Effects of ethylene inhibitors , silver nitrate ( AgNO 3 ) , cobalt chloride ( CoCl 2 ) and aminooxyacetic acid ( AOA ) , on in vitro shoot induction and rooting of banana ( Musa acuminata L . )

Significant increase in shoot regeneration, leaf chlorophyll content and rooting occurred when silver nitrate (AgNO3), cobalt chloride (CoCl2) or aminooxyacetic acid (AOA) were added to banana culture medium. The highest numbers of shoots per explants shoot length and leaf surface area was obtained when media were supplemented with 10 mgl -1 AgNO3. Number of shoots per explants increased three fold, shoot length and leaf surface area increased by an average of 4.5 and 2 cm 2 , respectively, in comparison to control. CoCl2 and AOA had less promotive effects on shoot generation with maximum shoot number per explant and shoot length achieved at 15 mgl -1 . Rooting of banana shoots in vitro was enhanced by these compounds. The highest number of roots per shoot (21.7) and the longest roots (12.68 cm) were observed when rooting media was supplemented with 10 mgl -1 AgNO3. For CoCl2 and AOA the maximum rooting occurred in media supplemented with 15 mgl -1 , although roots number and root length were lower than those achieved by AgNO3. Considerable increase in leaf total chlorophyll content occurred in shoots grown on media containing AgNO3 and AOA. The largest increase in leaf chlorophyll content (120%) was noted when shoots were grown in the presence of 10 mgl -1 AgNO3. This was followed by AOA which increased chlorophyll content by 35%. CoCl2 had no significant effect on leaf chlorophyll content. These findings suggest that application of ethylene inhibitors, particularly AgNO3, to culture media may be useful for improving in vitro growth performance of banana cultures.

Clonal propagation of banana (Musa acuminata L.) via in vitro culture techniques have been extensively studied using different explants sources, basal media components and phytohormones levels (Al-amin et al., 2009;Ngomuo et al., 2014;Devendrakumar et al., 2013).However, to the best of our knowledge, no information on the effect of ethylene or ethylene inhibitors on in vitro culture of banana is available despite the recognized positive effects of these inhibitors on plant regeneration and growth in vitro (Kumar et al., 1998).In the present investigation, we compare the efficacy of the ethylene inhibitors silver nitrate (AgNO 3 ), cobalt chloride (CoCl 2 ) and aminooxyacetic acid (AOA) to assess their effects on shoot and root development of invitro cultured banana plants.

MATERIALS AND METHODS
Shoot tip explants of banana (Musa acuminata L.) (cultivar Grand Nain) were excised from young suckers grown in pots.Explants were surface sterilized with 75% ethanol for 50 s followed by 30 min with 40% commercial bleach (Clorox 5.75% NaOCl) to which few drops of Tween-20 were added.After complete washing with sterile distilled water, explants were trimmed to final size of 10 to 15 mm in the laminar flow cabinet.For culture initiation, explants were cultured in screw-capped glass vessels containing 30 ml of initiation media composed of MS basal salts (Murashige and Skoog, 1962) supplemented with sucrose (40 g l -1 ), thiamine (0.1 g l -1 ), benzylaminopurine (BAP) (12 µM), indole-3-acetic acid (IAA) (3 µM) And cystein HCl (40 mg l -1 ).Medium was solidified with 2 g l −1 gelrite (Sigma Chemical Co., St. Louis) and its pH was adjusted to 5.8 before autoclaving at 121°C for 15 min.All cultures were incubated at 25°C under 16 h photoperiod for 4 weeks.Light intensity was 35 µmol s -1 m -2 .To evaluate the influence of AgNO3, CoCl2 and AOA on shoot multiplication and growth, banana shoot tip explants from in vitro initiated cultures were transferred to multiplication media.Multiplication medium contained MS basal salts, sucrose (40 g l -1 ), thiamine (0.1 g l -1 ), BAP (20 µM) and cystein HCl (40 mg l -1 ) supplemented with different concentrations (0 to 25 mg/l) of AgNO3, CoCl2 and AOA individually.Cultures were arranged in a randomized block design with 10 replicates per treatments (3 explants per culture bottle) and incubated at 25°C under16 h photoperiod for 4 weeks.Light intensity was 35 µmol s -1 m -2 .After 4 weeks of culture, the number of shoots formed per explant, shoot length(cm) and leaf surface area (cm 2 ) were determined.
For the determination of leaf chlorophyll content, 0.25 gfresh leaf material of individual treatments was extracted in 5ml 80% acetone (v/v) and total chlorophyll content was determined according to Lichtenthaler (1987).For evaluating the effect of AgNO3, CoCl2 and AOA on in vitro rooting, uniform banana shoots formed on multiplication media were excised and transferred to rooting medium.Rooting medium consisted of MS basal slats, sucrose (40 g l -1 ), 2-isopentenyladenine (2iP) (5µM) and indole-3-butyric acid (IBA) (0.1 µM) supplemented with different concentrations (0 to 25 mg/l) of AgNO3, CoCl2 and AOA individually.Medium was solidified with 1.8 g l −1 gelrite and its pH was adjusted to 5.8.Cultures, consisting of 10 replicates per treatment, were incubated at 25°Cunder16 h photoperiod.After 4 weeks the number of roots formed per shoot and root lengths (cm) were estimated.All data were expressed as means of all replicates ± standard error.Means were separated by Duncan's multiple range test (DMRT) (Duncan, 1955) at 5% significance level.

RESULTS
The effect of the ethylene inhibitors AgNO 3 , CoCl 2 and AOA on in vitro shoot regeneration of banana is presented in Table 1.In the control experiment low shoot regeneration (2.37 shoots/explant) with an average shoot length of 2.89 cm and mean leaf surface area of 3.74 cm 2 were observed.Presence of varying concentrations of AgNO 3 in the shoot multiplication medium had strong positive effect on shoot multiplicationand a maximum of 6.68 shoots/explant was achieved at10 mgl −1 AgNO 3 .This concentration was also the most effective in promoting shoot growth increasing shoot length and leaf surface area by 150 and 58%, respectively, compared to control.Addition of CoCl 2 or AOA to multiplication media was also beneficial to banana shoot multiplication although less effective than AgNO 3 .The highest number of shoots/explant was observed in media supplemented with 15mgl -1 CoCl 2 or AOA giving an average of 4.59 and 5.12 shoots/explants, respectively.Among these treatments only CoCl 2 increased shoot length to a maximum of 85% relative to control when added to the medium at15mgl -1 . Application of 5 to 15 mgl -1 AOA to multiplication media did not, however, influence shoot length and higher concentrations inhibited shoot elongation.Leaf surface area on the other hand, was not significantly affected by all concentrations of CoCl 2 tested whereas considerablereduction in leaf surface area was noticed in the presence of increasing concentrations of AOA in the medium.The data presented in Figure 1 shows that application of 5 to 20 mgl -1 AgNO 3 to banana multiplication media resulted in a significant increase in total leaf chlorophyll content.The highest amount of chlorophyll was observed at 10 mgl -1 AgNO 3 .At this concentration total leaf chlorophyll content increased by 120% compared to control.Treatment with AOA, however, resulted in a lower increase in total leaf chlorophyll content reaching a maximum of 35% over control at a concentration of 10 mgl -1 .On the contrary, CoCl 2 treatment had no significant effect on leaf chlorophyll content.The presence and concentration of AgNO 3 in the rooting medium had significant effect on rooting in banana (Figure 2).The highest number of roots/explant and the highest root growth were achieved on medium containing 10 mgl -1 AgNO 3 (Plate 1).At this concentration silver nitrate increased root formation and mean root length by 190 and 115%, respectively, relative to control.Incorporation of CoCl 2 and AOA in the rooting medium also stimulated rooting but proved to be less effective than AgNO 3 (Figures 3 and 4).The best concentrations of CoCl 2 and AOA for rooting (15 mgl -1 ) produced 85 and 115% more roots/ explant, respectively, compared to control.Contrary to CoCl 2 treatment which had no significant effect on root length, treatment with 15 mgl -1 AOA resulted in 80% increase in root growth relative to control.

DISCUSSION
In the present study the influence of ethylene inhibitors AgNO 3 , CoCl 2 and AOA on in vitro culture of banana (Musa acuminata L) was investigated.The results show that the use of ethylene inhibitors in culture media can  elongation and leaf expansion.The maximum number of shoots as well as shoot length and leaf surface area was achieved on media supplemented with 10mgl -1 AgNO 3 ; shoot number/expant were 3 times higher, shoots formed Mean number of roots/plant Mean length of roots (cm) were 4.5cm longer and leaf surface area were 2 cm 2 greater than those recorded on media without AgNO 3 .This result agrees with previously reported findings demonstrating the stimulative role of AgNO 3 on shoot organogenesis in many plants such as coffee sp., (Giridhar et al., 2003), strawberry (Qin et al., 2005), sweet potato (Gong et al., 2005), sesame (Abdellatef et al., 2010), tomato (Osman and Khalafalla, 2010) and turmeric (Dikashet al.,2012).For CoCl 2 and AOA treatments, higher concentration (15 mgl -1 ) were required to enhanced banana shoot development, although, lower number of shoots/explant were achieved compared to AgNO 3 treatment.While, shoot length was improved by CoCl 2 treatment, no positive effect on leaf surface area was noted by either of these compounds.
It is well known that AgNO 3 is a potent inhibitor of ethylene action (Beyer, 1979;Veen and Overbeek, 1989;Pua and Chi, 1993) whereas CoCl 2 and AOA are known to inhibit the enzymes ACC synthase and ACC oxidase involved in ethylene biosynthesis (Satoh and Esashi, 1980;Yang and Hoffman, 1984;Abeles et al., 1992).There is also accumulating evidence suggesting that in vitro tissue cultures produce ethylene in sealed containers (Chi et al., 1991).In addition there have been several reports indicating that ethylene produced during in vitro culture impairs plant growth and development and could limit in vitro propagation of several plants (Biddington, 1992;Pua and Chi, 1993;Chraibi et al., 1991).Accordingly, the findings of this study may suggest that ethylene inhibitors, particularly AgNO 3 alleviated the negative effects of ethylene on the growth of banana culture in vitro.Support for this suggestion comes from the finding that these compounds are also capable of increasing chlorophyll content in banana leaves (Figure 1).The association of ethylene with senescence of plant parts is well known (Jona et al., 1997) and its negative effect on chlorophyll content of plants was documented.For example, Jakob-Will et al. (1999) reported that ethylene induced expression of chlorophyllase genes (Chlase) in citrus fruits.There are also some reports suggesting that inhibition of ethylene action by silver ions increased leaf chlorophyll content (Ehsanpour and Jones, 2001;Perl et al., 1988).Apparently, addition of AgNO 3 and to a lesser extent CoCl 2 and AOA to banana culture media could improve shoot multiplication and promote the maintenance of green healthy in vitro tissue for long time periods.
In addition to their positive effects on in vitro shoot growth and development, AgNO 3 , CoCl 2 or AOA incorporated into banana rooting media also improved rooting of in vitro produced banana shoots.Among these compounds AgNO 3 resulted in the best rooting response.Shoots in rooting media supplemented with 10mgl -1 AgNO 3 produced 3 times more roots and increased root length by 7 to 8 cm.The optimal concentration of CoCl 2 and AOA for rooting was achieved at 15 mgl -1 .This concentration resulted in approximately two fold increase Tamimi 2515 in the number of roots formed per shoot, compared to control with limited or no significant influence on root elongation.These observations are in accordance with previous findings demonstrating improvement of in vitro rooting of plants by ethylene inhibitors such as Decalepis hamiltonii (Bais et al.,2000;Reddy et al.,2001), coffee (Giridhar et al.,2003) and apple (Ma et al.,1998).
In conclusionthe findings of this study demonstrate that ethylene inhibitors particularly AgNO 3 and to a lesser extent CoCl 2 and AOA enhanced vigour of banana shoots proliferation in vitro along with leaf expansion and shoot elongation; possessed the potential to protect developing banana leaves from ethylene induced senescence by maintaining high leaf chlorophyll content and increased the rooting capacity of in vitro grown banana shoots.Taken together, this study suggests that AgNO 3 , CoCl 2 and AOA may be used as important tools for improving protocols of banana cultures in vitro and for protecting in vitro cultured banana tissue from the possible negative effects of ethylene in culture vessels.

Figure 2 .
Figure 2. Effects of various concentrations of AgNO3 (mg/L) on rooting of in vitro grown banana shoots after 4 weeks of culture.Data are means of 10 replicates± SE.

Figure 3 .
Figure 3. Effects of various concentrations of CoCl2 (mg/L) on rooting of in vitro grown banana shoots after 4 weeks of culture.Data are means of 10 replicates± SE.

Figure 4 .
Figure 4. Effects of various concentrations of AOA (mg/L) on rooting of in vitro grown banana shoots after 4 weeks of culture.Data are means of 10 replicates± SE.

Table 1 .
Effects of various concentrations of AgNO3, CoCl2 and AOA on shoot regeneration from shoot tips of bananaafter 4 weeks of culture in vitro.
b Data are means of 10 replicates with 3 explants per replicate.Means followed by different alphabet denote significant differences within column based on DMRT (p = 0.05).Figure 1.Effects of various concentrations of AgNO3, CoCl2 and AOA on total chlorophyll content in leaves of banana shoots cultivated in vitro for 4 weeks.Data are means of 10 replicates ± SE.