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Vegetative propagation of Primulina tabucum Hance by petiole cuttings
Scientia Horticulturae 134 (2012) 163–166
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Vegetative propagation of Primulina tabucum Hance by petiole cuttings Jinfeng Lü a,b , Jaime A. Teixeira da Silva c , Guohua Ma a,∗ a b c
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, PR China Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken 761-0795, Japan
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 19 October 2011 Accepted 22 October 2011 Keywords: Germplasm conservation Adventitious shoots Root Regeneration
a b s t r a c t Primulina tabucum Hance is a first grade rare and endangered monospecific genus endemic to China. Petioles derived from one-year old potted plants (originally derived from in vitro culture) were used to study the effect of substrate and plant growth regulators on the induction of adventitious shoots and roots. Sand and perlite (1:1) was the most suitable substrate. Petiole explants dipped in 4.9 M indole-3-butyric acid for 30 min could induce the highest rooting percentage (90%) and an average of 8.8 adventitious shoots per petiole, establishing thus a simple, quick and efficient propagation pathway. This simple protocol would allow for mass production of ex vivo- or in vitro-derived germplasm conservation.
1. Introduction Primulina tabacum Hance (Gesneriaceae; monospecific genus) was first discovered in the Lianjiang limestone drainage mountain areas in Lianzhou, North Guangdong, China in 1881 (Hance, 1883), followed shortly thereafter by its disappearance for more than 120 years. Its recent rediscovery has important scientific value in the study of both ancient and recent climate, soil and co-evolutionary biology of animals and plants in South China in the Five Ridges Region (Ni et al., 2006; Wang et al., 2009; Liang et al., 2010). Now P. tabacum has been listed as a ‘first grade’ critically endangered species in China (Peng and Cheng, 2002). P. tabacum is usually reproduced by seed in nature. However, in fact, very few seeds germinate and grow successfully in the wild. To address this reproductive weakness, a tissue culture technique was recently developed allowing for the successful mass propagation of the species in vitro and re-introduction of plants into their natural habitats (Ma et al., 2010; Ren et al., 2010a,b). In this study, we report an asexual propagation method by petiole cuttings to induce adventitious shoots and roots.
© 2011 Elsevier B.V. All rights reserved.
One-year-old plants that had been propagated in vitro under the same conditions (Ma et al., 2010) were transplanted to pots (15 cm × 15 cm) containing sand and pond sludge (1:3) and placed under a shed covered with 80% shade film (maximum 150 mol m−2 s−1 ; natural light), at 20–30 ◦ C and 60–95% relative humidity. No fertilizers were applied. The 2–5 cm long petiole cuttings, including the whole leaf, served as the experimental explant, and were inserted obliquely into different substrates. All PGRs used in the study were purchased from Sigma–Aldrich (St. Louis, USA) and were used as an aqueous solution. 2.1. Effect of substrate on petiole cuttings Sand, perlite and vermiculite were selected as the basal substrates that were used singly or as a mixture (1:1 or 1:1:1, v/v) in plastic square trays (40 cm × 30 cm × 10 cm) (Table 1). The petioles were dipped in 14.8 M indole-3-butyric acid (IBA) solution for 30 min and then inserted obliquely into different substrates. The trays were sprayed with water twice daily in the morning and afternoon. Prior to inserting cuttings (but never after), carbendazime (a fungicide), was applied.
2. Materials and methods 2.2. Effect of plant growth regulators (PGRs) on petiole cutting The experiments were conducted between February and May, 2011. Petioles >10 cm long from healthy plants were used.
Abbreviations: BA, 6-benzyl adenine; IBA, indole-3-butyric acid; NAA, ␣-naphthaleneacetic acid; IAA, indole-3-acetic acid. ∗ Corresponding author. E-mail address: [email protected] (G. Ma). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.10.024
In the first experiment, petioles were either dipped in 14.8 M IBA solution for 30 min, then inserted obliquely into a substrate of sand and perlite (1:1, v/v) or inserted directly into the same substrate then sprayed with 10 ml of 14.8 M IBA solution twice a week for each tray. In the second experiment, petioles were dipped for 30 min in different concentrations of IBA, 6-benzyladenine (BA), indole-3-acetic acid (IAA), ␣-naphthaleneacetic acid (NAA), singly
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J. Lü et al. / Scientia Horticulturae 134 (2012) 163–166
Table 1 Effect of different substrates and IBA treatment on induction of adventitious shoots and roots from Primulina tabucum petioles. Substrates (v/v)
14.8 M IBA
Rooting (%)
No. of shoots/petiole
Sand Vermiculite Perlite Sand:vermiculite (1:1) Sand:perlite (1:1) Sand:vermiculite: perlite (1:1:1)
Dipping for 30 min
26.7 c 3.3 d 26.7 c 80.0 a 80.0 a 81.7 a
0c 0c 0c 3.6 b 8.5 a 7.9 a
Sand:perlite (1:1)
Spray twice a week
65.0 b
4.5 b
The same letter within a column indicates no significant difference using the post hoc LSD test (P < 0.05).
or in combination (Table 2), then inserted obliquely into a substrate of sand and perlite (1:1, v/v). 2.3. Transplanting of adventitious shoots Adventitious shoots that formed on the trays which were dipped in 4.9 M IBA solution were separated into individual shoots, transferred to the same trays with a substrate of sand and perlite (1:1, v/v) and watered daily. After 30 days, survival percentage was calculated. 2.4. Statistical analyses All experiments were repeated in triplicate. Each treatment consisted of six trays and 20 petioles per tray. Since rooting occurred after 4 weeks, data was recorded in the sixth week (at 50 days). Rooting rate was expressed as a percent response and adventitious bud formation was investigated by recording the number of adventitious buds per explant. All data were statistically analyzed by one-way ANOVA with a post hoc test (PLSD) used to separate treatment means at P ≤ 0.05. 3. Results 3.1. Effects of different substrates on cutting survival When used alone, none of the three substrates (sand, vermiculite or perlite) could achieve a rooting percentage after 50 days greater than 30% even after dipping in a solution of 14.8 M IBA. However, in a mixture of sand and vermiculite (1:1, v/v), the rooting percentage increased to 83.3% and an average of 3.6 adventitious shoots/petiole were induced while in mixture (sand and perlite, 1:1, v/v), the rooting percentage was 80.0% and an average of 8.5 adventitious shoots developed from each petiole base (Table 1).
In addition, adventitious roots also developed well in the rooting mixture (sand, vermiculite and perlite, 1:1:1, v/v). The rooting percentage was 81.7 and an average of 7.9 adventitious shoots formed per petiole.
3.2. Effect of different PGRs on petiole cuttings Both dipping and spraying with 14.8 M IBA solution could induce adventitious shoots and roots from petioles. In the former, the rooting percentage was 81.7% and an average of 8.5 adventitious shoots was induced; for the spray treatment, these values were 65% and 4.5, respectively (Table 1). The control (no PGRs) could induce few adventitious shoots (2.0) and roots (26.7%) from the cut petiole surface. As BA concentration increased (4.4 M, 13.3 M and 22.2 M), the number of adventitious shoots increased (6.0, 6.4 and 8.3, respectively) while rooting percentage decreased (63.3%, 51.7% and 48.3%, respectively) (Fig. 1A and B). Furthermore, as IBA concentration increased (4.9, 14.8, 24.6 M), there was no significant change in rooting percentage (90.0, 83.3 and 81.7%, respectively), although the number of adventitious shoots decreased (8.8, 8.5 and 6.8, respectively). We then assessed the effects of the auxins (IBA, NAA and IAA, at 4.9, 5.4 and 5.7 M, respectively) on rooting and adventitious shoot formation from petiole cuttings. IAA induced 71.7% rooting percentage and an average of 6.6 adventitious shoots per petiole (Fig. 1C). NAA resulted in achlorophyllous adventitious roots with low (45%) rooting percentage and few (3.0/petiole) adventitious shoots (Fig. 1D). IBA resulted in the greatest rooting percentage (90%), highest average number of adventitious shoots (8.8/petiole) and the longest roots (10.3 cm) (Fig. 1E). Petioles dipped into a solution containing NAA, IBA and BA formed few adventitious shoots (0.6) and a low percentage rooting (25%).
Table 2 Effect of different plant growth regulators (PGRs) on induction of adventitious buds and roots from Primulina tabucum petioles. Treatment with PGRs (M)
Rooting (%)
No. of shoots/petiole
Description of root development
Water (control) 4.4 BA 13.9 BA 22.2 BA 5.7 IAA 28.5 IAA
26.7 d 63.3 b 51.7 c 48.3 c 70.0 b 71.7 b
2.0 c 6.0 b 6.4 b 8.3 a 5.9 b 6.6 b
5.4 NAA 4.9 IBA
45.0 c 90.0 a
3.0 c 8.8 a
14.8 IBA
83.3 a
8.5 a
24.6 IBA
81.7 a
6.8b
4.4 BA + 5.7 NAA + 4.9 IBA
23.3 d
0.6 d
Late developing roots, thin short roots, swollen, no callus at base Thin and short, not swollen, callus at base Thin short roots, not swollen, no callus at base Short roots, not swollen, no callus at base Thin short roots, swollen, adventitious roots at base Thin and adventitious roots, side roots developed well, swollen, no callus at base Strong long roots, few side roots, no obvious swelling, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base Short roots, few side roots, not swollen, no callus at base
The same letter within a column indicates no significant difference using the post hoc LSD test (P < 0.05).
J. Lü et al. / Scientia Horticulturae 134 (2012) 163–166
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Fig. 1. Different PGRs induce adventitious shoots and roots from Primulina tabucum petioles. (A) Low concentration of BA (4.4 m l−1 ) induced adventitious shoots and roots; (B) high concentration of BA (22.2 m l−1 ) induced adventitious shoots and short roots; (C) 5.7 m l−1 IAA induced adventitious shoots and short roots; (D) 5.4 m l−1 NAA induced strong, long roots and few adventitious shoots; (E) 4.9 m l−1 IBA induced well developed adventitious shoots and roots; (F) Adventitious shoots were separated into individual shoots and transplanted successfully after culturing for 30 days.
3.3. Transplanting of shoots Shoots could develop new roots within 20 days since most of them formed many root primordia at the base of shoot stems. The rooting percentage reached 93.3% after culturing for 30 days (Fig. 1F). 4. Discussion In the traditional vegetative propagation of cuttings, the substrate is a key factor that influences cutting efficiency in many species due to the blends of propagation substrates that create suitable air and drainage characteristics while retaining moisture – without causing waterlogging – during the period of root initiation (Di Benedetto, 2007; Ragonezi et al., 2010). Different substrates have been successfully used to promote rooting in various species, herbaceous and woody (Santelices and Cabello, 2006; Mpeck and Atangana, 2007; Ragonezi et al., 2010; Pelizza et al., 2011). In this study, when sand, vermiculite or perlite were used alone as a substrate, rooting percentage was low. The main reason is that moist sand has poor aeration and is easy to be infected by pathogens. Vermiculite and perlite have better aeration but they are lighter than water. Thus, petiole cuttings were not so stable in the vermiculite or perlite substrate and rooting was erratic, which seems to be common among several plants cuttings (Hartman et al., 2002).
The effect of the vermiculite or perlite substrate was reported as the survival percentage of Arbutus unedo cuttings in perlite and perlite-peat was twice as high as in sand (Metaxas et al., 2008); cherry cuttings placed in perlite: peat (1:1) or perlite with bottom heat gave the best rooting percentage (Exadaktylou et al., 2009) and coir dust was superior to other commonly used substrates for the rooting and acclimatization of shoe flower (Seran et al., 2011). In our study, a mixture of sand and perlite (1:1, v/v) resulted in the highest rooting percentage. PGRs are another important factor influencing the vegetative propagation of cuttings. Commonly used PGRs for the rooting of cuttings include IBA, NAA, IAA and BA (Balestri and Lardicci, 2006; Felzener et al., 2007; Yoo, 2009; Murthy et al., 2010). Usually, IBA is applied for effective rooting, particularly in hardwood or tree species (e.g., Trueman and Richardson, 2011). IBA at 0.4% resulted in significantly higher rooting in the vegetative propagation and initial growth of Stereospermum suaveolens (Baul et al., 2009). IBA at 5.0 mM was most effective on root length and rooting percentage of Lavandula angustifolia cv. ‘Provence Blue’ microcuttings, despite a reduced number of roots with a wide range of survival (82–100%) among cultivars (Machado et al., 2011). In our test, IBA proved to be the best to induce adventitious shoots and roots from petiole explants of P. tabacum. BA as a cytokinin was used to induce adventitious shoots and roots. A low concentration of BA induces small adventitious shoots and some taproots. As the concentration of BA
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increased, the size of adventitious shoots also increased while the percentage rooting decreased. There are few reports on the effect of BA on cuttings (Wang, 1990; Shimada et al., 2005). Petiole cuttings have also been used successfully as an asexual propagule for some other Gesneriaceae and Begoniaceae members, for example, African violet (Bilkey et al., 1978; Cassells and Plunkett, 1984; Hartman et al., 2002), and Begonia tuberhybrida (Shimada et al., 2006, 2007). Our study has successfully induced adventitious shoots and roots from petiole explants on an endemic rare and endangered P. tabacum. New plantlets can be fully generated within three months. It is a simple, quick and efficient propagation method, allowing for an efficient propagation protocol and valuable tool for ex situ conservation. Since this plant is critically endangered, by being able to mass produce P. tabacum in vitro (Ma et al., 2010) and also ex vitro (this study) provides ample opportunities to revegetate natural parks or to create in vitro sanctuaries through non destructive means. Acknowledgement Support from the National Natural Science Foundation of China (30671711; 30972295) is gratefully acknowledged. References Balestri, E., Lardicci, C., 2006. Stimulation of root formation in Posidonia oceanica cuttings by application of auxins (NAA and IBA). Marine Biol. 149, 393–400. Baul, T.K., Mezbahuddin, M., Mohiuddin, M., 2009. Vegetative propagation and initial growth performance of Stereospermum suaveolens DC, a wild tropical tree species of medicinal value. New Forest 37, 275–283. Bilkey, P.C., McBrown, B.H., Hildebrandt, A.C., 1978. Micropropagation of African violet from petiole cross-sections. HortScience 13, 37–38. Cassells, A.C., Plunkett, A., 1984. Production and growth analysis of plants from leaf cuttings and from tissue cultures of disks from mature leaves and young axenic leaves of African violet (Saintpaulia ionantha Wendl.). Sci. Hortic. 4, 361–369. Di Benedetto, A., 2007. Alternative substrates for potted ornamental plants based on Argentinean peat and Argentinean river waste: a review. Floriculture Ornamental Biotechnol. 1 (2), 90–101. Exadaktylou, E., Thomidis, T., Grout, B., Zakynthinos, G., Tsipouridis, C., 2009. Methods to improve the rooting of hardwood cuttings of the ‘Gisela 5’ cherry rootstock. HortTechnology 19, 254–259. Felzener, L.T., Barreiro, A.P., Ono, E.O., Barros-Cardoso, S.A., Rodrigues, D., 2007. Effect of plant growth regulators on rooting of Poncirus trifoliata var. monstrosa (T. ITO) cuttings. Rev. Bras. Frutic. 29, 399–402. Hance, H.F., 1883. Primulina tabacum Hance. J. Bot. 21, 169. Hartman, H.T., Kester, D.E., Davies, J.F.T., Geneve, R.L., 2002. Plant Propagation: Principles and Practices, seventh ed. Prentice Hall, Upper Saddle River. Liang, K.M., Lin, Z.F., Ren, H., Liu, N., Zhang, Q.M., Wang, J., Wang, Z.F., Guan, L.L., 2010. Characteristics of sun- and shade-adapted populations of an endangered plant Primulina tabacum Hance. Photosynthetica 48, 494–506.
Ma, G.H., He, C.X., Ren, H., Zhang, Q.M., Li, S.J., Zhang, X.H., Bunn, E., 2010. Direct somatic embryogenesis and shoot organogenesis from leaf explants of Primulina tabacum. Biol. Plant 54, 361–365. Machado, M.P., Santos, G.D., Deschamps, C., Biasi, A., 2011. Rooting of Lavandula angustifolia microcuttings. Ciência Rural 41, 767–772. Metaxas, D., Syros, T., Economou, A., 2008. Factors affecting vegetative propagation of Arbutus unedo L. by stem cuttings. Prop. Orn. Plant 8, 190–197. Mpeck, M.N., Atangana, A., 2007. Rooting of leafy stem cuttings of Baillonella toxisperma. Forest Sci. 53, 571–579. Murthy, G., Umesha, K., Smitha, G.R., Krishnamanohar, R., 2010. Effect of growth regulators and bio-inoculants on rooting and growth of vanilla stem cuttings. Indian J. Hortic. 67, 90–93. Ni, X.W., Huang, Y.L., Wu, L., Zhou, R.C., Deng, S.L., Wu, D.R., Wang, B., Su, G., Tang, T., Shi, S., 2006. Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP). Genetics 127, 177–183. Pelizza, T.R., Damiani, C.R., Rufato, A.D.A., Souza, L.K., Ribeiro, M.D., Schuch, M.W., 2011. Microcutting in blueberry using branch from different positions and substrates. Bragantia 70, 319–324. Peng, S.L., Cheng, W.C., 2002. Rare and Endangered Plants in Guangdong. Science Press, Beijing (in Chinese). Ragonezi, C., Klimaszewska, K., Castro, M.R., Lima, M., Oliveira, P., Zavattieri, M.A., 2010. Adventitious rooting of conifers: influence of physical and chemical factors. Trees – Struct. Funct. 24, 975–992. Ren, H., Ma, G.H., Zhang, Q.M., Guo, Q.F., Wang, J., Wang, Z.F., 2010a. Moss is a key nurse plant for reintroduction of the endangered herb, Primulina tabacum Hance. Plant Ecol. 209, 313–320. Ren, H., Zhang, Q.M., Wang, Z.F., Guo, Q.F., Liang, K.M., 2010b. Conservation and possible reintroduction of an endangered plant based on an analysis of community ecology: a case study of Primulina tabacum Hance in China. Plant Spec. Biol. 25, 43–50. Santelices, R., Cabello, A., 2006. Effect of indolebutyric acid, bottom heat, substrate, and parent tree on rooting capacity of Nothofagus glauca (Phil.) Krasser stem cuttings. Rev. Chil. Hist. Nat. 79, 55–64. Seran, T.H., Meerashahib, N.B.M., Teixeira da Silva, J.A., 2011. Influence of coir dust on plant growth of shoe flower (Hibiscus rosasinensis) stem cuttings. Eur. J. Hortic. Sci. 76, 109–115. Shimada, Y., Mori, G., Katahara, Y., Oda, M., 2005. The influence of BA and NAA on adventitious bud formation in leaf cuttings of Begonia x tuber hybrida. In: Okubo, H., Miller, W.B., Chastagner, G.A. (Eds.), Proceedings of the Ninth International Symposium on Flower Bulbs, vols. 1 and 2. , pp. 537–541. Shimada, Y., Mori, G., Katahara, Y., Oda, M., 2006. Formation of adventitious buds on leaf pieces cutting of Begonia tuberhybrida Group. J. Jpn. Soc. Hortic. Sci. 75, 318–322. Shimada, Y., Mori, G., Oda, M., Ishida, G., 2007. Effects of BA and leaf piece orientation on adventitious bud formation in leaf cutting of Begonia tuberhybrida group. J. Jpn. Soc. Hortic. Sci. 76, 157–162. Trueman, S.J., Richardson, D.M., 2011. Propagation and chlorophyll fluorescence of Camptotheca acuminata cuttings. J. Med. Plant Res. 5, 1–6. Wang, Y.T., 1990. Growth substance, light, fertilizer, and misting regulate propagation and growth of golden pothos. HortScience 25, 1602–1604. Wang, Z.F., Ren, H., Zhang, Q.M., Ye, W.H., Liang, K.M., Li, Z.C., 2009. Isolation and characterization of microsatellite markers for Primulina tabacum, a critically endangered perennial herb. Conserv. Genet. 10, 1433–1435. Yoo, Y.K., 2009. Effects of plant growth regulators and several conditions on rooting and shoot growth in rhizome cutting of Acorus gramineus. Kor. J. Hortic. Sci. 27, 560–566.
Contents lists available at SciVerse ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Vegetative propagation of Primulina tabucum Hance by petiole cuttings Jinfeng Lü a,b , Jaime A. Teixeira da Silva c , Guohua Ma a,∗ a b c
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, PR China Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken 761-0795, Japan
a r t i c l e
i n f o
Article history: Received 18 August 2011 Received in revised form 19 October 2011 Accepted 22 October 2011 Keywords: Germplasm conservation Adventitious shoots Root Regeneration
a b s t r a c t Primulina tabucum Hance is a first grade rare and endangered monospecific genus endemic to China. Petioles derived from one-year old potted plants (originally derived from in vitro culture) were used to study the effect of substrate and plant growth regulators on the induction of adventitious shoots and roots. Sand and perlite (1:1) was the most suitable substrate. Petiole explants dipped in 4.9 M indole-3-butyric acid for 30 min could induce the highest rooting percentage (90%) and an average of 8.8 adventitious shoots per petiole, establishing thus a simple, quick and efficient propagation pathway. This simple protocol would allow for mass production of ex vivo- or in vitro-derived germplasm conservation.
1. Introduction Primulina tabacum Hance (Gesneriaceae; monospecific genus) was first discovered in the Lianjiang limestone drainage mountain areas in Lianzhou, North Guangdong, China in 1881 (Hance, 1883), followed shortly thereafter by its disappearance for more than 120 years. Its recent rediscovery has important scientific value in the study of both ancient and recent climate, soil and co-evolutionary biology of animals and plants in South China in the Five Ridges Region (Ni et al., 2006; Wang et al., 2009; Liang et al., 2010). Now P. tabacum has been listed as a ‘first grade’ critically endangered species in China (Peng and Cheng, 2002). P. tabacum is usually reproduced by seed in nature. However, in fact, very few seeds germinate and grow successfully in the wild. To address this reproductive weakness, a tissue culture technique was recently developed allowing for the successful mass propagation of the species in vitro and re-introduction of plants into their natural habitats (Ma et al., 2010; Ren et al., 2010a,b). In this study, we report an asexual propagation method by petiole cuttings to induce adventitious shoots and roots.
© 2011 Elsevier B.V. All rights reserved.
One-year-old plants that had been propagated in vitro under the same conditions (Ma et al., 2010) were transplanted to pots (15 cm × 15 cm) containing sand and pond sludge (1:3) and placed under a shed covered with 80% shade film (maximum 150 mol m−2 s−1 ; natural light), at 20–30 ◦ C and 60–95% relative humidity. No fertilizers were applied. The 2–5 cm long petiole cuttings, including the whole leaf, served as the experimental explant, and were inserted obliquely into different substrates. All PGRs used in the study were purchased from Sigma–Aldrich (St. Louis, USA) and were used as an aqueous solution. 2.1. Effect of substrate on petiole cuttings Sand, perlite and vermiculite were selected as the basal substrates that were used singly or as a mixture (1:1 or 1:1:1, v/v) in plastic square trays (40 cm × 30 cm × 10 cm) (Table 1). The petioles were dipped in 14.8 M indole-3-butyric acid (IBA) solution for 30 min and then inserted obliquely into different substrates. The trays were sprayed with water twice daily in the morning and afternoon. Prior to inserting cuttings (but never after), carbendazime (a fungicide), was applied.
2. Materials and methods 2.2. Effect of plant growth regulators (PGRs) on petiole cutting The experiments were conducted between February and May, 2011. Petioles >10 cm long from healthy plants were used.
Abbreviations: BA, 6-benzyl adenine; IBA, indole-3-butyric acid; NAA, ␣-naphthaleneacetic acid; IAA, indole-3-acetic acid. ∗ Corresponding author. E-mail address: [email protected] (G. Ma). 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.10.024
In the first experiment, petioles were either dipped in 14.8 M IBA solution for 30 min, then inserted obliquely into a substrate of sand and perlite (1:1, v/v) or inserted directly into the same substrate then sprayed with 10 ml of 14.8 M IBA solution twice a week for each tray. In the second experiment, petioles were dipped for 30 min in different concentrations of IBA, 6-benzyladenine (BA), indole-3-acetic acid (IAA), ␣-naphthaleneacetic acid (NAA), singly
164
J. Lü et al. / Scientia Horticulturae 134 (2012) 163–166
Table 1 Effect of different substrates and IBA treatment on induction of adventitious shoots and roots from Primulina tabucum petioles. Substrates (v/v)
14.8 M IBA
Rooting (%)
No. of shoots/petiole
Sand Vermiculite Perlite Sand:vermiculite (1:1) Sand:perlite (1:1) Sand:vermiculite: perlite (1:1:1)
Dipping for 30 min
26.7 c 3.3 d 26.7 c 80.0 a 80.0 a 81.7 a
0c 0c 0c 3.6 b 8.5 a 7.9 a
Sand:perlite (1:1)
Spray twice a week
65.0 b
4.5 b
The same letter within a column indicates no significant difference using the post hoc LSD test (P < 0.05).
or in combination (Table 2), then inserted obliquely into a substrate of sand and perlite (1:1, v/v). 2.3. Transplanting of adventitious shoots Adventitious shoots that formed on the trays which were dipped in 4.9 M IBA solution were separated into individual shoots, transferred to the same trays with a substrate of sand and perlite (1:1, v/v) and watered daily. After 30 days, survival percentage was calculated. 2.4. Statistical analyses All experiments were repeated in triplicate. Each treatment consisted of six trays and 20 petioles per tray. Since rooting occurred after 4 weeks, data was recorded in the sixth week (at 50 days). Rooting rate was expressed as a percent response and adventitious bud formation was investigated by recording the number of adventitious buds per explant. All data were statistically analyzed by one-way ANOVA with a post hoc test (PLSD) used to separate treatment means at P ≤ 0.05. 3. Results 3.1. Effects of different substrates on cutting survival When used alone, none of the three substrates (sand, vermiculite or perlite) could achieve a rooting percentage after 50 days greater than 30% even after dipping in a solution of 14.8 M IBA. However, in a mixture of sand and vermiculite (1:1, v/v), the rooting percentage increased to 83.3% and an average of 3.6 adventitious shoots/petiole were induced while in mixture (sand and perlite, 1:1, v/v), the rooting percentage was 80.0% and an average of 8.5 adventitious shoots developed from each petiole base (Table 1).
In addition, adventitious roots also developed well in the rooting mixture (sand, vermiculite and perlite, 1:1:1, v/v). The rooting percentage was 81.7 and an average of 7.9 adventitious shoots formed per petiole.
3.2. Effect of different PGRs on petiole cuttings Both dipping and spraying with 14.8 M IBA solution could induce adventitious shoots and roots from petioles. In the former, the rooting percentage was 81.7% and an average of 8.5 adventitious shoots was induced; for the spray treatment, these values were 65% and 4.5, respectively (Table 1). The control (no PGRs) could induce few adventitious shoots (2.0) and roots (26.7%) from the cut petiole surface. As BA concentration increased (4.4 M, 13.3 M and 22.2 M), the number of adventitious shoots increased (6.0, 6.4 and 8.3, respectively) while rooting percentage decreased (63.3%, 51.7% and 48.3%, respectively) (Fig. 1A and B). Furthermore, as IBA concentration increased (4.9, 14.8, 24.6 M), there was no significant change in rooting percentage (90.0, 83.3 and 81.7%, respectively), although the number of adventitious shoots decreased (8.8, 8.5 and 6.8, respectively). We then assessed the effects of the auxins (IBA, NAA and IAA, at 4.9, 5.4 and 5.7 M, respectively) on rooting and adventitious shoot formation from petiole cuttings. IAA induced 71.7% rooting percentage and an average of 6.6 adventitious shoots per petiole (Fig. 1C). NAA resulted in achlorophyllous adventitious roots with low (45%) rooting percentage and few (3.0/petiole) adventitious shoots (Fig. 1D). IBA resulted in the greatest rooting percentage (90%), highest average number of adventitious shoots (8.8/petiole) and the longest roots (10.3 cm) (Fig. 1E). Petioles dipped into a solution containing NAA, IBA and BA formed few adventitious shoots (0.6) and a low percentage rooting (25%).
Table 2 Effect of different plant growth regulators (PGRs) on induction of adventitious buds and roots from Primulina tabucum petioles. Treatment with PGRs (M)
Rooting (%)
No. of shoots/petiole
Description of root development
Water (control) 4.4 BA 13.9 BA 22.2 BA 5.7 IAA 28.5 IAA
26.7 d 63.3 b 51.7 c 48.3 c 70.0 b 71.7 b
2.0 c 6.0 b 6.4 b 8.3 a 5.9 b 6.6 b
5.4 NAA 4.9 IBA
45.0 c 90.0 a
3.0 c 8.8 a
14.8 IBA
83.3 a
8.5 a
24.6 IBA
81.7 a
6.8b
4.4 BA + 5.7 NAA + 4.9 IBA
23.3 d
0.6 d
Late developing roots, thin short roots, swollen, no callus at base Thin and short, not swollen, callus at base Thin short roots, not swollen, no callus at base Short roots, not swollen, no callus at base Thin short roots, swollen, adventitious roots at base Thin and adventitious roots, side roots developed well, swollen, no callus at base Strong long roots, few side roots, no obvious swelling, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base More and long adventitious roots, side roots developed well, not swollen, no callus at base Short roots, few side roots, not swollen, no callus at base
The same letter within a column indicates no significant difference using the post hoc LSD test (P < 0.05).
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Fig. 1. Different PGRs induce adventitious shoots and roots from Primulina tabucum petioles. (A) Low concentration of BA (4.4 m l−1 ) induced adventitious shoots and roots; (B) high concentration of BA (22.2 m l−1 ) induced adventitious shoots and short roots; (C) 5.7 m l−1 IAA induced adventitious shoots and short roots; (D) 5.4 m l−1 NAA induced strong, long roots and few adventitious shoots; (E) 4.9 m l−1 IBA induced well developed adventitious shoots and roots; (F) Adventitious shoots were separated into individual shoots and transplanted successfully after culturing for 30 days.
3.3. Transplanting of shoots Shoots could develop new roots within 20 days since most of them formed many root primordia at the base of shoot stems. The rooting percentage reached 93.3% after culturing for 30 days (Fig. 1F). 4. Discussion In the traditional vegetative propagation of cuttings, the substrate is a key factor that influences cutting efficiency in many species due to the blends of propagation substrates that create suitable air and drainage characteristics while retaining moisture – without causing waterlogging – during the period of root initiation (Di Benedetto, 2007; Ragonezi et al., 2010). Different substrates have been successfully used to promote rooting in various species, herbaceous and woody (Santelices and Cabello, 2006; Mpeck and Atangana, 2007; Ragonezi et al., 2010; Pelizza et al., 2011). In this study, when sand, vermiculite or perlite were used alone as a substrate, rooting percentage was low. The main reason is that moist sand has poor aeration and is easy to be infected by pathogens. Vermiculite and perlite have better aeration but they are lighter than water. Thus, petiole cuttings were not so stable in the vermiculite or perlite substrate and rooting was erratic, which seems to be common among several plants cuttings (Hartman et al., 2002).
The effect of the vermiculite or perlite substrate was reported as the survival percentage of Arbutus unedo cuttings in perlite and perlite-peat was twice as high as in sand (Metaxas et al., 2008); cherry cuttings placed in perlite: peat (1:1) or perlite with bottom heat gave the best rooting percentage (Exadaktylou et al., 2009) and coir dust was superior to other commonly used substrates for the rooting and acclimatization of shoe flower (Seran et al., 2011). In our study, a mixture of sand and perlite (1:1, v/v) resulted in the highest rooting percentage. PGRs are another important factor influencing the vegetative propagation of cuttings. Commonly used PGRs for the rooting of cuttings include IBA, NAA, IAA and BA (Balestri and Lardicci, 2006; Felzener et al., 2007; Yoo, 2009; Murthy et al., 2010). Usually, IBA is applied for effective rooting, particularly in hardwood or tree species (e.g., Trueman and Richardson, 2011). IBA at 0.4% resulted in significantly higher rooting in the vegetative propagation and initial growth of Stereospermum suaveolens (Baul et al., 2009). IBA at 5.0 mM was most effective on root length and rooting percentage of Lavandula angustifolia cv. ‘Provence Blue’ microcuttings, despite a reduced number of roots with a wide range of survival (82–100%) among cultivars (Machado et al., 2011). In our test, IBA proved to be the best to induce adventitious shoots and roots from petiole explants of P. tabacum. BA as a cytokinin was used to induce adventitious shoots and roots. A low concentration of BA induces small adventitious shoots and some taproots. As the concentration of BA
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increased, the size of adventitious shoots also increased while the percentage rooting decreased. There are few reports on the effect of BA on cuttings (Wang, 1990; Shimada et al., 2005). Petiole cuttings have also been used successfully as an asexual propagule for some other Gesneriaceae and Begoniaceae members, for example, African violet (Bilkey et al., 1978; Cassells and Plunkett, 1984; Hartman et al., 2002), and Begonia tuberhybrida (Shimada et al., 2006, 2007). Our study has successfully induced adventitious shoots and roots from petiole explants on an endemic rare and endangered P. tabacum. New plantlets can be fully generated within three months. It is a simple, quick and efficient propagation method, allowing for an efficient propagation protocol and valuable tool for ex situ conservation. Since this plant is critically endangered, by being able to mass produce P. tabacum in vitro (Ma et al., 2010) and also ex vitro (this study) provides ample opportunities to revegetate natural parks or to create in vitro sanctuaries through non destructive means. Acknowledgement Support from the National Natural Science Foundation of China (30671711; 30972295) is gratefully acknowledged. References Balestri, E., Lardicci, C., 2006. Stimulation of root formation in Posidonia oceanica cuttings by application of auxins (NAA and IBA). Marine Biol. 149, 393–400. Baul, T.K., Mezbahuddin, M., Mohiuddin, M., 2009. Vegetative propagation and initial growth performance of Stereospermum suaveolens DC, a wild tropical tree species of medicinal value. New Forest 37, 275–283. Bilkey, P.C., McBrown, B.H., Hildebrandt, A.C., 1978. Micropropagation of African violet from petiole cross-sections. HortScience 13, 37–38. Cassells, A.C., Plunkett, A., 1984. Production and growth analysis of plants from leaf cuttings and from tissue cultures of disks from mature leaves and young axenic leaves of African violet (Saintpaulia ionantha Wendl.). Sci. Hortic. 4, 361–369. Di Benedetto, A., 2007. Alternative substrates for potted ornamental plants based on Argentinean peat and Argentinean river waste: a review. Floriculture Ornamental Biotechnol. 1 (2), 90–101. Exadaktylou, E., Thomidis, T., Grout, B., Zakynthinos, G., Tsipouridis, C., 2009. Methods to improve the rooting of hardwood cuttings of the ‘Gisela 5’ cherry rootstock. HortTechnology 19, 254–259. Felzener, L.T., Barreiro, A.P., Ono, E.O., Barros-Cardoso, S.A., Rodrigues, D., 2007. Effect of plant growth regulators on rooting of Poncirus trifoliata var. monstrosa (T. ITO) cuttings. Rev. Bras. Frutic. 29, 399–402. Hance, H.F., 1883. Primulina tabacum Hance. J. Bot. 21, 169. Hartman, H.T., Kester, D.E., Davies, J.F.T., Geneve, R.L., 2002. Plant Propagation: Principles and Practices, seventh ed. Prentice Hall, Upper Saddle River. Liang, K.M., Lin, Z.F., Ren, H., Liu, N., Zhang, Q.M., Wang, J., Wang, Z.F., Guan, L.L., 2010. Characteristics of sun- and shade-adapted populations of an endangered plant Primulina tabacum Hance. Photosynthetica 48, 494–506.
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