LABORATORY REPORT

Plant Physiology and Tissue Culture

MERISTEM CULTURE

Marie Joy Gilian C. Calalang, Karla Hanriette Caña, Hemsly Austin Dela Cruz,
Gillier Dela Cruz and Ryza Camille Enriquez

CAS 02-701A, Department of Biology, College of Arts and Sciences,

Rizal Technological University, Mandaluyong City
Prof. Jovita Anit
December 25, 2023

ABSTRACT

Meristem culture has made significant progress in plant biotechnology, revolutionizing

crop improvement, conservation, and disease management. Recent advances in regulatory

genes and signaling pathways have refined meristem culture techniques, allowing researchers

to better understand the role of meristems in plant growth and development. Sterilization,

nutrient formulation, and automation advancements have increased success rates, making

meristem culture more efficient and scalable. The applications of meristem culture are

numerous. Crop yields and quality have increased due to the production of virus-free,

genetically uniform planting material. It is also helpful in the conservation of endangered plant

species and the preservation of genetic diversity. Despite successes, challenges such as

specific variation and optimization for recalcitrant species remain. Addressing these challenges

and investigating genetic modification within meristems are the next steps.

KEYWORDS: Meristem culture, Micropropagation, Shoot apex, Clonal propagation, In vitro

cultivation, Meristematic cells, Explant,Tissue culture techniques, Plant regeneration.
INTRODUCTION

such as Panama disease (Ortiz, 2018).

Bananas, which belong to the
Understanding genetic traits such as
genus Musa, are a staple food for
drought tolerance guides the
millions of people around the world,
development of bananas that are
providing vital nutrients as well as
suitable for changing environmental
economic sustenance. Among the
conditions and nutritional requirements.
various species in this genus, Musa

acuminata stands out as a key cultivar, In our most recent laboratory

playing an important role in the world's experiment involving meristem culture,

agricultural and culinary landscapes. we utilized bananas as our primary

component. Meristem culture, also

Musa acuminata, also known as
known as shoot tip culture, is a plant
the "sweet banana," has been a staple
tissue culture technique that entails the
in cultivated bananas since 5000 BCE in
isolation and cultivation of meristematic
Southeast Asia (Perrier et al., 2011).
tissues. Meristems are regions of
Genomic advances, particularly genome
actively dividing cells located in the
sequencing, have revealed the genetic
growing tips of roots and shoots, as well
basis for traits such as disease
as in the cambium layer of vascular
resistance and yield (D'Hont et al.,
plants. These tissues harbor
2012). This knowledge expedites
undifferentiated cells with the potential
targeted breeding and improves cultivar
to differentiate into various cell types,
development. Musa acuminata genomic
facilitating the regeneration of entire
diversity is critical for breeding resilient
plants.
varieties that can withstand challenges
MATERIALS AND METHODS

The materials used in this

laboratory activity were banana (Musa

acuminata), Detergent Solution,

Fungicide Solution, Distilled water and

Bleach Solution.

Media Preparation

Set A:
Figure 1 : Removal of leaves and roots.
● MS + 20 ppm BAP + 100 ppm
Ascorbic acid (5 bottles or test tubes per
group)
● MS + 20 ppm BAP + 1 g/L charcoal (5
bottles or test tubes per group)

Set B:
● MS + 10 ppm BAP + 100 ppm
Ascorbic acid (5 bottles or test tubes per
group)
● MS + 10 ppm BAP + 1 g/L activated
charcoal (5 bottles or test tubes per
group)

To begin the process, carefully

Figure 2 : Rinsing into tap water
remove any leaves and roots from the

items, ensuring a thorough cleaning. Once the initial rinsing is

Subsequently, place the items under a complete, prepare a detergent solution

stream of running tap water, allowing the by combining 7 tablespoons of

flow to dislodge and remove any detergent with 1 liter of water. Submerge

accumulated dirt or debris. the items in this solution for a duration of

5 minutes, allowing the detergent to For an added layer of protection

effectively break down and remove any against potential fungal growth, immerse

remaining contaminants. Following this the items in a 1% fungicide solution,

step, rinse the items once again under consisting of 10 grams of fungicide per

running tap water for an additional 5 liter of water. Allow the items to soak in

minutes to ensure the complete removal this solution for a period of 20 minutes,

of detergent residue. ensuring that the fungicide thoroughly

penetrates and disinfects the surfaces.

Afterward, rinse the items with sterile

distilled water to eliminate any residual

fungicide.

Figure 3:Submerge to Detergent Solution

Figure 5:Submerge into Fungicide Solution

Figure 4: Rinse with running tap water after
detergent removal
To further sanitize the items,

prepare a 40% bleach solution by

combining 400 milliliters of bleach with 1

liter of water. Submerge the items in this tissues. Meristem culture is an important

bleach solution for 15 minutes, allowing technique in plant tissue culture,

the bleach to effectively disinfect and especially for propagating disease-free

sterilize the surfaces. Conclude the and genetically uniform banana plants.

process by rinsing the items three times

After selecting healthy,
with sterile distilled water, ensuring the
disease-free banana plants as the
complete removal of any remaining
source of meristems. The meristem, a
bleach residue.
small region at the growing tip of the

Figure 6: Rinsing using sterile distilled water shoot, is carefully excised for culture

initiation. Please keep in mind that we

This comprehensive cleaning and
are still using an aseptic technique.
disinfection protocol not only removes
Excise the meristem from the shoot tip
physical impurities but also addresses
with a sterilized scalpel. The precision of
potential microbial threats, ensuring that
this step is crucial for the success of the
the items are thoroughly cleansed and
culture, as the meristem contains
ready for their intended use.
undifferentiated cells capable of giving
Preparation of Banana Meristem
rise to a whole new plant.
Culture.
Meanwhile, a nutrient-rich culture
Banana Meristem Culture
medium tailored to the specific needs of
preparation is a meticulous process
banana meristems is being prepared. A
involving several steps to ensure the
precise balance of macro and
successful initiation and growth of
micronutrients, vitamins, sugars, and
banana plants from meristematic
growth regulators such as auxins and
cytokinins is typically present in this

medium. The medium's composition is

intended to promote the proliferation

and differentiation of undifferentiated

cells within the meristem.

After cleaning the meristem and

preparing the culture medium, the

meristem is carefully placed onto the

Figure 7: Inoculation
culture medium in a sterile environment.

This process is known as inoculation,

and it marks the beginning of the

meristem culture. The culture vessels

are then sealed to maintain a controlled

environment, and the cultures are

placed in an incubator with controlled

temperature and lighting conditions.

Figure 8: Culture placed in a controlled
temperature/light room.
 RESULTS AND DISCUSSION Medium # of Shoot
# Cultures Formation
with Shoots Rate (%)

Mediu Initial # # of Survival M1A 5 100

m of Cultures Rate (%)
# Cultures Survived M2A 4 80

M1A 5 3 60 M1B 1 20

M2A 5 3 60 M2B 0 0

M1B 5 1 20
Table no. 3 Total number of culture with
M2B 5 0 0 shoots and shoot formation rate

Table 1. Total Number of Media and the

survival rate.

The table 1 presents outcomes

for four experimental conditions (M1A,

Medium # of Cultures Contaminatio M2A, M1B, M2B), each starting with five
# Contaminated n Rate (%)
cultures. M1A and M2A show a
M1A 2 40
consistent 60% survival rate, while M1B
M2A 2 40

M1B 4 80 has a lower rate of 20%. M2B, however,

M2B 5 100 results in a 0% survival rate, indicating

Table 2. Total number of Contaminated harsh conditions. The data emphasizes

cultures and the contamination rate
the influence of experimental conditions

on culture survival, warranting further

investigation into specific contributing

factors.
 The table 2 reveals indicating no shoot formation. The data

contamination levels in distinct highlights variability in shoot

experimental conditions (M1A, M2A, development across conditions,

M1B, M2B). M1A and M2A show a 40% underscoring the need for further

contamination rate, indicating a investigation, particularly in M2B where

moderate susceptibility. In M1B, the rate shoot formation was entirely absent.

increases to 80%, signaling a more

Several observations were made
significant contamination issue. Notably,
over a week after implementing a
M2B exhibits a 100% contamination
modified sterilization procedure and
rate, highlighting a severe problem in
inoculating banana explants of varying
this condition. Addressing specific
ages and sizes. Maintaining aseptic
contamination factors in each setting is
conditions in meristem culture is critical
crucial, particularly in M2B, where all
for experiment success, necessitating
cultures were affected.
meticulous attention to detail in the

The table 3 outlines shoot sterilization of banana explants. The

formation outcomes in different sterilization agents used and the

experimental conditions (M1A, M2A, duration of exposure are critical, with

M1B, M2B). M1A demonstrates a 100% adjustments required based on explant

rate, indicating all cultures produced age and size to strike a balance

shoots, while M2A has an 80% rate. between gentler methods for younger,

M1B shows a lower rate of 20%, with smaller explants and more rigorous

only one culture developing shoots. sterilization for older, larger explants to

Notably, M2B records a 0% rate,

prevent tissue damage and eliminate explants to the growth medium, is

contaminants. critical in meristem culture. Even with a

well-established sterilization protocol,

maintaining sterility throughout the

process poses challenges, necessitating

work in a laminar flow hood or

cleanroom environment. Handling

explants is delicate, especially in the

meristematic region, requiring a steady

hand and precision for successful

inoculation without compromising

Figure 9: Visible contamination in Meristem
Culture (M2B)
meristematic tissue integrity.

Observations after the first week

offer valuable insights into culture

success. Contamination symptoms such

as discoloration or abnormal growth

may be evident. On a positive note, the

development of healthy callus or the

initiation of shoot regeneration indicates

Figure 10: Visible contamination in Meristem
Culture (M1B) successful culture initiation. The rate of

growth and overall morphology of

The inoculation procedure, which explants provide valuable information

involves the careful transfer of sterilized about banana tissue responsiveness to

culture conditions, necessitating regular responsiveness of banana tissue to the

monitoring to detect deviations from established culture conditions. Given the

expected outcomes and make timely dynamic nature of plant cultures, regular

adjustments to the culture environment monitoring is required to detect any

if needed. modifications from expected results as

soon as possible. This data introduction

underscores the significance of the

observed indicators in evaluating the

overall success of the culture and

highlights the need for continuous

vigilance to make timely adjustments to

the culture environment.

TABLE 4: CLASS DATA MERISTEM

CULTURE CONTAMINATION RATE

Figure 11: Visible shoot formation in

Meristem Culture (M1A)

Furthermore, the explants' growth

TABLE 5: CLASS DATA MERISTEM
rate and overall morphology were CULTURE SURVIVAL RATE

closely examined. These parameters

provide critical information about the

 share an 80% rate, groups 4 and 7

share a 60% rate, group 2 has a 40%

TABLE 6: CLASS DATA MERISTEM rate, and groups 5 and 6 show no

CULTURE RESPONSE RATE
contamination (0%). Lastly, M4, groups

3, 4, and 7 have a 100% contamination

rate, groups 2 and 8 have an 80% rate,

group 5 has a 40% rate, and groups 1

and 6 have the lowest contamination at

20%

As shown in table 4, M1 groups Furthermore in table 5 M1,

1, 2, 7, and 8 exhibit the highest groups 5 and 6 display the highest
contamination rate at 100%, followed by survival rate at 100%, followed by
group 5 at 50%. Groups 3 and 4 share a groups 3 and 4 with a 60% rate, while
contamination rate of 40%, while group groups 2 and 7 show a 20% rate, and
6 shows no contamination (0%). The group 1 has the lowest rate at 0%,
overall mean for groups 1–8 is 67.5. For resulting in a final mean of 40. Similarly,
M2, groups 1 and 8 have a 100% in M2, groups 5 and 6 demonstrate an
contamination rate, groups 2 and 7 80% survival rate, with groups 3 and 4
share an 80% rate, and groups 3 and 4 at 60%, and groups 2 and 7 at 20%,
share a 40% rate. Groups 5 and 6 show while groups 1 and 0 have the lowest
the lowest contamination. The mean for rate at 0%, yielding a final mean of 40.
M2 is 60. In M3, group 1 has a 100% In M3, groups 5 and 6 exhibit the
contamination rate, groups 3 and 8 highest survival rate at 100%, groups 4
and 7 have a 40% rate, and group 2

shows a 60% rate, while groups 3 and 8

CONCLUSION
share a 20% rate, and group 1 has the
In conclusion, the successful
lowest rate at 0%, resulting in an overall
implementation of a modified
mean of 47.5. These findings
sterilization procedure, as well as
underscore variations in survival
meticulous attention to the inoculation
capabilities among different groups
process, are critical factors in achieving
across the three scenarios..
favorable results in banana meristem

Lastly, In table 6 The response culture. The findings highlight the

rates for shoot formation in M1 and M3 importance of adjusting sterilization

indicate notable differences among agents and exposure durations based

groups. In M1, groups 3 and 6 on the age and size of the explants,

demonstrate a 100% formation rate, striking a delicate balance between

while groups 1, 2, 7, and 8 show no gentler methods for younger specimens

response. The overall mean for M1 is and more rigorous sterilization for older

37.5. In M3, groups 5 and 6 achieve a ones. The importance of maintaining

100% formation rate, with group 2 aseptic conditions during inoculation

surpassing the average at 60%. Groups cannot be overstated, requiring a

1, 7, and 8 exhibit no shoot formation. controlled environment such as a

The overall mean for M3 is 40. These laminar flow hood or cleanroom.

findings underscore the varied

responsiveness of groups in different The observations made during

scenarios. the first week of culture provide valuable

insights into the experiment's overall Regular culture monitoring is critical for

success. The presence of contamination detecting deviations from expected

symptoms and abnormal growth serves outcomes in a timely manner. This

as a critical indicator, emphasizing the proactive approach enables adjustments

importance of maintaining a sterile to the culture environment to be made,

culture environment at all times. On the ensuring optimal conditions for meristem

plus side, the formation of healthy callus culture success. Finally, the findings

or the initiation of shoot regeneration emphasize the intricate nature of

indicates successful culture initiation. banana meristem culture, emphasizing

The explants' growth rate and the importance of precision, adaptability,

morphology emerge as critical and continuous monitoring for achieving

parameters, serving as key indicators of consistent and positive results in tissue

banana tissue responsiveness to culture culture experiments.

conditions.
REFERENCES

Perrier, X., De Langhe, E.,

Donohue, M., et al. (2011).
Multidisciplinary perspectives on
banana (Musa spp.) domestication.
Proceedings of the National Academy of
Sciences, 108(28), 11311–11318.

D'Hont, A., Denoeud, F., Aury, J.

M., et al. (2012). The banana (Musa
acuminata) genome and the evolution of
monocotyledonous plants. Nature,
488(7410), 213–217.

Ortiz, R. (2018). Banana Breeding:

Progress and Challenges. Annual
Review of Plant Biology, 69, 45–68.