Artículo original                                              Biotecnología Vegetal Vol. 15, No. 3: 163 - 175, julio - septiembre, 2015

ISSN 2074-8647, RNPS: 2154 (Versión electrónica)

Instituto de Biotecnología de las Plantas. UCLV. MES.

 

 

Novel explant for somatic embryogenesis in Sorghum bicolor (L.) Mohen

 

Silvio Martínez Medina^1,2*, Raúl Collado², Raúl Barbón², Mayelin Rodríguez², Marta Pérez², Miguel Tejeda³, Luis Rojas, Dion D. Daniels⁴, Berkis Roque², Damaris Torres², Luís O. Maroto⁵, Amanda L. Vitlloch⁵, Rafael Gómez-Kosky² *Autor para correspondencia

 

¹Centro de Investigaciones Agropecuarias, Universidad Central Marta Abreu de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Cuba. CP 54 830. e-mail: silviod@uclv.edu.cu

 

²Instituto de Biotecnología de las Plantas, Universidad Central Marta Abreu de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Cuba. CP 54 830 e-mail: silvio@ibp.co.cu

 

³Biofábrica MINAGRI, Cienfuegos. Carretera a Palmira km 1.5, Cienfuegos, Cuba.

 

⁴University of Belize, Belmopan, Belize C.A.

 

⁵Departamento de Biología, Facultad de Ciencias Agropecuarias. Universidad Central Marta Abreu de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Cuba. CP 54 830

 

ABSTRACT

This work was carried out with the objective to form somatic embryos of sorghum, cv. 'CIAP 132R-05' starting from callus obtained from sections of in vitro shoots. For the formation of callus, different concentrations of 2,4-D were studied as well as three concentrations of ascorbic acid to eliminate the phenolic oxidation. To increase the percentage of callus formation with embryogenic structures, different segments of the shoots were used. For the formation of somatic embryos, different concentrations of 2,4-D; 6-BAP and L-Proline were added to the culture media. The greatest callus formation (50%) was obtained in the culture medium with 18.1 |jM of 2,4-D. When 50 mg l^-1 of ascorbic acid was added to the culture medium, the percentage of callus formation increased to 67.5%, and was couple with absence of oxidation of the medium and the explant. The frequency of callus formation with embryogenic structures increased to 95% with the use of segment 1 of the shoot sections in vitro as explant. The greatest number of somatic embryos per callus was obtained when 2,4-D was reduced to 4.52 jM, combined with 22.2 jM of 6-BAP and 500 mg l^-1 of L-Proline (41.88). Histological analysis confirmed that structures formed in callus came from a somatic embryogenesis process. For the first time, the efficient formation of somatic embryos of sorghum section 1 of in vitro shoots in vitro was obtained.

 

Keywords: 2,4-D, shoots in vitro, callus, somatic embryos

 

Nuevo explante para la embriogénesis somática en Sorghum bicolor (L.) Mohen

 

RESUMEN

El trabajo fue realizado con el objetivo de formar embriones somáticos de sorgo, variedad 'CIAP 132R-05' a partir de callos obtenidos de secciones de brotes in vitro. En la formación de callos fueron estudiadas diferentes concentraciones de 2,4 D y tres concentraciones de ácido ascórbico para eliminar la oxidación fenólica. Para incrementar el porcentaje de formación de callos con estructuras embriogénicas fueron empleados diferentes segmentos de brote. Se añadieron diferentes concentraciones de 2,4-D, 6-BAP y L- prolina al medio de cultivo para la formación de embriones somáticos. La mayor formación de callos (50%) se obtuvo en el medio de cultivo con 18.1 jM de 2,4-D. Cuando se añadieron 50 mg l^-1 de ácido ascórbico al medio de cultivo, el porcentaje de formación de callos se incrementó en 67.5%, unido a la ausencia de pigmentos fenólicos en el explante. La frecuencia de formación de callos con estructuras embriogénicas aumento hasta 95% con el uso del segmento 1 como explante. El mayor número de embriones somáticos por callos se obtuvo cuando 2,4-D fue reducido a 4.52 jM, combinado con 2.22 jM de 6-BAP y 500 mg l^-1 de L-prolina (41.88). El análisis histológico confirmó que las estructuras formadas a partir de los callos provienen del proceso de embriogénesis somática. Por vez primera se obtuvo la formación eficiente de embriones somáticos de sorgo de a partir de la seccion 1 de brotes in vitro.

 

Palabras clave: 2,4-D, brotes in vitro, callos, embriones somático

 

Abbreviations

CIAP    Centro de Investigaciones Agropecuarias

2,4-D    2,4- dichlorophenoxyacetic acid

6-BAP   6- benzylaminopurine

 

INTRODUCTION

 

Sorghum (Sorghum bicolor (L.) Mohen) is a plant with physiological C-4 metabolism that adapts well to an agro-ecological environment that is hot and dry, in which it is difficult to cultivate other cereals (Antonopoulou et al., 2008; Liu et al., 2009).

 

This species is a native Poaceae of the tropical and subtropical regions of Africa (Sudhakar et al., 2009). It is used for animal feed, human nutrition, fiber production and biocombustible ethanol (Rooney et al., 2007). Its cultivation is widespread, ranking fifth after wheat, rice, maize and barley (Zhao et al., 2010). However, its production is affected by several factors such as bird attacks, diseases, insect pests, the tannin content that affects the quality and availability of protein in the grain, as well as its poor amino acid composition. Thus, it requires of breeding programs to develop tolerant and resistant cultivar, capable of face adverse factors without affecting their yield. (Pérez et al. 2010).

 

The use of traditional methods of breeding is limited by various factors, among them: long selection periods with the utilization of morphological markers, the complexity of the trait to be improved and the influence of the environment. So that the availability of biotechnological techniques, including genetic transformation, will be very useful as a method of genetic improvement of the crop (Kumar et al. 2011).

 

The establishment of a plant regeneration system from somatic cells constitutes a prerequisite of extreme importance within the process of genetic transformation (Brandao et al., 2012). This way of regeneration constitutes a rapid method to produce a large number of plantlets (Baskaran et al., 2006).

 

Tomas et al. (1977) and Gambor et al. (1977) did the first works on the regeneration of plants via somatic embryogenesis in sorghum. Various types of explants have been used for the formation of callus such as: apical buds (Maheswari et al., 2006); immature zygotic embryos (Gupta et al., 2006); immature inflorescence (Jogeswar et al., 2007); mature zygotic embryos (Zhao et al., 2008; 2010). However, with these expiants, the frequency of callus formation with embryogenic structures has been low (Zhao et al., 2010). Besides, due to photoperiod sensitivity, most of these types of expiants are not available in sorghum all year (Kishore et al., 2006). It is known that one of the problems described in the different protocols for plant regeneration in sorghum is the production of phenolic compounds that can affect the formation of callus and thereafter obtaining plants, especially in the red-seed genotypes (Nguyen et al., 2007).

 

Even though plant regeneration protocols via somatic embryogenesis exist for Sorghum bicolor, these have low regeneration efficiencies and are dependent on the genotypes. These factors are fundamentally related with the type of explants used, the influence of the genotype and the occurrence of phenolic compounds that cause a low frequency of callus formation with em bryogenic structures, which all affect plant regeneration. Although genetic transformation have been achieved in sorghum, to date persist inefficiencies in calli with embryogenic structures formation, big amount of initial explants (7000 immature zygotic embryos) are needed for obtaining low (848 calli) callus formation (Wu et al., 2014). These limitations make sorghum one of the most recalcitrant species for in vitro culture and genetic transformation (Gupta et al., 2006; Maheshwari et al., 2006).

 

The objective of this work was to form somatic embryos from callus developed from sections of in vitro shoot sections of Sorghum bicolor cv. 'CIAP 132R'. This paper constitutes the first report on obtaining somatic embryos of sorghum using this explant.

 

MATERIALS AND METHODS

 

Plant material

 

Mature seeds of sorghum cv. 'CIAP 132R-05' (red sorghum) were used as starting material. Donor plants were grown under controlled conditions (shade houses), whose panicles from emission were covered with wax paper bags. To obtain in vitro shoots, seeds were disinfected and germinated according to the methodology proposed by Baskaran and Jayabalan (2005). In vitro shoots regenerated via direct organogenesis and thickened using the protocol described by Martínez et al. (2012) were used for callus formation.

 

Callus formation

 

Effect of 2,4-D

 

Different concentrations of 2,4-D (9.05, 18.1 and 27.14 jM) were evaluated for callus formation. Cross sections above the base of the in vitro shoot of 0.5 cm long, were used as explants for callus formation (Figure 1 A, B). The explants were placed in a culture medium of Murashige and Skoog (1962) (Duchefa) MS (4.32 g l^-1), MS vitamins, supplemented with 100 mg l^-1 myo-inositol, 50 mg l^-1 L-Proline, and 3% sucrose. As gelling agent in the different culture media used it was, 8.0 g l^-1 Agar (BIOCEN, Cuba). For the experiments, glass culture vessels 250 ml total capacity with plastic cap (polycarbonate) were used and 30 ml of culture media were added in each flask. The pH of the different culture media was adjusted to 5.7 with NaOH or HCl before autoclaving.

 

The plant material was placed in a growth chamber at 27 ± 2^oC and constant darkness. Visual observations were performed every seven days for the presence of phenolic compounds in the culture media and the explant. After 30 days of culture, the number of explants forming callus, expressed as a percentage of callus per culture vessel was assessed; and the number of explants phenolized.

 

Effect of ascorbic acid on the phenolic oxidation

 

To reduce phenolic oxidation during callus formation, different concentrations of ascorbic acid (20, 50 and 80 mg l^-1) in the culture media were evaluated. The culture medium was composed of MS salts, MS vitamins modified with a thiamine concentration of 0.4 mg l^-1, 100 mg l^-1 myo-inositol and the concentrations of 2,4-D that gave the best results in the experiment above was used as control.

 

Observations were made to determine the onset of callus formation, presence of dark pigments in the culture media as well as on the explant. After 30 days of culture the number of explants forming callus and expressed in percentage was evaluated.

 

Influence of different sections of in vitro shoots on callus formation

 

The experiment was conducted with the objective of increasing the percentage of callus formation. For this, different types of thickened shoot sections obtained over 0.3 mm diameter were studied, whose shoot was cut and divided into three sectios as described below and shown in Figure 1C: I) section 1 (unlike the explant used in the experiment where the effect of 2,4-D was studied in the formation of callus, this section contains a part of the base where the meristematic zone of growth is located); II) section 2 (above 1, a cauline section of about 0.5 cm long was taken; III) section 3 (above 2, a cauline section of about 0.5 cm long was taken).

 

The sections were placed on culture medium containing MS salts, MS vitamins modified with a concentration of thiamine 0.4 mg l^-1, supplemented with 100 mg l^-1 myo-inositol, with concentrations of 2,4-D and ascorbic acid determined in previous experiments. After 30 days of culture, the number of explants forming callus was evaluated and expressed in percentage.

 

Formation and differentiation of somatic embryos

 

Influence of 2,4-D and 6-BAP

 

To form and differentiate somatic embryos, light yellow, compact and friable calli with four multiplication subcultures were used. Calli fragments of approximately 0.5 cm in size were transferred to callus culture medium containing MS salts, MS vitamins modified with a thiamine concentration of 0.4 mg l^-1, supplemented with 100 mg l^-1 myo-inositol, and 20 mg l^-1 ascorbic acid. The effect of different concentrations of 2,4-D (2.26, 9.05 and 18.1 uM) and 6-BAP (1.11, 2.22 and 4.44 uM) in the formation and differentiation of somatic embryos was studied. As a control the culture medium described previously, free of growth regulators was used.

 

For all assessment of in vitro responses of shoot sections or calli on the various media tested, a total of ten replicated culture vessels with four explants each, i.e. 40 replicated shoot sections or callus pieces, were used. Observations were made on all treatments for the presence of somatic embryos. After 30 days of culture, the number of somatic embryos per callus was evaluated and the somatic embryos formed by developmental stage were classified.

 

Observations made during the formation and differentiation of the somatic embryos were done using a Leica brand (WILD M8 model, Germany) with 100x magnification and a manual counter stereomicroscope.

 

Effect of L-Proline

 

With the objective of increasing the number and quality of somatic embryos, L-Proline was added to the culture medium. The concentration of 2,4-D and 6-BAP that gave the greatest number of somatic embryos formed were used. Light yellow, compact, friable calli with embryogenic appearance were used. Callus fragments were transferred to MS medium with the best combination of growth regulators in the above experiment, and the effect of three concentrations of L-Proline (250, 500, 750 mg l^-1) was evaluated. As a control the culture medium described previously, free of L-Proline was used.

 

For all assessment of in vitro responses of shoot sections or calli on the various media tested, a total of ten replicated culture vessels with four explants each, i.e. 40 replicated shoot segments or callus pieces, were used. Observations were conducted to determine the presence of embryogenic structures in all treatments at 30 days of culture. The following variables were evaluated:

- number of somatic embryos per callus,

- stage of development of somatic embryos formed.

 

Histological analysis of callus with embryogenic estrutures

 

Histological analysis was performed in order to check whether structures formed in calli were derived from a somatic embryogenic process in this monocotyledonous plant species. For this analysis calli with embryogenic structures that came from a culture medium containing 4.52 uM 2,4-D, 2.22 uM 6-BAP and 500 mg l^-1 L-Proline were used. These were placed for 24 hours in a fixing solution containing 37% formaldehyde (v/v), glacial acetic acid 100% (v/ v) and ethyl alcohol 70% (v/v), in a 1:1:18 ratio. Progressive dehydration of the samples was performed in ethanol solutions of increasing concentrations starting with 70% up to the absolute ethyl ethanol and the inclusion was made into paraffin blocks (Kraus and Arduin, 1997). The cuts were made at 10 um with a rotary microtome HM 320 Heidelberg and subsequently stained with safranin 0.1% and toluidine blue 0.1% for the general examination of the tissue (Jensen, 1962). The samples were examined with a microscope (Olympus SZX7, USA) and photographed with a Canon camera

(Power Shot A 630).

 

Experimental design and data analysis

 

The experimental design was completely randomized. The statistical analysis of the experimental data was performed using the software package SPSS version 18.0 in Microsoft Windows operating system. In all experiments evaluated, the data obtained underwent assumption checks of normality and homogeneity of variance (Lindman, 1974). The experimental data that did not meet the above assumptions were processed using the nonparametric Kruskal-Wallis test. The comparison of means was performed according to Mann Whitney nonparametric test with a level of significance p < 0.05. Experimental data that met the assumptions above were performed by analysis of variance (ANOVA) and two-factor simple classification. In each experiment, the tests carried out are outlined.

 

RESULTS AND DISCUSSIONS

 

Callus formation

 

Effect of 2,4-D

 

After 10 days of culture, the start of callus formation at the border closest to the base of in vitro shoot sections in all treatments tested (Figure 1D) was observed. After 30 days of culture, the callus had covered the base of the section. Two types of calli were formed; one compact, nodular, friable, light yellow with embryogenic appearance (Figure 1D) and the other watery, with dark and creamy yellow coloration, and non-embryogenic (Figure 1E).

 

In this context, and in line with our results, Zhao et al. (2010) described the formation of two types of calli during this stage of plant regeneration via somatic embryogenesis in sorghum; one compact, shiny yellow, friable and nodular with embryogenic appearance and the other watery and friable with non-embryogenic appearance.

 

At all concentrations of 2,4-D evaluated, there was callus formation. The highest percentage of callus with embryogenic structures (50%) was formed with 18.1 uM 2,4-D, with significant differences to the other treatments (Figure 2).

 

The lowest callus formation frequencies was obtained with 27.14 uM 2,4-D (30%). It was noted that many explants became dark brown and no callus formed in all treatments. The largest exudation by explant of dark brown pigments occurred with a concentration of 27.14 uM 2,4-D, observed in 100% of the explants and in the culture medium.

 

The low efficiency of the protocols for somatic embryogenesis in sorghum is one of the main difficulties in the development of genetic transformation in this species. In this study, various factors are optimized to improve the efficiency of somatic embryogenesis in sorghum. In the formation of callus, the growth regulator used plays a major role (von Arnold et al., 2002). The growth regulator most widely used in cereal for tissue culture in the formation of calli with embryogenic structures is 2,4-D (Bhaskaran and Smith, 1990; Vikra and Rashid, 2003; Jogeswar et al., 2007). This auxin has been proven to be very useful for the induction of calli with embryogenic structures in sorghum (Sudhakar et al., 2009; Zhao et al., 2010). In this work, the addition of 2,4-D to the culture medium favored the formation of callus from shoot segments grown in vitro. In the culture medium without growth regulator, no shoot segments with callus were observed.

 

The 2,4-D concentration for callus formation varied with the genotypes and explants used (Gupta et al., 2006; Arulselvi and Krishnaveni, 2009). In this work, a genotype and explant not previously studied, were used, and hence different concentrations of 2,4-D were added to the culture medium. Optimum callus formation occurred in the culture medium with 18.1 uM of 2,4-D. With concentrations of 9.05 and 27.14 uM there were low percentages of callus formation. In line with our results Lateefat et al. (2014) in a red grain mutant sorghum also achieved the highest frequency of callus induction with 18.1 uM 2,4-D in the culture medium, but used mature seeds as initial explant.

 

Effect of ascorbic acid on the phenolic oxidation

 

The addition of ascorbic acid to the culture medium reduced necrosis of explants. In the culture medium with this antioxidant, the formation of dark purple pigment was decreased. With the concentration of 50 mg l^-1 ascorbic acid, necrotic explants were not observed, neither were dark purple pigments in the culture medium (Figure 1F).

 

The use of this concentration of ascorbic acid significantly increased the frequency of callus formation up to 68%. This result may have been caused by the removal of phenolic compounds excreted from the explants, which should increase the availability of the components in the culture medium. With the concentration of 20 mg l^-1, though reduced, the formation of these compounds relative to the control was not adequate to achieve greater inhibition of the formation of the phenolic compounds from the explants. With the addition of 80 mg l^-1 to the culture medium, it was not possible to increase the control of phenolic oxidation. This may be given by the toxic effect of high concentrations of ascorbic acid produces to the tissue, which prevented the control of the activity of the polyphenol oxidase enzyme (Table 1).

 

Phenolic oxidation is one of the main obstacles for the development of somatic em bryogenesis in sorghum (Sadia et al, 2010; Brandao et al., 2012). In this study, the presence of phenols in the culture medium around the explant caused mortality and negatively influenced the formation of callus. Thus, the addition of ascorbic acid to the culture medium was necessary. The use of 50 mg l^-1 of ascorbic acid resulted in a significant reduction of phenolic oxidation, which increased callus formation by 17%. The concentration of this antioxidant, which resulted adequate (50 mg l^-1) in this experiment using 'CIAP 132R-05' cv., differs from that reported by Lu et al. (2009) in line P898012, which used 10 mg l^-1 of ascorbic acid for diminishing phenolic oxidation during callus induction stage. The need to vary the concentration of ascorbic acid for controlling phenolic oxidation when different varieties are cultured, shows that the emission of phenols by the explants to the culture medium is highly influenced by the genotype.

 

Influence of different sections of in vitro shoots on callus formation

 

Callus formation was achieved only in the treatments where section 1 (section containing part of the base where the meristematic zone of growth is located) was used as explant. This result was related to the presence of the vegetative meristem at the base of the explant. The use of this type of explant increased the frequency of embryogenic callus formation to 95%. Callus formation from section 1 began at the base of the explant where the zone of active cell division is located. After 30 days of culture, the callus formed covered the entire base of the explant (Figure 1D). Explants of sections 2 and 3 did not form callus. They became necrotic and also delivered phenolic compounds to the culture medium. This indicates that the presence of this zone of active cell division is critical for callus formation from in vitro shoot sections in sorghum, at least for the conditions established in this experiment. The different response of in vitro shoot sections suggests that the cells that make up the apical meristem are better able to achieve dedifferentiation than other cell types present in these explants.

 

An important factor to consider for the establishment of an efficient regeneration protocol is the type of initial explant. Different types of explants have been described for the development of somatic embryogenesis in sorghum. Among the explants most widely used are those resulting from the process of sexual reproduction such as inflorescences, zygotic embryos and apices of germinated seeds in vitro (Arulselvi and Krishnavenl, 2009).

 

According Zhao et al. (2008) a high calli induction frequency has been achieved using mature seeds and zygotic embryos. However, with these explants the frequency of callus formation was lower than 40%. The same researcher group increased the formation of embryogenic calli up to 67% using germinating seeds as explant (Zhao et al., 2010). In this work, calli were formed from sections of in vitro shoots, which have not been used previously for somatic embryogenesis in sorghum. The callus with embryogenic structure formation frequency that we obtained with this novel explant is high compared with the results previously reported for Zhao et al. (2008; 2010).

 

Formation and differentiation of somatic embryos

 

Influence of 2,4-D and 6-BAP

 

After 30 days of culture, somatic embryos were observed in various stages of development in all treatments. Groups of proembryos and somatic embryos in the globular stage with round and translucent appearance (Figure 1G) were observed. Some of the globular somatic embryos showed an intussusception, which represents a transition towards the scutellar stage (Figure 1H). Seventy two percent of the somatic embryos were in the globular stage and 25% in the scutellar stage. Also embryos in the coleoptile stage were observed in treatments with 4.52 uM 2,4-D, in combination with 2.22 uM 6-BAP (Figure Besides, some somatic embryos germinated in the same culture medium that were formed (Figure 1J).

 

The highest percentage of callus that formed somatic embryos were obtained in the culture medium containing 4.52 uM 2,4-D, in combination with 2.22 uM 6-BAP. Usually, in the treatments where the concentration of 2,4-D were superior to 4.52 uM, the most number of somatic embryos per callus was achieved. This value was significantly higher in treatment 11 with 13.84 somatic embryos per callus (Figure 3).

 

Seetharama et al. (2000) and Arulselvi and Krishnaveni (2009) described the regeneration of sorghum plants via somatic embryogenesis. However, the information on the formation of somatic embryos and the description of the different stages of development of these embryos is limited. Generally, to stimulate the regeneration of plants from embryogenic callus of sorghum, auxins and cytokinins are used, but the type and concentration of such growth regulators are varied. The use of certain growth regulators for regeneration of sorghum plants is subject to the genotype and the type of explant used for callus formation. A reduction of the concentrations of 2,4-D used during callus formation combined with low concentrations of 6-BAP favored the formation of somatic embryos of sorghum in this study.

 

It is known that the addition of plant growth regulators in the culture medium affects the auxin/cytokinin endogenous balance (Del Pozo et al., 2005). Arulselvi and Krishnaveni (2009) in four genotypes of Sorghum bicolor added kinetin as the growth regulator and it increased the formation of somatic embryos. In this study, the combination of 4.52 uM 2,4-D with 2.22 uM 6-BAP reacted with the endogenous balance of auxin/cytokinin causing an increase in the formation of somatic embryos. In other investigation, Sudhakar et al. (2008) obtained the highest number of somatic embryos when 6-BAP, at concentrations of 8.88 uM, was used as cytokinin.

 

Embryogenic cells develop when the experimental conditions allow them to express their potential. Generally, this occurs when the amounts of auxin-cytokinin ratio is reduced. This can be demonstrated with in vitro systems where plant somatic cells can regain their totipotency and form embryos through the development of somatic embryogenesis (Raghavan, 2000).

 

Souter and Lindsey (2000) mentioned that the phase of formation and differentiation of somatic embryos is crucial. It is here where the patterns of apical-basal polarity is determined and specified for developing the cauline shoots and root apices of the somatic embryo.

 

Lateefat et al. (2014) in a red grain mutant sorghum (Karandafi), observed after three weeks the formation of somatic embryos on most of the callus transferred to culture media containing low concentrations of 2,4-D. Reducing the amounts of 2,4-D combined with 6-BAP used in this experiment is apparently the appropriate auxin-cytokinin ratio for this sorghum variety and type of explant. The appropriate relationship between these growth regulators allows somatic cells of this variety to retain their totipotency and form somatic embryos through the development of somatic embryogenesis.

 

The combination of growth regulators needed to form somatic embryos depends on the type of cells present in the explant, embryogenic cell either predetermined (ECPDs) or embryogenic cell induced (ECIs). For ECIs tissues, the use of high concentrations of auxin is necessary to induce cell embryonic stage and subsequently decreasing the threshold of these auxins or cytokinins added to form and develop the embryos (Merkle et al.,1995). The results revealed that the calli formed in this study presented ECIs, because with only the addition of a cytokinin, somatic embryos (Figure 3) were formed. Besides, the formation of somatic embryos increased with the combination of auxin and cytokinin in the culture medium, which revealed that in most of the callus cells present ECIs.

 

Effect of L-Proline

 

In the present study, after 21 days of culture, groups of embryos at different development stages (Figure 1K), were observed. Some of the globular somatic embryos showed an intussusception, which represents a transition stage towards a more advanced stage in the development of somatic embryo (Jayasankar et al., 2003). After 30 days of culture, the formation of somatic embryos at various stages of development in all treatments was observed.

 

The greatest number of somatic embryos formed was in the treatment three in a culture medium with 4.52 uM 2,4-D, 2.22 uM 6-BAP and 500 mg l^-1 L-Proline (Figure 4).

 

The role of amino acids as an immediately available source of nitrogen to plant cells is well known. The uptake may be much faster than that coming from inorganic nitrogen in the same culture medium (Thom et al., 1981). Monnier (1990) noted that reduced nitrogen is required for induction of somatic embryo, as these young embryos lack the enzyme nitrate reductase, responsible for the reduction of nitrate to nitrite. Organic nitrogen can be built quickly, stimulating the growth of cells, even when the mechanisms of absorption of ammonium and nitrate are not fully functioning (Pola et al., 2007). As has been described by several authors, L-Proline promotes the formation of embryogenic callus and plant regeneration capacity of sorghum (Armstrong and Green, 1985; Rao et al., 1995).

 

Sorghum is a crop that requires high amounts of nitrogen. L-Proline is an important amino acid for the development of somatic em bryogenesis in this monocot. The presence of L-Proline in the culture medium can influence the formation and differentiation of somatic embryos of sorghum. Thus, Sudhakar et al. (2008) reported that L-Proline (1000 mg l^-1) promotes the development of somatic embryos of sorghum. In this study the addition of L-Proline in the culture medium up to 500 mg l^-1 caused an increase in the formation of somatic embryos. Other authors such as Neguyen et al. (2007) also achieved high efficiency plant regeneration with the addition of 500 mg l¹ of L-Proline to the culture medium. Similar effects were reported by Zhao et al. (2010) who achieved the highest efficiency of calli with embryogenic structures and plant regeneration via somatic embryogenesis of sorghum varieties 'Yuantian No.1' and 'M81E' with 650 mg l^-1 of L-Proline in the culture medium.

 

The role of individual amino acids or mixtures of them is controversial in the literature, and sometimes they were reported to have no effect or a different one dependent on the species studied (Thorpe et al., 2008). L-Proline is more stimulating compared with the other two amino acids such as L-glutamine and L-arginine (Amali et al., 2014). These authors, in their study, were able to increase the number up to 33.3 somatic embryos per callus in a growth medium where callus with embryogenic structure was subcultured on MS salts with 11.31 uM 2,4-D, 0.25 mg l^-1 kinetin, and 500 mg l^-1 casein hydrolysate and 500 mg l^-1 L-Proline. In the present investigation, the addition of 500 mg l^-1 L-Proline into a culture medium containing 9.05 UM 2,4-D and 1.11 uM 6-BAP caused a significant increase in the number of somatic embryos per callus (41.88), which showed the influence of this amino acid in the formation and differentiation of somatic embryos of sorghum.

 

Histology of embryogenic callus and somatic embryos

 

In S. bicolor cv. 'CIAP 132R-05', two types of calli with embryogenic and non-embryogenic structures were formed. It was observed that one type of callus also had the formation of embryogenic structures. These embryogenic calli were characterized as compact, friable and of a light yellow color. Histological analysis of this embryogenic callus was found to be composed mainly of meristematic cell type that is isodiametric cell with no well-defined nucleus and vacuoles.

 

A histological section of a callus with embryogenic structures revealed the presence of different cells characterized by their sizes and densities of the cytoplasmic, small well-defined intercellular spaces and cell walls are present (Figure 1L). In this type of callus, meristematic cells and others in smaller numbers, with parenchymatic characteristics were observed. The latter is located further inside the callus; while the meristematic cells are located on the surface regions of the callus (Figure 1M). The parenchymal cells (PC) could be derived from meristematic cell differentiation during the initial period of callus formation. It is characterized by a larger, more elongated shape or a higher level of cell differentiation (Figure 1M). While meristematic cells, of smaller size, were grouped into the surface region of the callus (Figure 1M).

 

In histological sections, the presence of somatic embryos in the coleoptile stage (Figure 1N) was observed. These somatic embryos had well-defined vascular bundles along the central axis of the somatic embryo, which is characteristic in the morphogenetic development phase (Figure 1O). It was observed in the histological analysis that somatic embryos that had formed coleoptile stage remained with suspending structures. Also in the cross section, a well-defined structure of the conductive tissue was observed (Figure 1P).The histological studies are important for the confirmation of somatic embryogenesis and have been used for this purpose. In Sorghum bicolor Kishore et al.

(2006) described meristematic and non-meristemetic cells, from embryogenic calli, with similar characteristics to the cells observed in this investigation.

 

CONCLUSIONS

 

The use of section 1 from in vitro shoots as explant that has not been utilized before for the somatic embryogenesis in sorghum was an important factor for the somatic embryos formation in this crop. The formation of callus with embryogenic structures was influenced by 2,4-D concentration, the antioxidant effect of ascorbic acid and the type of explant. The formation of somatic embryos depended on the growth regulators added to the culture medium. The rate of exogenous auxin/ citokinine played a determinant role on the increase of somatic-embryo formation. The L-Proline provoked a significant increase of the average number of somatic embryos per callus (41.88). It evidenced the influence of this aminoacid on the somatic embryos formation and differentiation in sorghum. The histological analysis confirmed that the structures formed from callus are derived of somatic embryogenesis morphogenetic process.

 

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Recibido: 14-7-2015

Aceptado: 07-10-2015