In vitro germination, seed viability and organogenesis of Anthurium schlechtendalii Kunth subsp. schlechtendalii

S. E. Rangel-Estrada1*; E. Hernández-Meneses2; J. Canul-Kú1; E. J. Barrios-Gómez1; M. C. G. López-Peralta3; L. Tapia-Jaramillo1

1. Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias-Campo Experimental Zacatepec. Carretera Zacatepec Galeana s/n, Centro, 62780, Zacatepec de Hidalgo, Morelos. Tel (01) (800) 0882222 Ext. 86608., Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias,

<postal-code>62780</postal-code>
<city>Zacatepec de Hidalgo</city>
<state>Morelos</state>
, Mexico , 2. Postgrado en Recursos Genéticos y Productividad (PREGEP)-Fisiología Vegetal, Colegio de Postgraduados, Campus Montecillo, Km. 36.5 Carretera Federal México-Texcoco, Montecillo, Texcoco, Estado de México. 56230. Tel (01) (595) 95 20200 Ext. 1542. , Colegio de Postgraduados, Postgrado en Recursos Genéticos y Productividad (PREGEP)-Fisiología Vegetal, Colegio de Postgraduados,
<city>Texcoco</city>
<state>Estado de México</state>
<postal-code>56230</postal-code>
, Mexico ,
3. PREGEP-Genética, Colegio de Postgraduados, Campus Montecillo., Colegio de Postgraduados, PREGEP-Genética, Colegio de Postgraduados, Mexico

Correspondence: *. Corresponding Author: Sandra Eloísa Rangel-Estrada, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias-Campo Experimental Zacatepec. Carretera Zacatepec Galeana s/n, Centro, 62780, Zacatepec de Hidalgo, Morelos. Tel (01) (800) 0882222 Ext. 86608. Email: E-mail:


Abstract

Anthurium schlechtendalii Kunth subsp. Schlechtendalii is a large sized anthurium with lustrous leaves which provides a high horticultural potential as an ornamental pot plant for interiors or landscape. Development of efficient propagation systems to avoid the extraction of their natural environment and use it commercially is required. The objectives of this research were to define the optimal conditions for in vitro seeds germination, to determine their viability and to establish plant regeneration via organogenesis from micro-cuttings of in vitro germinated seedlings. In vitro seed germination was of 96.0-98.8% in MS medium with 50% concentration of salts. Seeds viability was maintained for a maximum period of five months at 10 °C although it was drastically reduced after first month. Organogenesis was achieved in MS medium added with 7.5 μM BAP and 1 μM IAA where 16.3 shoots per explant were induced after eight weeks. Shoots multiplication was obtained at the same combination of plant growth regulators. Plants elongation and rooting was achieved in MS medium both at 50 and 100 % concentrations of salts added with 1 μM IBA. Plants acclimatization was efficient in peat moss + perlite (1:1) with a survival rate of 95 % after six weeks in greenhouse conditions.

Received: 2018 March 23; Accepted: 2018 July 25

revbio. 2018 Dec 18; 5(spe2): e475
doi: 10.15741/revbio.05.nesp.e475

Keywords: Key words: Anthurium schlechtendalii, micropropagation, anthurium, epiphyte, wild, native.

Introduction

Araceae family includes around 110 genera and 4,700 species distributed into tropical, subtropical and temperate regions of the Northern hemisphere of all continents. In Mexico, this family is represented by 13 genera and 109 species, between native and naturalized; moreover, 5 genera and 7 species have been introduced (Mayo et al., 1997; Croat & Acebey, 2015). The edible use of leaves, corms, mature fruits, soft spadix and seeds has been reported for some species. Its use as forage, medicinal, building material, fibres for basket making, handicrafts elaboration, packaging and/or as insecticide and pesticide has also been reported. However, the most important use of this botanical family is as ornamental plants (Croat &Acebey, 2015) where the most important genera are Aglaonema, Anthurium, Caladium, Colocasia, Dieffenbachia, Monstera, Philodendron, Spathiphyllum, Syngonium, Xanthosoma and Zantedeschia (Mayo et al., 1997).

Plants from the Anthurium Schott genus are characterized by being epiphytes, sometimes terrestrial or rupicolous; its stems present from short to extended internodes and simple leaves. The number of species originating from neotropical regions has been estimated between 1000 and 1300 (Croat & Acebey, 2015). The main use of Anthurium species is as ornamental plants for cut flowers and cut foliages or pot plant. Since last century the commercial cultivars dominating the market have been obtained by plant breeding between A. andraeanum Linden ex André and A. scherzerianum Schott (Kamemoto & Kuehnle, 1996; Kuehnle et al., 2001; Teixeira da Silva et al., 2015).

In Mexico, the diversity of Anthurium species is outstanding since between 41 and 48 species have been reported, most with a wide ornamental potential. Anthurium schlechtendalii Kunth subsp. Schlechtendalii is a wild species, distributed from Mexico to Nicaragua. They are characterized for being epiphytes plants with erected leaves in rosette and obovate and elliptic blades, measuring 30-112 cm long and 10-58 cm wide. The inflorescence is patent shorter than leaves; the spathe is green to nuanced-purple-violet green; the spadix is green and tapered. Infructescences are arched and pendant; the spathe is persistent and brown; berries are glossy and red (Croat, 1983; Croat & Acebey, 2015). The large size and brightness of the leaves are the main attributes that confer value to this variety as an ornamental pot plant for the decoration of interiors or landscape.

Incorporating this phytogenetic resource to the agricultural production at a commercial scale is limited by the way of propagation. It is mainly carried out from seeds, but they can be obtained only once a year and, based on producers’ opinion, they present viability issues. Moreover, the division of plants is not a method effective enough since plants have to reach at least five years to produce shoots. These restrictions require the development of effective propagation systems that favor the constant production of vegetative material along the year. In this sense, the current research aimed to determine optimal conditions for in vitro seed germination of Anthurium schlechtendalii subsp. schlechtendalii, to assess its viability and to develop a protocol for regeneration via organogenesis from in vitro germinated seedlings.

Materials and Methods

Source of seeds and disinfection. Seeds were extracted from mature infructescences collected from wild plants of the high evergreen forest in of the Sierra of Tabasco state, Mexico (Figure 1a-b). Seeds were washed with a commercial detergent for 5 minutes to remove the mucilage and were rinsed three times with tap water and then sterile distilled water. Then, they were submerged in a commercial sodium hypochlorite solution (NaOCl, Cloralex®; 40 % v/v) and Tween-20® (Sigma-Aldrich, 0.5 % v/v) for 15 minutes and were washed five times with sterile distilled water. Finally, seeds were immersed in Captan (N-triclorometiltio4-ciclohexeno-1,2 dicarboximida, 4 g L-1) for 10 minutes and were rinsed with sterile distilled water.


[Figure ID: f1] Figure 1.

In vitro seed germination and organogenesis of Anthurium schlechtendalii subsp. schlechtendalii. a) Adult plant; b) Infrutescence; c) Emergence of the radicle four days after sowing, d) Growth of first leaf and root after two weeks and e) Seedlings of eight weeks old obtained in half strength MS medium added with 20 g L-1 of sucrose; f) Shoots multiplication after eight weeks and g) Second cycle of multiplication with 7.5 μM of BAP and 1 μM of IAA; h) Plant elongation and rooting; i) Acclimatization of plants after six weeks.


Culture medium and incubation conditions. The culture medium was composed of the complete salts of the MS medium (Murashige & Skoog, 1962), added with sucrose (30 g L-1), myo-inositol (100 mg L-1), thiamine (0.1 mg L-1), pyridoxine (0.5 mg L-1), glycine (2 mg L-1), nicotinic acid (0.5 mg L-1) and solidified with agar-agar (Merck®, 7 g L-1). The pH of the culture medium was adjusted to 5.7 by means of a pH-meter (Hanna® HI 3220) with NaOH or HCl 1N and the culture medium was sterilized in a vertical autoclave (FELISA®, FE-405) at 121 °C and under 1.5 kg cm-2 of pressure for 20 min. Cultures were incubated at 25 ± 2 °C, with a 16/8 light/dark photoperiod and a luminous intensity of 45 µmol m-2 s-1.

In vitro seed germination and viability

Seeds were placed in the MS medium with 50 or 100% concentration of mineral salts and added with 10, 20 or 30 g L-1 of sucrose. Thirty mL of the culture medium were poured in 90 mL glass flasks. After four weeks, the percentage of germinated seeds, plant height (cm) and rooting (%) were measured. The experiment was established in completely randomized design with 25 repetitions per treatment and the experimental unit was 10 seeds placed in each culture glass flask. For viability tests, seeds were placed in paper envelops and stored at room temperature (25 ± 1 °C) and kept refrigerated (10 °C) for six months. Batches of 300 seeds were planted every month, divided into groups of 25 in each 90 mL glass flasks with 30 mL of the best response culture medium obtained in the germination phase.

Regeneration via organogénesis

Shoots multiplication. Dissected micro-cutting of 8 weeks old of in vitro germinated seedlings, with an average length of 1.5 cm and devoid of roots and leaves, were planted in MS medium added with four concentrations of 6-bencylaminopurine (BAP: 2.5, 5, 7.5 and 10 µM), alone or combined with indoleacetic acid (IAA: 1µM). After eight weeks, the number of explants that generated shoots (to obtain % of shooting), the number of shoots per explant and shoots length (cm) were quantified. A completely randomized design was used; each treatment was represented by 25 repetitions and the experimental unit was five seedlings per culture glass flask. To increase the number of plants, shoots of 2 cm length were selected and established in the best combination of BAP and IAA resulting from the previously assessed treatments. Two culture cycles of 8 weeks each were made and the number of shoots per explant was quantified at the end of each culture cycle.

Plants elongation and rooting. Shoots 3 cm in length were cultivated in MS medium with 50 or 100% concentration of mineral salts, added with 1 μM of indolebutyric acid (IBA) and without plant growth regulators. After four weeks, plant height (cm), rooting (%) and the number of roots were quantified. The experimental design was completely randomized where each treatment was represented by 25 repetitions and the experimental unit was five plants per culture glass flask.

Plants acclimatization. At this stage, 300 rooted plants of 7 cm height were planted in peat moss and perlite substrates (1:1) (Atak & Çelik, 2012) in 3 inches plastic pots and covered with a transparent dome. Plants were maintained in greenhouse conditions for 4 weeks at a temperature of 26 °C and watering with 50% concentration of mineral salts from the MS culture medium every third day. At the end of this period of time, the protector dome was removed and after 2 weeks the survival rate was quantified.

Statistical analysis

Data obtained in each experiment were submitted to analysis of variance with the statistic software SAS (SAS Institute, 2003). Values in percentage were squared root and the Tukey test (p ≤ 0.05) was performed to compare means.

Results and Discussion

In vitro seeds germination and viability

Treatments of culture medium and sucrose concentration showed a similar response in the germination and its effect was significant only on seedlings height and number of roots (p ≤ 0.05). The higher germination percentage (98.8%) occurred in the MS medium with 50% concentration of salts added with 20% of sucrose. Seedlings growth was higher in the MS medium with 100% concentration of salts added with 20 and 30% of sucrose. Rooting was present in every treatment, although the higher number of roots was obtainedreached with 20% sucrose in MS medium with 50% concentration of salts (Table 1).

Table 1.

In vitro seed germination and seedling growth of Anthurium schlechtendalii subsp. schlechtendalii after four weeks of culture.


MS (%) + sucrose (g L-1) Germination (%) Plant height (cm) Rooting (%) Roots (Num.)
50 + 10 97.2a 1.2d 100a 1.7c
50 + 20 98.8a 1.5cd 100a 1.8c
50 + 30 97.2a 1.7c 100a 1.9bc
100 + 10 98.0a 2.2b 100a 2.2ab
100 + 20 96.4a 2.8a 100a 2.4a
100 + 30 96.8a 3.1a 100a 2.3ab
MSD 1.9 0.3 0 0.4

TFN1Averages with equal letters within each column are not statistically different (Tukey, 0.05). MSD= Minimum significant difference. MS = Murashige y Skoog.


Germination started on the third day and ended after a week in all of the treatments (Figure 1c). After 2 weeks, the growth of the first leaf was observed and after 4 weeks, seedlings with leaves and roots were observed (Figure 1d-e). In wild species where the conservation is prioritized and where a clonal reproduction is not an objective, sexual reproduction is the most effective propagation method, because genetic variability is maintained and used. However, reproduction of Anthurium schlechtendalii by means of seeds in its natural habitat is complicated due to the need of birds for consuming fruits and scatter their seeds. In addition, because it is an epiphyte species, the seeds must be deposited and adhered on tree barks or rock crevices so they can germinate and grow.

The source of carbohydrates in the culture medium is indispensable for cells, tissues and organs explants because they are not completely autotrophic. However, in in vitro germination of seeds, sucrose effectiveness is associated to seeds maturity degree and genotype, as it has been reported in orchids (Deb & Pongener, 2011; Johnson et al., 2011; Udomdee et al., 2014). Normally sucrose is added because it contributes to maintain the osmotic potential and supplies a source of vital source of energy for the development of morphogenic processes (Yaseen et al., 2013).

In Anthurium schlechtendalii, sucrose concentrations of 10, 20 and 30 g L-1 did not have a significant effect on in vitro germination and a significant effect on seedlings growth. This response is due to the fact that the seeds used the reserve substances (lipids, proteins and starch) which allowed them to germinate. However, for plants to keep on growing, a source of carbohydrate and nutrients was necessary, in this case 30 g L-1 of sucrose from the MS medium with 100% concentration of salts. The growth of Anthurium schlechtendalii seedlings has to be vigorous for them to posteriorly serve as a source of explants in regeneration processes.

In literature, there are few scientific reports where seeds germination conditions of Anthurium wild species have been determined for purposes of conservation and commercial use. In all cases, MS medium with 50% concentration of salts favored germination with variations in sucrose concentration. In A. parvispathum Hemsl the germination was viable after six weeks, although sucrose concentration was not specified (Atta-Alla et al., 1998). In A. affine Schott germination was obtained too by adding 30 g L-1 of sucrose after four weeks of culture (Oliveira De Freitas et al., 2010). In A. antioquiense Engl, a threatened species, 20 g L-1 of sucrose favored germination after two weeks of culture (Murillo-Gómez et al., 2014); while in A. andreanum cv. Rubrun it was efficient with 30 g L-1 of sucrose (Maira et al., 2010). In the present study, germination was favored by both concentrations of salts of MS medium, although seedlings grew better with 100% concentration of salts and 20 and 30 g L-1 of sucrose.

Seeds viability

Six-months-stored seeds showed distinct responses in its capacity to germinate when they were planted in 50% concentration of salts of MS medium added with 20 g L-1 of sucrose. Independently from temperature storage, viability presented a reduction, consistent over time, until being null after five months. Stored seeds at 10 °C showed germination values slightly superior to those stored at room temperature; however, they maintained the same downward trend (Table 2). This viability response is the main limiting factor for Anthurium schlechtendalii subsp. Schlechtendalii propagation since its seeds can only be efficiently used during the first month of harvests. These results confirm what producers exposed about limited viability of this species.

Table 2.

Seed viability of Anthurium schlechtendalii subsp. schlechtendalii (%) during six months of storage.


Temperature Month 0 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6
10 ºC 98.8 81.3 52.3 27.7 9.3 2.3 0
25 ºC 98.8 76.3 49.7 23.3 5.7 0 0

This is the first study where seeds viability of a Anthurium species is reported. Viability loss in Anthurium schlechtendalii subsp. Schlechtendalii might be related to its tropical origin, since the seeds of many species growing in these climates showed a low tolerance to desiccation (recalcitrance), making its preservation difficult under conventional methods for germplasm conservation (Berjak & Pammenter, 2008; Bewley et al., 2013). Results of viability tests suggest the need for implementing new studies on conservation strategies for this kind of phytogenetic resource.

Regeneration via organogenesis.

Shoots multiplication. All concentrations of BAP, alone or combined with IAA, induced s formation in explants (100%); however, the higher number of shoots per explant (16.3) and shoots length (4.0 mm) were promoted by the combination of 7.5 µM of BAP and 1 µM of IAA (P ≤ 0.05) (Table 3). BAP concentrations also promoted the formation of calluses with a compact appearance at the base of the explants; its size increased as the dose in the culture medium increased, but it did not result morphogenic.

Table 3.

In vitro shoots multiplication in micro-cuttings of Anthurium schlechtendalii subsp. schlechtendalii cultured with benzylaminopurine (BAP) and idolacetic acid (IAA) after eight weeks.


BAP (µM) IAA (µM) Explants with shoots (%) Shoots per explant (Num.) Shoot length (cm)
2.5 - 100a 6.1f 1.9
5.0 - 100a 7.3ef 2.1cd
7.5 - 100a 9.2cd 2.4bc
10.0 - 100a 6.9f 2.3bc
2.5 1 100a 8.5de 2.0cd
5.0 1 100a 9.7bc 2.7b
7.5 1 100a 16.3a 4.0a
10.0 1 100a 10.9b 2.7b
MSD 0 1.2 0.18

TFN2Averages with equal letters within each column are not statistically different (Tukey, 0.05). MSD= Minimum significant difference.


The use of seeds as explants results important when studies focused on the rescue or the conservation of a phytogenetic resource of ecological importance (threatened or endangered species). It is also valuable when the purpose is the sustainable commercial use of some species without affecting natural populations.

Studies on in vitro germination of Anthurium species are limited in literature; the common objective in the studied cases is to obtain explants to establish regeneration via organogenesis. In the current study, the purpose of obtaining in vitro A. schlechtendalii seedlings was to get explants which would be assessed in the regeneration via organogenesis from micro-cuttings.

In A. parvispathum, shoots regeneration was induced from explants of dissected shoots apexes of seedlings obtained from seeds; MS medium with 50% concentration of salts supplemented with 8.9 µM of BAP and 1.1 µM of NAA produced the higher quantity of shoots per explant (4.1) after six weeks of culture (Atta-Alla et al., 1998). In the case of A. antioquiense, Murillo-Gómez et al. (2014) induced the shoots formation using seedlings obtained from in vitro germinated seeds as explants; the best response of shoots per explant (23.2) was obtained in the MS medium with 50% concentration of salts added with 4.4 µM of BAP after four weeks of culture.

Results obtained in A. parvispathum and A. antioquiense differed from those achieved in the present research with A. schlechtendalii where the best organogenetic response (16.3 shoots per explant) was induced with 7.5 µM of BAP and 1 µM of IAA in 100% concentration of salts of MS medium (Figure 1f). Regarding the quantity of shoots produced per explant, it was also appreciated that it was different and strongly defined by genotype. In A. andraeanum, differences in morphogenetic response have be proved to be present at the level of cultivars, that is the reason why more than a hundred of specific protocols for in vitro regeneration have been developed in this species to obtain the clonal propagation of commercial cultivars (Teixeira da Silva et al., 2015).

In wild species of Anthurium, research on in vitro propagation is insufficient and more studies are required to allow defining specific conditions of culture medium and optimal plant growth regulators since it was not possible to propose a standard methodology for the species of this genus.

To increase the quantity of A. schlechtendalii plants, two culture cycles of eight weeks each were performed in MS medium added with 7.5 µM of BAP and 1 µM of IAA. With this combination of plant growth regulators a multiplication rate similar to the obtained in initial explants of seedlings micro-cutting was maintained. Cultivated shoots produced in average 17.3 shoots in the first cycle (Figure 1g) and 15.6 in the second.

In A. schlechtendalii, as well as in others wild species of Anthurium, plants regeneration from seedlings micro-cutting represents a great advantage compared to commercial cultivars developed in A. andraeanum. In species of wild Anthurium, benefit is taken from genetic diversity, while in cultivars of A. andraeanum, clonal multiplication systems are required to guarantee genetic stability of agronomic traits of interest (George & Debergh, 2008). In addition many of these cultivars have lost the capacity to produce seeds.

Plants elongation and rooting. Rooting was induced in all of the treatments of culture medium; however, plant height was higher with 100% concentration of salts of MS medium; the number of roots was not significant, but the response was slightly higher in the MS medium with 50% concentration of salts added with 1 μM of IBA (p ≤ 0.05; Table 4; Figure 1h). Rooting initiated after the first week of culture and at the fourth week, plants showed a complex system of roots in all the treatments.

Table 4.

Height and rooting of plants of Anthurium schlechtendalii subsp. schlechtendalii in MS medium added with indolebutyric acid (IBA) after four weeks of culture.


MS (%) IBA (µM) Plant height (cm) Rooting (%) Roots (Num.)
50 - 5.4b 100a 5.0a
50 1 5.7b 100a 5.1a
100 - 7.0a 100a 4.8a
100 1 7.3a 100a 4.9a
MSD 0.7 0 0.9

TFN3Averages with equal letters within each column are not statistically different (Tukey, 0.05). MSD= Minimum significant difference. MS = Murashige y Skoog.


Results of rooting indicated that A. schlechtendalii showed capacity for regenerating roots in MS medium even without addition of auxins. This response also has been commonly observed in regeneration systems of cultivars of A. andraeanum plants (Teixeira da Silva et al., 2015). For the phylogenetic relationship between the species of the Anthurium genus, rooting responses obtained in A. schlechtendalii were similar to those in A. andraeanum.

Plants acclimatization.

Plants survival rate in the process of acclimatization was 98% in peat moss and perlite (1:1) substrate after six weeks (Figure 1i). In literature, it has been reported that this stage of the in vitro propagation process of Anthurium, and specifically of A. andraeanum, can be performed on various substrates as mixtures of vermicompost and sand (1:3), vermiculite and perlite (1:1), soil and organic humus (1:1) with survival rates of 60% to 98% (Atak y Çelik, 2012). The mixture of peat moss and perlite (1:1) resulted efficient for the acclimatization of 95% of the plants of A. schlechtendalii. Nevertheless, changing plants to another substrate allowing a better airing of roots is necessary for plants to keep growing, considering it is a species presenting epiphyte growing habits.

Conclusion

A protocol for in vitro propagation of Anthurium schlechtendalii subsp. schlechtendalii. was developed. In vitro germination is feasible in MS medium with 50 and 100% concentration of salts. Seeds viability can be maintain for five months maximum. Regeneration via organogenesis is established from micro-cutting of in vitro germinated seedlings. Shoots induction and multiplication was achieved with 7.5 µM of BAP and 1 µM of IAA. Plants elongation and rooting was obtained in MS medium with 50 and 100% concentration of salts and 1 µM of IBA. Survival rate of acclimatized plants (95%) was obtained in a mixture of peat moss and perlite (1:1) after six weeks of culture in greenhouse conditions.


fn1Cite this paper: Rangel-Estrada, S. E., Hernández-Meneses, E., Canul-Kú, J., Barrios-Gómez, E. J., López-Peralta, M. C. G., Tapia-Jaramillo, L. (2018). In vitro germination, seed viability and organogenesis of Anthurium schlechtendalii Kunth subsp. schlechtendalii. Revista Bio Ciencias 5(nesp), e475. doi: https://doi.org/10.15741/revbio.05.nesp.e475

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