Effect of different phytohormones on micropropagation of banana ( Musa sp . ) cultivars and their assessment through RAPD

A research was conducted to study the multiplication rates of banana shoot tips derived from different suckers under in vitro conditions during successive sub-culture of FHIA-23, KM5 and Basari and assessment of rapid amplified polymorphic DNA (RAPD) techniques of banana cultivars through molecular marker. Shoot tips of banana varieties were cultured on MS medium supplemented with different concentrations of Benzaline alkaline phosphate (BAP). MS 1⁄2 with Indole buric acid (IBA) 1 mgL + 5.0 g, 10 g, 20 g, 30 g and 40 g sugar L for root length, number of root and root weight were used for root induction. The results revealed that the best shoot initiation was observed in Basari using MS medium containing BAP 4 mg L. Maximum shoot length was observed in KM5 under the concentration of BAP 3.0 mg L. Maximum numbers of shoots and shoot weight were observed in KM5 followed by FHIA-23 and Basari variety. Maximum root length was observed in KM5 followed by FHIA-23 under the concentration of sugar 30 g L + IBA 1.0 mg L. Maximum numbers of roots were observed in KM5, followed by FHIA-23 and Basari variety under concentration of 30 g L + IBA 1.0 mg L. It was concluded that the KM5 variety was superior, followed by FHIA-23 and Basari. The concentration of 3.0 to 4.0 mg L of BAP were found better for shooting and 1.0 mg L of IBA + 30 and 40 g L sugars for root induction of banana micropropagation.


Introduction
Banana (Musa sp.) is an important fruit crop cultivated in Pakistan.The total cultivated area in Pakistan is about 348.0 thousand hectares with 154.8 thousand tons production annually.[1].Composition of edible portion of the fruit is 74.8% water, 1.2% proteins, 0.2% fats, 0.84% ash, 0.6% fiber, 19.2% sugar as invert, 445 calories per pound and 0.39% malic acid [2].It is rich in carbohydrates, calcium, phosphorus, and potassium.Blood pressure, ulcer and heat burn are also improved and controlled using banana [3].Cavendish variety locally known as Basari, is particularly cultivated in Sindh, which in result holds about 98% of the plantation.Banana is of major importance to food security which as well provides a valued source of income.Banana tissue culture propagation has gained attention because of their ability to provide uniform genetically and diseasefree planting materials.Rate of multiplication is a key factor by which the efficiency of the micro-propagation system is being affected.Israeli et al. [4]; Mendes et al. [5], observed and reported that multiplication rate of banana depends on the genotype, whereas cultures originated from same banana genotype cultured in vitro, have been observed with variable behaviour.Banana's low yield and production in Pakistan is caused due to some biotic and abiotic reasons where the virus is included as one of the most important problems.Traditionally clonal propagation method seems to be incapable to meet the growing demand for disease-free materials of banana.Virus disease has greatly reduced the productivity of vegetatively propagated banana [6].Banana production is being reduced by some internal and intracellular pathogens, where micropropagation under in-vitro is a possible way for the development of pathogen free plants [7].
Novak [8] have demonstrated banana tissue culture in liquid media through somatic embryogenesis.Whereas Muhammad et al.
[9] also found that banana multiplication rate depends on the genotype.An experiment was conducted for the standardization of in vitro culture technique for mass propagation of Musa acuminate, shoot tips were used as an explant and cultured on Murashige and Skoog (MS) medium.It was observed that highest results were obtained from MS medium supplemented with BAP + IAA at the concentration of 3.0 mg L -1 and 0.

Media preparation
To prepare 1liter media, initially 700 ml of distilled water was taken in 1liter beaker.Each chemical component was weighted accurately according to the required concentration and added in the beaker containing distilled water, which was then kept on magnet stirrer for complete mixing.After the process, the media was poured into bottles covered with caps.The medium was autoclaved at 121 O C, 15 psi for 20 minutes.After sterilization bottles were labelled with the name of the media.

Sterilization of the explants and culture
After removing the unnecessary portion of leaves, remaining distilled water was used for washing first, and then was sterilized with 70% ethanol for a minute and 10% sodium hypochlorite solution for 20 minutes.Explant material was rewashed with double distilled water two-three times after sterilization to remove any mark of disinfectant.Only the innermost leaves, which are supposed to be the infection free, were cut into 2-3 mm apical meristem.This apical meristem was cultured aseptically into the bottles of the media Micropropagation Four stages were adopted for micropropagation for banana.Stage 1: Plants were selected and maintained in a controlled environment.Explants were prepared, such as shoot tip or meristem and were transferred to a suitable culture medium.Stage 2: In the second stage, the explants were induced to form multiple shoots, generally each explant produces five to six shoots within 4 to 5 weeks.Stage 3: Multiple shoots produced in stage II were induced to produce rooting in a fresh nutrient medium.Rooted shoots were highly fragile and sensitive to moisture.
Stage 4: Hardening is a process by which rooted plantlets were prepared for soil conditions.The plants were acclimatized to face the field conditions.When plants were acclimatized over a period of time they were transferred to the field.

Isolation of DNA
Young and fresh leaves from each banana were used for DNA extraction using MTAB method.Two hundred milligrams of fresh leaves were grounded in liquid nitrogen.The ground sample was placed in a 15 ml tube, 3 ml of the cell lysis solution was added and the tube was incubated at 65°C for one hour.Fifteen micro-litters of RNase solution were then added to the cell lysate and incubated at 37 °C.Protein precipitation solution after 30 minutes was added to a tube which was then vortexed for 20 seconds and then placed on ice for 30 minutes.Mixture was centrifuged at 2000 x g for 10 minutes.The supernatant containing DNA was poured into separate 15 ml centrifuge tube adding five millimeters of isopropanol, which was then precipitated by centrifuging at 2000 x g.The pellet was washed with 70% ethanol and the DNA samples were then hydrated with TE buffer.DNA was quantified on BIOMATE 3 (spectrophotometer).

Days to initiation
The results indicated that cultivars, BAP concentrations and their interactions were highly significant at P > 0.05 probability level (Table 1).The results in (Table 2, Figure 1) shown for cultivars revealed that early days to initiation observed in KM5 was 16.06, FHIA-23 (17.58) and Basari variety (20.22).The results for BAP concentrations indicated that minimum early days to initiation (12.20) were observed under the concentration of BAP 4 mg L -1 , followed by concentration BAP 3 mg L -1 (14.06) and control (24.76).The results indicated for their interactions that early days to initiation were observed in KM5 (10.16), followed by FHIA-23 (12.20) under the concentration of BAP 4 mg L -1 .The maximum days to initiation (27.46) were observed in Basari variety under control.It was noted that early days to initiation were appeared when MS + 3.0 and 4.0 mg L -1 BAP treatments were applied.The present findings are supported by Goshu et al. [18], who also reported that MS + 5 mg L -1 BAP showed early days to initiation at BAP 4.0 and 3.0 mg L -1 , respectively.

Shoot length (cm)
The results indicated that cultivars, BAP concentrations and their interactions were highly significant at (P > 0.05) probability level (Table 1).The results for shoot length are shown in (Table 3, Figure 1) indicating that maximum shoot length (7.88 cm) was noticed in KM5 followed by FHIA-23 (7.34 cm), and minimum was noticed in Basari (6.60 cm) variety.The results for BAP concentrations indicated that maximum shoot length (9.89 cm) was in the concentration of BAP 4.0 mg L -1 followed by concentration of BAP 3.0 mg L -1 (9.77 cm), and minimum (3.43 cm) was observed in control.The results for their interactions showed that KM5 produced maximum shoot length (10.73 cm) under the concentration of BAP 3.0 mg L -1 , followed by concentration of BAP 4.0 mg L -1 (10.64 cm) and minimum shoot length (2.98 cm) was observed in Basari variety under control.On overall, it was found that increasing the concentration of MS + BAP 4.0 mg L -1 appeared to be better for shoot length.While further increase in concentrations of BAP did not prove to be feasible because shoot length was reduced when BAP concentration was increased.

Number of shoots
The results for number of shoots in banana obtained from micropropagation were proliferated under various concentrations of MS + BAP, their interactions were highly significant at P > 0.05 probability level (Table 1).Maximum number of shoots (6.41) were observed in KM5, followed by (5.33) in FHIA-23 where minimum with 5.32 were observed in Basari (Table 4, Figure 1).The results under various concentrations of BAP indicated that maximum number of shoots (9.58) were observed under the concentration of BAP 4.0 mg L -1 , followed by the concentrations BAP 3.0 mg L -1 (8.61) and control (2.49), respectively.The results indicated for their interactions were maximum (10.86) under the concentration of BAP 4.0 mg L -1 , followed by (9.

Shoot weight (g)
The results for shoot weight of banana obtained from micropropagation under various concentrations of MS + BAP were significant at (P > 0.05) probability level (Table 1).The results in (Table 5, Figure1) shows that maximum shoot weight with 7.52 g was observed in KM5, followed by (6.59 g) in FHIA-23 and minimum (5.32 g) in Basari variety.The results under various concentrations of BAP indicated maximum shoot weight (9.16 g) under the concentration of BAP 4.0 mg L -1 , followed by (8.57g) under the concentration of BAP 3.0 mg L -1 and minimum (3.49g) was observed in control.The results for their interactions showed that maximum shoot weight (10.26 g) was observed in KM5, followed by FHIA-23 (9.34 g) under the concentration of BAP 4.0 mg L -1 and minimum shoot weight (2.95 g) was recorded in Basari variety under control.
The overall result showed that MS + BAP concentrations of 3.0 and 4.0 mg L -1 proved to be excellent for shoot weight in KM5 variety.Similar results have also been reported by Kadota and Niimi [23], who reported that MS medium supplemented with 25 µM BAP treatments had prolonged the leaves in banana.

Root length (cm)
Results for differences in the length of root between the various concentrations of sugar (0 + 05 mg L -1 , 1.0 + 10 mg L -1 , 1.0 + 20 mg L -1 , 1.0 + 30 mg L -1 and 1.0 + 40 mg L - 1 ) + IBA were highly significant (P > 0.05) at probability level (Table 1).Maximum root length with 5.49 cm was observed in KM5, followed by FHIA-23 (5.26 cm) and minimum (4.87 cm) was observed in Basari variety (Table 6, Figure 2).For sugar concentrations + IBA, the results indicated that maximum with 6.41 cm was observed under the concentration of sugar 30 g L -1 IBA 1.0, followed by 6.14 cm under the concentration of sugar 40 g L -1 + IBA 1.0 whereas minimum (3.25 cm) was observed in control.For their interactions, maximum root length (6.84 cm) was observed in KM5, followed by (6.44 cm) in FHIA-23 under the concentration of sugar 30 g L -1 + IBA 1.0 and minimum root length (3.04 cm) in Basari variety under control.The overall average showed that best root length was recorded under MS½ + sugar 30 g L -1 + IBA 1.0.The results are supported by Molla et al. [24] where they observed 2.60-5.67cm root length in 0.5 mg L -1 IBA.The results of number of roots in banana obtained from micropropagation were proliferated for further study under various concentrations of Sugar (0 + 05, 1.0 + 10, 1.0 + 20, 1.0 + 30 and 1.0 + 40 g) + IBA, their interactions were highly significant (P > 0.05) at probability level (Table 1).Table 7 represents the results, where maximum number of roots (8.21) were observed in KM5, followed by (7.12) in FHIA-23 and minimum (6.60) in Basari variety (Fig. 2).The results under various concentrations of Sugar + IBA indicated that maximum number of roots (8.58) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (7.91) under the concentration of sugar 10 g L -1 + IBA 1.0 and minimum with 7.72 under the concentration of sugar 40 g L -1 + IBA 1.0.The results for their interactions indicated maximum number of roots (9.84) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (8.83) in KM5 under the concentration of IBA 1.0 + sugar 10 g L -1 and minimum number of roots (5.26) were observed in Basari variety under control.The overall average showed that best root length was recorded under MS½ + sugar 30 g L -1 + IBA 1.0.The present results are similar to the findings of Gubbuk and Pekmezci

Root weight (g)
The results indicated that root weight of banana obtained from micropropagation under various concentrations of Sugar (0 + 05, 1.0 + 10, 1.0 + 20, 1.0 + 30 and 1.0 + 40 g) + IBA were significantly at (P > 0.05) probability level in (Table 1).The results in (Table 8, Figure 2) displays that maximum root weight with 7.07 g which was observed in KM5, followed by 6.53 g in FHIA-23 and minimum with 5.86 g in Basari variety.The results showed that maximum root weight with 7.75 g was observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (7.43 g) under the concentration of sugar 20 g L -1 + IBA 1.0 and minimum root weight (4.58 g) in control.The results for their interactions showed that maximum root weight with 8.55 g was observed in KM5 under the concentration of sugar 30 g L -1 + IBA 1.0 and minimum root weight with 4.06 g in Basari variety under control.Rahman et al.
[27] in their study indicated that highest root weight was achieved under sugar 30 g L -1 + 1.0 mg L-1 IBA.This may be due to the genotype of their cultivar and the relatively low concentration of IBA used.
In our study, it was evident that sugar 30 g L -1 +1.0 mg L -1 IBA can increase the average root weight.This may have a positive influence on root weight Al-Amin et al. [28].

DNA quantification of banana by spectrophotometer
Extracted DNA was quantified by biomate spectrophotometer at 260/280.DNA absorbs ultraviolet light, with an absorption peak at 260 nm wavelength.A sample is exposed to the ultraviolet light spectrophotometer and a photo-detector measures the light that passes through the sample.The more absorbed by the sample, the higher the nucleic acid concentration in the sample.The protein concentration was determined at the 280 nm.The 260/280 ratio reflects the purity of DNA.Usually the ratio between 1.2-1.8 is considered to be the best for PCR amplification.The results of the Banana DNA extraction used in experiment are as follows: High molecular weight DNA was extracted from banana clones through MATAB method.Extracted DNA of 7 banana clones were quantified by spectrophotometer at 260/280nm.The highest DNA ratio (1.03) was observed in Basari-3 and highest DNA concentration (2724 ng/ul) was in the FHIA-23 (Table 9).

Conclusion
It was concluded that the days to initiation, number of shoots, shoot length and shoot weight were better under the concentration of 4.0 mg L -1 , followed by 3.0 mg L -1 .The results indicated that the root length, number of roots and roots weight were better under the concentration of 30 g sugar L -1 + IBA 1.0 mg L -1 .The BAP under the concentrations of 4.0 to 3.0 and 30 g sugar L -1 and IBA 1.0 mg L -1 should be applied to achieve beneficial results in tissue culture technique.

Figure 2 .
Figure 2. Roots of banana cultivar FHIA-23 KM5 and Basari as affected by different concentrations of Sugar + indole-3-butyric acid (IBA) Number of rootsThe results of number of roots in banana obtained from micropropagation were proliferated for further study under various concentrations of Sugar (0 + 05, 1.0 + 10, 1.0 + 20, 1.0 + 30 and 1.0 + 40 g) + IBA, their interactions were highly significant (P > 0.05) at probability level (Table1).Table7represents the results, where maximum number of roots (8.21) were observed in KM5, followed by (7.12) in FHIA-23 and minimum (6.60) in Basari variety (Fig.2).The results under various concentrations of Sugar + IBA indicated that maximum number of roots (8.58) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (7.91) under the concentration of sugar 10 g L -1 + IBA 1.0 and minimum with 7.72 under the concentration of sugar 40 g L -1 + IBA 1.0.The results for their interactions indicated maximum number of roots (9.84) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (8.83) in KM5 under the concentration of IBA 1.0 + sugar 10 g L -1 and minimum number of roots (5.26) were observed in Basari variety under control.The overall average showed that best root length was recorded under MS½ + sugar 30 g L -1 + IBA 1.0.The present results are similar to the findings of Gubbuk and Pekmezci [26]; Rahman et al. [19].
Figure 2. Roots of banana cultivar FHIA-23 KM5 and Basari as affected by different concentrations of Sugar + indole-3-butyric acid (IBA) Number of rootsThe results of number of roots in banana obtained from micropropagation were proliferated for further study under various concentrations of Sugar (0 + 05, 1.0 + 10, 1.0 + 20, 1.0 + 30 and 1.0 + 40 g) + IBA, their interactions were highly significant (P > 0.05) at probability level (Table1).Table7represents the results, where maximum number of roots (8.21) were observed in KM5, followed by (7.12) in FHIA-23 and minimum (6.60) in Basari variety (Fig.2).The results under various concentrations of Sugar + IBA indicated that maximum number of roots (8.58) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (7.91) under the concentration of sugar 10 g L -1 + IBA 1.0 and minimum with 7.72 under the concentration of sugar 40 g L -1 + IBA 1.0.The results for their interactions indicated maximum number of roots (9.84) were observed under the concentration of sugar 30 g L -1 + IBA 1.0, followed by (8.83) in KM5 under the concentration of IBA 1.0 + sugar 10 g L -1 and minimum number of roots (5.26) were observed in Basari variety under control.The overall average showed that best root length was recorded under MS½ + sugar 30 g L -1 + IBA 1.0.The present results are similar to the findings of Gubbuk and Pekmezci [26]; Rahman et al. [19].

Table 1 . Mean squares for various traits of Banana cultivars revealed by analysis of variance (ANOVA)
The results are in line with Rahaman et al. [19]; Arinaitwe et al. [20], they also observed a hard ball like structure being developed during shoot multiplication.

Table 4 . Effect of different concentrations of 6-benzylaminopurine (BAP) on number of shoots of banana cultivars
Habiba et al. [25]also got more or less same observation during the experiments.