EXERCISE 3: Callus InductionGroup 1

Gerald Aquino Abraham Cruz Erica Fortuno Ciara Lim Date Due: July 31, 2008

Date Submitted: July 31, 2008

ABSTRACTExplants, 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.

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.

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

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 Ingredients Amounts (grams) Side NotesMS MACRO(500 ml stock solution)

NH4NO3

KNO3

CaCl2-H2OMgSO4-7H2OKH2PO4

16.5019.004.4003.7001.700

MS MICRO(500 ml stock solution)

Solution A MnSO4-H2O H3BO3

ZnSO4-7H2O

1.1150.3100.430

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Solution B KI NaMoO4-2H2O Solution C

CuSO4-5H2OCoCl2-6H2O

0.4150.125

0.1250.125

0.0415 (for 50mL) 0.0125 (for 50 mL)

0.0025 (for 10 mL) 0.0025 (for 10 mL)

Fe-EDTA(250 ml stock solution)

FeSO4-7H2ONA2EDTA

0.6960.931

MS Vitamins(50 ml stock solution )

Nicotinic AcidPyroxidine HClThiamine HClGlycine

0.0500.0500.0500.050

Myo-inositol 0.100

Sucrose 30.00Agar 10.00

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, Fe-EDTA, 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 (sddH2O) 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.

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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.

Figure 3. Development of callus in vessel A after 9 days. Lumps were observed on the surfaces of the explants.

Figure 1. Development of callus in vessel A on the first day. This was the day of inoculation where no callus growths are observed.

Figure 5. Development of callus in vessel A after 23 days. Callus continued to grow and developed at one edge of the explant.

Figure 6. Development of callus in vessel A after 23 days (top view). Callus is seen to be localized at one corner only.

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.

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)

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Figure 8. Development of callus in vessel B after 9 days. No major changes in terms of callus formation were visible on the explants.

Figure 9. Development of callus in vessel B after 14 days. Formation of suspicious lumps were observed.

Figure 7. Development of callus in vessel B on the first day. No callus growth is yet observed.

Figure 11. Development of callus in vessel B after 23 days. Callus continued to grow and develop.

Figure 10. Development of callus in vessel B after 21 days. Callus formation is observed at a base corner of a single explant.

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.

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 & 6). The callus formed appears to be less opaque as compared to the appearance of explant.

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 &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.

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Figure 13. Development of callus in vessel C on the first day. No callus formation is visible.

Figure 14. Development of callus in vessel C after 7 days. Surface discoloration was observed in the explants. Lumps were formed on some explants.

Figure 15. Development of callus in vessel C after 14 days. Formation of callus was observed on the base corner of a single explant.

Figure 16. Development of callus in vessel C after 21 days. The observed callus continued to grow on the corner length of the explant.

Figure 17. Development of callus in vessel C after 23 days. Other callus formations were seen on other corners of the explant.

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.

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 17 & 18) gave the earliest and most extensive callus growth than those in vessels A and B. Overall, the frequency of callus formation in explants, 3 out of 15, is low after 23 days and might be due to different factors.

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Figure 19. Development of calli on portioned mung bean leaf. (A) No evident callus formation after two days of incubation. (B) Callus and adventitious roots (black arrow) started to form after 7 days of incubation. (C) Callus continued to grow and became massive (maroon arrow); discoloration (blue arrow) was apparent on a few explant after 14 days. (D) Callus formation and growth increased (maroon arrow) and evident growth of adventitious roots in some calli (yellow arrow) was observed after 16 days.

A B

C

DC

Development of Calli in Mung Bean Leaf Explants (July 1 to July 17, 2008)

Cotyledonary leaves from germinated mung bean seeds were incubated on MS basal medium for 16 days. Figure 19 illustrates the development of calli on the mung bean cotyledonary leaf explants. No callus formation was observed after two days of incubation (Figure 19A). Figure 19B shows apparent callus formation after 7 days as well as formation of adventitious roots. Calli were observed to initially form at the midrib of the cotyledonary leaves as marked in Figure 19B. Calli continued to grow and form after two weeks along with the increase in growth of adventitious roots as shown in Figure 19C. In the same figure, the discoloration of some calli is seen. More extensive callus formation in some leaf sections, mostly on the basal part, as well as further elongation of adventitious roots were visible after 16 days (Figure 19D). Callus that was initially formed at the midrib did not show any development after 7 days. Out of six explants, only two did not show any formation of callus after 16 days of incubation. The calli formed were white to cream in color and semi-transparent in appearance.

Development of Calli on Rice Grain Explant (July 3-July17, 2008)

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Figure 20. Callus induction in rice grain explants. Adventitious root growth in an explant as well as fungal contamination occurred after 5 days of incubation.

Callus formation in the rice grain explants cannot be seen clearly due to fungal contamination. However, adventitious root formation was evident on an explant in Figure 20. But due to the occurrence of the contamination, the observation period was discontinued and the set up was discarded.

DISCUSSION

Found in angiosperms, gymnosperms, pteridophytes and bryophytes (Purohit 2005), a callus is an amorphous aggregate of loosely arranged parenchyma cells that proliferate from the mother cells (Dodds and Roberts 1995). It has no meristematic tissues and is considered abnormal (Purohit 2005) because it has neither structural nor functional counterpart on any tissue from a normal plant (Dodds and Roberts 1995). However, it is beneficial because it has the potential to produce normal roots and embryoids, which can turn to plantlets and further develop into viable plants. Two types of calli can arise: 1) the more compact type that is usually embryogenic, and 2) the loose and friable type that is usually non-embryogenic.

Growth characteristics of callus involve the relationship among the plant material, composition of medium, plant growth regulators, and conditions during incubation. Hence, conditions for callus induction are highly variable among different species of plants. The conditions are often optimized for each type of tissue and plant.

The first consideration for callus induction is the source of explant. As in most cases, the explant has to be free from disease and contamination. It is also usually juvenile for better regeneration. For callus initiation, all multicellular plants can be potential sources of explants (Dodds and Roberts 1995). However, the best plants for callus initiation are those that are rich in nutrients and have endogenous hormones. Examples of these are storage organs (e.g. potato tubers, sweet potato, carrots) and cotyledon of seeds (e.g. soybean cotyledon). For this experiment, the explants used were from rice (Oryza sativa) grain, sweet potato (Ipomoea batatas), and mung beans (Vigna radiata).

Calli are natural growths resulting from wounding at the cut end of roots or stems. Initiation of callus is, therefore, usually performed by wounding the explant. In the experiment, this was done by dicing the sweet potatoes, by cutting the ends of the rice grain, and by slicing the cotyledonary leaves of the mung beans. Wounding increases the surface area of the explant and so further exposing it to the nutrient medium. Usually, a wounded plant material tends to

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take in more growth regulators in its environment to repair itself. However, since auxin is the only hormone present in the medium, instead of repair, cellular dedifferentiation or callus formation is induced.

Callus induction is stimulated by the presence of exogenous growth regulators added to the medium. The hormonal requirement for initiation depends on the origin of the explant (i.e. plant species). For callus initiation, some explants need: 1) auxin only, 2) cytokinin only, 3) both auxin and cytokinin, and 4) complex natural extract (Dodds and Roberts 1995). Auxin stimulates shoot cell elongation, formation of adventitious root and it plays a role in embryogenesis. The commonly used exogenous auxins are 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA). Cytokinin’s ability to promote cell division makes it initiate callus formation in tandem with auxin. The commonly used exogenous cytokinins include kinetin and 6-benzylaminopurine (BAP).

In this experiment, however, the MS basal medium was only added with 2,4-D. According to Dodds and Roberts (1995), it is one of the most effective auxins for callus proliferation and is often employed without exogenous cytokinin. It is also a powerful suppressant of organogenesis; therefore, it allows maintenance of the callus. On the other hand, regeneration of a plant from callus involves the transfer to a medium without 2,4-D and requires the addition of exogenous cytokinins (Hall, 1999).

Prior to establishment in medium, the explants were surface sterilized to remove dirt and possible microbial contaminants. Then, the explants were placed in the MS basal medium with 2,4-D. The explants, especially the wounded parts, were pressed against the medium to ensure exposure to the necessary growth requirements. After this, the explants were incubated at room temperature inside a dark cabinet. The dark is needed because it induces faster callus development and because light induces differentiation, which is unwanted for callus induction (Slater et al, 2003). Before the explants were incubated, its containers were sealed with plastic to avoid desiccation (Dodds and Roberts, 1995). The development of callus on each explant was then observed.

After two to three weeks of documentation and observation, only a few explants of the sweet potato were able to dedifferentiate into callus. However, in the leaf explants, most leaves have given signs of callus and adventitious tissue formation. Unfortunately, due to the contamination in the rice grain culture, this set up was discarded.

Before callus formation was evident in all of the sweet potato set ups, it was the growth of lumps or the development of a rough surface texture that first manifested. This according to Dodds and Roberts (1995) is an indication of the start of callus growth in the explants. Since most of the explants in the tuber showed this characteristic, it is possible that callus formation is occurring in the explant. However, due to time constraint the development of some of these explants cannot be observed. In the event of a reexperimentation, the incubation period for the sweet potato explants should be lengthened to give time for callus to grow on the explants. Commonly, callus cultures are given a maximum of six weeks to develop in a solid medium (Dodds and Roberts, 1995).

In some of the figures shown in the previous section, there were a few explants noted to have a brown discoloration in its tissue and in some in the callus itself. A brown color is an indicator of necrosis or cell death in those parts (Dodds & Roberts, 1995). The discoloration in the tissue may be caused by oversterilization of the explant such as overexposure to bleach or ethanol and thus cells died. This can be remedied by reducing the concentrations of the sterilizing agent

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(Phillips et al, 1995). Meanwhile, the necrosis in the callus may be caused by insufficient nourishment from the medium which can be solved by increasing the amount of the medium placed in the bottle.

The most evident problem in the sweet potato callus cultures is the low frequency and slow growth of the calli. This may be caused by insufficient wounding of the samples, the oversterilization of the explants and/or the displacement of the samples from the nutrient medium. By introducing more wounding sites to the explant, there is a strong tendency for callus formation in response to this (Phillips et al, 1995). Meanwhile, the oversterilization of the explants cause the death of the cells on the surface of the tissue and thus disallowing any form of growth. This is overcome by cutting of the surface of the tissue. Not only does this remove the dead cell layer, it also increases the number of wounding sites in the explant. Lastly, the displacement of the explant from the medium inhibits the tissue from nutrient uptake and so it cannot survive in an in vitro set up. For future studies, the explant must be properly planted on the nutrient media making sure that the entire face of its bottom has an interaction with the agar. Another factor that can influence this is the length of time in which the callus was allowed to grow.

In the mung bean leaf cultures, adventitious root formation is witnessed on the petiole section of the leaf and callus formation is shown on several of its sections. The discoloration of the callus in this context is not of necrosis since callus cultures of white to cream colors are still considered viable. Furthermore, this discoloration is a natural occurrence that may be attributed to the oxidase activity of some enzymes that the cell contains. Although, too much of this activity retards cell division. For security purposes in future experiments, an antioxidant mixture should be introduced to the sample (Dodds & Roberts, 1995).

Adventitious root formation in this set up is due to the auxin, 2,4-D in the medium which stimulates this kind of growth. The reason why not all of the sections manifested this response is because different cell parts (e.g. the petiole) react differently to varying auxin concentrations. Another possible reason is that the sections do not receive the same amount of auxin due to the varying exposure with the nutrient medium. During the experiment, some of the leaves curled up therefore decreasing the area by which they are exposed to the medium. This curling up can be prevented by ensuring the plantation of the section to the medium.

The last set up to be discussed is the rice grain callus culture. This is the unfortunate set up to have contained a contamination. Though one of the explants showed adventitious root formation, the explant was soon depleted due to the consumption and competition of the contaminating fungus. The surface sterilization of the explant needs to be more stringent and the sterilization of the materials used for the inoculation should be double-checked. Another reason for contamination is the probable nearness of the vessel to the opening of the laminar flow hood. This proximity may have allowed the entry of fungal spores into the medium. Therefore, inoculation should be done as farthest away from the flow hood opening as possible.

Studies involving callus usually do not involve callus induction alone. When the callus has developed, after some time, it is transferred to a new maintenance medium to replenish its nutritional requirements. Friable calli are often maintained in suspension media. To induce organogenesis, the calli are transferred into media with the appropriate auxin-cytokinin ratio. A high auxin to cytokinin ratio promotes root formation, while a low auxin to cytokinin concentration favors shoot formation. The combinations of plant regulators can then be manipulated to generate plantlets for further study or for micropropagation.

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CONCLUSION

Based on the results and discussion given it is evident that the quality of the explant, the medium in which the explant is placed and the incubation period of the culture influences callus growth.

The explant quality is affected by the effectiveness of the sterilization techniques in which it was exposed to. An oversterilized explant has dead surface cells and so disallows callus formation and rather promotes necrosis as characterized by the brownish tissue color. In contrast, an understerilized explant increases chances of contamination and therefore renders the set up invalid. Lastly, the nature of the explant determines how it reacts to the hormone present in the medium. It can either manifest as adventitious tissue growths or callus formation.

Secondly, the medium which holds the explant determines the longevity of the culture. The medium is the one that sustains the survival of the tissue by providing it nourishment. The hormones it contains such as auxin in the experiment is the one that stimulates the growth of callus and adventitious tissues.

Thirdly, the incubation period of the culture allows the callus to grow as it must. A short incubation period such as the one in the experiment did not allow for the completion of the callus development. Thus, the callus growth observed are only found in a few explants, as most of the other explants were only beginning callus formation as characterized by the lumps on its surface. Meanwhile, a very long incubation period slowly kills the tissue. This is because by this time, all the nutrients in the medium have been consumed and there is already a build up of toxic wastes from the tissue.

REFERENCES

Dodds, J. and L. Roberts. Experiments in Plant Tissue Culture. 3rd ed. USA: Cambridge University Press, 1995.

Hall, R.D. Plant Cell Culture Protocols. New Jersey: Humana Press, 1999.

Phillips, G.C., J. Hubstenberger and E. Hansen. “Plant Regeneration by Organogenesis from Callus and Cell Suspension Cultures.” Plant Cell, Tissue and Organ Culture: Fundamental Methods. Ed. Dr. Oluf Gamborg and Gregory Phillips. Germany: Springer-Verlag Berlin Heidelberg, 1995.

Purohit, S.S. Plant Tissue Culture. Jodhpur, India: Shyam Printing Press, 2005

Slater, A. Scott, N. Fowler, M. Plant Biotechnology: The Genetic Manipulation of Plants. Oxford: Oxford University Press, 2003.

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