An efficient plant regeneration system for elite genotypes is essential to apply biotechnological breeding methods. Somatic embryogenesis is a rapid and non-chimeric pathway for plant regeneration in sugarcane in this respect. However, it is limited by low regeneration efficiency and strong genotype-dependent effects. Even though many established protocols are available for sugarcane, not all are adapted to elite cultivars. Usually, biotechnological breeding procedures involve the use of genotypes that have good characteristics for in vitro culture but poor adaptation to Argentinian agro-ecological conditions. In the current study, we identified the in vitro culture response of five commercially grown Argentinian sugarcane cultivars through indirect somatic embryogenesis. These cultivars were developed by the Sugarcane Breeding Program of INTA and are considered elite genotypes. We based on Snyman [28] protocol for genetic transformation with some modifications according to other studies and our conditions.
A range of explants has been tried in somatic embryogenesis studies in sugarcane [11, 13]. Immature leaf roll and developing inflorescence are the best explants for embryogenic callus production. We used immature leaf explants and achieved a successful in vitro establishment in our genotypes. In Tucumán, where sugarcane flowering does not occur naturally, it is easier to work with leaf roll than immature inflorescence as explant, thereby we can dispense with inducing flowering. Non-flowering clones can also be found among commercially elite genotypes [29]. Furthermore, explant contamination was almost absent following a disinfection protocol, such as that proposed by Ho and Vasil [9].
Embryogenic calluses are the best target tissue for in vitro mutagenesis [30] and biolistic transformation [12, 28] as well as cryoconservation and synthetic seed production [8] in sugarcane. Therefore, the nature and type of callus are some of the most important factors to consider in sugarcane tissue culture. Explants of all genotypes formed the different types of calluses described by Taylor et al. [10] with the two protocols we assessed. A highly embryogenic callus was observed in four INTA genotypes, which showed a differential response to induction protocols. Our results are in agreement with other studies demonstrating that embryogenic capacity depends on the genotype and culture medium interaction [13, 31]. Genotype dependence for embryo formation has been reported for other elite sugarcane cultivars in Brazil [14, 15, 32], Venezuela [17], South Africa [12], India [19, 20], Pakistan [21], and the USA [18]. According to the results of the present study, the cultivars INTA CP 98-828, INTA NA 89-686, and INTA NA 91-209 could be classified as highly embryogenic, L 91-281 as moderate, and FAM 81-820 as recalcitrant cultivar with MS3/MS1 and M3 protocols. It is worth noting that other medium formulations, mainly with other growth regulators, could increase embryogenic capacity in L91-281 and induce somatic embryogenesis in FAM 81-281. Cytokines such as kinetin or 6-benzylaminopurine increased embryogenic response in some sugarcane genotypes [13, 19], whereas they decreased it in others [14]. Some cultivars showed a better response to picloram or dicamba than to 2,4-D [17, 21]. A recent study has shown that low concentrations of methylglyoxal enhanced somatic embryogenesis in Indian sugarcane cultivars [33].
A better embryos maturation was observed in MS3/MS1 protocol than MS3 protocol, most likely because of the lower concentration of 2,4-D during the last weeks of culture. The dedifferentiation process and the embryogenic-cell initiation require relatively high auxin concentration, whereas lower auxin concentration may be required for advanced embryogenesis stages [16]. Well-developed embryos are obtained from embryogenic callus when 2,4-D in the medium is lowered [9]. Dibax et al. [32] observed that a decrease in 2,4-D concentration doubled the number of embryogenic masses in Brazilian genotypes. However, we did not observe an increase in the percentage of embryogenic callus with MS3/MS1 protocol.
Light is a major factor affecting somatic embryogenesis and subsequent plant regeneration [34]. Darkness is required for the somatic embryo initiation to keep high auxin concentrations since the auxins are generally prone to breakdown in light [35]. In contrast, light conditions and low auxin levels are necessary for embryo maturation and when the plant becomes autotrophic. Also, the type of light affects these stages, since specific light spectral ranges are involved in specific plant responses. In sugarcane, fluorescent lamps were more efficient in embryo maturation and regeneration, whereas LED-light emitting diodes provided higher multiplication rates [34]. In the current study, we used only fluorescent lamps, but we will subsequently evaluate the multiplication rate of these cultivars using LED lights.
Several factors are considered for the selection of a genotype for biotechnological breeding, mainly its ability to regenerate plants. The germination of somatic embryos in sugarcane can be induced under light conditions and without auxins [9, 10]. We added IBA to promote root development in agreement with Mustafa and Khan [36], whereas other studies managed to regenerate plants without growth regulators [12, 17, 18, 32] or with some cytokine [13, 14]. The ability to form regenerable calluses is genotype-dependent in many species [16], including sugarcane [19, 37]. Our results, which are in agreement with these previous studies, confirm the genotype-dependent regeneration in sugarcane. Although the germination percentage of somatic embryos is very low in some crops [30], we obtained a good number of plants per genotype. In vitro regenerated plants were successfully acclimatized in the greenhouse and later under field conditions. Acclimatization in trays advantaged the stem girth increasing and avoided fungus proliferation when compared with pot culture conditions under high humidity. We found a positive correlation between variables related to embryogenic callus production and plant regeneration, thus suggesting that the regeneration potential of a cultivar can be assessed through the type of callus it produced. No correlation was found between regeneration capacity and establishment capacity because these pathways are controlled for different mechanisms [18].
