Morphogenic responses of three explants of Lupinus montanus (H.B.K.) cultured in vitro

 

Respuestas morfogénicas de tres explantes de Lupinus montanus (H.B.K.) cultivados in vitro

 

Gabriel Ramírez-González¹*; José L. Rodríguez-de la O²; Jesús G. Arreola-Ávila³; José G. Álvarez-Moctezuma^1†

 

¹ División de Ciencias Forestales, Universidad Autónoma Chapingo. km 38.5 Carretera México-Texcoco. C. P. 56230. Chapingo, Texcoco, Estado de México. MÉXICO. Correo-e: arzemir@gmail.com, tel.: 01 (55) 24531364 (*Autor para correspondencia).

² Departamento de Fitotecnia, Universidad Autónoma Chapingo. km 38.5 Carretera México-Texcoco. C. P. 56230. Chapingo, Texcoco, Estado de México. MÉXICO.

³ Unidad Regional Universitaria de Zonas Áridas, Universidad Autónoma Chapingo. km 38.5 carretera Gómez Palacio-Cd. Juárez. C. P. 35230. Bermejillo, Durango, MÉXICO.

 

Received: July 11, 2013.
Accepted: November 28, 2014.

 

ABSTRACT

Necrosis and morphogenic response of cotyledon, epicotyl and hypocotyl explants of Lupinus montanus (H.B.K.) seedlings germinated in vitro were evaluated to establish the basic conditions of micropropagation. Necrosis was evaluated in MS medium with 0.40 mg·liter^-1 thiamine, 100 mg·liter^-1 myo-inositol, 3 % sucrose and 7 g·liter¹ agar-agar and different doses of activated carbon (AC) and citric acid. Treatments with AC (50 mg·liter^-1 and 100 mg·liter^-1) were the best in the control of necrosis (P = 0.001); the cotyledon had a higher level of necrosis (< 60 %) than the epicotyl and hypocotyl (10 to 30 %). Morphogenic responses were analyzed on MS medium with 0.40 mg·liter^-1 thiamine, 100 mg·liter^-1 myoinositol, 3 % sucrose, 7 g·liter^-1 agar-agar, 100 mg·liter^-1 AC and five combinations of IAA (indole acetic acid) and BA (6-benzyladenine). The hypocotyl and epicotyl explants showed higher organogenic response (> 70 %), whereas the cotyledon response was primarily callogenic (50 %). The epicotyl cultured on MS medium with 3.0 μM IAA and 1.0 μM BA had the highest number of shoots (10) and height thereof (11.4 ±2.6 cm).

Keywords: Shoots, callus, necrosis, morphogenesis.

 

RESUMEN

El necrosamiento y la respuesta morfogénica de explantes de cotiledón, epicótilo e hipocótilo de plántulas de Lupinus montanus (H.B.K.) germinadas in vitro se evaluaron para establecer las condiciones básicas de micropropagación. El necrosamiento se evaluó en medio MS con 0.40 mg·litro^-1 de tiamina, 100 mg·litro^-1 de mioinositol, 3 % de sacarosa y 7 g·litro^-1 de agar-agar y distintas dosis de carbón activado (CA) y ácido cítrico. Los tratamientos con CA (50 mg·litro^-1 y 100 mg·litro^-1) fueron los mejores en el control del necrosamiento (P = 0.001); el cotiledón tuvo mayor nivel de necrosamiento (< 60 %) que el epicótilo e hipocótilo (entre 10 y 30 %). Las respuestas morfogénicas se analizaron en medio MS con 0.40 mg·litro^-1 de tiamina, 100 mg·litro^-1 de mioinositol, 3 % de sacarosa, 7 g·litro^-1 de agar-agar, 100 mg·litro^-1 de CA y cinco combinaciones de AIA (ácido indolacético) y BA (6-benciladenina). Los explantes de hipocótilo y epicótilo mostraron mayor respuesta organogénica (> 70 %) a diferencia del cotiledón, cuya respuesta fue primordialmente callogénica (50 %). El epicótilo cultivado en medio MS con 3.0 μM de AIA y 1.0 μM de BA tuvo el mayor número de brotes (10) y altura (11.4 ± 2.6 cm).

Palabras clave: Brotes, callo, necrosamiento, morfogénesis.

 

INTRODUCTION

Several recent studies have identified various potential applications arising from the cultivation and use of plants of the genus Lupinus, including agrochemicals (insecticides) (Bermúdez, Martínez, Figueroa, Legal, & Wink, 2009), food (protein isolates) (Lee et al., 2006) and even medicines (antimicrobial and hypoglycemic drugs) (Dove et al., 2011). In all the cases above, one of the major aims is to achieve a production system that ensures obtaining plants with specific characteristics based on the needs of the final product; for example, increased production of foliage for the extraction of alkaloids and seed production for obtaining protein isolates.

Currently, somatic clones can be obtained and whole plants regenerated with uniform characteristics through tissue culture techniques. As a result, valuable microorganism-free plant cultivars that are difficult to obtain by traditional farming methods can be established. However, establishing a protocol for large-scale micropropagation requires basic knowledge of the behavior of tissues cultured in vitro. Consequently, the objectives of this study were to determine the degree of necrosis in cotyledon, epicotyl and hypocotyl explants of Lupinus montanus (H.B.K.) seedlings under different concentrations of antioxidants (activated carbon and citric acid), and evaluate the morphogenic response under different concentrations of plant growth regulators (indole acetic acid and 6-benzyladenine).

 

MATERIALS AND METHODS

Plant material

The research was conducted in the Plant Tissue Culture laboratory belonging to the Department of Plant Science of the Universidad Autonoma Chapingo. As a source of explants, four- and six-week-old L. montanus seedlings, obtained by in vitro seed germination, were used. The seeds were obtained from plants collected in 2011 on Xipes Hill (19° 00' 48'' NL, 97° 21' 20'' WL), municipality of Libres, Puebla. The plants were identified using taxonomic keys and descriptions of Dunn (1979), were validated and subsequently deposited in the National Herbarium of Mexico (MEXU) of the Institute of Biology of the National Autonomous University of Mexico.

Germination of L. montanus seedlings

One hundred viable seeds with uniform morphological characteristics (shape, size, weight and color) were selected. Subsequently a random sample of 30 seeds was collected and subjected to a disinfection process consisting of a wash with Foca^® detergent and Tween 80® (Thermo Scientific, USA) for 2 min, followed by immersion in 70 % alcohol for 3 min and a second wash in 10 % sodium hypochlorite for 15 min, following the procedure described by Mroginski, Sansberro, and Flaschland (2010). Seeds were mechanically scarified by a lateral cut in the head, in order to break the physical latency due to its impermeability (Corona, Gómez, & Linding, 2013). The seeds were sown on MS basic medium (Murashigue and Skoog, 1962) supplemented with 0.40 g·liter^-1 thiamine, 100 g·liter^-1 myo-inositol, 3 % sucrose and 7 g·liter^-1 agar-agar, and incubated at 25 ± 1 °C and a photoperiod of 16:8 h (light/dark) for four weeks.

Evaluation of antioxidants on necrosis in L. montanus explants

After four weeks of growth, six L. montanus seedlings were harvested at random. Regions to be used as an explant source, namely the hypocotyl, cotyledon and epicotyl, were obtained from each seedling. These regions were sectioned into portions of approximately 3 x 3 x 2 mm and distributed (depending on the type of explant) randomly into five treatments with 12 replications, incubated in a photoperiod of 16:8 h (light/dark) for three weeks. Treatments consisted of the application of antioxidants in different doses: T0 = Control (no antioxidants), T1 = citric acid (CA, 100 mg·liter^-1), T2 = CA (150 mg·liter^-1), T3 = activated carbon (AC, 50 mg·liter^-1) and T4 = AC (100 mg·liter^-1). Antioxidants were applied on MS medium supplemented with 0.40 mg·liter^-1 thiamine, 100 mg·liter^-1 myo-inositol, 3 % sucrose and 7 g·liter^-1 agar-agar, at pH 5.7 ± 0.1. The antioxidant effect of each treatment was evaluated on the tissue of the explants using a visual scale with values from 1 to 3:1 = low level of necrosis (< 30 %), 2 = medium level of necrosis (≥ 30 % < 60 %) and 3 = high level of necrosis (≥ 60 %). The results were obtained using a stereomicroscope (Leica^® EZ4 [20X objective], Germany).

Morphogenesis induction in L. montanus explants by plant growth regulators

Morphogenesis induction was evaluated in 12 six-week-old seedlings harvested at random. The regions to be used as the explant source (hypocotyl, cotyledon and epicotyl) were cut into sections of approximately 3 x 3 x 2 mm. Subsequently, the explants with 10 replications were seeded on MS medium supplemented with 0.40 mg·liter^-1 thiamine, 100 mg·liter^-1 myo-inositol, 3 % sucrose, 100 mg·liter^-1 AC, 7 g·liter^-1 agar-agar and different concentrations of IAA (indole acetic acid) and BA (6-benzyladenine) as plant growth regulators (PGR). Treatments were as follows: T0 = Control (no PGR), T1 = IAA (0.1 μM) and BA (0.1 μM), T2 = IAA (0.1 μM) and BA (0.3 μM), T3 = IAA (0.3 μM) and BA (0.1 μM), T4 = IAA (1.0 μM) and BA (3.0 μM) and T5 = IAA (3.0 μM) and BA (1.0 μm).

Morphogenic responses of the explants were evaluated after a four-week incubation period under a light/dark period (16:8 h) at 25 ± 1 °C. The type of morphogenic response was evaluated based on a qualitative scale of 0 to 2:0 = explants that showed no response, 1 = response in callus formation, 2 = response in shoot formation. The number of shoots and their height were also evaluated.

Statistical analysis

Necrosis and morphogenic response were evaluated in a completely randomized design. Data were analyzed using the Kruskal-Wallis test (P ≤ 0.05). The number of shoots and their height were evaluated in a completely randomized design with a factorial arrangement, using the following model:

Y_ijk= μ + A_i + B_j + (AB)_ij + ξ_ijk

Where:

Y_ijk= Response in number of shoots or height

μ = Overall mean

A_i = Effect of the type of explant (hypocotyl, cotyledon and epicotyl)

B_j = Effect of the treatment (combinations of IAA: BA)

AB_ij= Interaction of the type of explant with the treatment

ξ_ijk = Experimental error

Data were subjected to an analysis of variance and Tukey's comparison of means (P = 0.05). All the analyses were performed with the SYSTAT V.13.1 statistical package (2009).

 

RESULTS AND DISCUSSION

Effect of antioxidants on the necrosis level of L. montanus explants

The explants evaluated three weeks after being planted showed different degrees of necrosis. In Figure 1 it can be seen that the highest necrosis percentage occurred in the control treatment (no antioxidants). In this treatment, the level of necrosis in the hypocotyl, cotyledon and epicotyl was high (≥ 60 %). The necrosis level in the treatments with CA at concentrations of 100 mg·liter^-1 and 150 mg·liter^-1 was also high (≥ 60 %). On the other hand, the treatments containing 50 mg·liter^-1 and 100 mg·liter^-1 AC induced a significant reduction (P = 0.05) in the oxidation level of the three explants cultured; the necrosis level in the hypocotyl and epicotyl was low (< 30 %).

The Kruskal-Wallis test (P = 0.05) showed the difference in the effect of at least one treatment in controlling necrosis in the explant (X²₍₄₎= 64.368 and P < 0.001). Table 1 presents the results of the pairwise comparison test. This test allowed determining the treatments with statistically different effects in controlling oxidation.

According to Table 1, the best treatments to control necrosis in the explants were those containing AC. This result agrees with the findings of Azofeifa (2009), who reported the use of AC in culture media for woody species, due to the antioxidant role of AC in the absorption of substances exuded by the explant, which kill the tissue cells by making the medium toxic. Similarly, Phoplonker and Caligari (1993) found that the addition of AC in the culture medium decreased the level of necrosis in explants of L. mutabilis Sweet.

Regarding the relationship between the degree of necrosis and the type of explant cultured, it was observed that the cotyledon explants had higher levels of necrosis (P < 0.001) than the hypocotyl and epicotyl explants (P=0.057). In other words, the degree of necrosis in the cotyledon under the evaluated antioxidants is associated with both the culture medium and the explant itself. This could be related to the findings reported by some authors with respect to the presence of a large amount of secondary metabolites (alkaloids, phenols and terpenes) in the cotyledons of the seeds of L. mexicanus Cerv. ex. Lag. (Zamora-Natera, García-López, Ruiz-López, & Salcedo-Pérez, 2008) and L. mutabilis (Ortega-David, Rodríguez, David, & Zamora-Burbano, 2010). Cotyledons, when subjected to the stress of cutting for in vitro planting, release these metabolites into the culture medium, adding to the increased production of reactive oxygen species, resulting in increased necrosis of the explant (Arauz, 1998).

Evaluation of the morphogenic response of L. montanus

In the control treatment and the treatment with the combination of 0.1 μΜ IAA and 0.1 μΜ BA, no morphogenic response was obtained in the explants evaluated. In treatments where one of the two PGR (IAA or BA) exceeded the 0.1 μΜ concentration, there was a greater response in shoot formation (> 55 %) followed by the response in callus formation (< 24 %).

Effect of the L. montanus explant on the type of morphogenic response

Figure 2 shows that hypocotyl and epicotyl explants cultured under different combinations of IAA-BA (except T1 = 0.1 μΜ IAA and 0.1 μΜ BA) presented a greater organogenic response (72 to 80 %), while the cotyledon explants were divided into those with no response (40 %) and those with callus formation (> 50 %).

Callus formation. Results show that the cotyledon explants are mainly induced to callus formation (> 50 %) when combinations of IAA and BA are applied in a range of concentrations from 0.1 μΜ to 3 μΜ. In all cases, the calluses generated in this research showed a compact structure and a green coloration (Figure 3). This information is similar to that reported by Rivera et al. (2008), who were able to induce the greatest callus formation (45 %) of L. aschenbornii, S. Schauera from cotyledon explants, under a combination of auxin (2,4-dichlorophenoxyacetic acid) and cytokin (kinetin) at concentrations of 1 mg·liter^-1 (4.4 μΜ) and 0.5 mg·liter^-1 (2.1 μΜ) respectively.

Shoot formation. The cotyledon explant caused four cases of organogenesis; however, after four weeks, the shoots failed to differentiate clearly. Therefore, the response variable number of shoots was only evaluated for hypocotyl and epicotyl. Figure 4 shows shoot formation in these explants.

Number of shoots. Figure 5 shows the effects induced by the factors PGR and explant type. Their interaction had no statistical significance (P = 0.74), so the analysis of the factors was made in isolation. The analysis of variance showed that there was a significant difference (P = 0.05) in the effect of the PGR factor (IAA-BA) on the average value of the number of shoots. Tukey's test indicates that T5 (3.0 μΜ IAA and 1.0 μΜ BA) differs significantly from the other treatments by explant type (except for T4 = 1.0 μΜ IAA and 3.0 μΜ BA in hypocotyl, P = 0.617). These differences can be seen in Figure 5, which shows that the number of hypocotyl and epicotyl shoots (5.9 and 10.1, respectively) is greater in T5 than in the other treatments.

Figure 5 also shows that the explant factor generates different average values for the number of shoots (P < 0.001), with a higher response in the epicotyl explants, except for what occurs in T4 where the standard deviations of hypocotyl and epicotyl (± 2.4 and ± 2.8, respectively) overlap.

Shoot height. The effect of the PGR factor on the average value of shoot height was statistically different (P = 0.012). In Figure 6 it can be seen that the epicotyl shoots cultured under the environmental conditions of the T5 medium (3.0 μΜ IAA and 1.0 BA) had greater height (P = 0.05) compared to the other treatments; however, hypocotyl shoots under T5 showed no significant differences in height with those obtained with T4 (1.0 μΜ IAA and 3.0 μΜ BA).

Results obtained for shoot height in hypocotyl cultured in medium supplemented with 1.0 μΜ IAA and 3.0 BA, and 3.0 μΜ IAA and 1.0 BA contrast with those reported by Rodríguez, Hechevarría, Rodríguez, and Rivera (2003). These authors worked with herbaceous species such as Artemisia absinthium L. and found that the increase in the concentration of indoleacetic or indolebutyric auxin and the reduction in the concentration of BA generate greater shoot height than those treatments in which the BA concentration exceeds that of the auxin.

 

CONCLUSIONS

The best necrosis control (< 30 %) in explants cultured in vitro was obtained with the addition of activated carbon (50 mg·liter¹ and 100 mg·liter¹) to the culture medium. Cotyledon explants were more susceptible to the process of necrosis in all treatments evaluated. Regarding the type of morphogenic response, hypocotyl and epicotyl explants showed higher organogenic response, which may be of great use in scaling up the in vitro propagation of the species. At the explant level, the epicotyl shows significant differences in the number and height of shoots relative to the hypocotyl. At the level of plant growth regulators, the combination of IAA (1.0 μΜ) and BA (3.0 μΜ) and that of IAA (3.0 μΜ) and BA (1.0 μΜ) were the best media for inducing a greater number of shoots in the hypocotyl and epicotyl, while with IAA (3.0 μΜ) and BA (1.0 μΜ) the greatest height in epicotyl shoots was obtained. On the other hand, cotyledon explants showed a greater tendency toward callus formation in media containing plant growth regulators.

 

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