Developing an in vitro optimized protocol to sweet potato landraces conservation

This study aimed to develop a protocol for the in vitro conservation of 30 accessions of sweet potato collected in the northern region of Rio de Janeiro State, Brazil, aiming to extend the period between subcultures for this species. An in vitro minimal growth experiment was established with accession UENF 1931 in a factorial arrangement consisting of four concentrations of mineral salts in MS medium and four concentrations of sucrose, at two temperatures. The others accessions were grown in medium containing 100% MS salts and 2% sucrose, which was the medium that allowed the in vitro conservation of accession UENF 1931 for a longer time period. For this second experiment, 30 accessions of sweet potato and two temperatures were used. Plant height, number of leaves, and survival rate were measured every 30 days for 12 months, in both experiments. In the last stage, the sweet potato plantlets originating from in vitro minimal growth were acclimatized and exhibited 100% survival. The recommended in vitro minimal growth conditions for the studied accessions are MS medium with 100% concentration of mineral salts and 2% sucrose, at a temperature of 27±2°C, with subculture performed every 180 days.


Introduction
The sweet potato is considered the seventh most important crop in the world and the fifth most important in developing countries (Loebenstein, 2009).In Africa and Asia, thousands of people depend on sweet potato for food safety (Zhang, Wang, Liu, & Wang, 2009).On the Latin American continent, Brazil is the main producer, with an estimated annual production of 505,000 ton.(Reetz, 2014).This vegetable crop can be used in home cooking or as a raw material in industrial processes for the production of sweets, flour, flakes, and starch, in addition to the production of biomass for biofuel (Ferrari, Guigou, & Lareo, 2013;Wang, Shi, Xia, Li, & Chen, 2013;Zhang et al., 2013).
In several areas of production, the sweet potato is a typical crop for small rural producers who are responsible for the maintenance of high genetic variability through the conservation of local varieties on their farms.However, some factors have jeopardized this so-called important form of conservation of genetic resources, e.g., the modernization of agriculture, which leads to rural exodus and contributes to the loss of genetic diversity of crops that were traditionally farmed by small farmers, such as the sweet potato.Another factor is the change in the eating habits of populations brought about by the emergence of new consumption options.In addition, landraces have been replaced by commercial varieties (Rodrigues & Santos, 2011).To prevent this loss of diversity, it is essential to preserve the germplasm so that it can be available for future use (Sá, Lédo, & Lédo, 2011).
In vitro plant culture is an alternative method for the conservation of germplasm, especially vegetatively propagated species, such as the sweet potato.This technique offers many advantages over the germplasm conservation process in the field.In vitro plant material is free from pathogens and weather conditions and risks present in the field such as theft, predation, attacks by pests and diseases, floods and droughts.In addition, the technique allows the maintenance of a large number of accessions in a small physical space, contributing to the reduction of financial costs (Withers & Williams, 1998;Cid, 2001).
One of the ways to preserve plant material in vitro is through the slow-growth or minimalgrowth technique, in which the plant metabolism is reduced, thereby increasing the interval between subcultures.To this end, alterations are made in the chemical conditions of the growth medium, such as the addition of growth regulators or a reduction in the concentrations of salt and organic components of the growth medium, as well as a reduction in the light intensity or temperature in the growth room.In this way, the labor and space necessary for the maintenance of the germplasm are reduced (Tahtamouni, Shibli, & Ajlouni, 2001;Islam, Leunufna, Dembele, & Keller, 2003;Sarwar & Siddiqui, 2004;Divakaran, Babu, & Peter, 2006;Santos, Lédo, Lédo, Souza, & Junior, 2011).Sweet potato subculture is performed on average every 90 days, and this is one of the most time-and costconsuming factors in the maintenance of in vitro collection (Teixeira & Nascimento, 1999).
Published studies on the preservation of sweet potato by minimal in vitro growth report an evaluation period of up to 90 days (Teixeira & Nascimento, 1999).However, for the creation of an in vitro sweet potato germplasm bank, several unanswered questions arise in the literature: (1) How will in vitro conditions, such as salt and sucrose concentrations and temperature, influence plant growth after these 90 days of minimal growth?(2) How long can I maintain this material in vitro?(3) What happens to the plant material after subculture?(4) What is the maximum period between subcultures?(5) How will the in vitro conditions affect the ex vitro response of plants after preservation?
This study describes a protocol to optimize the in vitro conservation of landraces of sweet potato by increasing the period between subcultures for this species.

Plant material
Twenty-eight accessions of sweet potato were collected from small farms in the northern region of Rio de Janeiro State (Brazil) and characterized by Moulin et al. (2012a and2012b), were established in vitro, kept in a growth room at a temperature of 27±2°C and irradiance of 25 µmol m −2 s −1 , provided by fluorescent lamps (OSRAM ® , daylight), under a photoperiod of 16:8 hours of light:darkness, for 90 days.Two commercial genotypes developed by Empresa Brasileira de Pesquisa Agropecuária (Embrapa), i.e., 'Brazlândia Rosada' (UENF 1997) and 'Princesa' (UENF 1994), were also used (Table 1).
For the in vitro establishment of the material, nodal segments of branches of sweet potato containing one bud each were utilized.A semisolid growth medium was used containing mineral salts of MS medium (concentration of 100%) and White's vitamins (Murashige & Skoog, 1962), 100 mg L −1 myo-inositol, sucrose at a concentration of 3%, and pH adjusted to 5.7.Next, the medium was solidified with 8.0 L −1 bacteriological agar Vetec ® and autoclaved for 15 min.at 121ºC and 105 kPa.
The experiments were conducted in the Setor de Horticultura of the Laboratório de Fitotecnia, in the Centro de Ciências e Tecnologias Agropecuárias of Universidade Estadual do Norte Fluminense Darcy Ribeiro, located in Campos dos Goytacazes, Rio de Janeiro State, Brazil.

Establishment of in vitro minimal growth conditions
A preliminary trial was conducted to establish the conditions for in vitro minimal growth, randomly using accession UENF 1931 as the model plant.In this experiment, nodal segments containing one bud from accession UENF 1931 obtained from plants established in vitro and maintained in a growth room under the aforedescribed conditions were used as explants.The experimental design was completely randomized, with a 4 × 4 factorial arrangement, which consisted of four concentrations of the mineral salts from the MS medium (0, 10, 50, and 100%), and four concentrations of sucrose (0, 1, 2, and 3%), at two temperatures (18±2°C and 27±2°C), resulting in a total of 16 treatments with three replicates.Each replicate consisted of three test tubes (25 × 150 mm) with 10 mL of medium containing one explant each.
After the explants were inoculated, test tubes were sealed with PVC film.Half of the tubes were kept in a B.O.D. growth chamber and the other half in a growth room, both under a photoperiod of 16:8 hours of light:darkness and irradiance of 25 μmol m −2 s −1 provided by fluorescent lamps (OSRAM ® ) similar to daylight.The temperature in the growth chamber was maintained at 18±2°C and that in the growth room at 27±2°C.Assessments were made every 30 days, for 12 months; plant height (mm), number of leaves, and survival rate were measured.
At 360 days, the plants of accession UENF 1931 in the treatment with 100% MS medium and 2% sucrose at both temperatures were explanted, and nodal segments containing one bud were transferred to a new medium with the same concentrations of salts and sucrose.Plant survival rate was measured after 90 days.

In vitro minimal growth of sweet potato accessions
The combination that allowed the in vitro conservation of accession UENF 1931 for the longest period was that which contained mineral salts from the MS medium at 100% concentration and 2% sucrose.This combination was then tested using all other accessions.For this purpose, nodal segments containing one bud each from plants retained in vitro under the previously described conditions were used.
The experimental design in this phase of the study was completely randomized, with 30 genotypes of sweet potato plants at two temperatures (18±2°C and 27±2°C), totaling 30 treatments, with three replicates.Each replicate consisted of three test tubes (25 × 150 mm) with 10 mL of medium containing one explant each.
After the inoculation of the explants, the test tubes were sealed with PVC film.Half of the test tubes were maintained in a B.O.D. growth chamber at a temperature of 18±2°C, and the other half in a B.O.D. growth chamber at a temperature of 27±2°C, both under a photoperiod of 16:8 hours of light:darkness and irradiance of 25 μmol m −2 s −1 provided by fluorescent lamps (OSRAM ® ) similar to daylight.
Evaluations were carried out every 30 days for 12 months; plant height, number of leaves, and survival At 360 days, the plants of the surviving accessions at both temperatures were explanted, and nodal segments containing one bud were transferred to a new medium with the same concentrations of salts and sucrose and the same experimental design.Plant survival rate was measured after 90 days.

Acclimatization of plants
The experimental design in this phase was completely randomized, consisting of 30 genotypes of sweet potato plants, totaling 30 treatments, with two replicates.Each replicate consisted of three test tubes (25 × 150 mm) with 10 mL of medium containing one explant each, maintained at 27±2°C.The medium contained 100% MS mineral salts and 2% sucrose.
After 180 days of in vitro conservation, plants of all 30 accessions tested that were approximately 9.0 cm in height were removed from the test tubes and placed in pots containing 1.5 L of the substrate Basaplant Hortaliças ® .Two pots were used for each accession, and each pot contained three plantlets.Next, plantlets were transferred to a greenhouse with a plastic cover (100 µm) and shade net 50% (Sombrite ® ).The temperature was monitored daily, and it ranged from 21 to 33ºC.Plant survival was assessed every 30 days, for 360 days.

Statistical analysis
To determine whether the data would meet the assumptions of analysis of variance (ANOVA), they were subjected to preliminary analyses to determine the normality and homogeneity of variances among treatments, for each factor (plant height, number of leaves, and survival rate), based on the Lilliefors (1967) and Bartlett, Bobko, and Mosier (1978) tests.Subsequently, the data were subjected to analysis of variance, and the means were compared according to the F test at 5% probability, utilizing SAEG 9.1 (2007) software.The variation in the parameters analyzed over time was evaluated using regression analysis.

Establishment of in vitro minimal growth conditions
Overall, the temperature of 27±2°C was more favorable for plant survival (66.29%) and provided the greatest height (2.20 cm) and lowest number of leaves (1.41) when the entire in vitro growth period was evaluated (Table 2).The plants from the treatments with total suppression of mineral salts and sucrose in the growth medium grew less (0.20 cm), produced fewer leaves (0.16) and had one of the lowest survival rates (59.82%),irrespective of the temperature.
The treatments with 100% MS medium and 1%, 2%, and 3% sucrose simultaneously displayed a greater number of leaves, reduced growth, and high survival rate and were thus the most indicative of minimal growth for the accession under study for subculturing every 360 days (Figures 1 and 2).After 360 days, the plants of accession UENF 1931 in the treatment with 100% MS medium and 2% sucrose at both temperatures were explanted, and nodal segments containing one bud were transferred to a new medium with the same concentrations of salts and sucrose.After 90 days of reculturing, it was observed that the plants at 18±2°C showed only 10% survival, whereas those maintained at 27±2°C had a survival rate of 100%.

In vitro minimal growth of sweet potato accessions
A temperature of 18±2°C resulted in the highest plant survival rate (77.06%), whereas a temperature of 27±2°C resulted in the tallest plant height (4.88 cm) and the greatest number of leaves (1.77) when the entire in vitro growth period was evaluated (Table 3).For most accessions, the greatest plant height was observed from 150 to 270 days under in vitro minimal growth at both temperatures.The most representative accessions at temperatures of 18±2°C and 27±2°C were UENF 1925 and UENF 1953, respectively (Figure 3).At both temperatures, the highest number of leaves was observed at 90 days of in vitro conservation.Accessions UENF 1932 and UENF 1962 were the most representative at the temperatures of 18±2°C and 27±2°C, respectively (Figure 3).
The percent survival was 100% for all accessions up to 180 days.After 180 days, the percent survival of most accessions started to decline, with accessions UENF 1922 and UENF 1917 being the most representative at the temperatures of 18±2°C and 27±2°C, respectively (Figure 3).

Plant acclimatization
The survival rate of the plants in the greenhouse was 100% for all accessions maintained at 27±2°C (Figure 5).

Discussion
In the treatments with total suppression of mineral salts and sucrose in the growth medium, it was observed that the plants grew less, produced fewer leaves, and had the lowest survival rates irrespective of the temperature.Therefore, mineral salts and sucrose are essential components for the growth and survival of sweet potato plants in vitro and should not be totally suppressed in the growth medium.
The highest survival rate, the greatest height, and the highest number of leaves were observed in the treatments with MS salts at a concentration of 100%, at both temperatures (except for the treatments with complete suppression of sucrose).Other researchers working with in vitro conservation of sweet potato also utilized MS salts at a concentration of 100%, despite not having tested other concentrations, such as 10% and 50% (Jarret, 1997;Jarret & Gawel, 1991a;Jarret & Gawel, 1991b;Hirosse, Creste, Custódio, & Machado-Neto, 2012; among others).The reduction of the concentration of the salts in the present study did not benefit the in vitro conservation of the studied accessions.Plants in the treatment with 100% MS medium and 2% sucrose, at both temperatures, showed the highest survival rates and the largest number of leaves.Jarret and Gawel (1991b) analyzed the effect of different levels of sucrose on the minimal growth of sweet potato.Sucrose concentrations reduced to 1.5% and 2% also resulted in higher survival rates, corroborating the results observed in the present study.However, these authors evaluated the development of plants for 90 days, unlike the period evaluated in the present study (360 days).
Plants from the treatment with 100% MS medium and 2% sucrose showed a higher number of axillary buds; i.e., shorter internodes, which generates a larger number of nodes.A higher number of nodes results in a higher number of explants and consequently, greater efficiency with respect to in vitro minimal growth.Until now, no studies have been conducted on in vitro minimal growth of sweet potato that have reported shortening of plant internodes.Some studies on the in vitro minimal growth of sweet potato utilized growth retardants such as abscisic acid (Arrigoni-Blank et al., 2014;Jarret & Gawel, 1991a), sorbitol, and mannitol (Jarret & Gawel, 1991b).Growth retardants were not utilized in this study, to reduce costs and to avoid somaclonal variation.Moreover, according to the obtained results, sweet potato requires mineral salts and sucrose in the growth medium for its survival in vitro.The reduction of these components and the use of growth retardants can result in increased mortality of sweet potato in vitro (Arrigoni-Blank et al., 2014).
The positive effect of the temperature decrease was not observed for the majority of the sweet potato accessions in this study after 360 days of in vitro growth.The temperature of 27±2°C was more efficient compared with the temperature of 18±2°C, since 90 days after the subculture of accession UENF 1931, the plants at 27±2°C showed 100% survival, whereas those maintained at 18±2°C showed 10% survival.Although Jarret and Gawel (1991b), who studied the effect of different temperatures on the minimal growth of sweet potato, reported a 50% decrease in plant growth with a temperature decrease from 21.1 to 15.6ºC, the evaluated period was only 90 days, and no subculturing was performed.In the present study, the evaluation period was 360 days, followed by another subculture, evaluated after 90 days.
Plants of accession UENF 1931 maintained at 18±2°C did not acclimatize after 360 days of in vitro conservation, resulting in plant death.However, the plants maintained at 27±2°C had 100% survival (data not shown).For the plants of all accessions acclimatized after 180 days of in vitro growth in 100% MS medium and 2% sucrose at a temperature of 27±2°C, there was 100% survival.According to Roesler, Gomes, Moro, Kummer, and Cereda (2008), sweet potato is a rustic, easily grown vegetable crop with great adaptation and high tolerance to drought.Moreover, the greenhouse temperatures (21 and 33ºC) did not vary widely in relation to the temperature at which the plants were grown in vitro (27±2°C), which is a factor that must have contributed to the high survival rate.
Although there were similar responses among all accessions studied, slight variations were observed among these genotypes.These different responses during the in vitro conservation of sweet potato may be related to the genetic traits of each accession.The results of different reactions of genotypes conserved in vitro were already reported in previous studies with Passiflora giberti (Faria, Costa, Junghans, Ledo, & Souza, 2006), Aechmea fasciata and A. miniata (Moreira, Costa, Souza, Bastos, & Rocha, 2008), among others.However, few studies have reported such differences in sweet potato (Manrique-Trujillo, Díaz, Reaño, Ghislain, & Kreuze, 2013;Arrigoni-Blank et al., 2014).
The results of the present study indicate it is possible to extend the period between subculturing of sweet potato from 90 (Teixeira & Nascimento, 1999) to 180 days, thereby reducing the working time of the curator and the amount of reagents utilized in the preparation of the growth medium, which can decrease the maintenance costs of the in vitro collection.The same growth medium can be utilized for all accessions because all of the accessions studied had a survival rate of 100% up to 180 days.In addition, the need for a temperature reduction may increase the maintenance costs of the collection, as this implies higher energy expenses to cool the environment, especially in tropical countries such as Brazil.For the sweet potato accessions evaluated in the present study, this temperature decrease was detrimental and is not necessary.
The main difference between this and other studies was that the in vitro conservation of sweet potato germplasm was evaluated for a longer time period (360 days).In addition, the survival rate after the first subculture of plants was maintained at 18±2°C and 27±2°C was presented.Although all accessions survived at both temperatures, after the subculture, the survival of explants at 18±2°C was only 10%.Thus, a temperature decrease is not recommended for the sweet potato accessions studied here.
In conclusion, it was possible to establish and maintain in vitro minimal growth of the sweet potato accessions collected on small farms from the northern region of Rio de Janeiro State.This methodology contributed to the reduction in the loss of genetic diversity of sweet potato in this region.

Conclusion
The recommended in vitro minimal growth conditions for the studied accessions are MS medium with 100% concentration of mineral salts and 2% sucrose, at a temperature of 27±2°C, with subculturing performed every 180 days.The results obtained in this study provide a technical basis for the establishment of a new in vitro germplasm bank of Ipomoea batatas.

Figure 1 .
Figure 1.Height (cm), number of leaves, and percent survival of sweet potato accession UENF 1931 conserved in vitro in 100% MS medium with sucrose at concentrations of 1%, 2% and 3% and temperatures of 18±2°C (left) and 27±2°C (right) as a function of time (every 30 days).

Figure 3 .
Figure 3. Height (cm), number of leaves, and percent survival of sweet potato plants conserved in vitro in 100% MS medium with 2% sucrose, at temperatures of 18±2°C (left) and 27±2°C (right) as a function of time (every 30 days).

Figure 4 .
Figure 4. Percent survival of in vitro sweet potato plants 90 days after the first subculture, in 100% MS medium with 2% sucrose, at temperatures of 18±2°C (left) and 27±2°C (right).

Table 1 .
Origin and characteristics of roots of 28 accessions of sweet potato collected in Rio de Janeiro State, Brazil, and two commercial genotypes grown in vitro (Adapted fromMoulin et al., 2012 a).

Table 2 .
Mean values for height, number of leaves, and survival of sweet potato accession UENF 1931 conserved in vitro for 12 months.

Table 3 .
Mean values for height, number of leaves, and survival of 30 accessions of sweet potato conserved in vitro for 12 months.