Expanding genetic and genomic analyses are needed to disentangle the subtle inter-species morphological differences

The jabuticabeiras were firstly taxonomically described into genus Myrtus Tourn ex. L. and Guapurium Juss. and were relocated within genus Myrciaria O. Berg (Berg, 1857). As some floral traits diverged from other species of genus Myrciaria, species of jabuticabeira were transferred to genus Plinia (Kausel, 1956; Sobral, 1985; Mattos, 1998). Currently, 80 species of Plinia are accepted (Govaerts et al., 2008).

Given the morphological variation observed in characteristics of fruits (Table 1, Table 2 and Table 3), the taxonomy of Plinia species is fairly a puzzle in the scientific literature. A miscellaneous of different species is suggested by diverging authors, making advances in selection, domestication, and breeding difficult.

Besides recognizing the morphological multiplicity, understanding the genetic divergence among species can be useful for the comprehension and conservation of the available genetic resources. Moreover, the morphological differences among some Plinia species are rather modest, making the species delimitation a great challenge for breeders and farmers.

Despite these needs, only a few studies using molecular markers were published, reporting the comparative analysis among different species of Plinia . Pereira et al. (2005) used 45 polymorphic RAPD markers in a study including P. jaboticaba, P. cauliflora, P. coronata, and P. phitrantha, while Vilela et al. (2012) used 37 polymorphic RAPD markers to study the relationship among P. cauliflora, P. jaboticaba, P. coronata, and P. peruviana. In both studies, clustering analyses were unsuited for defining the taxonomic groups, since the formed clusters comprised assortments of plants from different species and did not correspond to the taxonomic classifications based on morphological traits. Moreover, RAPD markers are not confident for such an analysis, due to several technical weaknesses.

The advances of the sequencing platforms have enabled researchers to generate comprehensive genomic data for several plant species, including also minor-crop species. Sequences of the complete chloroplast genome (plastome) of P. cauliflora, P. aureana, and P. trunciflora are available in the GenBank database. To test the usefulness of these molecular data for understanding the taxonomic relationship among Plinia species, the plastome sequences of P. cauliflora (KX527622; Machado et al., unpublished), P. aureana (KY392759; Machado et al., unpublished), P. trunciflora (KU318111; Eguiluz et al., 2017), Acca sellowiana (KX289887; Machado et al., 2017), Campomanesia xanthocarpa (KY392760; Machado et al., 2020), and Allosyncarpia ternata (KC180806; Bayly et al., 2013) were downloaded and a phylogenomic analysis was performed. This analysis (Figure 2) enabled more accurate analysis of the correlation among these Plinia species. Although the length, genetic composition, and structure of the plastomes are conserved among these species, the phylogenomic analysis based on the whole plastomes sequences resolved these three species with high support (Figure 2).

Thus, sequencing the plastomes of more Plinia species seems to be a meaningful strategy for resolving the remaining taxonomic uncertainties in this group, aiding the planning of conservation and breeding programs for Plinia species.

[Figure ID: f2] Figure 2..

Phylogenomic analysis based on the whole plastome sequences of Plinia cauliflora, P. aureana, P. trunciflora, Acca sellowiana, Campomanesia xanthocarpa, and Allosyncarpia ternata. The phylogenomic tree was obtained using the maximum likelihood algorithm, the GTR+G evolution model, and 1000 bootstrap replications for branch support, as implemented in the software RAxML, CIPRES Science Gateway V. 3.1 platform. Allosyncarpia ternata (Myrtaceae, Eucalypteae) was employed as an outgroup. All other species belong to the family Myrtaceae, tribe Myrteae.

Germplasm conservation and genetic improvement also need molecular genetic stu dies

Information about the genetic diversity of natural populations is also needed when aiming at the establishment of germplasm banks and genetic improvement of the species (Melo et al., 2015). Cruz et al. (2016) evaluated the genetic diversity of Plinia spp. in Northeastern Brazil. Thirty-five genotypes were characterized using ISSR (Inter Simple Sequence Repeats) markers. With a polymorphism of 99.65%, five groups were identified based on the genetic divergence among genotypes. Moreover, no correlation between geographical and genetic distances among genotypes was observed. This study revealed the existence of moderate genetic variability of the studied genotypes, an important insight regarding plant collection for breeding programs in the region.

The organization and distribution of population genetic variability of six populations of P. peruviana based on microsatellite markers were reported by Salla (2019). Ten microsatellite loci revealed significant genetic diversity for the six populations, with a high number of alleles and heterozygosis. The analyses of the molecular diversity partition revealed 17.6% of differentiation among populations, 2.6% within populations, and 72.2% within individuals. These results suggest that for conservation proposes, seed collections should maximize the number of seeds per matrix plant, lessening the number of individuals per population (Salla, 2019).

This scarcity of genetic studies and the small geographical amplitude of the two existing studies with Plinia natural populations hinders planning reliable programs of in situ or ex situ species conservation, seed collection, genotype selection, and genetic improvement. So, efforts towards characterizing the genetic diversity and structure of Plinia spp. natural populations using molecular markers and in a wider geographical perspective is needed.

Moderate to low rooting rates is the main limitation for traditional vegetative propagation of Plinia species

A review on the propagation of Plinia spp. by Silva et al. (2018) highlighted that the main form of seedling production is still carried out mainly by seeds, due to the greater ease and speed in the production of new plants. However, despite good germination rates, seed recalcitrance (Danner et al., 2011; Hössel et al., 2013) hampers the establishment of orchards through sexual propagation and studies about the conditions of seed germination of Plinia species (Andrade and Martins, 2003; Wagner et al., 2006; Alexandre et al., 2006; Rossa et al., 2010; Sartor et al., 2010; Dias et al., 2011; Wagner et al., 2011) are puzzling and inconclusive.

The development of methods for the asexual propagation of Plinia spp. is of great importance for obtaining seedlings. In addition to reducing the juvenile phase, vegetative propagation has some advantages such as maintenance of the genetic characteristics of the mother plant, greater productivity, and fruits of better quality (Danner et al., 2006; Hartmann et al., 2011). However, there is no established fully efficient methods of vegetative propagation of Plinia spp. that ensure the formation of commercial orchards in a short period.

In general, Plinia species are recalcitrant for the formation of adventitious roots, and layering seems to be a relatively effective method for vegetative propagation of the species. This method, mediated by indole-butyric acid (IBA) treatment provides the gathering of numerous rooting co-factors (Danner et al., 2006; Sasso et al., 2010; Cassol et al., 2015). However, for the establishment of a conclusive protocol, new studies must be carried out, considering the percentage of harvesting of the transplanted seedlings, the minimum period for the disconnection of the mother plant layering, the quality of the roots formed, the time between the planting of seedlings in the field, and the beginning of fruit production.

The few studies using cutting as a propagation method in Plinia species (Pereira et al., 2005; Fachinello et al., 2005; Sartor et al., 2010; Silva et al., 2019) revealed rooting rates lower than 50%. For commercial seedling production, the percentage of cuttings rooting obtained should be higher than 70% (Hartmann et al., 2011). The values reported for Plinia spp. are quite variable and usually lower than this threshold. The low rooting of the vegetative propagules seems to be correlated with the age of the tissue, the type and time of collection of the cuttings, the presence or absence of growth regulators, and cuttings cultivation conditions. Therefore, several adjustments are needed to increase the rooting percentage in Plinia species propagated through the cutting technique.

Grafting has supported somewhat more promissory results as a vegetative propagation strategy for Plinia species. Setting values reaching up to 90% were reported (Manica, 2000; Sasso et al., 2010, Franco et al., 2010; Malagi et al., 2012; Cassol et al., 2017), but these results revealed to be dependent on the cultivars used and the season. Despite the optimistic results obtained, long-term field monitoring of plants produced from grafting is necessary so that compatibility is verified. Besides, it is still necessary to verify the evolution of growth in the field, and the time elapsed from grafting to the beginning of the fruiting of the grafted seedling.

Protocols of in vitro propagation are promising strategies for Plinia spp.

In vitro micropropagation is the most practical application of tissue culture and the one with the greatest impact toward allowing conditions to obtain plants that are difficult to propagate and have long life cycles. This approach allows obtaining plants in an aseptic and controlled environment, faster than compared to conventional breeding. In vitro germination (Figure 3) can be the first step to obtain aseptic plants, which can be used as a source of propagules for micropropagation. In vitro germination of Plinia species also enables easy follow of the occurrence of polyembryony, since the number of embryos can reach up to five per seed (Figure 3C).

[Figure ID: f3] Figure 3..

In vitro germination of Plinia spp. (A) In vitro axenic germination of seeds. (B) Germination of a single plantlet from the seed. (C) Polyembryony with four plantlets germinated from a single seed.

The in vitro germination of P. jaboticaba has been performed in MS (Murashige and Skoog, 1962) culture medium (Picolotto et al., 2007; Santos et al., 2019) and agar:water medium (5-6%, w/v). However, fungal and bacterial contaminations are the most worrisome issues. Treatments of the seeds with sodium hypochlorite (Picolotto et al., 2007), and soaking for 24 hours in sterile water and antibiotics (Santos et al., 2019) have been the commonly used methods for seeds disinfection. Besides, the germination is also influenced by the temperature of culture, but not by the photoperiod (Picolotto et al., 2007), with the best germination rates at 25 °C.

Callogenesis was obtained in P. cauliflora leaf explants using MS medium with different combinations of plant growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.0, 1.0, 2.0, 4.0 mg l^-1 + 6-benzylaminopurine (BAP) at 0.0, 0.1, 0.2 mg l^-1 and naphthaleneacetic acid (NAA) at 0.0, 1.0, 2.0, 4.0 mg l^-1 + BAP (0.0, 1.0, 2.0 mg l^-1. All treatments presented callus formation, also in absence of plant growth regulators, not differing statistically (Cardoso, 2016).

Somatic embryos at the cotyledonary stage were obtained from seed explants of P. peruviana by Oliveira (2018). Pro-embryogenic masses were induced in 82.5% of explants using the two cotyledons detached and cultivated in the MS medium with 300 μM of 2,4-D and 1 g l^-1 of activated charcoal. During phase I of maturation, the most suitable medium for the conversion of somatic embryos at the torpedo stage was MS with 30 g l^-1 of polyethyleneglycol 4000 (77.5% of the formation of embryos in the torpedo stage). There was no conversion of somatic embryos into seedlings and the formation of embryos was possible up to the cotyledonary stage (3.05%), forming abnormal embryos. Anatomical studies showed the development of asynchronous somatic embryos.

Silveira et al. (2020) established a somatic embryogenesis protocol for P. peruviana using mature seeds (two separate cotyledons) as explant, inoculated in MS medium with the addition of 1000 mg l^-1 of glutamine, and 10 μM of 2,4-D. Larger embryos and in more advanced stages of development were obtained in a medium containing 60 g l^-1 of polyethyleneglycol 6000. There was no conversion of somatic embryos into seedlings and anatomical sections of the embryos revealed deleterious effects of the prolonged period of exposure to 2,4-D.

Although failing to convert somatic embryos into seedlings, the studies of Oliveira (2018) and Silveira et al. (2020) were pioneers in the development of a somatic embryogenesis protocol for Plinia spp. Thus, further studies are needed to determine the optimum exposure period to 2,4-D for the formation of somatic embryos with normal morphology, which drives conversion to seedlings.

How may biotechnology help to boost jaboticaba into the market?

The fruits of Plinia species have a high market value, good organoleptic characteristics, high nutritive content, and beneficial properties to health. Thus, there is a forecast of growth in demand for fresh consumption and use by industries. As the cultivation of Plinia spp. is practically restricted to domestic orchards and the establishment of commercial plantations demands a large amount of vigorous and uniform selected seedlings, it is necessary to develop protocols of vegetative propagation. Micropropagation seems to be the most promising technique providing seedlings on a large scale with high phytosanitary quality, regardless of the time of year.

Plastome based phylogenomic studies may be an important strategy towards understanding the taxonomic relationships among Plinia species, as well as to verify the validity of all proposed species. Also, genetic studies using molecular markers should be performed to characterize the genetic diversity of natural populations towards germplasm conservation and genetic improvement of the species. This biotechnological tool can also be used to evaluate the somaclonal variation of micropropagated seedlings since in clonal propagation the main purpose is the maintenance of the genetic characteristics of selected plants. Besides, to circumvent situations in which the multiplication from natural seeds is problematic, the encapsulation of vegetative structures into sodium alginate droplets and protocols for cryopreservation of embryos and cell cultures are also needed for the species.