Culture media for the multiplication of wild Manihot species Meios de cultura para a multiplicação de espécies silvestres de Manihot

The cassava propagation system is slow and favors disease transmission through successive generations. Micropropagation is an alternative to overcome the aforementioned limitations, besides allowing the generation of a larger number of pestand pathogen-free plants. Therefore, the aim of the present study is to investigate the effect of culture media on the multiplication in vitro of five wild Manihot species. The experiment followed a completely randomized design, at factorial arrangement 5 (wild Manihot species) x 6 (culture media), with 11 repetitions. Explants consisted in nodal segments (91 cm long and one lateral bud) of species Manihot flabellifolia, M. tristis, M. caerulescens, M. chlorosticta and M. jacobinensis, which were extracted in vitro from the collection of wild cassava species. One segment was placed in each test tube added with 10 mL of MS media 0.01, 17N, 12A3, 4E, 8S and WPM, and kept for 90 days in growth room under 30 μmol m-2 s-1irradiance, temperature 27 ± 1 °C and 16h photoperiod. Variables plant height (cm), number of green leaves, number of senescent leaves, number of shoots, number of microcuttings, fresh and dry shoot mass, fresh and dry root mass (mg) and callus mass (mg) were analyzed. Our results showed that the culture medium 12A3 was not responsive to any of the species; however, if one takes into consideration variables plant height and number of microcuttings, this medium can possibly be used in the micropropagation of other wild species belonging to genus Manihot.


INTRODUCTION
Choosing the most appropriate culture medium is an essential factor during multiplication, given the importance of its components in the regeneration process in vitro.Physical support for the explant and the supply of all nutrients participating in the growth and development of vegetal material stand out among functions performed by the medium.There are many culture medium formulations; however, the one formulated by Murashige and Skoog (1962) is mostly widespread.It is universally known as the MS medium and has been broadly applied in studies about tissue cultures, such as the nodal segment multiplication, somatic embryogenesis induction, and embryo rescue and cultivation.
Overall, tissue culture strategies are adopted when conventional sexual and vegetative propagation systems are not satisfactory.Propagation in cassava culture can happen through sexual reproduction.However, according to Cerqueira et al. (2016), the most often used propagation method lies on stakes or manivas from the mother plant.Nevertheless, this system contributes to pest and disease transmission, with emphasis on the systemic one, to the following generations, fact that can affect culture yield.
Thus, micropropagation becomes a promising alternative in comparison to the conventional cassava vegetative propagation method, since it allows the production of phytosanitary-quality plants, among other advantages.Culture medium 4E (ROCA et al., 1991) is applied to Manihot esculenta Crantz in the explant establishment stage in vitro; media 17N (CIAT, 1982) and MS 0.01 (Souza et al., 2008) are adopted during multiplication, and medium 12A 3 (Mafla et al., 2010) is recommended by CIAT for the micropropagation of wild species belonging to genus Manihot, whereas 8S is used to conserve germplasm in vitro (CIAT, 1984).Many studies have been performed in this field and they have shown the efficiency of this technique in cassava cultures (Demeke et al., 2014;Shiji et al., 2014;Kabir et al., 2015;Mongomake et al., 2015;Anjum;Shazia, 2015).
Accordingly, cultivation in vitro also emerges as a feasible strategy to the propagation of wild cassava species of great importance for cassava breeding programs.However, studies focused on multiplication methods applied to these species have not been enough.The quite heterogeneous behavior of each of the presented species, whenever they are micropropagated, has been one of the main barriers.Therefore, studies about the adjustment of methodologies that allow efficient multiplications have become essential.Thus, the aim of the present research was to investigate the effect of culture media on the multiplication in vitro of five wild species belonging to genus Manihot.These accessions were provided by the collection in vitro of Embrapa Cassava and Fruit Culture.Plants were sectioned in flow chamber in order to get the explants, which were cut into micropiles (at approximately 1cm length, with at least one gem).Subsequently, the micropiles were inoculated in test tubes (2.5cm x 15cm) with 10 mL of the assessed culture media.Next, the test tubes containing the explants were kept for 90 days in growth room under irradiance 30 µmol.m -2 s -1 , temperature 27± 1 °C and 16h photoperiod.Culture media MS 0.01 (Souza et al., 2008), 17N (CIAT, 1982), 12A 3 (Mafla et al., 2010), 4E (Roca et al., 1991), 8S (CIAT, 1984) andWPM (Lloyd;Mc Cown, 1980)], which were autoclaved for 20 minutes at 120 °C, are presented in Table 1.

MATERIAL AND METHODS
After this period, plants were subjected to the evaluation of the following traits: plant height (PH; cm), number of green leaves (NGL), number of senescent leaves (NSL), number of shoots (NS), number of micropiles 1cm (NMP), shoot fresh mass (SFM; mg), root fresh mass (RFM; mg) and callus mass (CM; mg).All plant material was identified and placed in forced air circulation oven at 70 °C for 48 hours.Subsequently, the shoot dry mass (SDM; mg) and the root dry mass (RDM; mg) were determined.
The study followed a completely randomized design at factorial arrangement 5x6 -5 wild species belonging to genus Manihot and 6 culture media, with 11 repetitions.Each repetition was composed of one explant (micropile) placed in a test tube.Data recorded after the evaluation were subjected to Scott-Knott test, at 5% probability level.The numbers of green leaves, senescent leaves, shoots and micropiles were assessed by transforming them into 0.5 x  , in order to fulfill assumptions based on analysis of variance.Statistical analyses were conducted in the 'ExpDes.pt'package implemented in the R software, version 3.4.2(R Development Core Team, 2017).

RESULTS AND DISCUSSION
It is necessary taking into account the interaction among some factors to achieve efficient development in vitro such as the case of plant physiological status, cultivation and genotype/species conditions, since they will contribute to crop yield.Genus Manihot presents wide genetic variability (Nassar;Grattapaglia, 1986); therefore, it is possible observing quite different behaviors in the species assessed in the current study.
According to the analysis of variance presented in Table 2, species and culture media, as isolated factors, had significantly influenced (p < 0.01) most of the assessed variables, except for variable 'number of shoots', which only had significant effect (p < 0.01) on factor 'species'.On the other hand, it is essential pinpointing the significant interaction between species and means in almost all variables, although there was no interaction in variable 'number of shoots', fact that did not influence the recorded results.
The cultivation in vitro of different wild plant species led to quite heterogeneous growth, as demonstrated by the respective coefficients of variation (CV), which ranged from 12.88% to 72.46% (Table 2) in variables 'number of shoots' and 'root fresh mass', in the correct order.These results are similar to results found in some other studies.Cardoso et al. (2018) recorded CV values ranging from 6.52% and 54.43% in the multiplication of Manihot esculenta Crantz varieties in vitro, whereas Miranda et al. (2016) found CVs between 12.25% and 99.53% in the multiplication of Eremanthus incanus Less in vitro.
According to Werner et al. (2013), there are few studies related to adequate CV values of variables assessed in experiments conducted in the plant-tissue culture field.Authors of these studies state that the accuracy of experiments conducted in tissue culture can be low due to the influence of some factors such as genetic material variability and plant physiological stage, which gave birth to explant type; and the chemical and physical characteristics of culture media, luminosity intensity and cultivation temperature.
Table 3 presents the results of interaction in variable plant height, as well as allows verifying how culture media MS 0.01, WPM and 8S helped finding higher values observed for the cultivation in vitro of the assessed species.According to Dezan et al. (2012), plant development is positively influenced since the MS medium is quite concentrated in macronutrients, micronutrients, vitamins and amino acids.Some studies about cassava (M.esculenta Crantz) (Mapayi et al., 2013), yam (Dioscorea rotundata Poir.) (Simões et al., 2017), tamarillo (Solanum betaceum Cav.) (Copatti et al., 2016) and peach [Prunus persica (L.) Batsch] (Reis et al., 2012) micropropagation based on MS evidenced the effectiveness of culture medium in the development in vitro of different species.
It is worth highlighting that the composition of media MS 0.01 and 8S has lower total nitrogen concentration than the MS medium, and it can cause late growth in explants.Thakur and Kanwar (2008) using MS and WPM medium in the in vitro multiplication of Pyrus pyrifolia (Burm F.) Nakai, did not observe statistical differences in shoot length.A similar result was found by Lencina et al. (2014) who, when using the WMP, MS and ½ MS medium, also did not observe statistical differences in shoot length.However, this behavior was not observed in the present study, fact that likely indicates a normal growth relation to cassava adaptation to less fertile soil in vitro.4 refer to results of interactions between factors 'species' and 'culture media' in variable 'number of living leaves'.Culture media MS 0.01, 4E, 8S and WPM were statistically higher than media 17N and 12A 3 in species M. flabellifolia and M. jacobinensis.

Values depicted in Table
Overall, the species M. jacobinensis was statistically higher than the other assessed species, and this outcome evidences higher NLL values (15.22, 15.20, 14.37 and 13.40), which were proportional through media MS 0.01, 8S, WPM and 4E, respectively (Table 4).According to Ribeiro et al. (2014), the largest number of leaves allows the formation of auxiliary gems and of internodes in Zantedeschia aethiopica (L.) Spreng., and this process reflects the increased number of nodal segments and, consequently, it increases the multiplication rate throughout the sub-cultivation procedure.
Based on Taiz and Zeiger (2008), leaf senescence is one of the physiological explanations enabling plant survival.Culture medium WPM led to lower values in variable 'number of dead leaves', with emphasis to the species M. flabellifolia and M. tristis, which did not show dead leaves (Table 5).Means followed by the same lowercase letter in the columns and by the same uppercase letter on the lines belong to the same group in the Scott-Knott at 5% significance level.Therefore, based on this result, the medium WPM was more responsive to leaf senescence reduction.According to Flores et al. (2015), data about senescence are used to determine that largest interval possible between subcultivations, which would end up in the production of the largest number of seedlings, however without harming the physiological quality of the plant.This larger interval between sub-cultivations is also relevant for the conservation in vitro of the germplasm when it is applied to explore the maximum vigor and feasibility.
The production of nodal segments is a relevant factor for micropropagation, since it reflects the generation of new plants at each sub-cultivation (Flores et al., 2009).All the assessed species presented similar behavior when they were cultivated in media 12A 3 , 4E, 8S and WPM, (Table 6).
However, the behavior of the species M. jacobinensis and M. chlorosticta was statistically higher than the other species in medium MS 0.01.Thus, they showed the highest NMP values, 6.11 and 5.14 respectively (Table 6).This result is similar to that found by Freitas et al. (2016), who developed a micropropagation protocol for Justicia pectoralis and found that MS was the most responsive medium among the assessed media in the multipropagation of nodal segments.
It is worth highlighting that, by associating these results with those presented in Table 4, the same culture media responsible for the highest NLL values were the ones allowing the highest NMP means, namely: MS 0.01, 4E, 8S and WPM.This relation between these two variables reinforced the previously mentioned statement by Ribeiro et al., (2014), who says that the larger number of leaves implies a higher multiplication rate due to the increased formation of nodal segments.
Table 7 shows the results related to the outcomes of interaction in variable 'fresh shoot mass', which evidenced that only species M. tristis did not statistically differ in the assessed culture media.
The opposite result was observed in species M. caerulescens, which recorded higher SFM value (400.00 mg) in medium 8S.This species was followed by M. chlorosticta and M. jacobinensis that recorded values 312.86 mg and 297.78 mg in medium MS 0.01, respectively (Table 7).The composition of these media are based on MS, which also stimulates greater shoot fresh mass production in mulberry multiplication in vitro (Villa et al., 2005), since these plants absorbed more water due to the increased osmotic potential of the medium.
It is important highlighting that the increased shoot fresh mass reflects the vigor of micropropagated plants and, consequently, the production of new betterquality explants.It is also known that more vigorous plants produced in vitro imply higher survival rates in the acclimatization stage.
Based on values of variable 'shoot dry mass' shown in Table 8, only species M. flabelifolia and M. tristis did not statistically differ in the culture media factor.On the other hand, the highest SDM values (52.28 mg and 34.61 mg) were observed in species M. caerulescens and M. jacobinensis in medium 8S.
The statistical differences in shoot dry mass between treatments indicate the possibility of different accumulation of photoassimilates due to differentiated absorption of minerals by part of the genotypes (Pereira et al., 2001).Overall, culture medium 8S led to the highest SDM, NLA, NDL and SFM means.
According to outcomes of the interaction between factors 'species' and 'culture media' for variables root fresh mass (Table 9), the species M. chlorosticta recorded the highest RFM value (285.11;mg) in medium MS 0.01 and it formed the statistically highest group in comparison to the other species in this medium.However, this value did not statistically differ from the result found when this species was cultivated in WPM medium (258.22 mg).Means followed by the same lowercase letter in the columns and by the same uppercase letter on the lines belong to the same group in the Scott-Knott at 5% significance level.According to Miyata et al. (2014), an efficient root growth process is related to high nitrogen, manganese and zinc concentrations in the composition of medium MS.The greater root formation presented by heterotrophic plants grown in vitro consisted in a desirable characteristic to improve acclimatization process efficacy, despite the low efficiency of the root system (Sousa et al., 2015).Means followed by the same lowercase letter in the columns and by the same uppercase letter on the lines belonged to the same group in the Scott-Knott test at 5% significance level.

Values shown in
Table 7: Mean shoot fresh mass (SFM; mg) of five plant species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.
Means followed by the same lowercase latter in the columns and by the same uppercase letter on the lines belong to the same group in the Scott-Knott test at %% significance level.there was root formation in culture medium 12A 3 (Tables 9  and 10), which had activated coal in its composition.This result does not comply with the statement by Chapla et al. (2009), who said that this substance simulates darkness, and it can favor root system development in vitro.However, Erig et al., (2004) investigated the root growth of Pyrus communis L. in vitro and found that coal addition to the culture medium can be a beneficial strategy.Moreover, Oliveira-Cauduro et al., (2014) suggest that the presence of this substance can help the absorption of other compounds found in the culture medium, among than one can find phytoregulators, fact that can compromise root system formation.
According with the results recorded for the outcomes of callus mass interaction (Table 11) shows that the culture media 4E, 8S and MS 0.01 recorded the highest values for this variable, with emphasis to species M. chlorosticta (84.76 mg), M. flabellifolia (83.40 mg) and M. jacobinensis (82.93 mg), in the media mentioned above, respectively.Contrasting result was found for the regeneration of Eucalyptus camaldulensis in vitro (DIBAX et al., 2010), in which the culture medium WPM indicated superior callus induction.
Overall, the assessed species presented quite different behaviors, and such outcome is likely related to the variability of species belonging to genus Manihot and to differences in the production of photoassimilates.Culture medium plays a key role in cell and tissue growth responses.Moreover, its composition can be changed based on the need of each explant type and on the species under study (Torres et al., 2001).Changes in the culture medium can be made either in the basic composition (minerals and vitamins) or in the types and concentrations of growth regulators.Accordingly, Faye et al. (2015) found that the responses from the assessed variables changed based on the type and concentration of growth regulators by using different phytoregulators in organogenesis conducted in vitro.
Variables 'plant height' and 'number of micropiles' were highly discriminating for culture medium determination in each species during the in vitro culture process.According to the recorded results, and by taking into account the aforementioned variables, it is possible saying that species M. chlorosticta and M. jacobinensis presented the best performance when they were cultivated in culture medium MS 0.01, whereas the species M. caerulescens and M. tristis were more responsive in media 8S and 17N, respectively.WPM and 8S were the most effective media for species M. flabellifolia in variables 'plant height' and 'number of micropiles'.
Accordingly, differences between these data and other experiments of the same nature can also result from the specification of culture media for each species, since different carbohydrate, minerals, vitamins and growth regulator combinations can stimulate, or not, the growth of organs, tissues or cells, and plant development (George et al., 2008).
These contrasting results reinforce the hypothesis that it is not possible generalizing a single culture medium for the in vitro cultivation of wild species belonging to genus Manihot.Therefore, the importance of adjusting a nutritional medium for each one, or at least to one group of species, is evident.
The experiment was conducted between November 2016 and February 2017 in the Tissue Culture Laboratory (LCT) of the Advanced Biology Center (NBA) at Embrapa Cassava and Fruit Culture (CNPMF), in Cruz das Almas County, Bahia State, Brazil.Accessions characterized as Manihot flabellifolia Pohl; M. tristis Müll.Arg; M. caerulescens Pohl; M. chlorosticta Standl.and M. jacobinensis Müll.Arg were used in the present study.

Table 1 :
Composition of culture media 4E, 17N, 8S, MS 0.01, 12A 3 and WPM, which were used in the multiplication of five wild species belonging to genus Manihot.

Table 3 :
The highest PH value (15.13cm) was recorded for species M. chlorosticta when it was cultivated in culture medium MS 0.01.This value did not statistically differ from the value observed in medium WPM (14.49).Mean plant height values (PH; cm) recorded for five species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.
Means followed by the same lowercase letters in the column and by the same uppercase letter on the line belong to the same group in the Scott-Knott test at 5% significance level.SV = Source of variation; DF¹ = Degree of freedom due to loss of treatments for RFM and RDM variables; DF² = Degree of freedom due to loss of treatments for CM variable; MS = middle squares; ns = non-significant; **and* = significant at 1% and 5% probability level, respectively, in the F test by ANAVA.

Table 5 :
Mean number of dead leaves (NDL) of five plant species belonging to genus Manihot in six nutritional media in the 90 th cultivation days in vitro.

Table 4 :
Mean number of living leaves (NLA) of five species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.

Table 6 :
Mean number of micropiles (NMP) from five plant species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.
Table 10 for the variable 'root dry mass' evidenced that the highest value (24.64 mg) was recorded for the species M. chlorosticta in medium WPM.However, this number did not present statistical difference from the response recorded for M. jacobinensis in the same medium, but it did differ from the numbers shown by the other species.It is worth highlighting that

Table 9 :
Means of root fresh mass (RFM; mg) of five plant species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.followed by the same lowercase letters in the columns and by the same uppercase letters on the lines belong to the same group in the Scott-Knott test at 5% significance level.

Table 8 :
Mean shoot dry mass (SDM; mg) of five plant species belonging to genus Manihot in six nutritional media at the 90 th cultivation days in vitro.
Dec. 2018 Agrotecnologia, 42(6):598-607, Nov/Dec.2018 Means followed by the same lowercase letter in the columns and by the same uppercase letter on the lines belong to the same group on the Scott-Knott test at 5% significance level.

Table 10 :
Means of root dry mass (RDM; mg) of five plant species belonging to genus Manihot in six nutritional culture media at the 90 th cultivation days in vitro.