Course name: Biotechnology I

Course Code: BIOT3113

Title: Introduction to Tissue Culture
Date: 26/09/2022

Title
Selection, surface sterilization, and initiation of explants

Aim

1. To select and extract the explant (pepper seeds) from Scotch bonnet pepper (Capsicum
chinence).
2. To investigate the effects of various surface sterilization methods on the explant (pepper
seeds) on the germination and growth of the pepper seed in culture media over three
weeks in tissue culture (initiation of explants).

Introduction

An explant is a portion of a plant used in the plant tissue culture procedure to create a
whole plant. An explant can be a shoot fragment, root fragment, leaf section, and in the case of
this experiment a seed from a pepper. In most cases, explants are removed by trimming or
cutting the explant form the plant. Explants are often the starting material for plant tissue culture
especially in micropropagation. Micropropagation is an in vitro vegetative propagation of plants
using a defined nutritional medium and high light intensity to create multiple identical clones of
a plant from the explant. Once the explant is carefully extracted to ensure that it is not damaged
the explant is then sterilized to remove any unwanted contaminants.

The aseptic technique is using an approach which involves taking precautions to reduce the
presents of unwanted microbes and other contaminating particles. During this experiment, the
sterilization method (a form of aseptic technique) which was used. It involved placing and
soaking the pepper seed in 10% bleach (for 10 minutes and 20 minutes respectively). Bacterial
pathogens and some viruses, like the tobacco mosaic virus, can be successfully eliminated from
the surfaces of seeds by treating them with a sodium hydrochloride commonly known as bleach.
It is important to sterile the seeds (or any explant) before placing them onto the culture media to
avoid unwanted growth of microorganism. The presents of contaminating particles and microbes
may hinder seed germination and plant growth. In addition to sterilizing the seed the entire
experiment was carried out in a sterile environment which includes the Lamina Flow and the
Mobile Micropropagation Unit. The Laminar Flow cabinet is intended to keep biological
samples, and other types of materials free of contamination. A HEPA filter is used to extract
contaminated air from the system. The sample is protected from the operator (person
manipulating the sample) due to the direction of air movement, but the operation is not protected
from the sample. Comparatively, the Mobile Micropropagation Unit a constructed cabinet uses
relatively the sample principle to keep the work area sterile.
Once the explant is sufficiently sterilized and rinsed with water, they can be introduced onto the
culture media. The culture media is defined which means it is specific combinations of nutrients
and other materials of known concentration and amounts used to support the plant growth. Once
the explant is in contact with the medium it used the available nutrients to germinate. Using this
method several plants can be produced from one plant i.e., several pepper seeds from the pepper
are micropropagated to produce several identical pepper plants.

Literature review

In the study entitled the ‘Effects of Rice Seed Surface Sterilization with Hypochlorite on
Inoculated Burkholderia vietnamiensis’ a combination of hydrogen peroxide and hypochlorite were
used to sterilize rice seeds. The article highlights that substances that are most frequently used for
sterilizing are oxidative ones, such as household bleach, which is made of sodium hypochlorite.
Further, the article made a point of how essential it was to rinse the seeds after sterilization and
before inoculation into the culture media was raised. This is done to prevent over exposure of the
explant to the oxidizing agent used to sterilize it which can cause the explant tissue damage
inhibiting it’s grown and may lead to death. The findings of this study thus show that the use of
sodium hypochlorite as a seed surface disinfectant to make gnotobiotic models; in addition to
killing surface pollutants, hypochlorite also kills most bacteria. This conclusion parallels with
many other studies of its kind which investigate the effects of surface sterilization on
contamination reduction, gemination and/or plant growth.

Another study with a similar aim is entitled ‘Sterilization factors affect seed germination and
proliferation of Achyranthes aspera cultured in vitro.’ The aim of that study was determining the
best the sterilization methods for in vitro culture of A. aspera. To get many sterilized seeds as
well as propagated explants for additional tests, it was crucial for the study to determine and
optimize a standard surface sterilizing technique using these various sterilant for seed explants.
After experimentation in the study which used various sterilant at different concentrations it was
determined that nystatin and flugal (antifungals) usually used to treat humans were must
effective at sterilizing the seeds which when place in culture media to germinate yielded the
greatest amount of progeny. Moreover, in the discussion and conclusion of the study it was
stated that both sodium hydrochloride and mercury chloride were the best substitutes for the
nystatin and fungal used. Once again, the use of sodium hydrochloride (bleach) as a sterilant for
the surface sterilization of seed indented to be used for tissue culture is proven to be a reliable
and effective method of sterilization.

Method and Materials

Materials and Equipment

Scotch bonnet pepper (Capsicum chinence)
70% isopropyl alcohol
10% Bleach
Distilled water
Test tubes
Test tube rack
Scalpel
Forceps
Tweezers
Petri dish
Test tubes
Parafilm
Timer
Lamina Flow
Mobile Micropropagation Unit

Method

 Selection of explants

1. Students at the respective benches (12 students) were grouped together and then split in
half with groups consisting of 5-6 persons. One of the two groups was assigned to sterile
area the Lamina Flow and the other to the Mobile Micropropagation Unit another sterile
area.

2. Fifteen test tubes were obtained. The five test tubes were labelled individually with PS
(for pepper seed), sterilization method (S1, S2, S3) this was triplicate (eg. 5 test tubes
labelled S1 etc.). The label included: the clean area used the Lamina Flow or Mobile
Micropropagation Unit (MMU or LF), group number, date, and the student’s initials, the
type of sterilization method used and the type of media.

3. Three sterilization methods include no sterilization (S1), 10% bleach for 10 minutes (S2),
and 10% bleach for 20 minutes (S3).

4. Students who worked under the Laminar Flow or Mobile Micropropagation Unit used the
sterilization method which involved using the 70% alcohol to sanitize the hands, the
surroundings inside the unit and the equipment and the materials used.

5. Using sterile forceps and the scalpel the Scotch bonnet pepper (Capsicum chinence) was
cut open on the petri dish it was contained in to expose the pepper seeds. Using a pair of
tweezers all the pepper seeds were removed.

 Surface sterilization
 6. A seed was individually placed into a test tube labelled S1 (no sterilization) using the
tweezers. This was repeated for the remaining S1 test tubes.

7. The remaining pepper seeds were then placed in a test tube which contained with 10%
bleach for 10 minutes then some of the seeds were removed and placed in another test tube
which contained water to be rinsed using tweezers. The remaining seeds in the test tube
were left in the 10% bleach for an additional 10 minutes (total time =20 minutes).

8. After the seeds that were left in the bleach for only 10 minutes were rinsed with water;
individual seeds were placed into test tube that contained solidified agar. One seed per test
tube labelled S2.

9. The seed that were soaked in the 10% bleach for 20 minutes were then washed in a test
tube containing water and then into test tube that contained solidified agar. One seed per
test tube labelled S3.

 Initiation of pepper seeds Effect of Sterilization method used & Clean area used

10. After the initiation of pepper seeds into culture (placed in respective test tubes) the test
tubes were removed from the sterile areas (LF and MMU) wrapped with parafilm and left
to germinate.

11. Over a three-week period, the test tubes were observed and checked for in-vitro growth or
contaminations. All observations were recorded via pictures and a weekly log.

Results
  Plant Selection

Fig.1
Photo of an Ackee (Blighia sapida) tree
branch.

Common name: Ackee

Scientific name: Blighia sapida
Fig. 2

Photo highlighting the nodes (potential explants) of the Ackee (Blighia sapida) tree.

 Pepper seed initiation Experiment

*A FEW SAMPLE
PHOTOS FROM
THE
EXPREMENT

Week 1
Fig. 3 MMU S1 Fig. 4 MMU S2

Descriptions

Fig.3 MMU S1 test tube with bacterial growth.

Fig. 4 MMU S2 test tube with no plant growth or

contamination.

Fig.3 MMU S3 test tube with no plant growth or

contamination.

Fig. 5 MMU S3
Week 2
Fig.6 LF S1 (growth) Fig.7 LF S2

Descriptions

Fig.6 LF S1 test tube with root growth.

Fig. 7 LF S2 test tube with funga; growth

(contamination).

Fig.8 Show the LF S3 test tube with no plant growth or

contamination.

Fig.8 LF S3

Week 3
Fig. 10 LF S1 Fig. 11 LF S2

Descriptions

Fig.10 LF S1 test tube with pepper seed plant growth.

Fig. 11 LF S2 test tube with no plant growth or

contamination.

Fig. 12
LF S3 test tube with no plant growth or contamination.

Fig.12 LF S3
Table 1

Observations of the tissue culture to investigate the contamination or in vitro in the grow of the pepper seed (explant) made after a
week of being subjected to various surface sterilization methods.
Table 2

Observations of the tissue culture to investigate the contamination or in vitro in the grow of the pepper seed (explant) made after two
weeks of being subjected to various surface sterilization methods.
Table 3

Observations of the tissue culture to investigate the contamination or in vitro in the grow of the pepper seed (explant) made after
three weeks of being subjected to various surface sterilization methods.
Key

Y = Yes
N = No
D = Discarded
LF = Lamina Flow
MMU = Mobile Micropropagation unit
S1 = No sterilization
S2 = 10% bleach for 10 minutes
S3 = 10% bleach for 20 minutes
Bac = Bacteria

Discussion

The aim of selecting and extracting the explant (pepper seeds) from Scotch bonnet pepper (Capsicum chinence) as well as to
investigate the effects of various surface sterilization methods used to sterilize the explant (pepper seeds) on the germination and
growth of the pepper seed in culture media over three weeks was successfully achieved. Over the three-week period, test tubes which
contained the explants were observed and checked for in-vitro growth or contamination. All observations were recorded via pictures
and a weekly log. Prior to the experiment contamination was expected to be seen in the S1 test tubes for both the Laminar Flow (LF)
and Mobile Micropropagation Unit (MMU). Reason for this is expectation is although the pepper seeds were extracted in these sterile
environments any contaminants (unwanted microbes and/or other particles) present in the pepper would have been introduced into the
medium. Furthermore, it was expected that most of the growth seen would have been seen in the S2 as they were sterilized using 10%
for 10 minutes thus most if not all contaminants would be removed. Additionally, exponential growth from the S2 was expected as
they were not sterilized (exposed to the harsh bleach) for as long as the pepper seed in the S3 test tubes which would have a high
exposure to the bleach therefore increasing the risk of potential tissue damage due to oxidation resulting in no growth. As a result of
that potential risk that is, over exposure to bleach and subsequent pepper seed tissue damage it was expected that the pepper seed in
the S3 test tube would have no combination or plant growth over the three-week period. Additionally, it antipathized that even if
contamination was present, the test tube the tubes that were manipulated in the LF would be less contaminated than those handled in
the MMU. This was the initial assumption that the LF has two areas of air flow were the “dirty” could leave the system while MMU
only has one. Additionally, the introduction of contaminants to the MMU seems more likely to occur as the plastic tarp used to
separate the inside unit environment and the outside is prone the lifting as the operator works; the LF does not have this issue.

Based on the observations there were discrepancies between what was expected, and the actual results (see tables 1-3). As expected,
there was no plant growth in any of the test tubes after one week because the seed needed sufficient time (at least 2 weeks to
germinated and produce a root). However, all the S1 test tubes handled in the MMU were contaminated with fungus. This was
expected as the seed were not sterilized, and they were placed inside of the medium instead of on the surface where oxygen is readily
available for the seed (oxygen would diffuse slower in the media) therefore the possibility of plant growth was low. There were no
contaminates in the S1 test tubes manipulated in the LF which was unexpected as the seed were also not sterilized. Further, the seed
were placed on the surface of the medium therefore the possibility of plant growth was high as oxygen was readily available and easily
accessible. As for the other test tubes no contamination or plant growth was observed. In week 2 of the experiment, seed germination
and root growth were observed in a LF S1 test tube and a MMU S2 test tube this was not unusual. The seed were given adequate time
and nutrients supplied by the nutrient media to germinate and produce root. As to why grow was not observed in the other test tube
there might have been manipulatable physiological seed characteristics that prevent or promoted slow seed germination and root
growth, the seed may have been damaged during manipulation (extraction from pepper). There was no contamination or plant growth
observed for the remainder of the test tubes. In the third and final week of this experiment, the lab demonstrator discarded the
contaminated test tubes as no plant growth would be possible under those conditions (presence of fungi) in the test tubes. In week
three, a MMU S3 test tube was contaminated with a bacterium; this was unexpected as the pepper seed where subjected to 10% bleach
for 20 minutes there all the microorganisms should have been killed and the those present when the seed was initially introduced to the
medium would have grown and been observed within the first two weeks of the experiment. Therefore, one can conclude that while
handling the test tubes at weeks 1 and 2 contaminant would have to be introduced into that test tube.

Precautions:

The bleach used to sterilize would need to be strong enough to remove all contaminants but gentle enough to allow future growth by an
implanted organism, which presents a paradoxical situation for this experiment. If the pepper seeds were left for too long in the bleach
tissue damage would have been induced due to oxidation thus killing the plant material. The sensitivity of the plant material calls for
extreme precaution and accuracy to be used when conducting the surface sterilization aspect of this experiment. Moreover, the biases
brought on by hypochlorite (sodium hydrochloride – bleach) and the risk of damaging plant material can be avoided in a number of
different ways. Utilizing rinse products that neutralize chloramines, such as peptone, dithiothreitol, ascorbic acid, or rinse sufficiently
with water is one option.
Conclusion
Scotch bonnet pepper (Capsicum chinence) seeds (explants) were extracted and the effects of various surface sterilization methods
used to sterilize the explant (pepper seeds) on the germination and growth of the pepper seed in culture media over three weeks in
tissue culture was investigated.

References
Kumer Sen, M., Abu, M., Mostofa, H., & Nasrin, S. (n.d.). Sterilization factors affect seed germination and proliferation of

Achyranthes aspera cultured in vitro. Eeb.Lu.Lv. Retrieved October 28, 2022, from

http://eeb.lu.lv/EEB/201309/EEB_11_Sen.pdf

Miché, L., & Balandreau, J. (2001). Effects of rice seed surface sterilization with hypochlorite on inoculated Burkholderia

vietnamiensis. Applied and Environmental Microbiology, 67(7), 3046–3052. https://doi.org/10.1128/AEM.67.7.3046-

3052.2001

Mitchell, S. (2022). BIOT3113 Biotechnology 1 Tissue Culture laboratory Manual. Plant Tissue Culture, 2-11.