Shoot regeneration from leaf explants of Brunfelsia calycina

Plant Cell Tiss Organ Cult (2010) 100:345–348 DOI 10.1007/s11240-009-9642-7 RESEARCH NOTE Shoot regeneration from leaf explants of Brunfelsia calycina Raya Liberman • Liat Shahar • Ada Nissim-Levi • Dalia Evenor • Moshe Reuveni • Michal Oren-Shamir Received: 8 July 2009 / Accepted: 15 November 2009 / Published online: 10 December 2009 Ó Springer Science+Business Media B.V. 2009 Abstract A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 lM BA and 2.85 lM IAA for young leaf explants, and at 8.88 lM BA, 2.85 lM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23– 2.46 lM indole butrytic acid (IBA), they developed roots. Keywords Regeneration
Solanaceae
Brunfelsia Abbreviations Aux Auxin BA 6-Benzyladenine Ck Cytokinin IAA Indole-3-acetic acid IBA Indole-3-butyric acid MS Murashige and Skoog (1962) NAA 2-Naphthaleneacetic acid TDZ Thidiazuron (N-phenyl N0 1,2,3-thidiazol-5-yl urea) Raya Liberman and Liat Shahar have equally contributed to this work. R. Liberman
L. Shahar
A. Nissim-Levi
D. Evenor
M. Reuveni
M. Oren-Shamir (&) Department of Ornamental Horticulture, ARO Volcani Center, P.O. Box 6, 50250 Bet-Dagan, Israel e-mail: [email protected] Brunfelsia (Solanaceae) is a genus of about 40 species of shrubs and small trees native to Brazil. The typical habitat for these plants is light woodland and thickets. Several Brunfelsia species contain medicinal and toxic alkaloids. For example, B. grandiflora is the source of the most important native remedies employed against rheumatism, arthritis and snake bites in the upper Amazon region (Plowman 1977). Other Brunfelsia species, among them B. calycina, have been cultivated as ornamentals. B. calycina has become a popular garden and pot plant due to its large blue flowers and pleasant fragrance (Heide 1963). A striking characteristic of B. calycina flowers is their rapid color change from dark purple on the day of opening to white within only 3 days, resulting in bushes with both purple and white flowers. Because of this rapid color change, their common name is Yesterday–Today–Tomorrow (Brenzel 2001; Halevy 1985; Heide 1963). We have shown that the color change in B. calycina flowers is due to degradation of anthocyanins and that this process is dependent on the synthesis of novel proteins (Vaknin et al. 2005). There is very little information about anthocyanin degradation in plants, thus B. calycina can serve as a model plant for understanding this process. A first step in testing the involvement of candidate genes in the anthocyanindegradation process is the development of a regeneration protocol for B. calycina. Here we present a method for regenerating B. calycina plantlets from leaf explants. To the best of our knowledge, this is the first published regeneration protocol for Brunfelsia. We tested the regeneration potential of several plant organs on a variety of media. Flower buds, axial buds, nonwoody green stems, young leaves that are still expanding (up to five leaves from the apical meristem) and fully expanded mature leaves were cut from B. calycina plants grown in pots in a glass greenhouse under controlled 123 346 Plant Cell Tiss Organ Cult (2010) 100:345–348 Table 1 Combinations of PGRs used for initial testing of regeneration Medium IAA (lM) A 2.85 B 2.85 C BA (lM) Kinetin (lM) TDZ (lM) Aux/Ck ratio 4.54 0.62 0.64 8.88 2.68 0.30 9.12 0.57 F G NAA (lM) 4.44 D E Zeatin (lM) 4.56 0.13 13.3 4.57 4.56 9.29 0.32 The table lists the different combinations of PGRs added to the MS salt, 3% sucrose and 0.75% agar Fig. 1 A typical leaf explant originating from young Brunfelsia leaves, incubated for 6 weeks in the different regeneration media. a–g Refers to the different media listed in Table 1 [bar = 2.5 (a), 3.5 (b), 3 (c), 3 (d), 4.5 (e), 3 (f), 2.5 mm (g)] temperature conditions (23°C/15°C day/night). The plant organs were washed with soap and running water for 1.5 h, sterilized with 70% ethanol for 5 min, mixed for 1 h in a solution containing 1 g/l Benlet (Du Pont de Nemours, France) and 1 g/l Marpan (Machteshim, Israel) and sequentially washed with sterile water. Then organs were surfacesterilized in a 30% (v/v) commercial bleach solution (1% sodium hypochlorite) for 15 min and sequentially washed twice with sterile water. To induce regeneration, flower buds and axial buds, 0.5cm long stems, and leaf sections of 1 9 1 cm were placed in Petri dishes on MS medium at pH 5.7, with 30 g/l sucrose, 7.5 g/l agar, and different combinations of plant growth regulators (PGRs) (Table 1). Explants were cultured under a 16 h photoperiod at 50 lmol m-2 s-1 supplied by coolwhite fluorescent lamps at a constant temperature of 25°C. After 6 and 9 weeks placement on the different MS media, explants were evaluated for initiation of regeneration and formation of shoot buds and shoots. Shoot buds and shoots formed only on leaf explants (Fig. 1), with faster and better regeneration on young leaves than on mature ones. Flower buds, axial buds and stem segments showed either no response or very poor 123 regeneration, with formation of either calli or shoot buds that did not continue to develop. The best regeneration response of young leaves after 6 weeks of incubation was on media A and B, containing 2.85 lM Indole-3-acetic acid (IAA) as an auxin source and 4.44 lM of 6-Benzyladenine (BA) or 4.54 lM Thidiazuron (TDZ) as a cytokinin source (Fig. 1). Medium A was chosen for subsequent work because after 9 weeks of incubation, the explants had more developed shoot buds and shoots on BA-containing medium than on TDZ-containing medium (results not shown). Leaf explants on media C-G dried up after several weeks of incubation without forming viable shoot buds or plantlets. In an effort to further improve the regeneration process, six additional media, all based on medium A, were tested using young and mature leaves. We increased BA concentration (4.44, 6.66, 8.88 lM) of medium A while keeping IAA constant (2.85 lM, Aux/Ck ratio 0.6–0.3), and increased IAA concentrations (5.70 7.95, 11.4 lM) while keeping BA constant (4.44 lM, Aux/Ck ratio 1–2). Media with a low concentration of IAA (2.85 lM) were advantageous for the regeneration of both young and mature leaf explants, even after 6 weeks incubation (Fig. 2). Increasing concentration of BA (from 4.44 to 8.88 lM) in Percentage of regeneration 60 347 6 weeks a Young Mature 50 ab 40 abc 30 bc abcd cde 20 de cde e 10 cde e e Percentage of regeneration Plant Cell Tiss Organ Cult (2010) 100:345–348 70 9 weeks a a 60 ab 50 ab 40 30 cd cd 20 4.44 BA 6.66 BA 8.88 BA 4.44 BA 4.44 BA 4.44 BA 2.85 IAA 2.85 IAA 2.85 IA A 5.7 IAA 8.5 IAA 11.4 IAA Hormon concentration (µM ) Hormon concentration (µM ) Fig. 2 Effect of supplementing different concentrations of the growth regulators BA and IAA on the frequency of regeneration. The percentage of regeneration was calculated from the number of explants with completely regenerated shoots per number of leaf explants in each Petri dish from Brunfelsia leaf explants, 6 and 6 weeks Mature a 1.2 ab abc 1.0 bc 0.8 cde 0.4 9 weeks bcde cde e de e e 0.2 0.0 3.0 Degree of bud development a 9 weeks after culture initiation. Values are the average mean ± standard error of 10 experiments, each consisting of a Petri dish with 10 explants. Letters (a, b, c, d, e) indicate statistically significant differences between means (P \ 0.05) using Student T-Test with JMP Statistical Discovery software (SAS Institute, Cary, NC, USA) Young 1.6 Degree of bud development cd d 10 4.44 BA 6.66 BA 8.88 BA 4.44 BA 4.44 BA 4.44 BA -10 2.85 IAA 2.85 IAA 2.85 IAA 5.7 IAA 8.5 IAA 11.4 IAA 0.6 bc bc bc 0 0 1.4 Young Mature ab Young a Mature ab 2.5 2.0 bc cd 1.5 bc cd cd cde cde 1.0 e 0.5 de e 0.0 4.44 BA 6.66 BA 8.88 BA 4.44 BA 4.44 BA 4.44 BA 2.85 IAA 2.85 IAA 2.85 IAA 5.7 IAA 8.5 IAA 11.4 IAA 4.44 BA 6.66 BA 8.88 BA 4.44 BA 4.44 BA 4.44 BA 2.85 IA A 2.85 IA A 2.85 IA A 5.7 IA A 8.5 IA A 11.4 IAA Hormon concentration (µM ) Hormon concentration (µM ) Stage 1 Stage 2 Stage 3 Fig. 3 Effect of BA and IAA concentration on degree of shoot bud development. Measurements were taken 6 and 9 weeks after culture initiation. The degree of shoot bud development was determined by comparison to a visual key of the developmental stages presented in the photographs. Only leaf explants that showed regeneration were considered in this evaluation. Values are the average mean ± standard error of 10 explants from 10 different Petri dishes (bar = 2 mm). Letters (a, b, c, d, e) indicate statistically significant differences between means (P \ 0.05) using Student T-Test with JMP Statistical Discovery software (SAS Institute, Cary, NC, USA) media with low IAA concentration (2.85 lM), had no significant effect on the regeneration of young leaves, but did increase the percentage of regeneration of mature leaf explants, both after 6 and 9 weeks (Fig. 2). Media A (2.85 lM IAA, 4.44 lM BA) was advantageous for young explants in comparison to those from mature leaves. In addition, media A had a slight advantage on the other media with low IAA concentration, though not statistically significant, in regeneration of young leaves after 6 and 9 week incubation (Fig. 2). The degree of shoot bud development was determined among the leaf sections that did regenerate (Fig. 3). Since shoot bud formation was very dense, a visual key is presented for the different developmental stages (Fig. 3). Leaf explants were scored according to the estimated number and length of the buds/shoots formed (Fig. 3). Shoot bud 123 348 Plant Cell Tiss Organ Cult (2010) 100:345–348 Fig. 4 Rooting of shoots from regeneration of Brunfelsia young leaf explants. a A shoot, about 1.5 cm long, before transferring to rooting medium. b Shoots in rooting medium. c Rooted plantlet at the stage of transferal to soil pots [bar = 2 (a), 3.5 (b) and 4.5 mm (c)] development was significantly more advanced in media with low IAA concentration (2.85 lM IAA), after 6 and 9 weeks, than in media with higher IAA concentrations (Fig. 3). The degree of shoot bud development was significantly higher in young leaves than in mature leaves after both 6 and 9 weeks incubation in medium A (2.85 lM IAA, 4.44 lM BA) with a similar trend in the other five media tested (Fig. 3). Clearly, low IAA concentration was crucial for both the initiation of regeneration from Brunfelsia leaves (Fig. 2) and shoot bud development (Fig. 3). The rate of bud/shoot development was more rapid in young leaves than in mature ones. Low IAA and BA (2.85 lM, IAA 4.44 lM BA) was best for young leaves, while mature leaves appeared to respond better to a higher concentration of BA in the presence of low IAA (2.85 lM IAA and 8.88 lM BA). After 9 weeks on the various media, the advantage of 8.88 lM BA for mature leaves was less pronounced. After 9 weeks, shoots that were about 0.5 cm long were separated from the leaf explants and transferred for elongation on medium A (4.44 lM BA, 2.85 lM IAA) for an additional 3 weeks until they reached a length of about 1.5 cm (Fig. 4a). These shoots were then transferred to rooting media containing MS medium, pH 5.7, 20 g/l sucrose and three different concentrations of indole3-butyric acid (IBA) (2.23, 2.46 and 4.92 lM). FeEDTA (0.12 mM) was added to the media to increase chlorophyll concentration in the developing plantlets (Mohamed and Aly 2004). Rooting efficiency was tested 12 weeks after transfer to rooting media and is summarized in Table 2. A concentration of 2.46 lM IBA was found best for rooting, with 100% survival and 72% rooting of the plants. After 12 weeks, plants were transferred to pots for further development and growth (Fig. 4). In conclusion, we established a regeneration protocol for B. calycina. The complete protocol until transfer of rooted plantlets to soil takes approximately 24 weeks. This is relatively long, and is probably due to the fact that Brunfelsia is 123 Table 2 Effect of IBA concentration on rooting of Brunfelsia shoots Concentration of growth regulator (IBA lM) Survival Rooting time Rooting efficiency (%) (%) (weeks) 1.23 2.46 12 12 33 72 100 100 4.92 12 0 0 Twelve shoots were tested in each medium a woody plant (Reuveni et al. 2003, 2005). Further refinement of the protocol is needed to increase efficiency and reduce duration. This study is the first step in developing Brunfelsia calycina as a model plant for functional testing of genes involved in the anthocyanin degradation process. 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