Enhancement of germination, hyperhydricity control and in vitro shoot formation of Vasconcellea stipulata Badillo Mejoramiento de la germinación, control de la hiperhidricidad y formación de brotes en Vasconcellea stipulata Badillo

Vasconcellea stipulata has great commercial importance because of its enzymatic activity and as a source for genetic impro- vement of papaya since it is resistant to the papaya ringspot virus. However, due to its low regeneration by seeds and limited knowledge of its genetic and pharmaceutical properties, this species is not widely cultivated. For propagation, in vitro culture of seeds has been used to address this problem, but hyperhydricity, a physiological disorder, mainly expressed in the developing embryonic axis and specifically associated with this species, is a significant constraint. In order to obtain elite material for culture of V. stipulata , the aim of this work was to increase germination, to control hyperhydricity in embryos and to evaluate the potential to induce morphogenic responses, i.e., shoot formation. Our results showed that it is possible to increase germination up to 53% under in vitro conditions within a short period in the presence of hydrogen peroxide. In addition, hyperhydricity was significantly reduced (50%) in vitro when gibberellic acid concentrations were included on a 1/2 Nitsch and Nitsch nutrient medium, resulting in approximately 80% recovery of viable seedlings. Finally, other plant growth regulators were evaluated and found to trigger shoot formation in axillary buds as well as induce the formation of callus in leaf sections derived of seedlings. Enhancement of germination, hyperhydricity and shoot formation of V. stipulata


Introduction
Vasconcellea stipulata B. (toronche) represents a genetic resource to be used for the improvement of common papaya due to its resistance to the papaya ringspot virus disease (Magdalita et al., 1997;Drew et al., 1998). This and other different species and varieties of the genus Vasconcellea are also important due to their potential to confer desirable characteristics to babaco (Vasconcellea × heilbornii 'Babaco'), i.e. phytosanitary problems, cold tolerance and organoleptic characteristics (Guerrero & Castro, 1999) of interest to the nutritional and pharmaceutical industries.
Furthermore, this species has commercial importance as a source of papain (Guerrero & Castro, 1999). This proteolytic enzyme, present in the Caricaceae, shows in V. stipulata enzymatic activity up to 17 times higher than that reported in Carica papaya (Scheldeman, 2002). However, despite its attributes this species is threatened by habitat loss due to deforestation and the conversion of native forest to cropland or pasture (IUCN, 2003).
The generative form in several Vasconcellea species shows limited responses due to seed dormancy, low germination rates and long-term germination up to 240 days with high variability of germination responses between sites (Jiménez et al., 1998;Scheldeman, 2002). Additionally, losses due to vulnerability of the sarcotesta to insects, pathogens and fungal attack are significant (Badillo, 1993). Therefore the establishment of efficient protocols is necessary for multiplication of new individuals, germplasm conservation and production of seedlings with better agronomic traits.
In this report, the specific aims were 1) To present some alternative protocols to increase the percentage of germination with the use of pre-germination treatments, 2) To reduce hyperhydricity tissue by modifying the culture medium and the application of gibberellins, 3) To stimulate shoot regeneration using growth regulators. The latter concerning sprouting of V. stipulata under in vitro conditions is reported here for the first time.

Materials and methods
Determination of viability. The presence and viability of embryos were compared from three provenances: Loja, El Oro (Ecuador), and Ayabaca (Perú), using the triphenyl tetrazolium salt test (TZ) (ISTA, 2005).

In vitro germination.
To evaluate the effect of pre-germination treatments, seeds from Loja were used due to the proximity and availability of plant material. The elimination and/or weakening of the sclerotesta were assessed by the application of different concentrations of chemicals and exposure times: hydrogen peroxide (10, 50, 100 % for 20, 30 and 40 min), sulfuric acid (20, 40, 80 % for 10, 15 and 20 min) and sodium hypochlorite (1, 3, 4 % for 15, 20 and 30 min). The concentrations of each chemical are not equal, because the effect of each is different. A total of 27 treatments and a control were applied. Table 2 shows the nine best results of all treatments in the results section. The seeds were then washed to remove residues of each of these substances by submerging them in water for 24 hours and were disinfected with 70% ethanol for 20 sec, followed by 1% sodium hypochlorite for 5 min, and then cultured in MS (Murashige and Skoog, 1962) medium under a photoperiod of 12 hours and photon flux density of 57 µmol -2 s -1 (white fluorescent light 40-W General Electric F40D-EX) and temperature of 21±2 °C, for a period of six months.
Control of hyperhydricity in germinated embryos. During germination, a high percentage of embryonic axes and seedlings showed hyperhydricity symptoms; this caused a substantial loss of plant material. In order to avoid or reduce this constraint, the embryos were extracted from seeds submerged for 24 h in water and then cultured in vitro. To disinfect the seeds the same procedure as for in vitro germination was performed. Embryo extraction was followed by liquid immersion in H2O2, (10 Vol.) for 1 min., and embryos were then cultured in MS and NN (Nitsch and Nitsch, 1969) nutrient media at different concentrations plus gibberellic acid (GA3) to evaluate embryo hyperhydration. Both media contained 2% sucrose and were solidified with 0.7% agar (Bacto TM Agar, BD). The light regime, lamps and photon flux density were the same as for in vitro cultured seeds.

Statistical analysis.
A factorial design was established for statistical analysis. Each treatment for germination, hyperhydricity and regeneration included five units (individual), five repetitions (flasks) and three replicates (total re-design). Data were registered periodically every five days. Variance analysis (ANOVA) was followed by comparison of the groups' means using the Duncan test at the p = 0.05 level using the R software (R Development Core Team 2012).

Results and discussion
Presence of embryos and viability. Seeds from Ayabaca showed the highest percentages of embryos and viability compared to those from El Oro and Loja (figure 1 a, b). Significant differences in the amount of empty seeds (without embryo) were found in the material collected at Loja, El Oro and Ayabaca (table 1). The high percentage of empty seeds may be due to the existing hybridization in the Vasconcellea genus, according to Kyndt et al. (2005 a, b) who stated that hybridization is common among species of the genus Vasconcellea, with evidence of introgression of V. cundinamarcensis into V. stipulata, both under natural conditions, in the province of Loja (Horovitz & Jiménez, 1967) and in controlled conditions in Venezuela (De Zerpa, 1980). The material from Loja evidenced the greatest amount of fruit with incomplete and non-viable seeds, possibly because this is the center of diversification and hybridization of highland papayas (Scheldeman, 2002).  Results from 75 seeds/treatment after 40 days. Statistically significant differences are indicated with different letters, with a significance level of 0.05 in the Duncan test. *The best nine treatments presented in this table were exposed to 20 minutes hydrogen peroxide and sodium hypochlorite, and 30 minutes to sulfuric acid, more a control.
Germination. The use of hydrogen peroxide increased the germination rate (figure 1c), evidencing significant differences in comparison with the other pre-germination treatments (sulfuric acid and sodium hypochlo-rite) (table 2). According to Scheldeman (2002), the presence of sulfuric acid affects the structure of the embryo and the sarcotesta, limiting responses. By contrast, hydrogen peroxide enabled maximum germination, equivalent to 52.6% by placing the seeds at a concentration of 100% for 30 minutes, after 40 days. This percentage was significantly higher than the control treatment and the results reported by Jiménez et al. (1998) and Scheldeman (2002) where germination was about 0-5% and 32% over 174 days, respectively. The application of hydrogen peroxide has been used to promote germination in several species (Dolatabadian & Modarres-Sanavy, 2008). Although its mechanism is not understood (Klein et al., 2008), it has been mainly attributed to a strong oxidant effect on organic matter (Moreno et al., 2007) coupled with the destruction of some inhibitors present in the outer layers of the seed, including phenols and other compounds, thus enabling gases and moisture to reach the embryo.
According to our results and several other studies, the low germination rates in the species of this genus is not a result of viability loss due to storage or to origin sites of V. stipulata; instead, it would be more attributable to the high site-specific variability existing in the genus (Horovitz & Jiménez, 1967). Carica and Vasconcellea seeds are very similar in structure (Badillo, 2000) and both groups require treatment to promote germination. In Vasconcellea the often-irregular germination could be improved by removal of the sarcotesta and application of GA3 (Scheldeman, 2002). In Carica papaya the sarcotesta and inhibitors present in the fruit can prevent germination, but the effect can be reduced by the application of gibberellic acid and/or potassium nitrate (Pérez et al., 1980;Yahiro & Oryoji, 1980).

Embryo germination and hyperhydricity control.
Despite having obtained a relatively high in vitro germination percentage in full seeds by the use of hydrogen peroxide, this was surpassed by isolated embryo culture. The highest germination percentage (80%) was obtained with ½ strength of NN salts in the culture medium in the presence of 1.44 µM GA3 (figure 1d). This is consistent with other reports about the application of GA3 promoting the germination of intact seeds in V. stipulata, V. cundinamarcensis and V. x heilbornii (Scheldeman, 2002). Pérez et al. (1980) also reported that GA3 significantly promoted germination in C. papaya seeds accelerating the transport of nutrients via the endosperm.
Hyperhydricity produces an abnormal anatomy in seedlings of many species, especially in young leaves and hypocotyls that appear swollen and translucent (Acram et al., 1996;Ziv, 1991) and is extremely frequent in seedlings and tissues of V. stipulata (figure 1e). Many factors cause this effect, including a high concentration of nutrient salts or nitrates (Ziv & Ariel, 1992;Ivanova & Standen, 2008). Thus, in C. papaya and some Vasconcellea species, the use of ½ MS salts (De Winnaar, 1988) and/or media with low nitrate levels as NN and woody plant medium (Jordán, 1986;Jordán and Piwanski, 1997) allowed survival and good quality of the plants, the same as in V. pubescens. Current results in V. stipulata apparently showed a lower hyperhydricity in the seedlings developed in presence of both, full MS nutrient medium and in more diluted media (NN, ½ NN salts), reaching levels of hyperhydricity between 6.7 to 13.3%. Another factor that can influence hyperhydricity is the type of agar (Ziv, 1991). In this case the Agar TM Bacto helped to keep hyperhydricity low, with only 13.3% of tissue affected (table  3). A similar result has been reported by Marga et al. (1997) and Ascencio et al. (2008), although the responses depend mainly on agar concentration as well as its preparation (Loreti & Pasqualetto, 1986).
Therefore, to establish a protocol for tissue culture in this species, it is recommended to use seeds from Ayabaca, Peru and El Oro, Ecuador, due to their high percentage of viability and germination. In addition, to start growing from seed or embryo, 1/2 NN with low concentrations of gibberellins can be used, as this favors a high percentage of germination and maintains controlled tissue hyperhydricity. These results could be used not only for growing V. stipulata but also for the in vitro culture of C. papaya, since the latest results show that even this crop has high percentages of hyperhydricity in leaves and roots of material from the somatic embryo's mature cotyledon (Clarindo et al., 2008, Koehler 2013.

Morphogenic responses.
The morphogenic responses of the various organs of V. stipulata are summarized in table 4. A wide range of inductive responses was observed in nodal segments: sprouting of axillary buds with up to 4 new buds per explant and profuse induction of callus. A relatively low level of NAA (on the order of 0.5 µM) and a similar concentration of BAP induced indirect shoot regeneration from leaves. According to results for C. papaya (De Winnaar, 1988) and in C. pubescens, the multiplication from axillary buds is similarly achieved in the presence of 0.1 mg l -1 BAP, 0.1 mg l -1 GA3 and 126 mg l -1 phloroglucinol (Jordán, 1992).
Reports on babaco showed shoot formation in leaf explants, although not on the leaf lamina but predominantly on the pre-existing nodular structures arranged in the central venation of the leaf (Jordán & Piwanski, 1997). However, for V. stipulata (this work) the nodular structures did not express any morphogenic responses under the various plant growth regulator combinations tested. The shoots exhibited a compressed structure, as it has also been reported for other Caricaceae (Mondal et al., 1994). Growth can be triggered subsequently by subculture on media with the application of 1.44 µM GA3 only.
Callus formation and regeneration of new tissues were low in leaf tissues. The opposite occurred with the hypocotyl where dedifferentiation of tissues was evident in most combinations of growth regulators, but these were not morphogenic. In other species and hybrids of the same family the formation of embryogenic callus was reported when cultured in medium with high concentrations of 2.4-D (Chen et al., 1991;Fitch, 1993).

Conclusion
Significant differences in V. stipulata seed viability between the three provenances were observed. The highest percentage of viability was observed in seeds from Ayabaca. The evaluation of different pre-germination treatments, determined that hydrogen peroxide at 100% promotes seed germination up to 53%. The culture of isolated embryos in 1/2 NN medium increases germination and controls hyperhydricity. Finally, to promote the production of shoots, nodal segments should be grown in 1/2 NN medium supplemented with NAA and BAP.  Results from 75 embryos/treatment, after 40 days. Statistically significant differences are indicated with different letters, with a significance level of 0.05 in the Duncan test.
Instituto de Biotecnología/Universidad Mayor. We also thank Mrs. Andrée Goreux for correction of the manuscript.