The effect of different nutrient media on in vitro shoot and root proliferation of Vanilla planifolia Jacks

Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW) nutrient media are among the most widely used for micropropagation of orchid species. Therefore, this study was conducted to evaluate the effect of these growth media on in vitro shoot and root proliferation of Vanilla planifolia by using nodal segment as explants. Three different media; Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW) were supplemented with 0.5 mg/L of α-naphthalene acetic acid (NAA) and 1.0 mg/L of 6-benzylaminopurine (BAP) respectively to enhance the plant growth. The findings showed that the explants developed in MS medium yield the highest increase in length of shoot and root, with the average of 2.37 ± 0.76 cm and 2.04 ± 0.95 cm respectively. Apart from that, the plantlets in MS medium have well-formed leaves and extended roots. On the other hand, plantlets in both KC and VW media showed lower average of shoot and root length as compared to those cultured on MS medium. The length of shoot and root initiated from the nodal segments of V. planifolia showed significant difference when cultured in different types of nutrient media.


INTRODUCTION
Tropical orchid, Vanilla planifolia belongs to the family Orchidaceae and is native to southeastern Mexico (Purseglove et al., 1981).The main producers of the vanilla plants are Madagascar, Indonesia, Mexico, Tahiti and India (Shruthi and Naira, 2013).Vanilla, the main product from this orchid plays a major positive role in the economy of several countries.V. planifolia are commercially cultivated for vanillin which is found in the beans.This substance is widely used as flavoring in foods, drinks and fragrance industries.Vanilla is also involved in the production of pharmaceutical and cosmetics (Hilda et al., 2008).Curing process which involves four stages which include killing, sweating, drying and conditioning is required to optimize the *Corresponding author.E-mail: noorhazira@umk.edu.my.Tel: +06-0193667581.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License development of aroma after the harvesting of vanilla beans (Renuga and Saravana, 2014).
The V. planifolia is conventionally propagated by stem cuttings (Kalimuthu et al., 2006).This technique however, will interrupt the growth of mother plants, thus lowering down the propagation capacity and lengthen the time for vanilla pod production.Moreover, the commercial propagation by stem cuttings in Vanilla plantation was regarded as uneconomical because it sacrifices the whole plant (Vijaykumar et al., 2016).Therefore micropropagation through tissue culture method was introduced to propagate V. planifolia, as this technique could overcome these shortcomings.
In micropropagation, selection of growth medium is very crucial.The composition of the culture medium is one of the important factors that effects in vitro growth and morphogenesis of plant tissue.Murashige and Skoog's (MS), Knudson C (KC) and Vacin and Went (VW) are examples of media widely used in micropropagation of Orchidaceae family.MS, VW and KC basal media was chosen in this study as they have been reported to show good response for in vitro regeneration of orchid.The efficacy of these media had been demonstrated for protocorm development of a Borneo orchid, Vanda dearei (Roslina et al., 2010).The potential of MS, VW and KC media for seed germination and seedling development of Cymbidium aloifolium (L.) Sw. orchid also had been studied (Bhowmik and Rahman, 2017).However, the effect of these media on shoot and root proliferation of V. planifolia has yet to be demonstrated.
Murashige and Skoog medium or MS media is a plant growth medium widely used in the cultivation of plant cell culture.MS was invented by plant scientists (Murashige and Skoog, 1962) and become the most commonly used medium in plant tissue culture experiments.Knudson C Modified Orchid Medium on the other hand is a nutrient solution for Orchid (Orchidaceae) seed germination and micropropagation of some orchid which was invented by Lewis Knudson in 1922 (Arditti and Ernst, 1993).Vacin and Went (VW) media was formulated by Emil Vacin and Professor Frits W. Went for proliferation of shoot and formation of protocorm-like bodies or PLBs.VW medium had been used in micropropagation of Dendrobium ovatum Lindl.(Akter et al., 2007) and micropropagation of Chang Daeng (Rhynchostylis rubrum) orchid by embryogenic callus (Te-chato et al., 2010).These three types of basal media differ in macronutrients and micronutrients composition.The major difference lies in the form and composition of ammonium.MS medium contains higher amount of ammonium (1650 mg/L) in the form of ammonium nitrate, while both KC and VW contains 500 mg/L of ammonium sulphate.MS medium also includes higher amount of potassium nitrate (1900 mg/L) as compared to VW (525 mg/L) and KC (0 mg/L).However, the composition of potassium phosphate monobasic in MS medium is slightly lower (170 mg/L) than both KC and VW media (250 mg/L) (Himedia Laboratories (n.d).The evaluation of basal media effect on shoot and root proliferation of V. planifolia is useful for selection of the optimum media for better explant regeneration.

Plant material and sterilization technique
The nodal segments from one year old V. planifolia plant were used as explant.The source of V. planifolia was obtained from the Forest Research Institute Malaysia (FRIM).The stem was cut from the mother plant and washed in dilute detergent for 15 min then rinsed under running tap water.Then the explants were immersed in 5% sodium hypochlorite solution with two drops of Tween-20 for 25 min then rinsed in sterile distilled water.After that, the explants were immersed again in 5% sodium hypochlorite solution added with one drop of Tween-20 for 15 min then followed by rinsing in sterile distilled water until no foam could be observed (Figure 1).

Media preparation
Three different types of media; MS, KC and VW media were prepared accordingly.0.5 mg/L of NAA and 1.0 mg/L of BAP were then added to all the media as plant growth regulator to support regeneration of the explants.Agar and sucrose were added at the concentration of 0.8% (w/v) and 3% (w/v), respectively in all media.The pH of the media was then adjusted to 5.8.MS media without plant growth regulators (MS 0) were used as control.

Initiation of culture
The nodal segment were cut from the plant in the size of 3 cm length and used as explants.Inoculation of the explants was carried out in laminar air flow cabinet under sterile conditions.All cultures were then grown at 25 ± 2°C under illumination of 16 h photoperiod.

Statistical analysis
The shoot and roots lengths produced in each treatment were measured every 2 weeks for a period of 6 weeks after culture initiation.The entire data collected were analyzed using statistical package for social science (SPSS 21.0).Data was subjected to one way Analysis of Variance (ANOVA).Degree of variations was shown by Standard Deviation (SD).Tukey's range test at 5% was used for comparing differences in means of various treatments.

The effect of plant growth media on shoots and root proliferation of Vanilla planifolia
The study showed that MS medium was the most effective for the development of shoots in V. planifolia, followed by KC (Knudson, 1946) and VW (Vacin and Went, 1949) media.In fact, VW showed no emergence of shoot after 2 weeks of initiation.Therefore, this finding suggested that VW is not recommended if the aim of the culture is to get fast shoot proliferation.Node culture Table 1.The effect of media on shoots and roots length after 2 weeks of initiation.produced elongated shoots when stimulated cytokinins (George et al., 2007).Therefore nodal segments are preferred in micropropagation as it offers clonal stability compared to other parts of the plant.The results showed that MS medium yield the highest mean of root length followed by KC and VW.No root was observed after 2 and 4 weeks in VW media.However, very short hairy roots only started to appear after 6 weeks.This pattern is similar to shoot growth, whereby MS showed the highest increase in length while VW showed the least increment (Table 1).

S/N Type of media 2 weeks
The overall results concerning the influence of growth media on the length of shoot and root proliferation of in vitro V. planifolia propagation showed that the difference was significant between MS media against control, KC and VW.The letter a, b and c represent the significant differences as revealed by Tukey's test, with p<0.05 value.Tables 1, 2 and 3 showed the mean length of shoot and root achieved after 2, 4 and 6 weeks respectively.
Figure 2 shows the shoot and roots emergence on nodal segments of V. planifolia upon inoculation in different nutrient media.Vigorous growth of shoots and extensive root were observed on explants in MS media.This was followed by the explants grown in KC media whereby tiny shoot tips and root emerged from the explants.On the other hand, the growth of shoot and root in VW medium were distorted, whereby only a small protrusion of shoot and root were formed on the explants.The result found that the shoot and root proliferation of V. planifolia was strongly dependent on the basal salt medium composition and its supplements.These also include type, concentration and ratio of macronutrient and micronutrient, ferum source and vitamin with suitable environment conditions such as photoperiod and temperature (Sokolov et al., 2014).Different media used in this research have different composition and concentration of minerals and therefore caused different response towards plant growth.
MS media contains higher content of macronutrients compared to KC and VW media.There are 5 components of macronutrients in MS media which includes ammonium nitrate, calcium chloride, magnesium sulphate, potassium nitrate, and monopotassium phosphate   ( Murashige and Skoog, 1962).Higher content of macronutrients such as potassium in MS media is responsible for rapid organogenesis in in vitro plants.
Most of the plant species responded authoritatively in MS nutrient media with slight modifications (Zhao et al., 2008).Nitrogen is one of the important macronutrient for plant growth and development which involved in production of proteins, nucleic acids, and secondary metabolites (Engelsberger and Schulze, 2012).The enzymatic activities in shoot and root indicated that organic nitrogen and ammonium (NH4 + ) were the most important nitrogen sources in plant which affects numerous in vitro responses (Meijer and Brown, 1987).
Studies showed that the balance between ammonium ion and nitrate needs to be adjusted accordingly for each plant species, and the total nitrogen content of the medium is important determinant of growth and morphogenesis (Phillips and Collins, 1979).A study conducted in pear species found that the nitrogen form in the growth media could be manipulated to produce different level of shoots and callus (Wada et al., 2015).Moreover, different iron (Fe) composition and source from MS, KC and VW media also may influence the shoot proliferation and root emergence.Iron (Fe) is one of the essential elements of plant growth media which is required in plants photosynthesis, chlorophyll biosynthesis and respiration.Ferrous sulfate (FeSO 4 ) has been used in most of basic media for in vitro culture (Gamborg et al., 1976).

Conclusion
Nutrient medium plays vital role in propagation of plants through tissue culture.From the result, it was observed that MS medium provides better response for shoot and root proliferation of V. planifolia when compared with KC and VW medium.All the explants in MS media treatment were able to show proliferation of shoot within 8 days after culture initiation while root are able to emerge within 10 days.Therefore, MS medium added with plant growth regulators are advisable for shoot and root regeneration from the nodal explant of V. planifolia.

Figure 1 .
Figure 1.V. planifolia plants used in the study.
(±SE)  with the same letter by the column are not significantly different.
(±SE)  with the same letter by the column are not significantly different.

Figure 2 .
Figure 2. Shoot and root proliferated from V. planifolia nodal explants after 6 weeks of inoculation in control medium (A I and A II), MS medium (B I and B II), KC medium (C I and C II) and VW medium (D I and D II).

Table 2 .
The effect of media on shoots and roots length after 4 weeks of initiation.

Table 3 .
The effect of media on shoots and roots length after 6 weeks of initiation.