3 LR 3 callus induction

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<p>Gerald Aquino</p> <p>EXERCISE 3: Callus Induction Group 1 Abraham Cruz Erica Fortuno Date Due: July 31, 2008 Date Submitted: July 31, 2008</p> <p>Ciara Lim</p> <p>ABSTRACT Explants, when cultured in a suitable medium can give rise to an unorganized mass of undifferentiated cells called callus. The experiment aims to generate calli from cultured explants and find out the different factors affecting callus induction. Three explants were used in the experiment: sweet potato root, mung bean cotyledonary leaves and rice grain. The explants were surface sterilized and subsequently cultured in Murashige-Skoog (MS) medium. Culture vessels were placed in a dark area and observations on callus growth or contamination were made. Calli were induced in each type of explant after 2-3 weeks of being in culture. However, the number of explants that generated callus is low and the callus formation was not that extensive. Contamination even occurred on the culture vessel containing the rice grain explants. Nonetheless, sufficient results have been acquired to point that the factors that influence the growth of callus include the quality of the explant, the nutrient medium used and the incubation period of the culture.</p> <p>INTRODUCTION Plant tissues are totipotent. This means that, given proper conditions, a cell has the capacity to develop into a whole organism (Purohit, 2005). Furthermore, plants exhibit a very high degree of plasticity, indicating that any tissue type of the plant can be initiated to grow into another type. Plant totipotency and plasticity are the basis of the many culture techniques that are used to segregate cells, tissues, and organs from the parent for subsequent study of the isolated biological units (Dodds and Roberts, 1995). A culture is basically a cultivation of cells or tissues on a solid gel medium or a liquid medium. It is usually initiated from the explants, which are sterile pieces of the whole plant and may be pieces of an organ (e.g. leaf) or specific cells (e.g. pollen). Then, the explants are transferred into a desired medium to allow differentiation and multiplication. This can result to the formation of callus (pl. calli), which is a mass of disorganized, mostly undifferentiated or undeveloped cells (Purohit, 2005). This callus can then be induced to redifferentiate to develop embryoids that can turn to plantlets. These plantlets can be used for studies of the plants as well as for mass production. Formation of callus can be divided into three stages: induction, cell division and differentiation. A very important step prior to callus culture is the induction of callus. It is usually initiated by wounding wherein a cut end of a stem or root results in the formation of callus. The hormones auxin and cytokinin are the stimuli involved in the initiation of callus. Then, maintenance of the callus is done by transferring it into another medium so that the nutritional requirements are replenished (Dodds and Roberts, 1995). Lastly, the calli can then be subcultured and induced to undergo organogenesis, which can lead to the generation of new plants. This experiment was done to generate calli from rice (Oryza sativa) grains, mung bean (Vigna radiata) leaves, and sweet potatoes (Ipomoea batatas) on Murashige and Skoog basal medium with 2,4- Dichlorophenoxyacetic acid. The factors affecting callus induction were determined.</p> <p>MATERIALS AND METHODOLOGY Materials Mung bean seeds Rice grains Sweet potato Gerber bottles Pipette tips Pipette racks for MnSO4 Mild liquid detergent Distilled water Cotton plugs Gauze Aluminum Foil Scratch paper Autoclavable bags Laminar Flow hood Sterile forceps Sterile beakers Sterile Petri dish Sterile paper discs Scalpel and scalpel blades Alcohol lamp MS media (components specified in table) 70% ethanol Sterile distilled water 50% bleach solution (2.75% sodium hypochlorite) 2% household liquid detergent</p> <p>Preparation of Glassware. Glasswares with no chips or cracks were first selected. They were washed with mild liquid detergent, then rinsed with tap water thrice and subsequently bathed with distilled water once. They were dried thoroughly by placing them on the tabletop open side down. Before autoclaving the materials, the mouths of the Erlenmeyer flasks, bottles, beakers, and the likes, were covered with foil or cotton plugs if necessary. For glass pipettes, a cotton plug was placed in the hole at the top and then the pipette was wrapped in paper. For petri plates, the dish was matched with its cover and wrapped in paper. These were then placed in autoclavable bags and were autoclaved for 15 minutes at 121 oC, 15 psi. The pressure gauge was allowed to return to zero and the temperature was brought down to 40 oC before the autoclave was opened and the glasswares were retrieved. Preparation of Supplies. The pipette tips were racked in their appropriate boxes. The tip boxes were then autoclaved for 15 minutes at 121 oC, 15 psi. The tips were then placed in the drying oven, upside down. The filter paper was cut to the size of a petri plate. The resulting paper discs were placed inside several Petri plates which were subsequently covered and wrapped with paper. The dishes were then placed in autoclavable bag and were autoclaved. These were then dried in the drying oven. Preparation of Murashige and Skoog (MS)Basal Medium. The components for MS medium were prepared by mixing the following amount of ingredients (as calculated). Components MS MACRO(500 ml stock solution) Ingredients NH4NO3 KNO3 CaCl2-H2O MgSO4-7H2O KH2PO4 Solution A MnSO4-H2O Amounts (grams) 16.50 19.00 4.400 3.700 1.700 1.115 Side Notes</p> <p>MS MICRO (500 ml stock solution)</p> <p>2</p> <p>Fe-EDTA (250 ml stock solution) MS Vitamins (50 ml stock solution ) Myo-inositol Sucrose Agar</p> <p>H3BO3 ZnSO4-7H2O Solution B KI NaMoO4-2H2O Solution C CuSO4-5H2O CoCl2-6H2O FeSO4-7H2O NA2EDTA Nicotinic Acid Pyroxidine HCl Thiamine HCl Glycine</p> <p>0.310 0.430 0.415 0.125 0.125 0.125 0.696 0.931 0.050 0.050 0.050 0.050 0.100 30.00 10.00 0.0415 (for 50mL) 0.0125 (for 50 mL) 0.0025 (for 10 mL) 0.0025 (for 10 mL)</p> <p>After mixing the ingredients to form the components, the components were then combined. The components were added in the following order: MS Macro, MS Micro, Sucrose, myo-inositol, FeEDTA, and finally H2O. The pH was then adjusted to 5.6 to 5.8 using KOH. Water was added before the agar was introduced. The prepared medium was now autoclaved and then cooled to 35 oC. The other components were then added in this order: 18.75 ml vitamins (40X) and hormone (40X), 0.75 Fungizone(1000X) and Pennstrep(1000X). After the preparation of MS medium, the medium was autoclaved for 20 minutes, 121 oC, 15 psi. After 20 minutes, the medium was allowed to cool and 30 ml of media was dispensed into each of the 10 autoclaved Gerber bottles. The bottles were then labelled 1 to 10 and marked with the group number and section. Each bottle was then covered with clear plastic and secured with a rubber band. The bottles were stored in clean place at room temperature. Preparation of Explants and Initiation of Callus Induction. Fifteen mung bean seeds were obtained. The seeds were then rinsed in sterile deionized distilled water (sddH 2O) to remove any soil or dirt adhering to the seeds. They were then soaked in 2% detergent for 5 minutes and it was ensured that they were immersed completely. Afterwards, the seeds were rinsed with sddH2O thrice. The seeds were then soaked in 70% ethanol for 30 seconds and were again washed thrice with sddH2O. Afterwards, the seeds were soaked in bleach for 30 minutes and rinsed with sddH2O thrice. Finally, the seeds were placed in sterile filter paper inside Petri dishes. The Petri dishes were then placed in an incubator to allow the seeds to germinate for a week. While the mung bean seeds were being germinated, explants from sweet potato were obtained. For the sweet potato, 15 small cubes were cut up from the sweet potato body. The sweet potato cubes were then sterilized with the same steps used for surface sterilization of mung bean seeds (except for duration in bleach which is only 15 minutes due to soft flesh of sweet potato). After surface sterilization, 3 bottles filled with medium were prepared. Five sweet potato cubes were placed with enough spacing between them in each of the 3 bottles. The bottles were then covered with clear plastic, secured with rubber bands and labelled A, B and C.</p> <p>3</p> <p>When the mung beans have been germinated, their leaves were obtained. The cotyledonary leaves were then cut into four parts: the tip, the middle section, the last section of the leaf and the petiole. Six cotyledonary leaf parts were then placed as explants in one medium filled bottle. The bottle was then covered with clear plastic and secured with a rubber band. After the inoculation of the mung bean leaves, rice grains were used as another explant source. For the rice grains, the husks were removed and then the grains were surface sterilized using the same methods used previously. Five grains were then placed with enough spacing between them in one bottle filled with medium. The bottle was then covered with clear plastic and secured with a rubber band. All the bottles with explants were then gathered and placed in a dark area. The bottles were inspected periodically for contamination, appearance of callus, the type and size of callus and other developments. All observations were recorded. RESULTS The following images were taken via a Sony Cyber-shot DSC-W100 digital camera with a Carl Zeiss lens. Images were taken during laboratory classes at no regular interval. Sweet potato, mung bean leaf and rice grain explants were inoculated on the following dates respectively: 24 th of June, 1st of July and 3rd of July. The explants were checked at different observation and incubation periods with respect to the time in which they were inoculated. The sweet potato, mung bean leaf and rice grain explants were inspected and documented within their respective incubation periods of nine, sixteen and twenty three days. Not all documented images taken are included. Only the images that showed evident development are displayed in this section Development of Calli in Sweet Potato Explants (June 24- July 17, 2008)</p> <p>Figure 1. Development of callus in vessel A on the first day. This was the day of inoculation where no callus growths are observed.</p> <p>Figure 2. Development of callus in vessel A after 7 days. No evident formation of callus in all the explants. Discoloration (browning) was apparent on some explants.</p> <p>Figure 3. Development of callus in vessel A after 9 days. Lumps were observed on the surfaces of the explants.</p> <p>Figure 4. Development of callus in vessel A after 21 days. Formation of callus was found on a single explant. Callus formed is less opaque than the explant.</p> <p>Figure 5. Development of callus in vessel A after 23 days. Callus continued to grow and developed at one edge of the explant.</p> <p>Figure 6. Development of callus in vessel A after 23 days (top view). Callus is seen to be localized at one 4 corner only.</p> <p>Figures 1 to 6 show the induction and development of callus of sweet potato explants placed in culture vessel A. No apparent changes were observed after a week of inoculation except for the browning of a few explants (Figure 2). Appearances of suspicious lumps were observed on the explants after nine days of incubation. Definite callus formation was then detected on a single explant after 21 days (Figure 4) and continued growth and development of the callus was observed after 23 days (Figure 5 &amp; 6). The callus formed appears to be less opaque as compared to the appearance of explant.</p> <p>Figure 7. Development of callus in vessel B on the first day. No callus growth is yet observed.</p> <p>Figure 8. Development of callus in vessel B after 9 days. No major changes in terms of callus formation were visible on the explants.</p> <p>Figure 9. Development of callus in vessel B after 14 days. Formation of suspicious lumps were observed.</p> <p>Figure 10. Development of callus in vessel B after 21 days. Callus formation is observed at a base corner of a single explant.</p> <p>Figure 11. Development of callus in vessel B after 23 days. Callus continued to grow and develop.</p> <p>Figure 12. Development of callus in vessel B after 23 days (top view). Callus growth is seen on the upper left corner of the explant.</p> <p>Figures 7 to 12 illustrates the callus growth and development of sweet potato explant in culture vessel B. Changes were first observed only after `14 days of incubation through the growth of lumps on the explant surfaces (Figure 9). However, only a single explant showed evident callus formation after 21 days as shown in Figure 10. The observed callus appeared to be less opaque than the explant. The callus formed mostly on the basal part of the explant and continued to develop until the 23rd day (Figures 11 &amp;12). Out of the five explants inoculated on culture vessel B, only one explant showed obvious formation of callus. However, some irregularities were observed at the corners and edges of the other explants in the same vessel.</p> <p>5</p> <p>Figure 13. Development of callus in vessel C on the first day. No callus formation is visible.</p> <p>Figure 14. Development of callus in vessel C after 7 days. Surface discoloration was observed in the explants. Lumps were formed on some explants.</p> <p>Figure 15. Development of callus in vessel C after 14 days. Formation of callus was observed on the base corner of a single explant.</p> <p>Figure 16. Development of callus in vessel C after 21 days. The observed callus continued to grow on the corner length of the explant.</p> <p>Figure 17. Development of callus in vessel C after 23 days. Other callus formations were seen on other corners of the explant.</p> <p>Figure 18. Development of callus in vessel C (top view). Callus formation is seen on two corners of the explant. Also, lumps were observed on the top surface of the explant.</p> <p>Figures 13 to 18 demonstrate the formation and development of callus on the sweet potato explants placed in culture vessel C. Appearance of suspicious lumps were detected as early as 7 days after inoculation on the first day. Along with this, subtle discolorations were observed on some explants (Figure 2). Callus formation was apparent on a single explant after 14 days of inoculation as shown in Figure 15. The callus started to form at the basal corner of the explant (figure 17) and continued to grow towards the upper surface of the explant (Figure 18) after 23 days of incubation. Only one out of the five explants inoculated showed apparent callus development. However, some irregularities in texture were seen on the surfaces of some explants. Only three explants, one on each culture vessel, were observed to show definite callus growth. Each explant differed on the time until callus growth as well as on the degree of growth of its calli. The explant on culture vessel C (Figure 1...</p>