In Vitro Long-Term Cultures of Papaya (Carica Papaya L.).

We present the data on proliferation corresponding to 10 years of continuous incubation in vitro of papaya shoots, and propose a reliable method for long-term micropropagation for papaya, using two types of explants: Microshoots from somatic embryos, and from axillary buds of papaya. Three different media were assayed. The proliferation medium (PPRM) allowed to maintain papaya shoots under continuous proliferation during 20 years, maintaining a consistent behaviour. Most of the shoots developed in PPRM rooted during the incubation, and after acclimated easily, maintaining the morphological characteristics of the parental plants, owering and setting fruits normally. The PPRM medium consist in MS medium supplemented with NAA (0.1 mg l -1 ), BA (0.5 mg l -1 ), GA 3 (0.5 mg l -1 ) and Adenine sulphate (40 mg l -1 ). The average multiplication rate was higher than 20 shoots per explant along the long-term assay. The elongation medium (PELM), was designed to recover shoots with a poor growth, and allowed the development of high quality shoots ready for rooting, and consist in a MS basal medium supplemented with NAA (0.1 mg l -1 ), Kin (0.5 mg l -1 ) and GA 3 (1 mg l -1 ). The rooting medium (PROM) was designed to induce high quality roots from non-rooted shoots and consist in a half strength MS medium plus IBA (1mg l -1 ). On PROM, agar can be exchanged for expanded vermiculite. Acclimation took place inside an acclimatization tunnel under progressive hydric stress. After 4 weeks, the plant recovery rate was 90% for plants maintained under continuous proliferation during ten years.


Introduction
Papaya (Carica papaya L.,) is an economically important fruit crop species belonging to family Caricaceae cultured worldwide in tropical and subtropical areas due to the nutritive and medicinal value of his fruits. World production is estimated in 1329320 tonnes, India, Brazil, Mexico, Dominican Republic, Indonesia and Nigeria being the major producers (FAOSTAT 2018).
Papaya plants can be propagated vegetatively by classic methods such as cutting (Reuveni and Shlesinger 1990) and grafting (Chandler 1958; cited by Allan et al 2010), but these methods are inappropriate for long scale production and normally papayas are propagated by seeds, but again this method is problematic due to the high heterozygosis of the seeds and the dioecious character of the papaya, resulting in strong differences in yield, fruit quality and pest and disease tolerance (Saker et al. 1999).
Micropropagation by tissue culture offer advantages over conventional propagation methods and it has been widely applied to papaya species. Different approaches have been follow such as axillary shoot culture (Drew 1988(Drew , 2003 In vitro reliable methods for papaya micropropagation adequate to be used during several years were also required for germplasm preservation in vitro of elite genotypes/mother plants, able to maintain elite lines of pathogen-free plants obtained through meristem cultures, after breeding processes or even to maintain an economically viable collection of mother plants, protected from environmental stresses or suitable for plant transport across frontiers.
All the methods developed until date have been successful in different degree, depending on papaya cultivars and physiological conditions of the plants, but it is usual that these protocols do not work properly when clonal lines must support continuous culture for long periods of time, as normally happen with transgenic or elite cell lines. After some months of incubation, it appears a progressive degeneration of the papaya vitro-shoots, resulting in limited or null growth, dwar ng, defoliation, abnormal callus proliferation, loose of rooting capabilities, apical necrosis, degeneration and nally the shoots died.
Studies (Larkin and Scowcroft, 1981 depending on the plant genotype and the culture approach and conditions, these changes can preclude further stages of the in vitro plant development or even in the nal product at the end of the in vitro culture, generating plants with undesirable or useful ploidy changes, somaclonal variations, epigenetic variations and morphological or functional changes at different levels ( owers, fruits, size, shape, color).
Micropropagation through development of axillary buds is considered the in vitro plant regeneration system with the lower level of genetic instability and somaclonal variation (Clarindo et al. 2008).
There are few studies about in vitro long-term assays in plant species: Chaudhuri and Jha (2008) work during 12 years with Ipecac (Cephaelis ipecacuanha) to maintain mother plants under conditions of reduced growth, succeeding in the regeneration of viable and stable plants from this medicinal species.
Other species such cherimoya (Annona cherimola) have been also maintained in vitro during 250 subcultures (more than 20 years) in our laboratory, maintaining the shoot multiplication rate but loosing most of the rooting capabilities (Encina, unpublished results). To date, we do not found even in the last In this article we propose a reliable method which allowed the maintenance on active proliferation and full development for long time periods of in vitro cultures of papaya, overcoming the above indicated problems, with the nal goal of recovery high quality plants of papaya.

Material And Methods
Plant material and disinfection.
Micropropagation of papaya (Carica papaya L.) was initiated from 2 years old mature papaya plants obtained from seeds of the type ´Solo´, growing at glasshouse at IHSM "La Mayora" (CSIC-UMA), located in Malaga, Spain. Two types of explants were used: a) Regenerated shoots obtained from somatic embryos of papaya obtained following the procedures developed by Fitch et al. (1994), and b) In vitro micropropagated shoots obtained from axillary nodal sections.
This long-term micropropagation study starts at 1998, and the papaya cultures were maintained under continuous proliferation during 20 years, data were scored for 13 years (1998-2010) and a plant stock was maintained in proliferation until 2017 in PPRM medium.
The axillary nodal sections used as explants, were obtained from actively growing young shoots, induced by local application of a solution of BA (100 mg l − 1 ), as sticky gelled drops, over the axillary bud areas of the main shoot, after the elimination of the main apical meristem of the mother plant. Nodal explants were surface sterilized for 20 min by immersing in 0.5% sodium hypochlorite solution containing a few drops of Tween-20, rinsed three times with sterile distilled water (5 minutes each), and cultured under aseptic conditions. Culture media and culture conditions. All media consisted of MS salts formulation (Murashige and Skoog, 1962) supplemented with (mg l − 1 ): thiamine-HCl (100), pyridoxine-HCl (50), nicotinic acid (50), glycine (200), myo-inositol (100), sucrose (30000) and agar (8000) (Sigma, A-1296). The rooting medium also consist on MS salts but at half strength. The pH of all the culture media was adjusted to 5.7 before autoclaving. For all experiments, 25 ml aliquots of medium were distributed into 150x 25 mm test tubes, covered with polypropylene tops and autoclaved for 20 min at 121°C and 1.05 Kg cm − 2 . One explant was cultured per tube. Cultures were incubated at 25 ± 1°C under a 16-h photoperiod with a light intensity of 45 µmol m − 2 s − 1 (400-700 nm) PAR (Photosynthetic Active Radiation) provided by cool white uorescent tubes (F40 tubes Gro-lux, Sylvania).

Culture initiation.
Both types of shoot explants, obtained from somatic embryos and from axillary nodal sections, were cultured during two years on ve media based on MS mineral formulation differing on the supplements added (C1, C2, PPRM, PELM, PROM). 100 explants of each type were cultured in each medium. These media were designed for active growth of the papaya vitro-shoots, searching for different balances between axillary proliferation, shoot elongation and rooting. Shoot proliferation and elongation were regarded as quality parameters being our main objective the production of high quality plantlets and/or shoots adequate for rooting, acclimatization and recovery of a high number of normal papaya plantlets. After evaluation of the results obtained during the two rst years, and never detecting signi cant differences for growth parameters between both types of explants we decided to select as source of plant material the shoots obtained from nodal sections in order to extend the assays for a longer period of time. Also. shoots explants regenerated from somatic embryos were maintained in culture for three more years in PPRM medium, to obtain data about their development in vitro (data not showed). Data obtained during the last 8 years correspond exclusively to shoot explants developed from nodal section explants.
The preliminary assays allowed us to design a proliferation medium PPRM able to generate an active growth of multiple shoots or rooted shoots, in a pattern of rosette.

Shoot elongation.
After preliminary assays using as explants small shoots of transgenic papaya (already in culture for 3-4 years) sometimes these shoots were unable to develop correctly, showing dwarf shoots without roots growing in clusters or individual non-rooted shoots with null growth, we design a medium in order to induce shoot elongation and produce shoots or plantlets with enough quality to be rooted or acclimated. This medium includes gibberellic acid and a softer cytokinin such as KIN instead BA to modulate the sprouting of axillary shoots.
Shoots for in vitro rooting experiments were obtained from actively growing shoots from clusters or elongated good quality shoots. Apical shoots (2 cm long) with a minimum of 3 fully expanded leaves were used for rooting. We studied the effects on rooting when MS basal medium was supplemented with 1mg l − 1 IBA, a dose obtained after some preliminary tests.
The last rooting assays for root development include the substitution of agar by inert substrates such as expanded vermiculite, able to act as physical support for the shoots under rooting. The percentage of rooted shoots and the number and length of roots were recorded every 8 weeks.
Foliar mineral composition analysis.
Values for mineral composition of chlorotic and non-chlorotic papaya leaves, were determined following the protocols of MAPA (1986). Phosphorus total through spectrophotometry with Vanadate-Molybdate reactive. Boron through spectrophotometry using Azomethine H. Nitrogen total through digestion and distillation. Calcium, Magnesium, Potassium, Iron, Copper, Zinc and Manganese through atomic absorption spectrophotometry. The leaves, chlorotic and non-chlorotic, were collected from shoots growing in PPRM medium. The values obtained for non-chlorotic and chlorotic leaves were compared with adequate values for mineral composition of papaya leaves in vivo.
Root system morphology analysis.
The root system morphology of plantlets in vitro rooted in each media (PPRM, PELM, PROM) was analysed using a developmental model (Rose, 1983). Roots produced directly from the base of the plants were referred to as primary or rst-order roots, those arising from the rst-order as second-order roots, and so on. Roots were removed from the substrate by soaking them in water, after which the roots were gently washed and excess water was removed with paper towels. Detailed measurements of root length were made for ve plantlets rooted in each media. Roots were separated into branching orders and the length of the roots in each order measured using video images (Digital Image Analysis system IBAS-2000 (IPS)).
The following morphometric parameters were determined: number of adventitious roots, number of roots of each order, and length of the roots present in each order, and compared between media. The plantlets used in this analysis belonged to subculture number 62, beyond the 60 subcultures which micropropagation data are shown in this work due to the destructive nature of the method applied to analyse the root structure.

Plantlets acclimation.
Micropropagated plantlets were thoroughly washed in tap water and transplanted into polyethylene seedbed trays with 51 cells (4 cm wide x 4 cm deep) containing a mixture of autoclaved peat: vermiculite (1:1). Potted plantlets were maintained inside a polyethylene tunnel with 90% initial relative humidity. The average temperature was 25ºC. Afterwards, the plastic cover was temporary and gradually removed every day, in an open-close cycle increasing daily the time with the plastic cover open, reducing drastically the RH for a limited period of time and recovering the initial RH afterwards by closing the tunnel. In this acclimatization system plantlets were subject to controlled shocks of hydric stress to activate growth and development of roots and shoots. At the end of the experiment the %RH was about 70%. During the experiment, plants were periodically watered and fertilized with ®Ficote (Scott O.M., Spain) containing 15%N, 6%P, 12%K, 2%Mg, 0.22%Fe, 0.08%Mn, 0.03Zn, 0.01Cu. During this period the PAR (400-700 nm) at the top of the plants was in average 200-300 µmolm 2 s 1 and the photoperiod of 12/12. A total of 30 plantlets were acclimatized, ten obtained from each media used during ten years (PPRM, PELM, PROM). Survival rate, shoot length, number of leaves were recorded after 2 months of acclimation and compared between the plantlets obtained from each media.

Somaclonal variation analysis
Due to the early loss of the original mother plant, and that at 1998 it was a lack of availability of reliable and sensitive molecular markers (EST-SSR) to detect somaclonal variations on papaya plants after multiple subcultures in vitro, we cannot offer any studies of veri cation of the "trueness-to-type" of the papaya progenies after 120 subcultures, we just evaluate the morphology and owering of the acclimated plants, and the Nuclear DNA ploidy level at the end of assay. Ploidy analysis by ow cytometry The ploidy level of twenty micropropagated papayas was determined by estimating the relative DNA content using ow cytometry (Ploidy Analyser PA-I; Partec GmbH, Münster, Germany). For analysis, 0.5 cm 2 of young leaves or tips of papaya shoots were chopped with a razor blade for 30-60 s to release nuclei (Galbraight et al., 1983) in a Petri dish containing 0.4 mL of nuclei isolation buffer (commercial Partec CyStain UV precise P, high resolution DNA staining kit 05-5002, extraction buffer). The homogenate was ltered through a 50-lm nylon mesh (Partec 50-lm CellTrics disposable lter), and subsequently nuclei were stained with uorescent dye (commercial Partec CyStain UV precise P, high resolution DNA staining kit 05-5002, staining buffer, about 1.6 mL). Finally, the samples were analysed after 30 s of incubation. Carica papaya type "Solo" (2n = 2x = 18) was used as an external standard. The nuclear DNA ploidy level of the samples was determined by using channel values corresponding to the average G 0 /G 1 peaks of sample and standard plants. The peak relative to the standard nuclei was set to channel 50. Three independent repetitions were performed, with over 4,000 nuclei being analyzed in each.

Statistical analysis.
All data were analyzed using SPSS software package (version 19.0; SPPS INC., Chicago, IL, USA). Normally distributed variables were analyzed by one-way ANOVA, using a HSD-Tukey test in the post-hoc analysis for comparisons between different culture medium. Plant survival percentages were analyzed by Generalized Linear Models using Logit as the link function and Binomial as the probability distribution.

Results And Discussion
In our preliminary tests the best results on papaya shoot proliferation were obtained either when low levels of BA (0.5 mg l − 1 ) plus low levels of NAA (0.1 mg l − 1 ) or when high concentrations of Kinetin (1 mg l − 1 ) plus relatively high concentrations of NAA (2 mg l − 1 ), were present in the medium, but this last combination, used by Litz and Conover (1977) induce after few subcultures an excessive callus proliferation and poor quality shoots. So, we opted by use 0.5 mg l − 1 of BA plus with 0.1 mg l − 1 of NAA in our proliferation media (PPRM). In our culture conditions papaya nodal explants were established in vitro without problems with a low level of contamination by microorganisms and bud sprouting of nodal explants was over the 85%.

Micropropagation.
The results obtained during the two rst years of incubation showed minimal differences between both types of explants (Table 1), but also was detected a clear and progressive loss of quality in both media used as control, C1 and C2 (Fig. 1), treatments were eliminated after 2 and 3 years of incubation respectively, because the shoots and plantlets showed progressively clear signs of degeneration (defoliation, lack of growth, necrosis, death) and nally we were obliged to discard both controls due the minimal number of survival explants in culture. The other media PPRM, PELM and PROM do not show the problems of growth and development occurring in the control media (Table 1, Fig. 1). Table 1 Comparison between micropropagation data obtained for explants issued from axillary shoots of mature papayas and shoots obtained from regenerated somatic embryos. Average data corresponding to the rst 2 years (12 subcultures). Data recorded every 8 incubation weeks. Different letters indicate signi cantly differences by HSD-Tukey at α = 0.05 in each parameter between different culture media and initial explant.
It is clear that the proliferation media (PPRM) promotes a proliferation rate, with the axillary shoot growing in a cluster pattern, signi cantly higher (24 shoots/explant) than other media assayed, such as The elongation medium PELM offer a good rate of axillary shoots development also ranking high in growth of leaves, which give to the cultures an aspect of very good quality. The rooting medium PROM give the best results on induction and development of roots (Table 1, Fig. 1).

Long-term micropropagation assays
Along the ten years of culture the data obtained for all proliferation parameters showed an acceptable homogeneity during the incubation in PPRM, PELM and PROM media, with the exception of the 4th and 8th years in which all the cultures and parameters especially the number of axillary shoots promoted in PPRM and PELM decreased notoriously, for unknown reasons. Figure 2 shows the results (No. of axillary shoots, Length of axillary shoots and No. of leaves) obtained along the long-term process of micropropagation.
We were able to con rm the ploidy stability of ours in vitro papayas progenies after 20 years incubating in PPRM medium. Through Flow Cytometry, we detect that the diploid level (2n = 2x = 18) of the mother plant remains diploid all along the long-term culture. The analyzed in vitro plantlets obtained at the end of micropropagation process are also diploid (2n = 2x = 18). (Fig. 3).

Proliferation
As we can see in Fig In general, PROM medium is better for root induction and development, and PPRM and PELM media are better for shoot development (Fig. 4). The advantage of PPRM is that in just one step can regenerate plantlets of good quality, reducing costs and shortening the time in vitro. These results make this medium PPRM an excellent option for maintenance of active collections of elite cell lines of papaya (transgenic new genotypes, selected breeding lines, mother plants, classic or endangered genotypes).
Additionally, we maintain shoot explants regenerated from somatic embryos in incubation for three more years in PPRM medium, and the nal data scored of ve-year-old cultures still showed similar developmental values (data not shown) to the values recorded for shoots developed from nodal sections after 5 years of incubation.
Foliar mineral composition analysis.
Along the long-term assays, some chlorosis problems with an unknown origin, were detected in the leaves of papaya when were incubated in PPRM medium (Fig. 5). Some authors as Hidaka et al. (2009) also detected chlorosis in leaves when papaya explants were incubated in media with high doses of BA and NAA (4 mg l − 1 ), origin that we must discard in our studies, due the low levels (≤ 0.5 mg l − 1 ) of these growth regulators supplementing the media PPRM. Trying to explain this chlorosis, we carried out foliar analysis over non-chlorotic and chlorotic leaves collected from shoots growing in PPRM medium and compare the adequate values for mineral composition of papaya leaves in vivo with the in vitro chlorotic and non-chlorotic leaves ( Table 2). Table 2 Data obtained in foliar mineral composition analysis of chlorotic and non-chlorotic papaya leaves obtained from in vitro plantlets after 4 weeks growing in PPRM. As control, adequate values for mineral composition of in vivo papaya leaves (Tamimi et al. 1997) are showed. The iron is present in leaves with high values at the beginning of the period of incubation and at the end of the incubation period decreased dramatically just when the leaves start to fade showing discoloration and chlorosis. We do not know the reason for this decrease of iron, but perhaps could be due to the exhaustion of the iron reserves in the culture medium, due to the huge uptake of iron for these fast growing clusters of papaya plantlets, or/and by the light degradation of the iron chelate making di cult the uptake by the plantlets, or could be due to some kind of interaction or inhibition caused by the very high level of Zinc found in non-chlorotic in vitro leaves. Castillo et al. (1997), indicate that a mix of iron chelates (EDDHA plus EDTA) improved shoot proliferation in papaya, and after some assays changing the type of iron chelate we cannot con rm this increase in shoot proliferation, perhaps because we never supplement the culture medium with both types of iron chelates together (EDTA and EDDHA), we just detected that changing the standard iron chelate EDTA by EDDHA in the mineral formulation MS, that EDDHA-Ferric form delayed for some time the discoloration and chlorosis of papaya leaves respect the EDTA-Ferric form normally used in MS formulation. Another possibility for discoloration and chlorosis could be a de ciency of magnesium as can be inferred by the low level detected in non-chlorotic and chlorotic leaves.

Type of papaya leaves
Elongation.
The PELM medium was designed to recover and develop most of the axillary shoots until a size adequate for rooting. Small shoots obtained from proliferating clusters or regenerated from callus, normally too short to be useful for rooting, can be developed using this medium until reach the suitable size to be considered a high quality shoot adequate for rooting. In PELM a percentage of shoots develop roots Rooting.
The percentage of rooting in PPRM uctuates along time between 84% and 38%, the lowest values corresponding to the years in which the general growth and development was poor (years 4th and 8th), in average a 63.1% of shoots develop roots (Fig. 6). In the case of PELM the rooting ranged between 60% and 15%, and in average a 35.7% of shoots rooted. In PROM medium, the rooting rate ranged between 84% and 52%, showing in average a 70.3% of success. In general, the root induction and development is better in PROM medium but the values corresponding to PPRM are also good enough to compete with the obtained with PROM. These results suggest that an inert substrate such as expanded vermiculite is a very good option for growth and development of roots in papaya vitro shoots, but the rooting in PPRM in just one step, also appear as a very good method to obtain plantlets able to be acclimated with success, due to the shortening of the incubation time and the lower costs. The vermiculite protocol appears more interesting for rooting recalcitrant shoots or for shoots lacking roots after incubation in PPRM, both rooting systems are clearly complementary and can be used together to obtain the maximum percentage of plantlets ready for acclimation (Fig. 7).
Root system structure analysis.
As we can see in Table 3 the higher root number for rst and second order roots correspond to proliferation medium (PPRM) with signi cant differences respect PELM, no differences between PPRM and PROM were detected on number of rst and third order roots. Respect the root length the best results correspond to PROM, with a global growth for all orders of roots scoring clearly higher, compared with the results obtained for the rest of media assayed (PPRM and PELM).  The results showed small differences respect the root number between PPRM and PROM media. In PPRM the number of second order root is higher than in PROM, but considering the root length the PROM medium is clearly superior than the PPRM medium. Even if the rooting induction rate is not too different for both media, the differences in root quality (root length) are important on plant recovery, but surprisingly we do not detect differences on acclimation and survival rate at transplanting between these two treatments. Apparently, both types of roots are functional and allow the plantlets growth and develop normally after transplanting. Only during the rst month of growth at glasshouse the plantlets obtained from PROM seems to develop quicker than the originated from PPRM but at the end of the second month these differences disappear and both types of plants showed similar size and development.
The morphology of the shoot in explants incubated and developed in PELM is also different from the obtained when explants are incubated in PPRM, in general PELM shoots in vitro are longer and harder, mostly without axillary development and growth generally as individual shoots, in contrast with the rosette pattern of growth showed by the shoots incubated in PPRM.
No differences were detected in survival rate, ranging in the 90% in all the media assayed. Best results in development correspond to plantlets obtained from explants obtained from PPRM and PROM media. These plants showed a signi cant difference in the average shoot length and leaf number respect the plants incubated in PELM (Table 4). . We also reported some thick roots, but in general for all media studied normal roots well branched are frequent (see Table 3) even in a medium with agar substrate and when plants with these roots were acclimated and transplanted the survival rate was high (90%), higher than When we change the rooting medium and the substrate to MS liquid plus 1 mgl − 1 IBA in expanded vermiculite we reach a 100% success in acclimation, even working with shoots maintained for years in vitro.
Acclimation allowed nally the recovery of plants after long-term incubation in vitro, showing a normal morphology and development, owering and fruiting normally after one year (Fig. 8).

Conclusion
We present a reliable method for maintenance and development of papaya plantlets in vitro starting from different sources of explants, able to preserve during more than twenty years in vitro actively growing papaya cell lines, maintaining the original ploidy level of progenies, just incubating the shoot explants in PPRM medium, and also we offer alternative methods to control e ciently morphogenesis (elongation, rooting, acclimation) of papaya plants during long periods of time. We suggest the use of PPRM medium to obtain a continuous proliferation of papayas and also as one-step method to produce full plantlets with good rates of success.    Flow cytometry traces for: A) Carica papaya type "Solo" used as mother plant (2n = 2x = 18). B) Micropropagated papaya after 120 subcultures (2n = 2x = 18).

Figure 4
In vitro papaya plantlets growing in different media: A) PPRM B) PELM C) PROM.

Figure 5
Chlorotic leaves on in vitro papaya plantlets after 4 weeks incubation in PPRM.