Effect of adenine sulphate, casein hydrolysate and spermidine on in vitro shoot multiplication of two banana varieties (FHIA-21 and PITA-3)

1 Laboratoire Central de Biotechnologies (LCB), Centre National de Recherche Agronomique (CNRA), KM 17 Route de Dabou, 01 BP 1740 Abidjan 01, Côte d’Ivoire. 2 Laboratoire de Physiologie Végétale, UFR Biosciences, Université Félix Houphouët-Boigny, 22 BP 582 Abidjan 22, Côte d’Ivoire. 3 Programme Palmier à huile, Centre National de Recherche Agronomique (CNRA), Station de Recherche de La Mé, 13 BP 989 Abidjan 13, Côte d’Ivoire.

second place after cassava (Manihot esculenta Crantz) in sub-Saharan Africa where they provide diet to millions of people (FAOSTAT, 2005).Plantains account for about 32% of total Musa production, from mostly Central and West Africa (Lescot, 2008).It represents an essential source of nutrients for millions of people, particularly in tropical and subtropical regions, as well as a cash crop in many developing countries.
Bananas are multipurpose plants with parts that can be used in various ways, depending on the species.Nutritionally, banana's fruit is rich in carbohydrate, vitamins (A, B, and C), and potassium (Aurore et al., 2009).
Despite the importance of bananas and plantains, the crop is threatened by many pests and diseases (Viljoen, 2010).Based on the high contribution of bananas and plantains to food security and human's health, there is great need for optimization of their production.
Basically, bananas are cultivated by conventional methods and in vitro cultivation.Banana propagation through the conventional method of using young shoots (suckers) or part of the tuber is not an ideal method.This method takes a long growth period and prone to various diseases (Matsumoto et al., 2003).Moreover, 5 to 10 suckers can be obtained per plant over a year.The traditional clonal propagation method appears to be unable to supply the increasing demand for disease free and healthy planting materials of banana.In order to increase conventional propagation and to avoid pathogens related constraints, in vitro approach has been considered (Tripathi, 2003).Methods developed using this approach offers more effective and better controlled condition for banana cultivation.
Positive role of different growth additives such as adenine sulphate, casein hydrolysate and spermidine has been reported extensively in the in vitro propagation of several species (Walia et al., 2007;Sanghamitra and Satyabrata 2011;Siwach et al., 2012).Earlier, it was reported that spermidine is essential for shoot multiplication in cucumber (Vasudevan et al., 2008), sugarcane (Shankar et al., 2011) and Wrightia tomentosa (Joshi et al., 2014).
Additives have been used in banana micropropagation media.However, there is no study on their specific effect.Hence, the present study was conducted to understand the influence of these growth additives such as adenine sulphate, spermidine and casein hydrolysate in the in vitro shoot multiplication from shoot-tip explants of two banana varieties.

Explant source
Suckers (Figure 1a) as explants source were collected from the banana collection of Azaguie Research Station of National Agronomic Research Center (CNRA) of Côte d'Ivoire.All the experiments were conducted at the Central Laboratory of Biotechnology of CNRA at Adiopodoume.

Selection of explant and surface sterilization
Healthy meristematic shoot tip from sword suckers were initially washed thoroughly with soap water and then were surface presterilized with 15% (v/v) calcium hypochloride containing few drops of 0.01% tween-20 for 10 to 15 min.In the laminar air flow cabinet, shoot tip were sterilized with ethanol (98% v/v) for 5 min followed by three time rinsing in sterile distilled water.Then, explants were again sterilized with commercial bleach (3.8% of active chloride) containing few drops of 0.01% tween-20 for 20 min.At the end, shoot tips were rinsed thoroughly with sterile distilled water to remove any traces of commercial bleach.

Influence of different cytokinins on shoot multiplication
For shoot induction, explants of 2 cm long (Figure 1b) were placed in 200 mL glass bottles (Figure 1c) containing 30 mL of induction medium consisted of Murashige and Skoog (MS, 1962) (Sigma-Aldrich Chemie Gmbh Munich, Germany) medium supplemented with BAP (5 mg /L) (Sigma-Aldrich Chemie Gmbh Munich, Germany) and sucrose (30 g/L).The pH of the media was adjusted to 5.7 prior to adding agar (7 g/L).One month later, microshoots were isolated and inoculated in media supplemented with different cytokinins BAP, Kin (Sigma-aldrich Chemie Gmbh Munich, Germany) and 2-iP (Sigma-aldrich Chemie Gmbh Munich, Germany) at various concentrations (1.0, 2.0, 3.0, 4.0 and 5.0 mg/L) for shoot proliferation.After 4 weeks, mean number of shoots per explant and shoot length of shoot per explant were recorded.All the cultures were kept on dark at 25 ± 2°C.

Shoots multiplication in MS media supplemented with different additives
In order to increase shoot multiplication rate, adenine sulphate (Sigma-aldrich Chemie Gmbh Munich, Germany), casein hydrolysate (Sigma-aldrich Chemie Gmbh Munich, Germany) and spermidine (Sigma-aldrich Chemie Gmbh Munich, Germany) were added to the multiplication media.Microshoots were isolated and cultured in 500 mL glass bottles containing 150 mL of shoot multiplication media.This multiplication medium consisted of MS medium supplemented with adenine sulphate (Ads), casein hydrolysate (CH) at various concentrations ranging from 0 to 50 mg/L and spermidine (Spd) (50 to 200 mg/L).These components were individually or in combination added to the medium, with the best concentration of cytokinins in order to determine their individual and combined effects on shoot induction and multiplication.After 4 weeks, the mean number and the mean length of shoots were recorded.

In vitro rooting of microshoots
For root induction, microshoots were transferred into MS medium containing indole butyric acid (IBA; Sigma-Aldrich Chemie Gmbh Munich, Germany) or naphthaleneacetic acid (NAA ; Sigma-Aldrich Chemie Gmbh Munich, Germany) (0.5 mg/L) in combination with 1 mg/L of BAP or Kin or 2-iP.Two concentrations (25 and 50 mg/L) of casein hydrolysate were individually added to the most suitable cytokinin-auxin combination to enhance rooting.After three weeks of culture, rooting frequency, mean number of roots, and mean lenght were recorded.

Acclimatization of regenerated plants
In vitro plants with well-developed roots and three new leaves were carefully removed from culture vessels and washed thoroughly with distilled water to remove the remaining gelled media.Plants were then transfered to plastic bag containing five different substrates: S1 (compost), S2 (forest soil), S3 (sand), S4 (forest soil and sawdust from dead tree in 1:1 ratio), and S5 (sand and sawdust from dead tree in 1:1 ratio).Cultures were transferred to greenhouse.Three weeks later, rate of surviving plants was calculated.Pseudostem growth (length and circumference) and the appearance of new leaves (number, length and width) were also observed.After one month in greenhouse, cultures were transfered to field and survival rate was assessed two months after.

Culture conditions
All explants were cultured on different types of media and transferred to growth room in dark for shoots multiplication or under 16 : 8 h (light : dark) photoperiod at 25 ± 2°C and with light intensity of 20 µmol m -2 s -1 for rooting.

Statistical analysis
Experiments were carried out using completely randomized design.Each treatment was repeated three times with ten replicates per repetition.Each replicate consists of one explant per glass bottles.Data were registered four weeks after inoculation and were analyzed using the analysis of variance (ANOVA).Differences among treatment means were compared by using Least Significance Difference (LSD) test at 5% probability level.Data statistical analysis was carried out using STATISTICA 7.1 version.

Influence of different cytokinins on shoot mutiplication
Microshoots cultured onto MS medium supplemented with various cytokinins were able to regenerate multiple shoots of the two banana varieties.The mean number of shoots per explants and the mean length of shoot in each tested medium are shown Table 1.Amongst various concentration, BAP (3 mg/L) gave the highest mean number of shoots per explants both for FHIA-21 (6.00 shoots) and PITA-3 (5.80 shoots) (Figure 1d and e).The longest induced shoots from shoot-tip explants were obtained in the presence of 4 mg/L BAP.The mean shoot length was 3.40 cm (FHIA-21) and 3.57 cm (PITA-3).

Shoots multiplication in MS media supplemented with different additives
Significant differences were observed when Ads, CH and   2).
Results showed that all additives supplemented individually or in combination, influenced shoot multiplication significantly.Supplied individually, Ads (25 mg/L), CH (50 mg/L) and Spd (100 mg/L) improved shoot multiplication in variety FHIA-21, while Ads (25 mg/L), CH (25 mg/L) and Spd (200 mg/L) were found to improve shoot multiplication in PITA-3.Additives combination was found to be better than their individual effects (Figure 1f).Dealing with FHIA-21 variety, the highest shoots mean (12.50 cm) per explants were observed on MS medium supplemented with Ads (25 mg/L) in combination with CH (25 mg/L) and Spd (100 mg/L).In respect with PITA-3 variety, Ads (25 mg/L) in combination with CH (50 mg/L) and Spd (200 mg/L) gave the highest shoots average (11.12 cm).Additives in combination had significant positive effect on shoot elongation (Figure 1f).The higher concentration of Spd (200 mg/L) was found more appropriate for shoots elongation.The highest mean shoot lengths were 3.35 cm (FHIA-21) and 2.97 cm (PITA-3) (Table 2).

In vitro rooting of microshoots
Except control, all media induced roots and the best results were observed on medium supplemented with NAA (0.5 mg/L) in terms of number of root per shoot with 11.20 in FHIA-21 and 13.80 in PITA-3.While, in terms of root length, IBA (0.5 mg/L) was found to be the best with 4.26 cm (FHIA-21) and 4.65 cm (PITA-3).
Addition of CH was found to be effective in enhancing the shoot number and length per explant (Table 3 and Figure 1g to h).In FHIA-21, the number of roots per explants was high in the medium supplemented with NAA (0.5 mg/l) + CH (50 mg/L); an average number of 20.80 roots and average root length of 6.67 cm were obtained.Whereas, in PITA-3, medium supplemented with NAA (0.5 mg/L) + CH (25 mg/L) was found to be the most suitable with 22.40 number of roots and 6.39 cm root length average (Table 3).

Acclimatization of regenerated plants
One month acclimatization in greenhouse showed that survival rate that ranged from 96 to 100% in both varieties and with all substrates.Addition of sawdust from dead tree to forest soil Mean values within a column followed by the same letters are not significantly different at p < 0.05 according to least significance difference (LSD).
Table 3.Effect of different concentrations of casein hydrolysate (CH) in combination with NAA and IBA number and mean length of roots after three weeks culture.or sand improved plantlets growth though different response on compost substrate was significantly higher.Pseudostem growth (length and Mean values within a column followed by the same letters are not significantly different at p < 0.05 according to least significance difference (LSD).circumference) was less important on sand substrate for the two varieties compared to the other substrates (Table 4 and Figure 2a).The mean (number, length and width) of leaves produced on compost substrate was also significantly higher than on others substrates.Six weeks later in the greenhouse, plantlets were finally transferred to the field (Figure 2b) and 2 months later the survival rate of the plants was 100%.

DISCUSSION
Various types and concentrations of cytokinins were used to study their effect on shoot multiplication using shoot tip explants of two banana varieties (FHIA-21 and PITA-3).Amongst the cytokinins, BAP is the most widely used, most effective and affordable for the proliferation of multiple shoots (Johnson and Manickam, 2003).BAP performance over other cytokinins has also been reported for some banana varieties.In our study, BAP (3 mg/L) was found to be suitable cytokinin for shoot proliferation.This result is in accordance with those obtained in banana varieties Dward and Poyo (Asmare et al., 2012;Kalimuthu et al., 2007).The same result was reported by Bikram and Bikram (2016) who stated that 3 mg/L BAP gave a maximum number of shoot buds.Similar results have been reported in banana with 4 mg/L BAP (Muhammad et al., 2007;Shiv et al., 2014).In addition, Shirani et al. (2011) mentioned that BAP (5 mg/L) was optimal for shoot proliferation as well as for shoot elongation from excised scalps of banana cultivars.Inclusion of Ads and CH in culture medium improved the frequency of multiple shoot production.The results of this study were similar with earlier observations made in Citrus reticulata Blanco (Siwach et al., 2012).In the study, 25 mg/L of Ads produced the highest average number of shoot 8.50 (FHIA-21) and 7.00 (PITA-3) shoots per explant.Similar results were reported by Sanghamitra and Maiti (2011) who stated that inclusion of Ads (25 mg/L) in the culture medium improved the frequency of multiple shoot production in Chlorophytum arundinaceum (Liliales : Liliaceae).Polyamines have been reported to play a vital role in cell division and differentiation (Yamada et al., 1986;Basu et al., 1989) and to help in the regulation of plant growth and development (Tisi et al., 2011).In the current study, 100 mg/L of spermidine in the medium increased the shoot multiplication in FHIA-21 (9.12 shoots per explant), while 200 mg/L of spermidine gave maximum number of shoot in PITA-3 (8.87 shoots per explant).Spermidine is essential for shoot multiplication, as reported in cucumber (Vasudevan et al., 2008), sugarcane (Shankar et al., 2011) and W. tomentosa (Joshi et al., 2014).Supplementation of CH to culture medium was effective in shoot multiplication.The number of shoots was found to be enhanced with 50 mg/L in FHIA-21 and 25 mg/L in PITA-3.Similar results have also been found with CH in Anogeissus pendula, Anogeissus latifolia (Saxena and Dhawan, 2001) and in Crataeva nurvala (Walia et al., 2007).Media components such as amino acids have demonstrated a profound effect on tissue culturing systems of several species (Benson, 2000;El-sharabasy et al., 2016).The mixture of amino acids like CH is frequently used as sources of organic nitrogen in culture media.On the other hand, it has been found that the combined effect of Ads, Spd and CH was more suitable for shoot multiplication when used in culture medium than their individual effects.
Rooting is a crucial step.Success of the protocol also depends on frequency of root induction (e.g., number roots and root length).In the present study, all media used were able to induce roots and exhibited good response in terms of number and length of roots produced.The inclusion of casein hydrolysate improved the number and length of roots.These results corroborate with those of Shereen et al. (2016) who found that casein hydrolysate was more effective for rooting in terms of number and length of roots in Phoenix dactylifera L. In contrast to this finding, Marvin et al. (2012) showed that casein hydrolysate did not have any significant influence on root production in Hyoscyamus niger L.
Transfer of in vitro plantlets to the ex vitro environment is extremely important as it can result in significant loss of propagated material.Loss could be the fact that, plantlets were produced under high humidity and low light intensity conditions.
In this study, acclimatization was successfully carried out in the greenhouse.The survival rate varied from 96 to 100% for all the tested substrates.Similar type of response was observed in terms of survival rate with different substrates mixture by various authors.One hundred percent of survival rate was obtained with the mixture soil : sand : farm yard manure (FYM) (2:1:1) in banana cv.Grand naine (Shahnawaz et al., 2014) and with sand, soil and vermicompost in banana cv.Matti (Lohidas and Sujin, 2015).In Eurycoma logifolia plants grown in potting media, jiffy 7 showed 100% survival (Muhammad et al., 2015).In the current study, compost was found to be more suitable for acclimatization process followed by the mixture of sawdust from dead tree and forest soil.Although, compost is a very good substrate, it is expensive and could not be accessible to small farmers.Its substitution by local substrates as a mixture of sawdust from dead tree and forest soil or sawdust from dead tree and sand could be more profitable to small farmers.In this study, all acclimatized plants were finally transferred to field conditions and grew normally in the natural environment.No phenotypic variability was observed in plants in this experiment.

Conclusion
The study showed advantages of the inclusion of Ads, CH and Spd in in vitro micropropagation of two banana varieties using shoot tip explants.A highly efficient micropropagation system, capable of sustainable multiplication of shoots, was obtained for two banana varieties.Inclusion of Ads, CH and Spd is most effective for shoot multiplication.MS medium supplemented with

Figure 1 .
Figure 1.In vitro shoot multiplication and rooting of two banana varieties.(a) Sword shaped suckers of banana.(b) Healthy meristematic shoot tip.(c) Initiation of healthy meristematic shoot tip on MS + 5 mg/L BAP medium.(d-e) Shoot multiplication on MS + 3 mg/L BAP medium after 4 weeks.(f) Shoot proliferation on MS + 3 mg/L BAP + Ads + CH + Spd medium after 4 weeks.(gh) In vitro rooting of regenerated shoots on MS + 1 mg/L 2-iP + 0.5 mg/L NAA + CH medium after 3 weeks.
alone or in combination (Table within a column followed by the same letters are not significantly different at p < 0.05 according to least significance difference (LSD).

Figure 2 .
Figure 2. Hardening of in vitro rooted plants in greenhouse and transplantation in the field.(a) Plantlets on different substrates (S1) compost, (S2) forest soil, (S3) sand, (S4) Forest soil + sawdust from dead tree, (S5) sand + sawdust from dead tree.(b) Acclimatized plants in the field after two months of transplantation.

Table 1 .
Effect of cytokinins on in vitro multiple shoots regeneration of two banana varieties cultured on MS medium after 30 days of culture.

Table 2 .
Different adenine sulphate, casein hydrolysate and spermidine concentrations effects on shoot multiplication of two banana varieties after 4 weeks of culture.

Table 4 .
Effect of different substrates on plant growth after one month of acclimatization of the two banana varieties