Introduction

Saccharum spp. (sugarcane) was brought to Brazil during colonial times and has since become one of the country’s key economically important crops. Brazil is currently one of the world’s leading producers of sugar and ethanol (Brasil 2016). For the 2017–2018 harvest, the cultivated area for sugar and ethanol production has been estimated at 8.73 million ha in the Brazilian producer states, with an average productivity of approximately 72,000 kg ha−1 (Conab 2018).

Greatly heterogeneous, sugarcane plants are normally propagated by cuttings of stalks with one or more buds (Tiwari et al.2010; Singh et al.2013), or, within a recently adopted modality, by implementing a system of planting pre-sprouted plantlets (Landell et al.2012). Because of the demand for sugarcane propagation is coupled with limited availability of seeds, a variety of diseases affect sugarcane, and world pressure for increased sugarcane productivity, the need to exploit biotechnological tools to obtain first-class, large-scale, and high-yield material is evident (Tiwari et al.2010).

Tissue culture includes several techniques likely to be important allies for the production of economically significant species, among which somatic embryogenesis should be highlighted. Somatic embryogenesis in sugarcane is a process that has been under study for a number of years by researchers worldwide. Nonetheless, established protocols currently available are not always clearly disclosed and/or do not apply to every sugarcane variety. Further, there is scant knowledge on the persistence of regeneration capacity by embryogenic calluses during in vitro cultivation. Most protocols involve immature leaves taken from stem tips, gathered from field plants (Taparia et al.2012; Alcantara et al.2014; Kaur and Kapoor 2016), which are usually not physiologically uniform among the specimens and/or at different seasons, impairing the method’s reproducibility. Moreover, several inconveniences are extant, such as a high rate of contamination by endophytic microorganisms and the release of large amounts of phenolic compounds into the culture medium (Garcia et al.2007). Evaluating the efficiency of various sterilizing agents and the exposure of explants to heat, Moutia and Dookun (1999) verified that none of the tested methods could completely eliminate endogenous microorganisms and indicated that other resources must be discovered to increase micropropagation success.

The current study defined several cultivating conditions that ensured the efficient and quick regeneration of 14 Brazilian sugarcane varieties from embryogenic callus. Somatic embryogenesis has a number of practical applications, particularly massive propagation. It may also be employed for the production of synthetic seeds, cryopreservation of germplasm, and genetic modification by transformation (von Arnold et al.2002).

In fact, substantial efforts have been made over the last decade to develop an efficient genetic transformation system for sugarcane (Sengar et al.2011). Different transformation techniques, such as electroporation (Seema et al.2001), bombardment with particles (Taparia et al.2012), and gene transfer mediated by Agrobacterium tumefaciens (Manickavasagam et al.2004; Joyce et al.2010), have been studied to employ genes of agronomic and industrial interest for the improvement of the genus Saccharum. However, before genetic transformation can be routinely successful, the type of explant employed and the morphogenic regeneration method used must necessarily be well defined. Under the totipotency theory, any living part of a plant may be used as material for in vitro growth (Krikorian and Berquam 1969). In general, embryogenic calluses have been a preferred tissue, chiefly due to their greater proportion of cells capable of clonal proliferation and subsequent regeneration under adequate cultivating conditions (Basnayake et al.2011).

The current study improved the protocol and analyzed the embryogenic potential of 14 Brazilian sugarcane varieties through the use of in vitro plants as a source for explants. Moreover, the present study not only described the morpho-anatomical stages involved in the process, but also assessed the morphogenic capacity and regeneration frequency of consecutive subcultures.

Materials and Methods

Plant material

Sugarcane plants with 8-mo-old were used as source of stem cuttings. In the laboratory, stem tips were disinfected with 70% (v/v) alcohol for 1 min, with sodium hypochlorite (NaClO; 2.5% (w/v) active chlorine) for 20 min, and rinsed three times in autoclaved distilled water, for the preparation of in vitro explants. After surface sterilization, shoot tips with 2 or 3 leaf primordia and approximately 1–2 mm were extracted with the aid of a stereomicroscope, and pre-cultured for 72 h in the dark in 25 × 150 mm test tubes containing 10 mL of MS (Murashige and Skoog 1962) salts and vitamins, supplemented with 0.2 mg L−1 6-benzylaminopurine (BAP), 0.1 mg L−1 kinetin (Kin), 20 g L−1 sucrose, and solidified with 2.3 g L−1 of Phytagel™, according Nogueira et al. (2013). After 40 d of cultivation at 25 ± 2°C with a 16-h photoperiod and irradiance of 30 μmol m−2 s−1 provided by Universal 40 W cool white fluorescent lamps (Osram, Alphaville Barueri, SP, Brazil), the regenerated 5-to 7-cm long shoots were transferred to 250-mL glass flasks containing the same culture medium used to establish the plants, where, after one 30-d subculture cycle, multiplied shoots were used as a source of explants for somatic embryogenesis experiments. The media pH was adjusted to 5.8 ± 0.1 with KOH prior to autoclaving at 121°C and 101.3 kPa for 20 min.

Determining explant size for induction of somatic embryogenesis

To determine the influence of explant size on induction of somatic embryogenic callus, shoots from two sugarcane varieties (RB85-5453 and IAC86-2210) were used as explant sources. Shoots, roughly 3.0-cm long, were isolated in a laminar flow cabinet and cut transversely into 1.0-, 3.0-, 6.0-, 9.0-, 12.0-, or 15.0-mm segments. Regardless of length, one shoot was used for each explant. Explants were placed on an induction culture medium containing MS salts and vitamins, supplemented with 3.0 mg L−1 2,4-diclorophenoxyacetic acid (2,4-D; Ho and Vasil 1983), 30 g L−1 sucrose, and solidified with 2.3 g L−1 of Phytagel™. The medium pH was adjusted to 5.8 ± 0.1 with KOH prior to autoclaving at 121°C and 101.3 kPa for 20 min. After transfer to induction medium in a laminar flow cabinet, the explants were kept in the dark at 25 ± 2°C. Average primary callus formation rate was assessed every wk over a total of 40 d of cultivation.

The experiment was conducted in an entirely randomized design. For replication, three 15 × 90 mm Petri dishes were employed, each with five shoot explants placed horizontally onto the medium for each length group and sugarcane variety.

Assessing the embryogenic potential of Brazilian sugarcane varieties

Fourteen Brazilian sugarcane varieties (RB83-160, RB99-395, RB86-3129, RB95-1541, CB45-3, SP85-4594, SP70-1143, SP13-3804, SP81-3250, SP85-1431, SP71-6949, VAT90-61, VAT90-212, and VAT90-186), retrieved as stem cuttings from the Germplasm Bank at the Embrapa Coastal Tablelands, Rio Largo, AL, Brazil, were used to confirm sugarcane embryogenic potential. Simões Neto (2009) and Sindaçucar (2018) may be consulted to verify the varieties’ agronomic characteristics and agronomic-industrial performance.

After the varieties were established in vitro, the stem segments were isolated and cut according to the results obtained in the preceding experiment and inoculated onto embryogenic callus induction medium.

Replication comprised of five 15 × 90 mm Petri dishes each with five explants placed horizontally on the culture medium for each variety. Cultures were kept in a growth chamber in the dark, at 25 ± 2°C, for a 40-d period. The formation of primary and embryogenic calluses was then assessed. The calluses were subsequently divided according to their morphological types: primary and embryogenic portions. Portions of primary callus were transferred to fresh induction medium and kept in the dark, whereas the embryogenic callus portions were inoculated onto a modified regeneration medium from Taparia et al. (2012), containing MS salts and vitamins, supplemented with 1.86 mg L−1 NAA and 0.09 mg L−1 BA, 30 g L−1 sucrose, and solidified with 2.3 g L−1 of Phytagel™, and transferred to light conditions in a growth chamber with a 16-h photoperiod and irradiance of 30 μmol m−2 s−1 provided by Universal 40 W cool white fluorescent lamps (Osram), at 25 ± 2°C. All chemicals above were obtained from Sigma-Aldrich®, St. Louis, MO.

The callus selection process, based on morphological types, was performed over three consecutive subcultures of 40 d each. After the end of each 40-d period, the primary callus portions were assessed with regard to the production of new calluses with the previously mentioned embryogenic features. On the other hand, the embryogenic calluses were assessed with regard to the regeneration percentage (percentage of green area containing somatic embryos at different development stages) and the number of plants (≥ 0.5-cm height) formed obtained by counting.

Morpho-histological analysis of somatic embryogenesis in sugarcane

The histological analysis comprised the collection of primary and embryogenic callus samples and their fixation in formaldehyde, acetic acid, and 70% (v/v) ethanol at a 1:1:18 (v/v) ratio (FAA 70; Johansen 1940). The samples were subsequently dehydrated in an increasing ethanol series [70–100% (v/v)], infiltrated and embedded in Histo-Resin (Leica, Heidelberg, Germany), following manufacturer’s recommendations. Crosswise and lengthwise serial sections (7–10-μm thick) were obtained by manual microtome (model RM 2125; Leica) and sections were then smoothed and placed on microscope slides on a hot plate heated to 40°C.

The samples were stained with toluidine blue (O’Brien et al.1964), followed by permanent mounting with Entellan® (Merck, Darmstadt, Germany). Results were recorded under a light microscope (BA300; Motic, Xiamen, PR China), coupled to a digital image capturing system (ImagePro Plus 4.5 software, Media Cybergetics, Rockville, MD).

Development and acclimatizing of regenerated plants

Regenerated in vitro sugarcane plants were transferred to 250-mL glass flasks containing MS culture medium to which 30 g L−1 sucrose and 2.3 g L−1 of Phytagel™ were added to improve the development of both the shoot and root systems. After inoculation, the flasks were closed with plastic lids and sealed with transparent plastic film, and shoots were maintained in the growth chamber with a 16-h photoperiod and irradiance of 30 μmol m−2 s−1 provided by Universal 40 W cool white fluorescent lamps (Osram), at 25 ± 2°C, for 30 d.

The pre-acclimatization process started while the plants were still in the growth chamber. Initially, at the end of the 30 d, the plastic wrap used to seal the flasks was removed. After 72 h, the flask covers were also removed. After a 48-h period without any kind of cover had elapsed, the shoots were rinsed in running water to remove culture medium residues and taken to the greenhouse, where they were transplanted into 60-cell trays containing a 1:1 ratio (v/v) substrate (Bioplant, Nova Ponte, MG, Brazil) and washed sand mix. Survival percentage was assessed after 21 d of acclimatizing in the greenhouse. In the greenhouse, plantlets were irrigated manually every 24 h but not fertilized. Greenhouse conditions were 75 ± 5% relative humidity, 30 ± 3°C, with photosynthetic photon flux density of 450–500 μmol m−2 s−1 and a 12-h photoperiod.

Data analysis

Data obtained in the current study were submitted to analysis of variance (ANOVA), while means were compared by Scott-Knott test at 5% probability (P ≤ 0.05), with the Sisvar 4.4 (Ferreira 2011) statistical analysis program. Data expressed as percentage were transformed by arcsine square root [(0.01x ÷ 100)1/2].

Results and Discussion

Determining explant size for induction of somatic embryogenesis

Results (Fig. 1) showed that the length of the initial stem segment explant had a close relationship with primary callus formation and proliferation. The formation of calluses in the two tested sugarcane varieties (RB85-5453 and IAC86-2210) was noted by the seventh day of cultivation in the more internal leaf layers, chiefly in the basal part close to the explants’ meristem region. After 40 d, the formation of primary callus with a brittle aspect (Fig. 2A) was more pronounced and reflected a clear influence of explant length on responses. Specifically, explants exceeding 9 mm in length were the most responsive, averaging roughly 76% callus formation (Fig. 1). Similar to the current findings, when Brisibe et al. (1994) used explants measuring 2 or 3 mm in length from leaf segments of in vitro plants for somatic embryogenesis in a sugarcane variety, an average of 48% of initial explants exhibited callus formation when inoculated on an induction medium. The lower callus induction rates of the shorter explants might reflect the strong phenolic oxidation found on the cut surface that might have damaged the development of callus-forming masses, as oxidation took place in larger explants on the most extreme portion and likely failed to interfere with the proliferation of callus at the opposite end. Owing to these results, the present investigation used explants with an initial length of 12.0 mm, measured from the basal part (meristem), excluding the first two most external leaves of the foliar segments, prior to inoculation.

Figure 1.
figure 1

Influence of Saccharum spp. hybrids (sugarcane) stem explant length on primary callus formation rates on MS (Murashige and Skoog 1962) medium supplemented with 3.0 mg L−1 2,4-dichlorophenoxyacetic acid for 40 d. Means (± SE) with the same letter belong to the same group, by Scott-Knott test at P ≤ 0.05.

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Figure 2.
figure 2

Depiction of typical somatic embryogenesis induced from stem segments of Saccharum spp. hybrids (sugarcane) varieties previously established in vitro. (A) Primary, brittle, mucilaginous callus formed after 40 d on MS (Murashige and Skoog 1962) induction medium supplemented with 3.0 mg L−1 2,4-dichlorophenoxyacetic acid; (B) whitish nodular compact embryogenic callus formed on mucilaginous tissue; (C) asynchronous embryogenic process following transfer to regeneration medium containing 1.86 mg L−1 1-naphthaleneacetic acid and 0.09 mg L−1 6-benzylaminopurine. (D) Plant formation on almost the entire callus surface. (E) Regenerated plantlets from somatic embryos. (F) Acclimatized plants in the greenhouse. MC, mucilaginous callus; WC, white compact nodular callus; GE, globular embryo structure; SE, somatic embryos; RP, regenerated plant. Scale bars A, B, C, D, and E = 0.5 cm; scale bar F = 1.0 cm.

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Embryogenic potential of Brazilian sugarcane varieties

The 14 sugarcane varieties (Table 1) tested in the current study responded significantly to primary callus induction, with an average 94% formation rate (data not show). As in the preceding experiment, cell proliferation took place during the first weeks of cultivation from the explants’ basal portion, which developed and gave rise to brittle calluses of yellowish color and mucilaginous aspect (Fig. 2A). However, a compact nodular callus of a whitish color was detected over time, especially in the first subculture, above or adjacent to the mucilaginous tissue (Fig. 2B). According to Ho and Vasil (1983), the mucilaginous callus features a brittle and brilliant aspect. It is not embryogenic and basically consists of slender and dissociated cells. On the other hand, the whitish compact calluses consist of small cells with a dense cytoplasm, which under ideal cultivating conditions will generate the embryoids of the species. Despite the efficiency in primary callus induction of the varieties in the present study, it became apparent that the evolution from the primary callus to the embryogenic stage was genotype-dependent and varied significantly in the course of three consecutive subcultures (Table 1). In general, the monitoring of calluses over time revealed that primary calluses of a brittle and mucilaginous aspect gave rise to embryogenic nodular portions on their tissue during the first subculture in 13 of 14 tested varieties (Table 1), with SP85-1431 being the only exception. In most cases embryogenic callus production recurred in consecutive subcultures, i.e., the primary calluses preserved their morphogenic ability when kept on induction medium (Table 1). It should be recalled that during subcultures, the calluses were separated in portions according to their morphological features. Consequently, primary callus portions remained in the medium containing auxin to form new embryogenic calluses. The embryogenic callus portions were transferred to the regeneration medium. Among the sugarcane varieties studied that displayed a constant frequency in producing embryogenic calluses during the three subcultures under analysis, SP70-1143, VAT90-61, SP71-6949, and VAT90-212 maintained averages of 32.3, 33.3, 36.0, and 39.0%, respectively (Table 1).

Table 1. Influence of cultivating time on the frequency of embryogenic callus formation, regeneration rate, and average number of shoots formed, using stem segments from in vitro plants of Brazilian Saccharum spp. hybrids (sugarcane) varieties as the initial explant source
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When transferred to the regeneration medium, the nodular embryogenic calluses produced during the three subcultures multiplied and soon showed their ability to regenerate by means of an indicative appearance of globular stage embryos (GE; Fig. 2C). The embryos started differentiating from the border of the white compact nodular structures after approximately 10 d of cultivation, followed by coleoptile development and somatic embryo germination (Fig. 2D). During this stage, an asynchronous embryogenic process of the sugarcane varieties under study was indicated by the emergence of somatic embryos (SE) with scutellum and coleoptile, green regeneration areas containing somatic embryos at different stages of development, and regenerated plants (RP) on the same callus globular structures (Fig. 2C).

Garcia et al. (2007) confirmed the formation of globular embryos after 15 d, mature embryos after 25 d, and the start of plant conversion after 30 d from transferring calluses to the regeneration medium lacking growth regulators. Although these authors also employed in vitro explants as a source of explants, their regeneration process was more belated than that observed in the current study, since in the present study, it was possible to see the formation of the first sugarcane plants after 15 d on regeneration medium.

Indeed, the current protocol proved to be efficient: in addition to precocity in starting regeneration of the sugarcane varieties, the frequency reached 100% when embryogenic calluses from the first subculture were transferred to a regeneration medium and exposed to light conditions (Table 1; Fig. 2D). Nonetheless, the average number of plants formed per explant was significantly different among the varieties; for example, although VAT90-61, VAT90-212, SP70-1143, SP81-3250, SP81-3804, RB99-395, and SP85-4594 averaged 19 plants per embryogenic callus from the first subculture, their individual values ranged from 15.3 to 23.3 shoots formed (Table 1).

The embryogenic callus regeneration rate stayed above 60% until the third subculture in 7 of the 14 sugarcane varieties assessed. Nonetheless, VAT90-186 and SP81-3250 lost their regeneration ability by the end of the experiment (Table 1). These data demonstrated the influence of time on somatic embryogenesis in sugarcane. Singh et al. (2008) confirmed maximum regeneration efficiency in 34.6% of calluses when inoculated on MS medium with the addition of 0.5 mg L−1 of BAP, whereas Dibax et al. (2011), studying two Brazilian sugarcane varieties (RB93-1003 and RB98-710), reported rates between 70 and 80% in inducing shoots based on embryogenic masses, also in the presence of BAP. In another study, Basnayake et al. (2011) obtained over 50% regeneration rates for embryogenic calluses with a nodular appearance inoculated on a basal MS medium without growth regulators.

An aspect of the current study revealed that, despite preserving the regeneration ability of embryogenic calluses from a number of varieties, the number of shoots taller than 0.5 cm was significantly reduced over cultivation time. This was demonstrated by the observation that both shoot formation and growth slowed with aging of calluses. The ability to maintain a callus culture over extended periods of time is a unique feature of a species and a constant effort among researchers, as it allows such materials to be employed quickly in experimental processes (Pola et al.2009). However, this type of extended culture becomes feasible only when the ability to regenerate is also supported throughout the course of cultivation time. In general, the calluses of 13 sugarcane varieties in the present study maintained their regeneration abilities between the induction of embryogenic callus and the third subculture on regeneration medium for over 22 wk of cultivation (Fig. 3), with a notable exception of the cultivar SP85-1431 that in none of the subcultures presented plant regeneration (Table 1).

Figure 3.
figure 3

Diagrammatic representation of stages involved in establishment of Saccharum spp. hybrids (sugarcane) embryogenic callus culture and regeneration process, started from shoot tips pre-established in vitro. Each stage described, whether on induction or regeneration medium, was equal to a 40-d cultivation period.

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Apparently, no morphological variations were detected in the regenerated materials that could be related to somaclonal variation. In sugarcane, the detection of phenotypic variations due to the occurrence of somaclonal variation is relatively easy to observe in vitro, due to the reduced internodal length of the shoots, giving it a rosette appearance (Ramage and Williams 2004; Nogueira et al.2015), or due the regeneration of albino shoots, which occurs due to lack of chloroplast (Gosal et al.1998). Some shoots can have extremely narrow leaves, while others have wide, short, thick leaves (Ahloowalia and Maretzki 1983). Possibly, the fact that the number of induction subcultures was purposely limited to only three influenced this apparent lack of phenotypic variation, since it is quite common to find in the literature reports of somaclonal variation occurrence in sugarcane due to the excessive number of subcultures (Lee 1987; Sobhakumari 2012; Hsie et al.2015; Nogueira et al.2015; Martínez-Estrada et al.2017).

Histological analysis

Anatomically, the mucilaginous type of callus had a large portion of cells in an elongated format with large intercellular spaces (Fig. 4A). Nonetheless, meristematic cells containing dense cytoplasm and a voluminous nucleus could be detected in the aggregate (Fig. 4A, arrow). These meristem cells developed to form compact nodular masses, which consisted of small, juxtaposed cells undergoing intense cell division (Fig. 4B), as usually found during the initial formation of embryogenic calluses. These explant cell features of dedifferentiation and subsequent development of meristematic regions was similar for all varieties under study, and corroborated previous findings by such pioneers in the study of somatic embryogenesis of sugarcane as Ho and Vasil (1983) and Chen et al. (1988).

Figure 4.
figure 4

Histological analysis of Saccharum spp. hybrids (sugarcane) somatic embryogenesis. (A) Aspect of mucilaginous calluses containing vacuolated and meristematic (arrow) cells; (B) nodular callus (NC) formed on mucilaginous tissue; (C) linear stage pro-embryos; (D) globular pro-embryo with suspensor cells (SP); (E) somatic embryo with radicular pole (RP) and protoderm (PT, arrow); (F) globular embryo (GE, right) and cotyledonary embryo (left) containing scutellum (ES), coleoptile (CL), procambium (PC), and radicular tip (RP); (G) nodular callus (NC) with embryos in different stages of development; (H) adventitious bud with apical meristem (AM), leaf primordia (LP), and procambium (PC) formed from the nodular callus (NC). Scale bars A, B, D, E, F, and G = 100 μm; H = 50 μm; C = 10 μm.

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When transferred to a regeneration culture medium, compact nodular embryogenic calluses displayed pro-embryo formation in a linear stage with two to four cells (Fig. 4C), particularly in the peripheral region. Pro-embryos were apparently enveloped in a fine layer of cell wall (Fig. 4C). Intense cell divisions of the pro-embryonic apical cell gave rise to globular stage embryos (Fig. 4D). At this stage, a suspensor cell (SP; Fig. 4D) was seen in the globular pro-embryo’s base, connecting the embryo to the compact nodular callus. The existence of the suspensor cell existence pointed to the embryos’ single-cell origin, although multi-cell origin somatic embryos had also been detected (data not shown). According to Sané et al. (2006), the single-cell origin is a typical form of embryogenesis development where the competent cell actively divides within an external polysaccharide layer that provides physical isolation from adjacent cells. Nonetheless, the simultaneous occurrence of single-cell- and multi-cell-origin embryos has already been reported for other sugarcane varieties (Alcantara et al.2014) in several scientific texts (Almeida et al.2012; Scherwinski-Pereira et al.2012).

It was subsequently found that somatic embryos, formed after the globular stage, had a cylindrical morphology and an initial development of the radicular pole and protoderm (RP and PT, respectively; Fig. 4E). Visualized embryos at the cotyledonary stage displayed an apical region containing scutellum (ES; Fig. 4F) and coleoptile (CL; Fig. 4F) formation, and a basal region where the radicular tip lay (RT; Fig. 4F). This feature was consistent with the cytological description of Brisibe et al. (1993) during the process of somatic embryo differentiation in sugarcane. The initial development of procambium bundles was also detected (PC; Fig. 4F).

As a rule, somatic embryos at different stages of development were found in the same nodular tissue, typifying an asynchronous embryogenic process (Fig. 4G). Further, the formation of adventitious buds from nodular calluses simultaneously with the development of somatic embryos was detected. This fact revealed the occurrence of indirect organogenesis during the embryogenic process (Fig. 4H) that, although not quantitatively determined, was of low frequency due to the types of callus used, which only followed to the stage of regeneration when presenting embryogenic characteristics. These results agreed with those by Falco et al. (1996), who analyzed the formation of single-pole adventitious buds, evidenced by the ample vascular connection with the originating tissue jointly with the formation of somatic embryos. Dibax et al. (2013) disclosed that the origin of regenerated shoots in two sugarcane cultures (RB92-579 and RB 93-509) took place solely through indirect organogenesis, even after detecting a nodular callus formation with structures similar to somatic embryos.

Development of regenerated plants and acclimatization

Regenerated sugarcane plantlets developed and produced a larger number of adventitious roots when transferred to culture medium lacking growth regulators (Fig. 2E), and acclimatized after having grown taller than 5.0 cm. Furthermore, survival rates above 80% were recorded in a greenhouse for the regenerated genotypes, seven of which achieved 100% plant survival after a 21-d acclimatization (Fig. 2F).

The current study described an efficient sugarcane somatic embryogenesis protocol featuring high plant regeneration rates, reduced culture time, and contamination-free plantlet production. In this protocol, in vitro plants were employed as a source of initial material to induce embryogenic calluses. The meristem region was the most responsive and the best callus induction rates arose from explants of roughly 12 mm in length. Further, the 13 sugarcane varieties assessed displayed embryogenic potential for regenerating complete plants after 6 wk of cultivation, and several varieties continued differentiating somatic embryos for as long as 22 wk, provided that appropriate development conditions and monitoring of materials were complied with during the different stages of cultivation. Sugarcane somatic embryogenesis as established in this current report should be useful for future efforts involving Saccharum spp., including plant mass production and/or genetic transformation of the species.