Title: Effects of surface sterilisation treatment and manipulation of plant growth regulator on the contamination rate and organogenesis

of explants. Abstract Aseptic culture preparation is the most crucial part prior to any micropropagation, different combinations of Auxin and Cytokinins as plant growth regulator plays an important role in organogenesis of explants of Crassula Argentea. This study focuses on the optimal surface sterilization treatment to reduce the contamination rate in the culture. Also, shoot, root formations are induced by combination of plant growth regulators. In this experiment, it was found that the using 10% concentration Commercial bleach for 20 minutes provides the lowest bacterial contamination of 14.04% and fungal contamination of 5.26%. Shoot formation was highest in high Cytokinins medium, while shoot and root formation was highest in high Auxin medium. Reasons for organogenesis in growth regulator free medium, no growth and browning were also discussed. Introduction Plant Tissue Culture involves growing of plant cells, organs and tissue in an aseptic or sterile conditions, it also has a tightly control on the environment, nutrients and hormones levels for growth. (Hartman et al., 2011; George et al., 2009; Purohit, 2004; Trigiano and Gray, 2000; Ponmurugan and Kumar, 2012). According to Hartman et al (2011). In this experiment, direct organogenesis method is used to regenerate the explants to form shoots or roots directly without involving callus formation. Surface sterilization treatment is a very important step in tissue culture which the success of micropropagation largely depends on the good sterilization method to obtain an aseptic culture. Microbial contamination typically fungus and bacteria can be a huge problem that will cause a huge lost for the tissue culture grower. This experiment investigates the types of surface sterilization treatment such as Ethanol and commercial bleach and the duration of explants exposed to the sterilant. Auxin and Cytokinins concentration has been found to be influential on the shoot and root formation on explants. Throughout this experiment, investigation on the growth of the explants based on manipulating the media content has been done. Reason for no growth and browning of media were also studied. Materials and Methods 3 leaves were picked out and washed under running tap water. Three equal size pieces were then excised by using a scalpel from each of the leaves. 9 pieces of leaves were taken to the laminar flow clean air bench. Three surface sterilization methods were employed: 70% alcohol for 2 minutes, 10% commercial bleach solution for 20 minutes, 10% commercial bleach solution for 30 minutes. Sterilants were poured into separate small jars respectively. 3 pieces of the leaves were placed into ethanol containing jar while the others were placed into bleach containing jar. The timer was started immediately.

Next, Sterile purified water was used to remove traces of surface sterilant from the plant tissues. Two rinsing were done to the plant tissues with sterile water, 1 minute for each. The tissues were then transferred to a sterile surface and sterile instruments were used to trim the excised surface of the tissues, removing the damaged edges that have been done by chemical sterilant. Final trimming was done to 1cm3 and aseptically, inoculation was done one explant per vessel into the medium containing culture vessels. Each piece out of three from each sterilization treatment was inoculated in three different medium: Medium A (MS medium + 0.1mg/l IBA + 2.0mg/l BAP), Medium B (MS medium + 2.0mg/l IBA + 0.1mg/l BAP), Medium C (MS medium without plant growth regulators). The tops of the cultures were sealed by using parafilm and were incubated in the light at 25C and examined in a later session. Presence of contamination and growth of explants were recorded after the cultures were incubated for 4 weeks.

Results: Based on (Figure 1), it showed that the most effective sterilization method was using bleach for 20 minutes, while 2 minutes ethanol sterilization was the least effective method employed. Based on the result, the 20 minute bleach treatment had the least contamination with only 5.26% of fungal and 14.04 % of bacterial contamination. With 10.53% of fungal infection and 35.09% of bacterial contamination, ethanol showed the most contaminated cultures among the others.

Figure 1
40 35
Percentage of Contamination

30 25
20 15 10 5 0 Ethanol Bleach 20 Types of Sterilant Bleach 30 Bacteria contamination(%) Fungal contamination (%)

Figure 1: Effects of surface sterilization treatment based on the percentage of fungal and bacterial contamination.

From (Figure 2), it showed that each kind of medium used were able to show a certain level of organogenesis. The shoot formation was the highest in medium A with a leading percentage of 17.54%, followed by 10.53% in medium B while the lowest shoot formation was demonstrated by the value of 3.51% in medium C. Root formation in medium B and C showed the same value of 5.26% followed by medium A with the least percentage of root formation which was 3.51%. Both root and shoot formation was shown to be highest in medium B with a percentage of 56.14%. The least growth of both root and shoot was shown to be 3.51% in medium A, while between both of the extreme, medium C showed an intermediate of 35.09%.

Figure 2
60

50
40

Percentage (%)

30 20 10 0 Shoot formation (%) Root formation (%)

Medium A Medium B Medium C

Organogensis

Shoot and root formation (%)

Figure 2: The difference in organogenesis was shown as a result of inoculation in different medium

Based on (figure 3), the highest no growth level was shown in medium A with a percentage of 75.44% followed by medium C which has a no growth percentage of 56.14%. The lowest no growth percentage was shown in medium B with 28.07%. Browning of tissues was shown to be the most in medium C with a percentage of 17.55% while medium A and B have the same level of tissue browning of 12.28%.

Figure 3
80 70 60 Percentage 50 40 30 20 10 No growth (%) Browning (%)

0
Medium A Medium B No growth/ Browning Medium C

Figure 3: inoculation in different types of medium showed a difference in the level of no growth and browning of tissues.

Discussion: Microbial contaminations in the cultures are one of the major challenges faced by many scientific and commercial laboratories. One of the most common treatments is by using Sodium hypochlorite. (Cronauer and Krikorian 1984; Mendes et al., 1996; Muhammad et al., 2004). Ethanol is also used for sterilization purpose by a number of researchers. (Silva et al., 1998; Rahman et al., 2002; Jalil et al., 2003). In this experiment, it was shown that the most effective sterilization treatment was using 10% bleach for 20 minutes, and ethanol was the least effective sterilant used. Although many researcher reports that ethanol is a very effective sterilizing material, in this experiment it is in contrast. This might be due to that bacteria is able to survive on the surface being sterilize the ability to produce spores that can withstand harsh environment and still able to germinate in suitable environment.(Purohit, 2004) Another reason for low effectiveness is probably due to the existents of bacterial endogenously in the plant tissue (Ponmurugan and Kumar, 2012). The third reason for ethanol to have the most contamination is due to the phytotoxicity to the plant tissue. According to Ponmurugan and Kumar (2012), explants are normally exposed ethanol for a short period. It might due to long exposure of explants to the ethanol, cause damage to them. This is also shown in the browning of the tissue, which the more serious that the plant is injured, the higher quantity of phenolic compound is released, causing the plant to become brown. Compared to the other sterilant, which also has certain phytotoxicity to the plant tissue, ethanol is most probably the detrimental one. Organogenesis of explants is largely dependent on the types of plant growth regulator that presence in the medium. There are many types of hormones combination used in direct organogenesis (George et al., 2009). Shoot formation can be induced by having a low Auxin to a high Cytokinins ratio, which is according to the result of the experiment. This proves that Cytokinins in a higher concentration does promote the shoot formation. In order to induce the root formation, a higher Auxin to a lower Cytokinins ratio should be used. For medium B which had a higher Auxin (IBA) to Cytokinins ratio, root formation should be induced. However, the result was in contrast, that the shoot formation is higher than the root formation. Although the root formation is low, it can be seen that media B had the highest level of shoot and root formation, suggesting that the root formation might be high according to the theory. The root growth enhanced the absorption of the medium and promoted more shoot growth, this proves the effects of high concentration of Auxin. Temperature is shown to be one of the reasons to affect the efficiency of the plant growth regulators. Auxin are found to be heat labile and tend to decompose if being autoclaved, it is also unstable at room temperature in culture media. (Campbell and Sutter, 1986; Nissen and Sutter, 1988). Oxidation of plant growth regulator is also one of the reasons which affect the result of the experiment, Auxin tends to undergo

oxidative degradation when oxygen is present. (George et al., 2009). According to Dunlap et al (1986), the level of Auxin tends to decrease in the presence of light and in the presence of MS salts, the degradation is even accelerated. It can be seen that from the result, the explants from media C are able to exhibit organogenesis without the presence of plant growth regulator. Sunderland and Wells (1968) believe that explants might be able to produce plant growth regulators endogenously, three types of natural occurring Cytokinins can be isolated from the explants. This is also true when juvenile explants are used, as stated by Clare and Collin, (1974), might have a higher concentration of natural growth factors due to the proximity from the meristematic cells. The reason that the level of no growth is the highest in media A might be due to the browning of the explants tissue. According to (Christianson and Warnick, 1988), which suggested a model for the process of organogenesis, it consisted of three phases, the dedifferentiation phase, induction and the differentiation phase. In dedifferentiation phase, which involves the reversion of the plant cell to a more plastic developmental phase, in the end of this phase, the plant achieve a state of competence, which is the ability of the plant tissue to respond of a stimulus such as plant growth regulator which generate organogenesis. Browning of tissue due to several reasons such as microbial infection, damaged by trimming, can cause the plant to lose its ability to become competent. In this case, the explant is not able to proceed to the next stage (induction stage), to achieve the determination to produce primordial. Browning of media is due to the diffusion of phenolic compound into the media caused by microbial infection. It depends on the situation whether the phenolic compound diffuse out of the explant, sometimes, the phenolic compound might just be oxidised around the edge of the explants, causing it to turn brown without turning the media brown. Yet, the reason for why browning is the highest in media C is unknown. In conclusion, there is no one best way of surface sterilization treatment and type of medium that will work for all plant. Different plant perform differently on the same culture and sterilant, even the same plant may perform differently. Therefore, further studies need to be done in order to improve the micropropagation techniques and to improve the economic efficiency in propagation through tissue culture. (1799 Words)

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