Labwork of plant biotechnology lesson 1 cultural medium and plant sample sterilization

LABWORK OF PLANT BIOTECHNOLOGY Lesson 1 CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATION Supervisor M Sc... 3 Labwork Of Plant Biotechnology CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATI

LABWORK OF PLANT BIOTECHNOLOGY

Lesson 1 CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATION

Supervisor M Sc Tran Quoc Tan

GROUP 7

Name of student 1: HUỲNH VĨNH THOẠI | 20187080

Name of student 2: LÊ MINH TUẤN | 20187215

Name of student 3 NGUY: ỄN THỊ ÁNH TUY T 20187218 Ế | Representative student email: minhtuan.lmtcns@gmail.com

Table of Contents

I OBJECTIVE 3

II MATERIAL AND METHOD 3

1 Material 3

2 Method 3

III RESULT 6

IV DISCUSSION 11

1 Seed fail 11

2 Infected seed 12

3 Differences in development between samples 14

V REFERENCES 15

List of Figures Figure 1: Procedures performed outside the Biosafety cabinet 4

Figure 2: Procedures performed inside the Biosafety cabinet 5

Figure 3: The cultured sample did not germinate 11

Figure 4: The culture sample was found to be contaminated 12

Figure 5: Developmental differences between cultured sample 14

List of Tables Table 1: Experimental results 6

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Labwork Of Plant Biotechnology

CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATION

I OBJECTIVE

To understand and know the aseptic technique during culture Additionally, to be capable of preparing some plant culture mediums

II MATERIAL AND METHOD

1 Material

Plant material: 10 peanut seeds (in dried fruit)

Plant culture medium: Murashige and Skoog medium (MS) culture peanut seeds in vitro The medium is autoclaved at 1 atm, 121oC for 15 minutes

Equipment: Erlenmeyer flasks (250 ml), measuring cylinder (25, 100, 1000 ml), Becher 250 mL, short test tubes with a rack, forceps, scalpel handles, disposable scalpel blades, alcohol lamp, sterilized surface of paper or glass

Chemicals and reagents needed:

• Sterilization experiment: Javel solution (1:9), dish-washing liquid, ethanol

96 , sterilized water o

• Medium chemical: Macronutrients and micronutrients, mineral salts and vitamin of MS medium, sucrose, agar, distilled water

2 Method

Purpose: Understand how to manipulate aseptically under the hood and plant tissue sterilization process

Procedure:

Figure 1: Procedures performed outside the Biosafety cabinet

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Labwork Of Plant Biotechnology

CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATION

Figure 2: Procedures performed inside the Biosafety cabinet

Observation criteria: Samples were cultured for 14 days then recorded: number of infected tubes that did not germinate, infected tubes that germinated, and uninfected tubes that germinated

III RESULT

Table 1: Experimental results

Status: Germinated, not contaminated

by microorganisms, strong root

development

Status: Germinated, not contaminated

by microorganisms, strong root development

Height: 9.8 cm Height: 9.3 cm

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Labwork Of Plant Biotechnology

CULTURAL MEDIUM AND PLANT SAMPLE STERILIZATION

Status: Germinated, not contaminated

by microorganisms, weak root

development, the stem of the plant sho

very weak development

Status: Germinated, not contaminated

by microorganisms, strong root development, the stem of the plant sho quite weak development

Height: 2.5 cm Height: 6.3 cm

Status: Germinated, not contaminated

by microorganisms, plants are growing

well, strong root development, the stem

of the plant shows vigorous developme

Status: Germinated, not contaminated

by microorganisms, plants are growing well, strong root development, the stem

of the plant shows vigorous developme

Height: 15 cm Height: 13.1 cm

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Labwork Of Plant Biotechnology

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Status: Germinated, not contaminated

by microorganisms, slow growth rate,

weak root development, the stem of th

plant shows very weak development

Status: Germinated, not contaminated

by microorganisms, plants are growing well, strong root development, the stem

of the plant shows vigorous developme

Height: 2.7 cm Height: 12.8 cm

Status: Not germinated, not

contaminated by microorganisms

Status: Not germinated, contaminated

by microorganisms

Height: 0.8 cm (seed height) Height: 0.9 cm (seed height)

The results of our group after the experimental period are as follows:

Number of germinated seeds: 8 seeds (germination rate 80%)

Number of seeds not contaminated: 9 seeds (contamination-free rate 90%)

Number of contaminated seeds: 1 seed (contamination rate 10%)

Among these, there was one seed that neither germinated nor was co ntaminated

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Labwork Of Plant Biotechnology

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IV DISCUSSION

1 Seed fail

Figure 3:The cultured sample did not germinate

Ignoring s uitable environmental conditions, seeds fail to germinate either because they are dead or dormant This can happen because, during the preparation of the seeds, they may have been washed too vigorously or bumped during the shaking of the Erlenmeyer flask, leading to damage or death Damage to the embryo can lead to several consequences that directly impact the seed's ability to germinate For example,

a damaged embryo may lose its vitality, interrupting the necessary metabolic processes and causing cell death or the inability to resume growth This results in a direct negative impact on germination success

In another scenario, if the test tube was accidentally sealed too tightly during the experiment, the bean seeds might not receive enough oxygen Plants require oxygen to live, and it is crucial for seed germination Oxygen enables the resumption of respiration and reactivation of metabolic processes during seed imbibition, resulting in

the production of reducing power and ATP, which are essential for survival and growth Some studies indicate that the state of oxygen availability (hypoxia) slows down ABA degradation in the embryo (Corbineau, 2022) This can lead to increased sensitivity of the embryo to ABA, hindering germination The presence of a seed coat (glumellae) seems to be linked to this effect GA promotes germination, while ABA inhibits it Hypoxia can make seeds insensitive to GA, further preventing germination There seems to be some disagreement on how hypoxia affects embryo sensitivity to

GA depending on the dormancy type (primary vs secondary)

Therefore, it can be concluded that our seeds failed to germinate because either the germination conditions were not optimal or the seeds were dormant

2 Infected seed

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is infected by bacteria (Ali et al., 2018) Bacteria and plants have developed mutualistic relationships over millennia that have influenced the evolution of both groups Bacteria reside on the surfaces of most plants and are also introduced into many plant organs These bacteria can have positive, neutral, or negative impacts on their host plants Beneficial probiotics can improve plant nutrition or increase resistance to biotic and abiotic stresses In contrast, pathogenic bacteria can destroy or diminish the vitality of the host plant Additionally, some bacteria live on plants and utilize excess metabolites

or shelter without harming the plant Beneficial bacteria can improve plant nutrition, increase tolerance to biotic and abiotic stresses, and have positive impacts on plant growth, proliferation, rooting, and organ/embryo formation, especially for difficult- -to culture genotypes Endophytic bacteria living within plant tissues can also affect plant physiological processes in a manner similar to the plant's own genetics They can provide metabolites different from those produced by the plant The plant microbiome, characteristic of each plant species and growth conditions, is actively shaped by the plant through root exudates and other factors The rhizosphere microbiome is typically more abundant than the aboveground microbiome On the negative side, pathogenic bacteria can devastate and kill plant organs, tissues, or the entire plant Some parasitic bacteria compete for nutrients with the host plant Other negative bacterial impacts may involve the secretion of toxins or the inhibition of essential plant physiological and biochemical processes (Orlikowska et al., 2017)

In the actual sample, our group believes two scenarios are possible The first is that the seeds did not germinate due to poor viability or other extrinsic factors (insufficient oxygen, seeds in dormancy, etc.), and the bacterial growth is unrelated to seed germination, instead resulting from contamination during handling or sample processing The second scenario is that the bacterial growth and lack of seed germination are closely correlated, suggesting the bacterial development inhibited seed germination However, due to experimental limitations, the group cannot definitively determine if the bacterial growth impacted germination, so they propose both possibilities

3 Differences in development between samples

Figure 5: Developmental differences between cultured sample

By working as a group, out group found that the uneven development among the samples came from various causes Since the culture environment was uniform, out group categorized the causes into two groups: internal factors and external factors For the internal factors, the genetic traits of the seeds were the key reason Different seed varieties have different growth and development capabilities Some varieties have strong growth capacity, so the seeds will grow tall, while other varieties have weaker growth capacity, so the seeds will grow shorter Some seed varieties have genetic characteristics that are beneficial for development, such as: genes related to the growth

of stems, leaves, and roots are strongly expressed, processes like photosynthesis, nutrient and water absorption occur efficiently, and they have good environmental stress tolerance These favorable genetic traits allow these seed varieties to grow and

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Labwork Of Plant Biotechnology

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efficiently, and they have poor environmental stress tolerance These unfavorable genetic traits make these seed varieties have weaker growth capacity, leading to the seeds only growing short in the same culture environment Therefore, genetics play a crucial role in determining the growth and development capability of each seed variety during cultivation Furthermore, seed size is also an important factor Larger seeds usually contain more food reserves and energy like starch, protein, and lipids These reserves help provide more nutrients for the development of the embryo and young roots when the seed germinates As a result, larger seeds can germinate faster and accelerate the early growth of the seedlings In addition, the maturity of the seeds cannot be overlooked The more mature the seeds (completing the development process), the more perfect their structures and physiological functions Mature seeds will be able to absorb and utilize nutrients more efficiently compared to immature seeds This helps their germination, growth, and development to occur more rapidly and vigorously Therefore, larger and more mature seeds usually have an advantage over smaller and immature seeds in terms of faster growth in the early stage This is a clear example of how the genetic characteristics of seeds can influence the growth and development of the plants The external factor that out group identified was the lack of oxygen due to the rubber bands being wrapped too tightly The tightness of the rubber bands was different for each tube It can be said that when the rubber bands were wrapped too tightly, it led to insufficient oxygen for the plants to develop normally, and vice versa

V REFERENCES

1 Ali, M., Boonerjee, S., Islam, M N., Saha, M L., Hoque, M I., & Sarker, R H (2018) Endogenous Bacterial Contamination of Plant Tissue Culture Materials: Identification and Control Strategy Plant Tissue Culture and Biotechnology, 28(1), Article 1 https://doi.org/10.3329/ptcb.v28i1.37202

2 Corbineau, F (2022) Oxygen, a key signalling factor in the control of seed germination and dormancy Seed Science Research, 32, 1 11 – https://doi.org/10.1017/S096025852200006X

3 Orlikowska, T., Nowak, K., & Reed, B (2017) Bacteria in the plant tissue culture environment Plant Cell, Tissue and Organ Culture (PCTOC), 128(3), 487 508 – https://doi.org/10.1007/s11240-016-1144-9