Somatic Embryogenesis in Agave Tequilana Weber Cultivar Azul

By Portillo, Liberato Santacruz-Ruvalcaba, Fernando; Gutierrez-Mora, Antonia; Rodriguez-Garay, Benjamin

Abstract Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(gamma-gamma- dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 [mu]M BA) compared to auxin (4.5 [mu]M 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 [mu]M 2,4-D and 1.3 and 4.0 [mu]M BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.

Keywords Plant regeneration * Unicellular origin * Tequila * Auxin * Cytokinin * Indirect somatic embryogenesis

Introduction

Agaves are native to the American Continent and show their greatest diversity in Mexico, where 75% from a total of 166 species occur (Garcia-Mendoza 1995). The genus Agave has been used in Mexico since ancient times for the production of food, fibers, sapogenins, and beverages. During the Colonial period, distilled alcoholic beverages started to be produced along the New Spain territories, with “tequila” being the most known. Tequila is obtained from Agave tequilana Weber cultivar azul, a xerophytic species which is the only plant accepted by the Mexican Official Norm to be used for the production of tequila (Diario Oficial de la Federation 1993).

A. tequilana plantations encompass more than 84,000 ha along five Mexican states, and its cultivation generates an important economic source of employment for more than 36,000 families and represents millions of dollars of income from taxes and exportation sales (Consejo Regulador del Tequila 2006).

This species is commercially propagated by asexual rhizomatous shoots, a procedure which allows for the increase of genetically elite clones with remarkable qualities, although it also facilitates the propagation of undesirable characteristics such as disease susceptibility. In the last decade, pathogenic bacteria and fungi have been an important problem causing economic losses to the agave growers, with the greatest infestation in 1997 when 20% of plants were affected to varying degrees (Fucikovsky 2002).

Since agaves have a long life span (A. tequilana life cycle requiring 6 to 12 yr), limited fertility (Ruvalcaba-Ruiz and Rodriguez-Garay 2002), and several degrees of ploidy (Castorena- Sanchez et al. 1991), conventional genetic improvement is a difficult and time-consuming task. In vitro culture techniques have been used to mass propagate several species of the genus Agave, and efficient protocols for plant regeneration have been developed either by axillary shoot proliferation (Madrigal-Lugo et al. 1989; Robert et al. 1992), organogenesis (Robert et al. 1987; Nikam 1997; Hazra et al. 2002; Valenzuela-Sanchez et al. 2006), or somatic embryogenesis (Rodriguez-Garay et al. 1996; Martinez-Palacios et al. 2003; Nikam et al. 2003). However, somatic embryogenesis for A. tequilana has not been reported. Here, we describe the production of indirect somatic embryos originated from dedifferentiated cells (callus) in A. tequilana Weber cultivar azul. This procedure will be useful for in vitro genetic improvement of this species.

Materials and Methods

Plant material. seeds and rhizomatous shoots from A. tequilana Weber cultivar azul were kindly provided by Instituto de Botanica, Universidad de Guadalajara. The plant material was rinsed under running water. All leaves of rhizomatous offshoots were removed, and only a segment of the basal part (stem cylinder of 1-cm height) was used. seeds and stems were dipped in 96% ethanol for 30 s, sterilized with 3% (v/v) sodium hypochlorite for 10 min and rinsed three times with sterile distilled water.

Young leaf blades of A. tequilana harvested from in v/fro- maintained plantlets following procedures reported for other Agave species (Santacruz-Ruvalcaba et al. 1999), originated through axillary shoot proliferation from six genotypes, (three from germinated seeds, S3, S7, SI3; three from offshoots, PI, P2, P8) were used as explants.

Experimental methods. Explants were placed in disposable sterile plastic Petri dishes (100 x 15 mm) containing 25 ml of freshly prepared MS medium (Murashige and Skoog 1962), supplemented with L2 vitamins (Phillips and Collins 1979), 30 g l^sup -1^ sucrose, and gelled with 8 g l^sup -1^ agar (A-1296; Sigma Chemical, St. Louis, MO), as it was described by Rodriguez-Garay et al. (1996). Additionally, in all experiments, several growth regulator combinations were used, and all media were adjusted to pH 5.8 prior to autoclaving. All cultures were incubated at 27+-2[degrees]C with a 16-h photoperiod under fluorescent light (16 pmol s^sup -1^ m^sup – 2^). Two experiments were conducted:

Experiment 1. In previous experiments, genotypes S7, SI3, PI, P2, and P8 did not respond to somatic embryogenesis with high cytokinin concentrations, but S3 did respond (Santacruz-Ruvalcaba, unpublished results). This experiment was conducted to study the responsiveness of A. tequilana (genotype S3) to different cytokinins for the production of somatic embryos. A 4 x 4 bifactorial design was carried out with four different cytokinins at four concentrations: 6- benzylaminopurine (BA): 4.4, 22.2, 44.4, and 66.6 [mu]M; 6-gamma- gamma-dimethylaminopurine (2ip): 4.9, 24.6, 49.2, and 73.8 [mu]M; 6- (gamma,gamma-dimethylallylamino)purine (KIN): 4.6, 23.2, 46.7, and 69.7 [mu]M; whereas 1-phenyl-3 (1,2,3-thia-diazol-5-yl)urea (TDZ) was added at 0.4, 2.3, 4.5, and 6.8 [mu]M because it has been reported that around ten times lower concentrations of TDZ have similar effects to other cytokinins (Huetteman and Preece 1993). 2,4- Dichlorophenoxyacetic acid (2,4-D) at 4.5 [mu]M was combined with all the above treatments. Ten dishes per treatment were used with four leaf segments per dish.

Experiment 2. To study somatic embryo production over a range of genotypes, one experiment using six genotypes was carried out: three obtained from germinated seeds (S3, S7, and SI3) and three from field axillary offshoots (PI, P2, and P8). These genotypes were cultured on three combinations of 2,4-D and BA (9.0/1.3, 13.6/1.3. and 13.6/4.0 pM, respectively), chosen from previous experiments under a 3 x 6 bifactorial design with eight dishes (four leaf segments per dish). In addition, in the same previous experiments, treatments with auxin alone did produce callus; however, no embryos were produced (Portillo, unpublished results).

For both experiments, the controls without plant growth regulators and cytokinin alone in this work were omitted because the production of callus was not achieved in preliminary experiments (Santacruz-Ruvalcaba, unpublished results). For the maturation and germination of somatic embryos, calli were transferred after 40 d from the induction media to freshly prepared modified MS expression medium (NH^sub 4^NO^sub 3^ reduced to 5 mM; CastroConcha et al. 1990), supplemented with L2 vitamins, 500 mg F1 L-glutamine (Sigma Chemical G-3202), 250 mg F1 casein hydrolisate (Sigma Chemical C- 7290; Santacruz-Ruvalcaba et al. 1998) and without growth regulators. Embryos started to appear 30 d after subculture in expression medium, and the number of produced embryos was recorded after 90 d.

Microscopic observations. To know the origin of somatic embryos, cells from embryogenic calli were processed by means of a double staining protocol (Gupta and Durzan 1987). Callus samples of about 8 mm^sup 3^ were suspended in 0.5-ml distilled water to which three drops of 0.5% acetocarmine were added and heated at 50[degrees]C in a water bath for 30 s. Thereafter, cells were washed with distilled water to remove excess stain. Two drops of 0.5% Evan’s blue were added to the acetocarmine stained cell suspension for 30 s and immediately washed twice to remove excess stain. The double-stained cell suspension was resuspended in 0.5-ml distilled water and observed with an inverted Olympus(TM) microscope (Mod. CK-2; Tokyo, Japan) equipped with an automatic Olympus(TM) camera (Mod. PM- 10ADS, 35 mm).

Histological analysis was carried following Alemanno et al. (1997). Samples that consisted of friable embryogenic callus containing all stages of somatic embryogenesis were fixed in a phosphate buffer (0.2 M, pH 7.2) containing 4% paraformaldehyde, 1% glutaraldehyde, and 1% caffeine. After dehydratation through a graded alcohol series, samples were embedded in Kulzer 7100 hydrosoluble resin (Leica, Wehrheim, Germany), and cut to 3 pm thick using a LKB Historange microtome. The double stain PAS (periodic acid-Schiff)-naphthol blue-black was used to stain polysaccharides and both soluble and insoluble proteins (Fisher 1968). Bright field observations were made under a BH-2 Olympus(TM) microscope. Figure 1. Somatic embryogenesis in A. tequilana Weber cultivar azul. (a) First embryogenic cell and suspensor cells of embryo. Sar=0.05 mm. (b) Embryos from genotype S7 obtained with 9.0/1.3 [mu]M 2,4-D and BA, 90 d after subculture in expression medium. Bar= 1.5 mm. (c) Creamy friable calluses from genotype S7. Bar = 1 mm. (d) Embryos from genotype S3 obtained with 4.5/ 66.6 uM 2,4-D and BA, 90 d after subculture in expression medium. Bar=2 mm. (e) Somatic embryos from genotype S7 were similar to those obtained from zygotic embryos upon germination. Bar=2 cm. (f) Plantlets obtained from somatic embryos 150 d after germination, (g) Potted plant obtained from germinated somatic embryo after 40 wk in greenhouse. Bar=5 cm. ac Apical cell; sc suspensor cells; se somatic embryo; ze zygotic embryo.

Statistical analysis. Quantification of the number of somatic embryos was carried out for the maturation and germination media. Statistics were calculated using the Statgraphics 6.0 software (Statistical Graphics, Rockville, MD) and results were analyzed by a two-way analysis of variance. Means were compared by the least significant difference (LSD) range test with a family error rate of 0.05.

Table 1. Means of somatic embryos from Agave tequilana Weber cultivar azul genotype S3 induced with four concentrations of four different cytokinins

Results and Discussion

Embryogenic calli were friable containing elongated and small globular creamy cells from which the somatic embryos arose (Fig. Ic). This differed to that reported for the monocot species Gasteria verrucosa and Haworthia fasciata, closely related to agaves where the somatic embryos were originated from yellow and compact calli (Beyl and Sharma 1983); also, these observations differ from those references on indirect somatic embryogenesis in other Agave species, where the embryogenic calli were snowy, compact, and nodular (Martinez-Palacios et al. 2003; Nikam et al. 2003).

Table 2. Means of somatic embryos from six genotypes of Agave tequilana Weber cultivar azul using tiiree combinations of 2,4-D and BA (9.0/1.3, 13.6/1.3, and 13.6/4.0 [mu]M, respectively)

Experiment 1. Statistical analysis showed a high significant difference for each of the studied factors: type of cytokinin (p<0.0000), concentration (p<0.0002), and interaction between these two factors (p<0.0000), with regard to embryo number. The LSD test detected three cytokinin groups that were statistically different, with BA giving the highest number of somatic embryos (Table 1). In general, increasing the cytokinin concentration led to an increase in somatic embryos, although not for TDZ. The increase was significant for BA at 44.4 and 66.6 [mu]M (p<0.0210) with the highest yield of somatic embryos being produced at the highest concentration tested (Table 1).

Table 3. Means of somatic embryos from Agave tequilana Weber cultivar azul obtained from 18 treatments (interactions of three induction combinations of 2,4-D/BA and six genotypes)

Somatic embryos developed with a high concentration of cytokinins were of a translucid green color (Fig. Id). This characteristic is most likely because of the effect of high concentrations of cytokinins that enhance chlorophyll synthesis in plants (George 1993; Zaffari et al. 1998). It is important to note that, in this experiment, high concentrations of cytokinins could induce high numbers of somatic embryos, as opposed to a large number of reports on several species where auxins are the main growth regulator to induce somatic embryos (George 1993; Khalil et al. 2002; Strosse et al. 2006).

Figure 2. Early stages of somatic embryogenesis in A. tequilana Weber cultivar azul. (a) Callus formed by highly vacuolated noncompetent cells and embryogenic cells containing large amounts of starch granules. Bar=40 [mu]m. (b) Polarized embryogenic cells containing large amounts of starch granules. Bar=20 [mu]m. (c) Polarized embryogenic structures resulting from first and second division of embryogenic cells. Bar=30 [mu]m. (d) Four-celled proembryo with a suspensor-like structure. Bar= 10 [mu]m. (e) and (f) Globular embryos with vestigial suspensor. Bars-50 and 100 [mu]m. (g) Globular somatic embryo without suspensor. Bar=15 [mu]m. (h) Torpedo stage embryos showing procambial initials. Sar=350 [mu]m. (i) Close-up of the vestigial suspensor of one of the embryos in (h; arrow head). Bar= 20 urn pzc Polarized cell; vc vacuolated cell; sg starch grain; n nucleus; tw thick wall; eh embryo head; s suspensor; ac apical cell; be basal cell; ge globular embryo; vs vestigial suspensor; p protoderm; pc procambial strands; rm root meristem; am apical meristem.

Experiment 2. No significant differences in the 2,4-D/BA induction combinations were observed (p<0.1095); however, statistical significant differences were observed for genotypes (p<0.0001) and also for the interactions between genotypes and induction combinations (p<0.0084). The most responsive genotype was S7 (Table 2), and the best combinations tested for embryo induction were 9.0/1.3 and 13.6/4.0 [mu]M 2,4-D and BA, respectively (Table 3). For S7, there was a significant increase in the embryo number when the concentration of 2,4-D was reduced with respect to BA (Table 3).

The production of somatic embryos was strongly influenced by the genotype, which has been previously mentioned by other authors for a large number of plant species (Chen et al. 1987; Tokuhara and Masahiro 2003; Sato et al. 2004). Indirect somatic embryos were obtained from all genotypes; however, the number was higher in some from germinated seeds (S3 and S7) than in those from offshoots, probably because of the higher genetic variation expected from the cross pollination than from vegetative propagation usual for this species (Gil-Vega et al. 2001, 2006). In this work, the S7 genotype was the best producer of somatic embryos with high doses of auxin and low concentrations of cytokinin, whereas genotype S3 showed a better performance with high doses of cytokinin. The possibility of selecting a highly embryogenic genotype is increased by evaluating a large number of genotypes (Compton 1994).

Somatic embryos produced with high concentrations of auxin were distinct in color from those obtained with high concentrations of cytokinins (compare Fig. lb, c versus d). In this case, embryos did not produce chlorophyll and were creamy yellowish in color. Once the maturation process was completed, somatic embryos acquired the normal green pigment and were similar to zygotic embryos (Fig. 1e).

Microscopic observations

Callus formed on leaf explants 20 d after the placement on the induction medium. This result differed from that reported by Valenzuela-Sanchez et al. (2006) where leaf explants did not develop callus even when 2,4-D was used. Callus growth was enhanced when transferred to maturation and germination media.

Sections of the callus varied with some parts having more somatic embryos. This behavior could be associated to the spatial position of some groups of competent cells (Fig. 2d). The relationship among cells and their physiological response is important to induce and maintain the cellular polarity (Figs, la, 2b) essential to develop the somatic embryogenesis (Schnepf 1986).

When embryogenic cells were double-stained, it was possible to distinguish the incipient forms of unicellular somatic embryos with putative suspensor cells stained with blue color and the first embryogenic head cell stained with red color (Fig. la). Additionally, the unicellular origin of the somatic embryos was also observed by means of histological analysis. Figure 2b to d show the polarity of embryogenic cells and also from the first to the third cell division of me embryo head. These results were different from the multicellular origin of somatic embryos found in A. sisalana (Nikam et al. 2003). Further embryo development resulted in characteristic globular and torpedo stages (Fig. 2e to h). It is important to note that, in some of the later stages, a vestigial suspensor was observed (Fig. 2e, f, h, and i). In addition, the histological analysis of somatic embryos revealed no vascular connection between the somatic embryos and the mother callus, which confirmed the individuality of somatic embryos described by Haccius (1978; Fig. 2h).

Germinated somatic embryos resembled their zygotic counterparts (Fig. 1e), giving rise to plantlets of a normal morphology (Fig. If g). The conversion efficiency from germinated embryos to established plants was as high as 95-100%.

Finally, the presented protocol demonstrates for the first time indirect somatic embryogenesis in A. tequilana Weber cultivar azul, which is the world’s most widely cultivated Agave species. This protocol is a further tool for the commercial mass propagation of the species, and the unicellular origin of the regenerated plants lends itself to perform genetic improvement.

Acknowledgments We would like to thank N. Michaux and D. Triare (CIRAD-France) for their technical assistance in the histological work, H. Gutierrez-Pulido (CUCEI-Universidad de Guadalajara) for his help in statistical analysis, and R. Barba-Gonzalez for his help with the artwork of Fig. 2.

L. Portillo is a graduate student (Posgrado en Procesos Biotecnologicos, Universidad de Guadalajara) financially supported by CONACYT-Mexico.

Received: 17 December 2006/Accepted: 10 April 2007 / Published online: 31 August 2007 / Editor: M.C. Jordan

CD The Society for In Vitro Biology 2007

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L. Portillo

Departamento de Botanica y Zoologia-CUCBA,

Universidad de Guadalajara,

Guadalajara, Mexico

F. Santacruz-Ruvalcaba

Departamento de Production Agricola-CUCBA,

Universidad de Guadalajara,

Guadalajara, Mexico

A. Gutierrez-Mora * B. Rodriguez-Garay (*)

Unidad de Biotecnologia Vegetal, Centro de Investigation y

Asistencia en Tecnologia y Diseno del Estado de Jalisco,

A. C. Av. Normalistas 800,

Guadalajara, Jalisco 44270, Mexico

e-mail: brodriguez@ciatej.net.mx

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