. 1D) in poinsettia micropropagation. The impact of cytokinins on poinsettia in vitro bud development was reported previously by De Langhe et al. (1974) who observed faster bud development with higher levels of the cytokinine 2-isopentenyl adenine, which is a cytokinin. This finding was similar to our research in which cytokinin (BA) helped produce additional micro-buds.

Table 1.

Red callus initiation from 1-month-old poinsettia ‘Prestige Red’ explants (apical buds and axillary buds) on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA).

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Explants grown in media supplemented with both BA and IAA produced red callus at the base of the explant showing a significant (α = 0.05) difference among treatments; treatments with BA greater than 8 μM produced more callus than treatments with 8 μM BA or less (Table 2). Continuous growth in a medium supplemented with BA (4 to 12 μM) alone resulted in a greater number of micro-buds and micro-shoots compared with medium supplemented with both IAA (4 μM) and BA (4 to 12 μM) (data not shown). Uchida et al. (2004) reported the impact of cytokinins on adventitious bud formation of E. tirucalli L.; adventitious buds were efficiently induced when the medium was supplemented with thidiazuron (TDZ) compared with a medium with both TDZ and IAA. We observed similar results in which BA alone induced micro-buds efficiently compared with a medium with both BA and IAA. Incorporation of IAA and BA into the medium generated additional red callus, but the BA:IAA ratio is critical in generating red callus. The greater the BA:IAA, the greater the potential for red callus production when the medium was provided with both BA and IAA. Equimolar concentrations of cytokinins to auxins are generally used to sustain callus, whereas higher cytokinin:auxin stimulates shoot development (Pickens et al., 2005).

Table 2.

Red callus initiation from one mo. old poinsettia ‘Prestige Red’ explants (apical buds and axillary buds) on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA) + 6-benzylaminopurine (BA).

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Rooting study.

Four-month-old poinsettia (40 micro-shoots) transferred into rooting media rooted irrespective of IAA concentrations. Incorporation of BA had an inhibitory effect on rooting; plants treated with BA developed callus at the base of the plant instead of rooting. In our study, the best medium for poinsettia in vitro rooting was half-strength MS salts and vitamins with 28.5 μM IAA because it increased rooting efficiency (Table 3). Higher IAA concentrations had a negative impact on rooting leading to callus development at the base of the plant (data not shown).

Table 3.

Root initiation from four mo. old poinsettia ‘Prestige Red’ micro-shoots (4 months old) on Murashige and Skoog (MS) medium supplemented with plant growth regulators (PGRs).

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In summary, BA (4 to 12 μM) can be used effectively to generate red callus, micro-buds, and micro-shoots from apical and axillary buds of poinsettia ‘Prestige™ Red’. Incorporation of IAA and BA into the medium generates red callus but BA:IAA is critical in generating red callus; the greater the BA:IAA ratio, the greater potential for red callus production. Red callus is more important for poinsettia proliferation than white callus. The best medium for in vitro rooting of poinsettia was half-strength MS salts and vitamins with 28.5 μM IAA for rooting efficiency. Results from this research can be used as an in vitro micropropagation and proliferation protocol for poinsettia ‘Prestige™ Red’. This protocol can be used in both poinsettia in vitro proliferation and as the first step in transgenic poinsettia production. Further research is necessary to determine how to minimize development of white callus during in vitro poinsettia culture.