Somatic embryogenesis in the commercial papaya hybrid UENF/Caliman 01 relying on plantlet production from sexed adult hermaphrodite donor plants

Abstract

Abstract: Somatic embryogenesis from explants from hermaphrodite papaya mother plants is an alternative for the production of true-to-type plants without the need for sexing. This study aimed to analyze hormonal and osmotic inducers in different somatic embryogenesis stages in the commercial hermaphrodite hybrid papaya UENF/Caliman 01. Leaf disks from in vitro shoots originated from ex vitro hermaphrodite plants were cultured in induction medium supplemented with different concentrations of 2,4-D (6, 9, 12, 15, and 18 μM) and 4-CPA (19, 22, 25, 28, and 31 μM). After 90 days, the formation of somatic embryos was verified. The 2,4-D induced the formation of light brown calli with low frequency (20%) of somatic embryogenesis. However, 4-CPA (25 μM) induced 96% of embryogenic calli, which were transferred to maturation medium (MM) and cultured for 30 days. The MM contained ABA (0.5 μM) and AC (15 g L^-1) and produced 36.6 somatic embryos callus^-1, mainly on cotyledonary stage. Cotyledonary embryos were transferred to germination medium supplemented with gibberellic acid (GA₃) (0.0, 1.44, 2.88, and 4.32 µM), and the conversion into plantlets was enhanced with GA₃ at 2.88 µM.

Key words
abscisic acid; auxins; Carica papaya L.; gibberellic acid; hermaphrodite explants; polyethylene glycol

INTRODUCTION

The culture of papaya (Carica papaya L.) is exploited by several countries, Brazil being its second largest producer and exporter, producing 1.5 million tons, in an area of 32,000 hectares, with average yield of 50 tons per hectare, second only to India, with production of five million tons. Among the main consuming countries of the fruit produced in Brazil are the United States (USA), England, Germany and Portugal (CnaBrasil 2016). Brazil generates a revenue of47.1 million USD with exports of 33.7 thousand tons a year, in which the state of Espírito Santo ranks first, averaging 12,000 tons exported per year, in which 25% are directed to the USA (Brapex 2016).

Among the hermaphrodite papaya plants of the group Solo, the cultivars Golden and Golden THB stand out. In the group Formosa, the hybrids Tainung 01 stand out worldwide and, UENF/Caliman 01 (Calimosa) and cv. Rubi Incaper 511 stand out in Brazil (Costa et al. 2013).

Sex determination is a crucial practice in the culture of papaya, and this technique is carried out during flowering, moment in which reproductive structures are visible. Plant sex can also be identified by means of molecular markers, a technique still not viable on a large scale basis (Abreu et al. 2015). Due to this hurdle, it has been recommended to plant three to four papaya plantlets per hole, aiming at future sex determination, leaving only one hermaphrodite plant after thinning the others. However, this practice leads to higher production costs, including the purchase of seeds - for example UENF/Caliman 01 (US$ 3,225.00 kg^-1) - and those related to fertilization, irrigation, weed management and roguing. Accounting the problems generated by seminiferous propagation, such as gene segregation and the absence of a practical and cost-effective method for early sexual determination, it is necessary to search for alternatives for the large-scale production of C. papaya hermaphrodite plants (Senthilkumar et al. 2014).

Biotechnological techniques have been investigated and used as alternatives to seed propagation. In that respect somatic embryogenesis (SE) has been induced in many different species; many of them crops of commercial interest (Cardoso et al. 2017, Kumaravel et al. 2017, Márquez-López et al. 2018), including papaya (Estrella-Maldonado et al. 2017, Cipriano et al. 2018). The SE relies on the exploitation of totipotency of somatic cell to be reprogrammed into embryonic pathways, leading to bipolar structures and sharing the verys same developmental stages of the zygotic embryogenesis (Oliveira et al. 2017, Yumbla-Orbes et al. 2017). And ultimately, the great potential of scaling-up the processes if coupled with liquid-based culture systems on bioreactors (Posada-Pérez et al. 2017).

Among the several factors influencing somatic embryogenesis, genotype choice is the most remarkable, since there are different embryogenic potentials for the same species. In addition to the genotype, each in vitro culture stage is precisely regulated by many factors, including the level of sucrose in the medium, the type and concentration of growth regulators, photoperiod, gelling agents, exposure time and induction and maturation media (Elmeer 2013).

Many difficulties are inherent to the development of a protocol for somatic embryogenesis, for instance, the lack of synchronization in the maturation process, appearance of anomalies, production cost, among others, making it necessary to analyzepotential embryogenic responses in vitro for each genotype. Therefore, considering the economic relevance of the UENF/Caliman 01 hybrid, the present work aimed to evaluate adult hermaphrodite tissue somatic embryogenesis controlled by different hormonal and osmotic inducing agents.

MATERIALS AND METHODS

The study was carried out at the Laboratory of Plant Tissue Culture, at the North University Center of Espírito Santo, Federal University of Espírito Santo, located in the municipality of São Mateus-ES and at the Laboratory of Tissue Culture II, at the Institute of Biotechnology Applied to Agriculture, Department of Plant Biology, Federal University of Viçosa, in Viçosa-MG.

PLANT MATERIAL

Five-month-old mother plants of the hybrid Carica papaya L. UENF/Caliman 01, F1 generation, were submitted to sexing, maintaining only the hermaphrodites that had apices pruned (breaking of the apical dominance). After six months, 0.5 cm apical segments from lateral shoots were used as explants.

IN VITRO ESTABLISHMENT AND MULTIPLICATION

Apical segments of lateral shoots were individually cultured in test tubes containing 15 mL of full-strength MS medium (Murashige and Skoog 1962), with salts, vitamins, myo-inositol (100 mg L^-1), sucrose (30 g L^-1), agar (6.5 g L^-1, Merck®), 25 μM kinetin (6-furfurylaminopurine) and 0.5 μM NAA (α-naphthalene acetic acid) (Schmildt et al. 2007). The test tubes with the explants were kept in a growth room, temperature of 27 ± 2 °C and 16-hour photoperiod, under a 90 μmol m-² s^-1 photosynthetic photon flux obtained with blue/red (3/2) LED lamps (LabPAR: LL-HR/DB-01, Lablumens®), during a period of 30 days.

Responsive explants were reculturedin flasks containing 30 mL of multiplication medium: MS supplemented with 0.5 μM NAA, 2 μM BA (6-benzyladenine) and 90 μM adenine sulfate; and kept in the growth room for 30 days and subcultured twice, at 30-day intervals. In the preparation of the culture media, the pH was adjusted to 5.7 before the addition of agar and subsequently autoclaved at 121 °C and 1.5 atm for 20 minutes.

INDUCTION OF SOMATIC EMBRYOGENESIS

For the induction of somatic embryogenesis, under aseptic conditions in a laminar flow chamber, leaf disks (five mm in diameter) were extracted from in vitro axenic shoots of UENF/Caliman 01 hybrid, and subsequently cultured in 90x15 mm polystyrene Petri dishes (Kasvi®) containing 40 mL of induction medium (IM) per plate. The IM consisted of full-strength MS medium with salts, myo-inositol (100 mg L^-1), sucrose (30 g L^-1), agar (6.5 g L^-1, Merck®), 2,4-D (2,4-dichlorophenoxyacetic acid) (6, 9, 12, 15, and 18 μM) or 4-CPA (4-chlorophenoxyacetic acid) (19, 22, 25, 28, and 31 μM).

Five Petri dishes were used per treatment, containing five explants on each plate. The explants were incubated in the dark at 27 ± 2 °C for 90 days. After this period, the percentage of calogenesis (%); formation of embryogenic callus (%) and absolute frequency of somatic embryos (SE callus^-1) were analyzed.

MATURATION OF SOMATIC EMBRYOS

The embryogenic calli were transferred to the following maturation media (MM): MS medium without growth regulators; MS medium + activated charcoal (AC, 15 g L^-1); MS medium + abscisic acid (ABA, 0.5 μM); MS medium + ABA (0.5 μM) + AC (15 g L^-1); MS + ABA (0.5 μM) + polyethylene glycol (PEG, 60 g L^-1). The cultures were maintained in a growth room with 16-hour photoperiod, temperature at 27 ± 2 °C and a 90 μmol m-² s^-1 photosynthetic photon flux obtained with blue/red LED lamps, during a period of 30 days. The following were analyzed: absolute frequency of SE callus^-1 (120 days in MM) and number of SE throughout the various development stages (globular, heart, torpedo and cotyledonary).

GERMINATION OF SOMATIC EMBRYOS

Normal cotyledonary somatic embryos were selected in the most efficient maturation medium (MS + ABA (0.5 μM) + AC (15 g L^-1)) and transferred to germination media (GM): MS media supplemented with different concentrations of GA₃ (gibberellic acid) (0.0, 1.44, 2.88, and 4.32 μM). Plates were maintained in the growth room until rooting of the embryos. The percentage of embryo germination, indicated by primary root protrusion, and percentage of normal plantlets were analyzed.

STATISTICAL ANALYSIS

In the embryogenic induction experiment, treatments were set in a completely randomized design (CRD), with five replicates and five explants per replicate. The maturation experiment was carried out in a completely randomized design with five treatments and five replicates, containing five explants per replicate. The germination experiment was performed in a completely randomized design with four GA₃ concentrations and five replicates, containing five explants per replicate.

Data on the induction of somatic embryogenesis and embryo maturation were submitted to analysis of variance and the means were compared by the Scott-Knott (1974) cluster test at a 5% significance level, using the Genes software (Cruz 2016). Exceptionally, descriptive statistics of the data were performed for the germination experiment.

RESULTS

INDUCTION OF SOMATIC EMBRYOGENESIS

Apices from lateral shoots (Figure 1a) were developed in vitro through the organogenic process in multi-stem shoots, with expanded leaves (Figure 1a-c). Somatic embryogenesis (SE) was initiated from axenic leaf disks of in vitro shoots (Figure 1c) and indirectly via a callus phase (Figure 1d) at different concentrations of 2,4-D and 4-CPA.

Figure 1
Somatic embryogenesis from axenic leaf explants of in vitro shoots of the hybrid hermaphrodite papaya UENF/Caliman 01. (a) Six-month-old mother plant. (b) Apical segments of lateral shoots. (c and d) In vitro established shoots. (e) Calli formed in induction medium (40 days). (f) Growth of friable callus. (g) Embryogenic callus induced with 2,4-D in maturation medium (MM) consisting of ABA (0.5 μM) + AC (15 g L^-1). (h) Somatic embryos in torpedo stage. (i) Somatic embryos at different development stages. (j) Normal cotyledonary embryo. (k) Embryogenic callus induced with 4-CPA in MM consisting of ABA (0.5 μM) + AC (15 g L^-1). (l) Somatic embryos in MM. (m) Normal somatic embryo with developed cotyledonary leaves. (n and o) Morphologically normal cotyledonary embryos. (p) Germinated abnormal embryo - monocotyledons. (q) Conversion of cotyledonary embryo into plantlet. Abbreviations: cot. cotyledons; ea. embryonic axis; Cse. Cotyledonary-shape somatic embryo; Hse. Heart-shaped somatic embryo; Tse. Torpedo-shape somatic embryo. Bar = 10 mm (a, b, c and d) and 1.0 mm (e-o).

Calogenesis responses from leaf explants were more efficient induced at 15 (88%) and 18 μM (100%) 2,4-D, whereas in all 4-CPA concentrations, no significant differences were verified, with means ranging from 84 to 100% (Table I). However, 4-CPA induced the highest percentages of embryogenic calli, at the concentrations of 25 (96%), 28, and 31 μM (94%), with no statistical differences among them (Table I). The highest frequency of SE callus^-1 (17.2) was obtained with 25 μM 4-CPA (Table I).

TABLE I
In vitro morphogenic responses of leaf disks from axenic shoots of the hybrid hermaphrodite papaya UENF/Caliman 01, after 90 days of culture in induction medium supplemented with 2,4-D or 4-CPA.

Auxin	Concentration (µM)	Characteristics analyzed
		Calogenesis (%)	Embryogenic calluses (%)	Absolute frequency of SE callus-1
	6	36 b(1)	4 c	1.0 d
	9	44 b	8 c	2.0 d
2,4-D	12	56 b	16 c	1.6 d
	15	88 a	12 c	2.4 d
	18	100 a	20 c	0.6 d
	19	96 a	28 b	6.4 c
	22	84 a	56 b	10.6 b
4-CPA	25	100 a	96 a	17.2 a
	28	96 a	84 a	10.0 b
	31	100 a	84 a	6.2 c

• ⁽¹⁾Means followed by the same letters do not differ statistically, at p ≤ 0.05, by the Scott-Knott mean clustering test. 2,4-D. 2,4-dichlorophenoxyacetic acid; 4-CPA. 4-chlorophenoxyacetic acid; SE. somatic embryo; MS. Murashige and Skoog.

MATURATION OF SOMATIC EMBRYOS

The maturation medium (MM) supplemented with ABA or ABA + PEG induced a low frequency of SE-callus^-1 (9.6 and 6.6, respectively) (Table II), even in embryos obtained from the 4-CPA treatment. However, in the presence of ABA (0.5 μM) + AC (15 g L^-1), after 120 days, a higher frequency of SE-callus^-1 (36.6) was observed, with an increase of 73.77% in the SE frequency response, when compared to the absence of AC in the MM (Table II; Figure 1k, 1).

TABLE II
Average absolute frequency SE callus-1 and number of somatic embryos at different development stages of the hybrid hermaphrodite papaya UENF/Caliman 01 obtained from leaf explants of in vitro axenic shoots induced with 2,4-D and 4-CPA, after 120 days of culture.

Auxinic induction SE	Maturation medium (MM)	Absolute frequency SE callus-1	Number SE at different stages
			Gse	Hse	Tse	Cse
	MS without growth regulator	4.0 e(1)	0.2 d	0.0 d	0.2 d	0.0 d
	MS + ABA (0.5 µM)	2.0 e	1.2 d	0.0 d	0.4 d	0.0 d
2,4-D	MS + AC (15 g L-1)	1.6 e	0.8 d	0.0 d	1.2 d	0.0 d
	MS + ABA (0.5 µM) + AC (15 g L-1)	2.4 e	0.6 d	0.6 d	1.2 d	0.0 d
	MS + ABA (0.5 µM) + PEG (60 g L-1)	6.0 e	0.0 d	0.0 d	0.6 d	0.0 d
	MS without growth regulator	9.4 c	1.6 d	2.6 c	4.0 c	1.4 c
	MS + ABA (0.5 µM)	21.4 b	2.8 c	0.8 d	4.2 c	1.6 c
4-CPA	MS + AC (15 g L-1)	9.6 c	5.0 b	5.0 b	6.6 b	4.8 b
	MS + ABA (0.5 µM) + AC (15 g L-1)	36.6 a	10.4 a	8.6 a	10.0 a	7.6 a
	MS + ABA (0.5 µM) + PEG (60 g L-1)	6.6 d	2.6 c	0.6 d	2.2 d	1.2 c

• ⁽¹⁾Means followed by the same letters do not differ statistically, at p ≤ 0.05, by the Scott-Knott mean clustering test. 2,4-D. 2,4-dichlorophenoxyacetic acid; 4-CPA. 4-chlorophenoxyacetic acid; ABA. abscisic acid; AC. activated charcoal; Cse. Cotyledonary-shaped somatic embryo; Gse. Globular-shaped somatic embryo; Hse. Heart-shaped somatic embryo; MS. Murashige and Skoog; PEG. polyethylene glycol; SE: somatic embryo; Tse. Torpedo-shape somatic embryo.

Embryogenic calli of the hybrid papaya UENF/Caliman 01 from 4-CPA induction and cultured in MM (ABA (0.5 μM) + AC (15 g L^-1)) showed enhanced embryo development in all stages, in addition to larger numbers of somatic embryos per callus (Table II, Figure 1 k, 1), reaching the highest average of cotyledonary embryos (7.6), when compared to the medium supplemented with only AC (15 g L^-1) (4.8) (Table II).

GERMINATION OF COTILEDONARY EMBRYOS

In the embryo germination phase, the absence of growth regulator did not promote germination. In the presence of GA₃, it was observed a 20% germination of somatic embryos at 1.44 μM and 40% at 2.88 μM, after 30 days, and conversions of 25% and 66% into plantlets, respectively (Table III).

TABLE III
Percentage of germination and plantlet conversion from cotyledonary somatic embryos of the hybrid hermaphrodite papaya (UENF/Caliman 01), obtained from MM (MS + ABA (0.5 μM) + AC (15 g L^-1), in germination medium (GM) with different concentrations of GA₃.

GM	SE germinated (%)	SE not germinated(%)	Plantlets (%)
MS without growth regulator	0	100	0
MS + GA3 (1.44 µM)	20	80	25
MS + GA3 (2.88 µM)	40	60	66
MS + GA3 (4.32 µM)	0	100	0

• ABA. abscisic acid; AC. activated charcoal; SE. Somatic embryos. GA₃. gibberellic acid; MM. maturation medium; MS. Murashige and Skoog.

DISCUSSION

INDUCTION OF SOMATIC EMBRYOGENESIS

Auxins are important in the process of embryogenic induction, since they regulate the cell cycle and promote cell differentiation (Rocha et al. 2012). In the present study, 2,4-D presented a negative effect, promoting low responsiviness in calli (Figure 1g; Table I). Also, abnormal embryos were observed when analyzing embryogenic induction in different concentrations of 2,4-D and 4-CPA (Figure 1). On the other hand, 4-CPA had a positive effect, especially at the concentration of 25 μM (Figure 1k; Table I).

The induction of zygotic embryos in Jarilla heterophylla (Caricaceae) with 2,4-D (18.09 μM) promoted a negative effect on callus production, similar to the control (0 μM), and a large number of embryos with multiple cotyledons was observed, suggesting that this auxin is probably unfit for this species under the conditions studied (Nuño-Ayala et al. 2012). However, if provided in pulses for certain periods, this phytohormone can promote cell polarity and normal embryo development (Moon et al. 2015).

The addition of auxins to the culture medium may increase the totipotency of cells with the ability to proliferate somatic embryos (Garcia et al. 2011). According to Guerra et al. (1999), both genotype of the explant donor mother plant and the auxin type play essential roles in the embryogenic competence. In the medium supplemented with 2,4-D, immature zygotic embryos of C. papaya L. were sufficient to produce highly embryogenic callus (Clarindo et al. 2008, Abreu et al. 2014).

Reports on the use of 4-CPA in somatic embryogenesis in papaya are still scarce. Nonetheless, Cipriano et al. (2018) have successfully reported the induction of cotyledonary leaves in papaya cv. Golden THB. According to these authors, auxins may trigger different induction responses in tissues of the same genotype, even among auxins with very subtle differences (presence of a chlorine atom in the carbon in position 2 of the 2,4-D phenol ring). In addition, cell response to the inducer is related to the number of receptors present on the cell surface, and may vary according to the type of tissue induced (Loschiavo et al. 1991).

The results obtained in the present work are in agreement with those reported by Cipriano et al. (2018), allowing to infer that papaya cv. Golden THB shows greater sensitivity and responsiveness to 4-CPA, as compared to 2,4-D.

By testing different auxins on embryogenic responses in immature wheat embryos, the application of 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) led to the formation of visible sites of embryogenic calli with several embryos, a pattern similar to that found for the 4-CPA induction (Miroshnichenko et al. 2013). According to the authors, although the use of 4-CPA generated a lower number of embryos, it promoted a more rapid development. The use of 2,4-D stimulated the formation of denser and more compact embryogenic calli, and the embryos developed in a more synchronized pattern, when compared to the other auxins.

The induction of cotyledonary leaves of papaya cv. Golden THB with 2,4-D presented low efficiency, unlike 4-CPA (25 μM), which provided high percentages of embryogenic calli (91.67%) and average number of embryos (65.75) (Cipriano et al. 2018). However, using 2,4-D (9.04 or 45.24 μM), Almeida et al. (2001) observed 41.7% of embryogenic calli in the hybrid Tainung 01. Sun et al. (2011), from immature seeds of papaya cv. Sunrise, obtained 68.7% callus frequency in MS medium containing 2.4-D (6.0 μM), NAA (2.5 μM) and kinetin [6-furfurylamino purine] (4.0 μM).

Somatic embryogenesis from zygotic embryos of papaya cv. Co7, with 2,4-D (9.05 μM), resulted in a high percentage (87%) of calli, 75.12% of which were embryogenic (Anandan et al. 2012). However, the authors observed a decrease in calogenesis when the 2,4-D concentration was increased from 18.09 to 72.36 μM (47.4%). Koehler et al. (2013) verified a high correlation between the low concentration of 2,4-D (9.05 μM) and the high frequency of friable embryogenic calli (74%) from leaf explants of Carica papaya L., after 30 days of culture.

In the induction of Jarilla heterophylla zygotic embryos, the presence of adenine sulphate in the previous medium had inhibitory effects on callus generation from initial explants, resulting in 14 somatic embryos, whereas in the absence of this cytokinin, 74 somatic embryos were obtained (Nuño-Ayala et al. 2012). Similar effects may have been induced in the present work. The transfer of shoots induced in adenine sulphate medium (Figure 1c) to the growth regulator-free medium and their maintenance for a certain period of time for lessening endogenous levels of this cytokinin may have contributed for the low frequency of somatic embryos of the hybrid hermaphrodite papaya UENF/Caliman 01, induced in a 2,4-D-supplemented medium.

Cells may become competent for somatic embryogenesis provided that the interaction among endogenous and exogenous factors, such as the concentration and type of growth regulator, are balanced, triggering a series of molecular events that may affect gene expression and determine the differentiation into somatic embryos (Nolan et al. 2014). It is known that in plants, auxin is distributed directly from cell to cell through the polar auxin transport (PAT) driven by auxin influx (AUX1/LAXs) and efflux (PINs) proteins. In Arabidopsis thaliana, PAT plays an important role in the formation of stem and root apical meristems during somatic embryogenesis (SE). In this regard, Estrella-Maldonado et al. (2017) found in papaya that auxin influx transport (AIT) genes (CpLAX ₁, ₂, ₃) are apparently more related to the development of somatic embryos in the torpedo and cotyledonary stages. In contrast, auxin efflux transduction (AET) genes (CpPIN ₁, ₃, ₄) appear to be related to the development of SEs at different stages (from globular to cotyledonary). Furthermore, when the mature somatic embryo is converted into plantlet, the expression of the transporter genes CpAUX₁, CpLAX₁, CpLAX₂, CpLAX₃, CpPIN₁, CpPIN₃ and CpPIN₄ become higher.

MATURATION OF SOMATIC EMBRYOS

Maturation is an important phase in somatic embryogenesis, and the addition of maturation promoters, such as polyethylene glycol (PEG), abscisic acid (ABA) and activated charcoal (AC) is crucial for the conversion of embryos into their respective stages of development (Calic-Dragosavac et al. 2010).

In the present work, the highest frequency of SE callus^-1 was obtained in ABA (0.5 μM) + AC (15 g L^-1) maturation medium (Table II). Considering AC supplementation, its effect is associated with the adsorption of toxic substances, such as phenolic compounds, among other metabolites, which are responsible for the inhibition of morphogenesis, a process that results from cell division and differentiation (Fridborg et al. 1978, Martínez et al. 2015). According to Cipriano et al. (2018), embryogenic calli of papaya cv. Golden THB in medium supplemented with ABA (0.5 μM) + AC (15 g L^-1) showed a higher frequency of SE callus^-1 and inhibition of early germination. This is associated with the fact that ABA aids in the formation and maturation of a large number of high-quality somatic embryos capable of developing into plantlets (Guerra et al. 1999). However, in the absence of ABA, the maturation phase results in poorly developed, often abnormal, somatic embryos presenting low capacity for germination and plantlet growth (Cipriano et al. 2018). ABA accumulates in the higher plants as a result of water stress (Pacheco et al. 2011) and is involved in various physiological functions, such as stomatal movement (Lim et al. 2015) and dormancy (Kermode 2005).

In papaya, transcription factors (TFs) related to the WRKY gene are involved in the response to water stress and are controlled by a family of proteins that increase TF807.3, TF43.76, TF5.242 and TF21,156 by 14.1, 19.2, 13.2, and 13.1 fold, respectively (Pan and Jiang 2015). In Solanum chacoense Bitt., the ScWRKY1 protein is expressed at almost undetectable levels during seed development, low levels in stems, roots and petals, high levels in leaves, and is strongly expressed transiently in fertilized egg cells bearing embryos at the final torpedo stage, indicating a specific role during embryogenesis (Lagacé and Matton 2004). In Gossypium raimondii and Gossypium hirsutum under abiotic stress (water and flooding), GhWRKY expression was very high during fiber development, foliar senescence, anthers and tissues (roots, stems, leaves and embryos), which implies that WRKY genes have a role in stress response (Dou et al. 2014). The expression pattern observed for ScWRKY1 in Solanum chacoense embryos at the torpedo stage suggests that transcription factors of the family may be WRKY, involved in crucial events during embryogenesis and seed development into seedlings (Lagacé and Matton 2004).

Krassimira and Alexandrova (2002) found out that DGE1 and DGE2 genes transcripts, containing a WRKY domain, related to somatic embryogenesis in Arabidopsis thaliana with possible regulatory nuclear functions. Pan and Jiang (2015) suggest that the high homology of WRKY in papaya may be related to similar functions of homologous genes in other species.

In the conifer Picea abies L. Karst., the combination of ABA and AC was beneficial, increasing the yield of cotyledonary somatic embryos, the number of genotypes forming cotyledonary embryos, and reducing the cost of embryo production. These embryos presented increased size, had larger apical regions, became more similar to zygotic embryos in shape, and showed higher percentage of epicotyl development during germination (Pullman et al. 2005).

The maturation medium supplemented with ABA (0.5 μM) + AC (15 g L^-1) from calli induced with 25 μM 4-CPA produced the highest number of embryos at all maturation stages (Table II). Anandan et al. (2012), using liquid medium supplemented with ABA (39.7 μM), obtained superior yield of embryos in the cotyledonary stage, while in the absence or lower concentrations of ABA, this yield was lower and with a high frequency of abnormal embryos.

Crocus explants (Crocus oliveri ssp.) exhibited higher proliferation and development of embryos in the cotyledonary phase in MM supplemented with IAA (11.4 μM) + thidiazuron - TDZ (9.08 μM) + ABA (378.3 μM) (Verma et al. 2016). Similarly, Ju et al. (2014) observed that the addition of AC (0.5%) in the MM increased the maturation percentage (87.52%) in Cucumis anguria L., in relation to the absence of AC (35.51%). According to Martínez et al. (2015), the supplementation of the basal MS medium with AC (0.4%), sucrose (3%) and silver thiosulfate (20 μM) was the most adequate for the production of cotyledonary embryos and subsequent germination (88.9%) in Quercus rubra.

The maturation medium with PEG 6000 (60 g L^-1) caused reduction in the development of somatic embryos at all stages (Table II). In mango, Pliego-Alfaro et al. (1996) observed that the further development from SE at the cotyledonary stage was significantly inhibited by ABA and mannitol treatments. Osmotic agents, such as high molecular weight PEG, reduce the water potential of the culture medium and caused a substantial water stress in cells.

The maturation of immature zygotic embryos of the hybrid papaya UENF/Caliman 01 in medium with PEG 3350 (60 g L^-1) resulted in higher maturation responses (83.3%) (Heringer et al. 2013). Vale et al. (2014), adding PEG (6%) to the maturation medium, obtained 40 cotyledonary somatic embryos per callus in the hybrid UENF/Caliman 01, in which the proteins in the range of 34-87 KDa were more abundant, suggesting that these proteins can be fundamental in the maturation process, acting as biochemical markers. Chagas et al. (2018) found that the increase in PEG 6000 concentrations reduced the normality of somatic embryos of papaya cv. Golden THB by approximately 54% to the maximum concentration tested (70 g L^-1), when compared to its absence.

The formation of morphological abnormalities in somatic embryos was observed in all media investigated in the present study, such as monocotyledons (Figure 1p), multi-cotyledons and fused cotyledons (images not shown). Variations like these were also observed in the development of somatic embryos of papaya cv. Golden (Anandan et al. 2012), cv. Golden THB (Cipriano et al. 2018) and cv. Golden as well as the F2 generation of the hybrid UENF/Caliman 01 (Vale et al. 2014). This is due in part to the stress caused by the culture of these embryos in sealed flasks, aggravating the lack of aeration, accumulation of ethylene and drought of the culture medium.

GERMINATION OF COTILEDONARY EMBRYOS

No somatic embryos were able to germinate in the medium without growth regulator, but in the presence of GA₃, there was low germination at the concentrations studied. The low efficiency of GA₃ is probably associated with the accumulation of growth regulators, such as ABA in papaya tissues in the maturation phase, which induces the production of LEA (Late Embryogenesis Abundant) proteins, conferring tolerance to embryo desiccation, and occasionally establishing physiological dormancy. The genes codifying these proteins are expressed in late stages of embryogenesis; however, the production of LEAs can be induced when somatic and zygotic embryos are treated with ABA (Ikeda-Iwai et al. 2002). In Acer platanoides L., the transcription of ABI5 and RGL2 proteins, which is mediated by ABA, probably blocked germination by inhibiting meristem activity and preventing procambial cell differentiation, respectively (Staszak et al. 2017).

In Arabidopsis thaliana, 51 genes encoding LEA proteins were identified and classified into nine distinct groups, in response to ABA signaling and/or low temperature (Hundertmark and Hincha 2008, Yoshida et al. 2015). Negin and Moshelion (2016) have confirmed, after treatment with ABA, which genes are upregulated, including those encoding LEAs, transcription factors and osmoprotectants. Proteins can be used as markers for somatic embryogenesis; therefore, associating embryonic stages and their changes in protein profiles with embryo development (Campalans et al. 2000).

A series of studies in plants of different species suggest that GA₃ stimulates genes that are mainly involved in elongation and cell division (Zimmerman et al. 1993). When studying hormonal transporters in plants, Park et al. (2017) found that multiple members of the NPF family, such as NPF3.1, that import GA₃ are dependent on the pH of the medium. According to Chen et al. (1987), germination is followed by increased regulation of a number of stress response genes and membrane transporter, and greening associated with the over-regulation of several genes encoding the photosynthetic components and chloroplasts. In this sense, Posada-Pérez et al. (2017) verified maximum germination (100%) of cv. Maradol in culture medium in the temporary immersion system RITA® with combination of GA₃ (2.90 μM) and BAP (0.02 μM).

In somatic embryos of papaya, low germination and callus production are observed at the base of the radicle, as reported in the present work (Figure 1o), also hyperhydric plantlets are commonly observed (Ascencio-Cabral et al. 2008). When testing gelling agents on the culture medium, these authors found an alternative to minimize the problems mentioned above, producing plantlets free of anomalies with the use of Bactoagar® (7.5 g L^-1) and obtaining 41% germination, which is superior to that obtained with Agar® (7.0 g L^-1) and Fitagel® (3.0 g L^-1): 10 and 23%, respectively. The results of the present study are in accord with those obtained by Ascencio-Cabral et al. (2008), who observed that the use of agar as a solidifying agent in the MS medium and supplementation with GA₃ resulted in low germination and formation of abnormal embryos, despite the absence of embryo hyperhydricity (data not shown).

In many embryogenic systems, SE transfer to growth media free of growth regulators increases the SE development and its conversion to seedlings (Ju et al. 2014). Embryo maturation in agar-solid medium resulted in a high germination rate (48%) when compared to maturation in the presence of gellan gum (30%), produced by the bacterium Sphingomonas elodea (Márquez-Martín et al. 2009).

Embryos require radicle stimulation to grow and, for this reason there was low efficiency with their maintenance in MS medium with absence and presence of GA₃ at the concentrations studied (Table III), suggesting that the explanation may rely on the type and concentrations of the growth regulator tested. Wu et al. (2012) observed in shoots of papaya cv. Meizhonghong highest percentage (90%) and quality of rooting in MS + IBA (indole-3-butyric acid) medium (1.44 and 2.88 μM for three and one day, respectively), prior to transfer to medium 3/2 MS + AC (0.5 g L^-1) + sucrose (5 g L^-1), and the exposure of these explants to IBA (4920.3 μM) for 10 seconds resulted in 84.7% rooting and with normal roots. Therefore, it is relevant to study the effect of other auxins on embryos of the hybrid hermaphrodite papaya UENF/Caliman 01, such as IBA, in different concentrations, exposure times in solid medium and pulses in liquid medium for a better development of the root system.

CONCLUSIONS

Embryogenic efficiency of axenic leaf explants from in vitro shoots of the hybrid hermaphrodite papaya UENF/Caliman 01 is higher when using 4-CPA (25 μM).

Maturation and conversion of the somatic embryos are higher using MS medium supplemented with ABA (0.5 μM) + AC (15 mg L^-1).

Germination of cotyledonary somatic embryos presents a higher average with GA₃ (2.88 μM).

ACKNOWLEGMENTS

Dissertation presented by the first author to the Universidade Federal do Espírito Santo as part of the requirements of the Programa de Pós-Graduação em Agricultura Tropical, to obtain the Master’s Degree in Tropical Agriculture. The authors are grateful for the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) to finance this work. The company Caliman Agrícola S/A, on behalf of Geraldo Antônio Ferreguetti, for the removal of explants from parent plants of UENF/Caliman 01.

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Publication Dates

• Publication in this collection
  19 Aug 2019
• Date of issue
  2019

History

• Received
  19 May 2018
• Accepted
  14 June 2018