OPTIMIZATION OF TISSUE CULTURE PROTOCOL FOR REGENERATION AND DIRECT MULTIPLICATION OF MORINGA OLEIFERA BY SEED GERMINATION

Asfandyar Chaudhary, Kashif Ali, Neha Farid And Nosheen Maqsood. Shaheed Zulfikar Ali Bhutto Institute of Science and Technology (SZABIST) Department of Biosciences, Karachi 75600, Pakistan. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 08 February 2019 Final Accepted: 10 March 2019 Published: April 2019

) gave almost similar results. Initially, seed coats were removed inside the Laminar Flow Hood but this was affecting the sterilization negatively. Seed sterilization was then modified by removing seed coats and sterilizing them outside the Laminar Flow Hood. The seeds were then transferred to the LFH for further sterilization, and then cultured on the growth media. For the sterilization of the explant for direct multiplication and callus induction, firstly Ethanol was used which caused bleaching effect. Later, Ethanol was removed from the protocol, reducing the bleaching effect to zero, and sterilization was maximized.

ISSN: 2320-5407
Int. J. Adv. Res. 7(4), 547-555 548 Around the world, especially countries that have an old cultural background, such as Pakistan, India, and China (Stephenson and Fahey, 2004), a significant percentage of population depend on the plants for the cure for certain type of ailments, ranging from slight allergic reaction of the skin to the serious conditions such as cancer (Förster et al., 2013). Through advance testing techniques, modern medicine has validated those plants, which have been previously known to have certain medicinal effects (Kinghorn, 2001;Mathur et al., 2014). Main active ingredients have also been identified by biochemical testing, and those compounds have been used to make medicines used for diagnosis and treatment in today's time (Wagner and Bladt, 1996;Balunas and Kinghorn, 2005).
Moringaceae family has total of 13 species, which grow in tropical and sub-tropical climates, and range in size from small herbs to gigantic trees. The trees of this family grow very quickly, and are drought resistant so they can withstand harsh environments. Native to the Sub-Himalayan tracks of Pakistan and India, and widely cultivated in the Philippines, South Africa, Sudan and Latin America (Verdcourt, 1985;Palada, 1996), Moringa oleifera (G.H. No.: 95436, Dr. Muneeba Khan, Centre for Plant Conservation, University of Karachi) is a fast-growing perennial tree, with a history of traditional medicine and culinary uses. Almost all parts of Moringa tree are edible and useful to humans (Fuglie, 1999;Anwar et al., 2007;Ferreira et al., 2008;Banerji et al., 2009). Seeds can be eaten raw green, powdered or roasted or steeped for use in curries or tea (Gassenschmidt et al., 1995). Seeds of Moringa oleifera have also been used as a natural flocculants to clarify water for drinking purposes (Bichi, 2013). The impressive range of medicinal uses and high nutrition makes it a plant of interest for the international pharmaceutical and agricultural community. It is highly nutritious for humans and animals, especially in life stock to increase their milk production (Shahzad et al., 2013). It is a potential food source, especially the leaves which are rich in nutrients, vitamins and as well as minerals (Fuglie, 1999). Previously, reported to have contained certain metabolites, having hypotensive activity (Faizi et al., 1995), anti-tumor activity (Bennett et al., 2003;Murakami et al., 1989), hepatoprotective activity (Pari and Kumar, 2002), Moringa oleifera has tremendous opportunities for sustainable agriculture and development as a cash crop. Thus, preservation of this plant is of great importance for dietary and pharmacological perspectives.
Tissue culture is the growing of living tissue in an artificial medium, away or apart from the source of isolated tissue (Ahloowalia et al., 2004). Micropropagation can be defined as the type of tissue culture that uses plant material such as leaves or other meristematic tissues as explant to grow the plant under aseptic environment (Debergh and Read, 1991). Micropropagation can either be direct or indirect. Direct multiplication initiates rooting and shooting in the explant, whereas in the indirect micropropagation callus formation is initiated followed by morphogenesis (Kadhimi et al., 2014). All this transformation of the plant is controlled by the manipulation of concentration and combination of different plant growth regulators.
Based on the concept of totipotency, tissue culture plays a major role in plant micropropagation and conservation (Jaskani et al., 2008). Techniques of tissue culture are important in applied and fundamental research, proving helpful in understanding gene structure, genetic resource conservation, functions in molecular biology and plant improvement through transgenic technology. Moreover, tissue culture has previously been used for mass propagation of elite and economically important plants (Preece and Read, 1993), also for the endangered species on the verge of extinction (Islam et al., 2005). Previously, Moringa oleifera has been cultured using nodal explants from non-aseptic sources from either mature plants or young seedlings (Stephenson and Fahey, 2004;Islam et al., 2005;Marfori, 2010).
Moringa is traditionally propagated by stem cutting. This method adversely affects the mother plant's growth, yield and even causes death (Islam et al., 2005). Tissue culture is an alternate method for its regeneration and multiplication. The objective of the research is focused on tissue culture of Moringa oleifera through callus induction, direct multiplication as well as from seed culture. The study is also focused on optimization of sterilization protocol and media for the tissue culture processes.

Seed Initiation:
The seed initiation of Moringa oleifera was done as per described below:

Media Preparation:
The basal media used for seed initiation was the Murashige & Skoog Medium (Murashige and Skoog, 1962) was prepared using the stock solutions of MS Macro, MS Micro, MS Vitamins and Fe-EDTA as per the recipe. Two types of media were prepared: one with the plant growth regulators (PGRs) and the other without PGR i.e. Basal media.
The PGRs used were: Indole 3-Acetic Acid (1-3 mgL ). The media was autoclaved at 121°C at 20 psi for 15 min. The sterilized media was stored in the tissue culture lab at 25±2°C until further use.

Sterilization
In Protocol 1, surface sterilization of seeds was done in Laminar Flow Hood with 0.1% Mercuric Chloride solution for 2 min. Then the seeds were transferred into 20% Sodium Hypochlorite solution and kept for 5 min. Then the seeds were thrice rinsed with sterilized distilled water. The seed coats were removed, and again surface sterilized by immersion in 20% Sodium Hypochlorite Solution for 2 min. The seeds were then rinsed again thrice with sterilized distilled water (Shahzad et al., 2014). In Protocol 2, changes were made in the second step in which the timing of sterilization with 20% Sodium Hypochlorite was increased to 10 min, and in fourth stage where after the removal of the seed coats surface sterilization by immersion in 20% Sodium Hypochlorite Solution was increased to 5 min (Saini et al., 2012). In Protocol 3, the outer hard coats of the Moringa oleifera seeds were manually removed under normal bench top environment. The seeds were then transferred into the Laminar Flow Hood. Inner seeds (white in color) were then first washed in sterilized distilled water. The remaining sterilization was done as per the steps of protocol 2 with the omission of the second use of sodium hypochlorite for seed coats' surface sterilization.

Initiation:
All the seeds after sterilization were transferred to the MS Basal media and MS media with different PGRs aseptically. The cultured seeds were kept in dark at 25°C± 2 for 15 days. On germination, the seeds were shifted to light zone of 2000 lux intensity with photoperiod of 16/8 (light/dark) hours.

Direct Multiplication and Callus Induction:
The direct multiplication and callus induction of Moringa oleifera was done as per described below:

Media Preparation:
For direct multiplication and callus induction, MS Basal media was used as a control, and MS Media supplemented with plant growth regulators was used for experimental purpose. The plant growth regulators used were: Indole 3-

Acetic
Acid (1-3 mgL ). The media was autoclaved at 121°C at 20 psi for 15 min. The sterilized media was stored in the tissue culture lab at 25±2°C.

Sterilization:
In Protocol A, the explants (stalks with leaves) were washed with tap water, and then transferred to sterilizing solution, which consisted of 20% sodium hypochlorite. It was kept on shaker for 15 minutes. After 15 minutes, the jar was taken into Laminar Flow Hood. The explants were transferred to sterilized distilled water and washed for 2 -3 minutes. Again, they were transferred to another bottle of sterilized distilled water and washed for 2 -3 minutes. The explants were then transferred to 70% ethanol for 1 min and then again in sterilized distilled water. UV was turned on for 10 minutes. After this, they were washed again for the last time with sterilized distilled water for 2 to 3 minutes. In Protocol B, the timing of sterilization with ethanol was increased to 5 min. In protocol C, no ethanol was used for sterilization of the explants.

Direct Multiplication:
For Direct Multiplication, the leaves were carefully removed from the stalks. The stalks were then cut at both ends diagonally at the meristematic regions. They were then planted in the media supplemented with different

Statistical analysis:
Ten replicates were produced for all the tests using different media. The results were observed after one week's interval. For statistical analysis, the standard deviation was set as ±1 and error bars were used for the analysis.

Optimization of Sterilization Protocol:
For the sterilization of seeds and explants, three different protocols were used for each of them namely as Protocol 1, 2 and 3 for seed and protocol A, B and C for the explants. Ten bottles were cultured with each protocol separately and rate of contamination was checked for each method carried out. Protocol 3, in case of seed sterilization, gave the best result as it gave less contamination rate as compared to the other two protocols used. The removal of seed on bench top prior to transfer of seed in the hood proved to be beneficial and gave promising results. Similarly, for direct multiplication and callus induction, the explants were sterilized using different protocols. Protocol A gave high rate of contamination every time when used. Protocol B although helped to reduce the rate of contamination to some extent but it also produced bleaching effect in leaves and stalks which were to be used for callus induction, turning them from green to yellow and eventually colorless leading to complete death of the explant. Protocol C helped in sterilization and reducing the bleaching effect. Less bottles were contaminated and the bleaching effect was totally reduced and the explant grew into desired part i.e. callus, and produced roots and shoots in case of direct multiplication.

Optimization of Sterilization Protocol:
For the sterilization of seed, different protocols were used. Each protocol was giving different contamination rate. The protocol 1 gave a contamination rate of 100%. Total ten seeds were cultured by using protocol 1 and none of the seed survived. High bacterial contamination was seen. After these results, the protocol 1 was modified by increasing the time for first washing with sodium hypochlorite solution from 5 minutes to 10 minutes, and for the second wash from 2 minutes to 5 minutes. This was protocol 2. Same contamination rate was also observed by this protocol i.e. 100%. All the three seeds sterilized with protocol 2 showed high rate of contamination. After all these results, protocol 3 was designed in which the seed coats were removed on the bench top before transferring the seeds into the laminar flow hood. Another change made was that the washing of seeds with sodium hypochlorite solution was done once after the washing with mercuric chloride solution. Initially, three seeds were sterilized using this protocol and no contamination was observed. Later on, all the seeds were sterilized using the protocol 3. Contamination rate observed in protocol 3 was 20%. This might be possible due to the removal of source of contamination from internal soft seed coat. Thus, protocol 3 proved to be optimum for sterilization of the seeds.
The sterilization of explants for callus induction and direct multiplication was done by performing three different protocols namely: Protocol A, B and C. Different rate of contamination and bleaching effect was observed in all the protocols. For all the three protocols, ten bottles of each concentration for every PGR were cultured. Protocol A gave a contamination rate of 100% and bleaching effect of 80%. After these results, the protocol B was used in which the time for ethanol was increased to 5 min to avoid any further contamination. The results obtained from this protocol were that the contamination rate decreased by 75% but bleaching effect increased by 20%. It was proposed that the bleaching effect was due to the use of ethanol for extended time, which led the explants towards chlorosis. To avoid contamination and bleaching effect, Protocol C was used. In this case, no ethanol was used. The contamination rate reduced to 5%. No bleaching effect was seen in this case and the explants remained green after sterilization. The results thus supported the Protocol C as the optimum sterilization method for explant without any bleaching effect.

Optimization of Media for Seed Initiation:
For seed initiation, MS Basal media and MS media supplemented with IAA, NAA and 2, 4-D (1 mgL ). As both the media gave the same results evidently, so it can be proposed that MS Basal media should be used for this purpose as addition of any PGR did not produce a significant increase in number of roots and shoot observed after germination.

Optimization of Media and Photoperiod for Callus Induction:
For the callus induction, the explant was cultured on different media. First of all, the optimum media was checked.
The MS Media supplemented with 2, 4-D (2 mgL ) gave better results than light conditions. MS Basal Media was used as control, which gave no callus production. Callus induction was done as it has many beneficial uses i.e. it can be used for biotransformation, secondary metabolites production and analysis, and genetic engineering.

Optimization of Media for Direct Multiplication:
The direct multiplication was done by culturing the explant on media having IAA, NAA, 2, 4-D (1 mgL . Surprisingly, it was observed that the lowest concentration of PGR such as BAP was suitable for the direct multiplication of Moringa oleifera. Whereas, the higher concentrations of BAP 552 inhibited the process of direct multiplication. Similar inhibition of direct multiplication was seen when the explant was grown in the MS media supplemented with other PGRs such as IAA, NAA, 2, 4-D and Kinetin. Direct multiplication was done as it is an important part of plant tissue culture and can further be used for acclimatization.   ----2, 4-D (2mg L -1 ) + +++ + + + +++ 2, 4-D (3mg L -1 ) + + ++ + + + + Table 1

Author contributions:
This work was carried out in collaboration between all authors. AC, KA and NF designed the experiments and did the result analysis. AC and NF carried out the lab experiments. NF and NM contributed to writing the paper.