Effects of some growth regulators on tissue culture of hardy chrysanthemums (chryssanthemmum x koreanum)

Treatment of experiment on the effect of 6-BA on callus formation in Striped Dwarf variety.... Treatment of experiment on the effect of 2,4-D on callus formation in Striped Dwarf variety

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF AGRONOMY

UNDERGRADUATE THESIS

TOPIC: EFFECTS OF SOME GROWTH

REGULATORS ON TISSUE CULTURE OF HARDY

CHRYSANTHEMUMS (Chrysanthemum × koreanum)

COMMITMENT

I hereby declare that the data and research results in the undergraduate thesis "Effects of some growth regulators on tissue culture of hardy

chrysanthemums (Chrysanthemum × koreanum)" are honest without being

copied or used in any other research All references are fully cited and traced I take full responsibility to the department, faculty, and the university for this commitment

ACKNOWLEDGE

In order to finish this thesis, I would like to sincerely thank to:

- The Faculty of Agronomy of Vietnam National University of Agriculture for creating the most favorable conditions for me to work on the thesis

- Dr Pham Phu Long for his direct guidance to me

- Dr Phung Thi Thu Ha and my fellow at the Lab of Plant Biotechnological Applications for supporting me on this thesis

- Teachers of Vietnam National University of Agriculture for teaching and imparting valuable knowledge as the foundation for me to complete this thesis

Hanoi, February 2021

Sincerely,

LIST OF CONTENTS

COMMITMENT i

ACKNOWLEDGE ii

LIST OF CONTENTS iii

LIST OF TABLES v

LIST OF FIGURES vii

LIST OF ACRONYMS viii

ABSTRACT ix

PART I INTRODUCTION 1

1.1 Introduction 1

1.2 Objective and scope of study 2

1.2.1 Objective 2

1.2.2 Scope of study 2

PART II LITERATURE REVIEWS 3

2.1 General introduction of chrysanthemum 3

2.1.1 Origin and classification 3

2.1.2 Botanical characteristics 4

2.1.3 Values and benefits 5

2.2 Chrysanthemum production and cultivation techniques 6

2.2.1 Chrysanthemum production in the world 6

2.2.2 Chrysanthemum production in Vietnam 7

2.2.3 Chrysanthemum cultivation techniques 9

2.3 The scientific basis of tissue culture 11

2.3.1 Scientific basis of plant cell culture 11

2.3.2 Plant cell culture techniques 12

2.3.3 Culture medium 12

2.4 Research situation in the world and Vietnam 13

2.4.1 Research situation in the world 13

2.4.2 Research situation in Vietnam 16

PART III MATERIAL AND METHOD 19

3.1 Plant material 19

3.2 Time and location 19

3.3 Researching contents 19

3.3.1 Evaluating the callus formation 19

3.3.2 Evaluating the rapid multiplication 22

3.3.3 Evaluating the shoot regeneration 24

3.3.4 Evaluating the shoot growth 26

3.3.5 Evaluating the root formation 27

3.4 Researching method 28

3.4.1 Sample sterilization 28

3.4.2 Experiment design 28

3.4.3 Parameters 28

3.4.4 Data processing 29

PART IV: RESULTS AND DISCUSSION 30

4.1 Striped Dwarf variety 30

4.1.1 Evaluating the callus formation 30

4.1.2 Evaluating the rapid multiplication 34

4.1.3 Evaluating the shoot regeneration 37

4.1.4 Evaluating the shoot growth 39

4.1.5 Evaluating the root formation 40

4.2 Yellow Dwarf variety 42

4.2.1 Evaluating the callus formation 42

4.2.2 Evaluating the rapid multiplication 45

4.2.3 Evaluating the shoot regeneration 48

4.2.4 Evaluating the shoot growth 49

4.2.5 Evaluating the root formation 50

PART V CONCLUSION AND RECOMMENDATION 53

5.1 Conclusion 53

5.2 Recommendation 54

REFERENCES 55

DATA ANALYSIS 57

LIST OF TABLES

Table 3.1 Treatment of experiment on the effect of 6-BA on callus formation in

Striped Dwarf variety 19

Table 3.2 Treatment of experiment on the effect of 2,4-D on callus formation in Striped Dwarf variety 20

Table 3.3 Treatment of experiment on the effect of 6-BA on callus formation in Yellow Dwarf variety 21

Table 3.4 Treatment of experiment on the effect of 2,4-D on callus formation in Yellow Dwarf variety 21

Table 3.5 Treatment of experiment on the effect of 6-BA on rapid multiplication in Striped Dwarf variety 22

Table 3.6 Treatment of experiment on the effect of 2,4-D on rapid multiplication in Striped Dwarf variety 23

Table 3.7 Treatment of experiment on the effect of 6-BA on rapid multiplication in Yellow Dwarf variety 23

Table 3.8 Treatment of experiment on the effect of 2,4-D on rapid multiplication in Yellow Dwarf variety 24

Table 3.9 Treatment of experiment on the effect of 6-BA on shoot regeneration in Striped Dwarf variety 25

Table 3.10 Treatment of experiment on the effect of 6-BA on shoot regeneration in Yellow Dwarf variety 25

Table 3.11 Treatment of experiment on the effect of 6-BA on shoot growth 26

in Striped Dwarf variety 26

Table 3.12 Treatment of experiment on the effect of 6-BA on shoot growth 27

in Yellow Dwarf variety 27

Table 3.13 Treatment of experiment on the effect of NAA on root formation in Striped Dwarf variety 27

Table 3.14 Treatment of experiment on the effect of NAA on root formation in Yellow Dwarf variety 28

Table 4.1 Effect of 6-BA on callus formation in Striped Dwarf variety after 4 weeks30 Table 4.2 Effect of 2,4-D on callus formation in Striped Dwarf variety after 4 weeks 32

Table 4.3 Effect of 6-BA on rapid multiplication in Striped Dwarf variety after 4 weeks 34Table 4.4 Effect of 2,4-D on rapid multiplication in Striped Dwarf variety after 4 weeks 36Table 4.5 Effect of 6-BA on shoot regeneration in Striped Dwarf variety after 4 weeks 38Table 4.6 Effect of 6-BA on shoot growth in Striped Dwarf variety after 4 weeks 39Table 4.7 Effect of NAA on root formation in Striped Dwarf variety after 2 weeks 41Table 4.8 Effect of 6-BA on callus formation in Yellow Dwarf variety after 4 weeks 43Table 4.9 Effect of 2,4-D on callus formation in Yellow Dwarf variety after 4 weeks 44Table 4.10 Effect of 6-BA on rapid multiplication in Yellow Dwarf variety after 4 weeks 45Table 4.11 Effect of 2,4-D on rapid multiplication in Yellow Dwarf variety after 4 weeks 46Table 4.12 Effect of 6-BA on shoot regeneration in Yellow Dwarf variety after 4 weeks 48Table 4.13 Effect of 6-BA on shoot growth in Yellow Dwarf variety after 4 weeks 50Table 4.14 Effect of NAA on root formation in Yellow Dwarf variety after 2 weeks 51

LIST OF FIGURES

Figure 4.1 Striped Dwarf variety’s callus after 4 weeks of callus formation stage in

6-BA 1.5 ml/l medium 34 Figure 4.2 Striped Dwarf variety’s callus after 4 weeks of rapid multiplication stage in 6-BA 1.5 ml/l medium 37 Figure 4.3 Striped Dwarf variety’s regenerated shoot from callus after 4 weeks of shoot regeneration stage in 6-BA 1.0 ml/l medium 39 Figure 4.4 Striped Dwarf variety’s shoot after 4 weeks of shoot growth stage in 6-BA 1.0 ml/l medium 40 Figure 4.5 Striped Dwarf variety’s complete plant after 2 weeks of root formation stage in NAA 2.0 ml/l medium 42 Figure 4.6 Yellow Dwarf variety’s callus after 4 weeks of rapid multiplication stage in 6-BA 1.5 ml/l medium 47 Figure 4.7 Yellow Dwarf variety’s regenerated shoot from callus after 4 weeks of shoot regeneration stage in 6-BA 1.0 ml/l medium 49 Figure 4.8 Yellow Dwarf variety’s complete plant after 2 weeks of root formation stage in NAA 2.0 ml/l medium 52

LIST OF ACRONYMS

6-BA/BA: 6-Benzylaminopurine NAA: 1-Naphthaleneacetic acid 2,4-D: 2,4-Dichlorophenoxyacetic acid IAA: Indole-3-acetic acid

IBA: Indole-3-butyric acid MS: Murashige and Skoog mg/l: milligram/liter

g/l: gram/liter g: gram kg: kilogram m: meter cm: centimeter Co: coconut water AC: active charcoal conc: concentration

ABSTRACT

This study aimed to evaluate the effects of some growth regulators on in

vitro propagation method of two varieties of hardy chrysanthemums

(Chrysanthemum × koreanum) and figure out the optimal medium for in vitro

propagation of two varieties of hardy chrysanthemums Plant materials included

two varieties of Chrysanthemum × koreanum Experiment design was a

complete randomized design was employed in all experiments with 3 times replication After collecting the experimental results, the optimal composition of the tissue culture medium to achieve the best results of the two chrysanthemum varieties was determined For Striped Dwarf variety: The optimal medium for callus formation is 1/2 MS + 6-BA 1.5 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l + Co 150 ml/l, pH = 5.7, with the largest callus mass of 127.96 ± 5.76 mg; The optimal medium for rapid multiplication is 1/2 MS + 6-BA 1.5 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l + Co 150 ml/l, pH = 5.7, with maximum multiplier of 13.74 ± 0.60 times; The optimal medium for shoot regeneration is 1/2 MS + 6-BA 1.0 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar

6 g/l + Co 150 ml/l, pH = 5.7, with the optimal number of shoot per callus was 8.90 ± 0.81 shoots; The optimal medium for shoot growth is MS + 6-BA 0.5 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l, pH = 5.7, with the maximum height increase of 2.19 ± 0.14 cm; The optimal medium for root formation is MS + 6-BA 1.0ml/l + NAA 2.0 ml/l + sucrose 30 g/l + agar 6 g/l + AC 0.2 g/l, pH = 5.7, with a root number of 10.11 ± 0.17 For Yellow Dwarf variety: The optimal medium for callus formation is 1/2 MS+ 6-BA 1.5 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l + Co 150 ml/l, pH = 5.7, with the largest callus mass

of 110.93 ± 1.71 mg; The optimal medium for rapid multiplication is 1/2 MS + 6-BA 1.5 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l + Co 150 ml/l, pH = 5.7, with maximum multiplier of 7.97 ± 0.23 times; The optimal medium for shoot regeneration is 1/2 MS + 6-BA 1.0 ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l + Co 150 ml/l, pH = 5.7, with the optimal number shoot per callus was 6.93 ± 0.28 shoots; The optimal medium for shoot growth is MS + 6-BA 0.5

ml/l + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l, pH = 5.7, with the maximum height increase of 1.99 ± 0.05 cm; The optimal medium for root formation is MS + 6-BA 1.0ml/l + NAA 2.0 ml/l + sucrose 30 g/l + agar 6 g/l + AC 0.2 g/l, pH = 5.7, with a root number of 8.90 ± 0.18

PART I INTRODUCTION 1.1 Introduction

Flowers' importance in nature is everywhere - they can feed insects, birds, animals, and humans; provide natural medicines for humans and some animals; and aid in a plant's reproduction For many centuries flowers occupied

an important place in human life Flowers are an indispensable product, bringing both spiritual value and great economic value Since ancient times, people have had the need to use flowers for decoration and food Today, with the development of society, the demand for flowers is increasing That is why flower production has become a lucrative part of agriculture The worldwide market for flower and ornamental plants is expected to grow roughly 6.3% over the next five years, reaching $57.4 billion in 2024, up from $42.4 billion in

2019

Chrysanthemum is one of the five most popular cut flowers in the world Chrysanthemum attracts consumers with its abundance of colors: white, yellow, green, red, purple, orange, etc Besides, the flower shape and size are also very diverse; along with the ability to control flowering all year round, they are the reasons why chrysanthemum the second most consumed flower in the world market (after rose) (Dang Ngoc Chi, 2006) Chrysanthemum in Vietnam is widely used for its beauty and durability, and also because of Vietnamese customs and habits, using the white and yellow varieties as a symbol of grief Chrysanthemum can also be used as medicinal herbs It is used in tea production, functional foods production, and many other products Because of these spiritual values and health benefits, chrysanthemum is popular with consumers, so the demand for chrysanthemum is always high

Currently in Vietnam, chrysanthemum is grown all over the country There are some specialized cultivation areas such as Da Lat, Ha Noi, and Quang Ninh with many different varieties Among the chrysanthemum varieties, hardy chrysanthemums are very popular for ornamental or decorative purposes, so the demand for healthy seedlings, disease-free, good resistance to pests and

diseases, and adverse environmental conditions with competitive prices is very high However, with traditional propagation methods such as sowing seeds, cutting branches, or taking young shoots from mother plants, the propagation coefficient is not high: the quality of seedlings is not uniform, the growth time is long and it is difficult to control the quality of the products To overcome these

shortcomings, in vitro culture methods and growth regulators are used to create

a very large number of disease-free, healthy seedlings to reduce production costs Several growth regulators are used widely in tissue culture but can be toxic to plants at inappropriate concentrations Besides, how to use growth regulators to reduce production costs to the lowest while ensuring the optimal amount of healthy and qualified products is still a problem

In summary, under the direct guidance of Dr Pham Phu Long, the topic

"Effects of some growth regulators on tissue culture of hardy chrysanthemums

(Chrysanthemum × koreanum)" is conducted to research on the optimal culture

medium for hardy chrysanthemums

1.2 Objective and scope of study

Determining the suitable media for shoot regeneration from callus

Determining the suitable media for shoot development

Determining the suitable media for root formation

PART II LITERATURE REVIEWS 2.1 General introduction of chrysanthemum

2.1.1 Origin and classification

The scientific name Chrysanthemum is defined from Greek words “Chiysos”

(yellow) and “themum” (flower) Chrysanthemum is native to China, Japan, and

some European countries Originated from some wild species of Dendranthema, the

chrysanthemum was grown in China 3000 years ago Undergone the process of cultivating, breeding and selecting, it has become modern varieties (Dao Thanh Van and Dang Thi To Nga, 2007) Chrysanthemum is proved to be used in celebrations and they appear in ancient paintings and sculptures From the 1930s, the cultivation

of chrysanthemums was appreciated In 1982, China held its first chrysanthemum exhibition in Shanghai with more than one thousand varieties of chrysanthemum, marking an important milestone in chrysanthemum cultivation In the following years, Chinese scientists collected photographic descriptions of thousands of varieties and continued to hold exhibitions of chrysanthemums In Japan, chrysanthemum is brought from China, it is highly appreciated and is often used in important ceremonies In 1843, British botanist Fortune brought from China the Chusan variety In 1889 Edsmit succeeded in breeding many varieties of chrysanthemum and he named more than 100 varieties of chrysanthemums of subsequent generations, some are still maintained and cultivated today In 1789, France imported 3 varieties of chrysanthemum from China Until 1927, Bemct succeeded in breeding new chrysanthemum varieties, leading to a strong improvement of chrysanthemum varieties in Europe (Dang Van Dong, 2004) In America, chrysanthemum has been grown widely since the early eighteenth century

In Vietnam, the chrysanthemum was imported in the fifteenth century Vietnamese people consider chrysanthemum to be a symbol of nobility It is one of four species classified as the precious quartet (Truong Huu Tuyen, 1979)

Chrysanthemum is a eudicot that belongs to Plantae kingdom, Magnoliophyta

phylum, Magnoliopsida class, Asterales order, Asteraceae family, Chrysanthemum

genus The Asterales order has only one family, the Asteraceae, which includes

nearly 1000 genera and 20,000 species They distribute widely on the land, survive in

many different climates and soils The genus Chrysanthemum is commonly grown as

a potted flower or cut flower all over the world with diverse colors, shapes and sizes

2.1.2 Botanical characteristics

Chrysanthemum has fibrous roots, shallow roots grow horizontally Many

secondary roots and root hairs creating a large root system, so the ability to absorb water and nutrients is strong These roots grow at the base of the stem, in parts close to the ground (Nguyen Xuan Linh, 1998)

Chrysanthemum stem is herbaceous with strong branching ability, the

height of the plant depends on variety characteristics and light Plant height, degree of branching, softness of stem depend greatly on the genetics of the variety The height of chrysanthemum plant depends largely on the genetics of the variety The lowest variety is only 20 - 30cm high, while the highest one can

be over 3m high The short varieties with many branches are suitable for planting in pots or growing flower beds The long stem varieties, often with low branching ability are suitable for planting on the ground for cut flowers or on high trusses The variety with many branches, small and soft branches that are

suitable for bonsai growing (Van Ruiten et al., 1984)

Chrysanthemum leaves are simple leaves, lobed, large and deep serrated,

the lower leaf surface is covered with a soft fuzz layer, the upper surface is smooth, and the veins are palmate Leaves are flat and thin, maybe slightly tilted upwards or slightly folded The closer to the base, the smaller the leaf The leaf arrangement is alternate Leaf sizes usually vary according to the external conditions and growing techniques Poorly growing plants have small, thin, hard leaves that oblique slightly upwards, with a light green or yellowish color Healthy plants have large and soft leaves, thick leaf blades, slightly curved

downwards leaf tips with dark green color Chrysanthemum leaves usually live

for 70 - 90 days, the photosynthetic efficiency of the leaves is strongest in the fourth leaf from the top Each leaf axillary usually produces a bud (Cockshull, 1972)

The flower shape of Asteraceae family isvery specific The inflorescence

is radially symmetrical, which is very typical, the main axis of the cluster expands into a flat or convex disc, on which there are no-peduncle, tightly arranged flowers, the leaves are arranged around outside of the inflorescence, the whole cluster is shaped like a flower (Dang Van Dong, 2005) Flowers

diameter varies in the range from 1.5 to 12cm Chrysanthemum flowers are

plentiful in colors: white, yellow, orange, purple, etc Usually, there are many

flowers on a stem, which grow from the axillaries Chrysanthemum often cannot

pollinate within flower In order to get seeds, conducting artificial pollination is necessary (Vo Van Chi, Duong Duc Tien, 1988) Based on the arrangement of petals, chrysanthemum flower can be divided into two forms: single flower and complex flower

The Chrysanthemum fruit is very small, each fruit contains only 1 seed

The fruit is dry, brown with a spherical shape The weight of a thousand seeds is

about 1g (Dang Van Dong, 2005) The Chrysanthemum seed has an embryo and

no endosperm (Le Kim Bien, 1984)

2.1.3 Values and benefits

Chrysanthemum is one of the precious quartet (Tung, Cuc, Truc, Mai), which is considered as the four precious ornamental plants of Eastern culture and carries many different meanings In China, the meaning of the chrysanthemum is a symbol of longevity In Japan, the chrysanthemum is a symbol of wealth, luxury, and nobility And in Vietnam, the chrysanthemum is associated with filial piety and love of children to parents Depends on the color, chrysanthemum also symbolizes nobility, honesty, love, joy, optimism, cheerfulness or nostalgia The chrysanthemum flower shape is beautiful, the fragrance is gentle and discreet, so chrysanthemums are used a lot to make flower vases in decoration and make gifts Chrysanthemums are often placed in the house on the Tet holiday because chrysanthemum is the symbol of eternity According to feng shui, chrysanthemum also brings the family fortune as well as joy in the new year Small pots or vases of chrysanthemum can help stabilize

your home's well-being Besides, many species of chrysanthemum are medicinal materials for the production of functional foods that help improve human health Chrysanthemum tea helps improve cardiovascular health, lose weight, improve eyesight, prevent cancer, treat insomnia, lower blood pressure, detox, reduce menstrual cramps, help wounds to heal quickly, nourish skin, treats inflammation, etc These benefits explain the reason why chrysanthemum becomes one of the most productive cut flowers in the world

2.2 Chrysanthemum production and cultivation techniques

Flower production has brought great benefits to the economies of many countries Over the past 20 years, the production of cut flowers has grown at a median annual rate of 7.5% In 2019, the Netherlands retains a key role in the global trade of cut flowers accounting for 48.9% of all global export volume, with a revenue of $4.6 billion Columbia comes second with a revenue of $1.4 billion, accounting for 14.9% Also in 2019, Kenya has grown to become the world’s third-largest exporter of fresh cut-flowers supplying close to 40% of all flower sales today in Europe The United States is the biggest consumer of cut flowers globally spending $1.83 billion annually The next is Germany ($1.28 billion), United Kingdom ($921 million), and then Russia ($554 million)

The Netherlands is one of the world's largest exporter of flowers and plants in general and chrysanthemum in specific Chrysanthemum growing area of the Netherlands accounted for 30% of the total planted flowers area Every year, the Netherlands has produced a great number of chrysanthemum branches and flower pots to serve markets in over 80 countries worldwide In 2006, 4 countries that produce the most chrysanthemum are the Netherlands with 1.5 billion branches, Columbia with 900 million branches, Mexico and Israel with 300 million branches (Erik Van Berkum, 2007)

Japan is currently the leader in Asia in the production and consumption

of chrysanthemum Chrysanthemum is the most important flower in Japan, accounting for 36% of agricultural products, each year Japan produces more

than 200 million branches for domestic demand Chrysanthemum growing area accounts for 2/3 of the total flower area However, Japan still has to import

a large amount of chrysanthemum from the Netherlands and some other countries around the world such as China, Taiwan, Malaysia, Thailand, Colombia In Malaysia, chrysanthemum accounts for 23% of the total flower production In some other countries, such as Thailand, chrysanthemums are grown all year round with the amount of 50 million branches (Oradee Sahavacharin, 1998)

In China, the chrysanthemum is one of the 10 most important cut flowers after rose and carnation, accounts for about 20% of all cut flowers on the wholesale market in Beijing and Kunming The main chrysanthemum production areas are Guangdong, Shanghai, Beijing, including summer, autumn, early winter and late spring varieties with a single flower, the most popular colors are yellow, white, and red (Nguyen Thi Kim Ly, 2001)

The annual export-import turnover of chrysanthemum in the world is estimated to be $1.5 billion (Dang Van Dong, 2003)

2.2.2 Chrysanthemum production in Vietnam

In Vietnam, the chrysanthemum was introduced in the fifteenth century, until the beginning of the nineteenth century, some specialized production areas were formed to serve the domestic market The growing areas are Hanoi with

450 ha, Ho Chi Minh City with 380 ha, Da Lat with 200 ha, Hai Phong with 150

ha, most of the provinces grow chrysanthemum with an area of several to several tens of ha Vietnam is a country that both exports and imports chrysanthemum due to the variety and weather, the production of off-season chrysanthemum will cost higher than that of importing from other countries Before 1997, the area of roses was the highest in all types of flower (31%), but

in 1998, the chrysanthemum area has surpassed (42%, the rose area was reduced

to 29.4%) Hanoi's total production of chrysanthemum in 1999 was 41.5 billion VND and the export amount to China was 3.8 billion VND, the annual growth rate of about 10% (Nguyen Xuan Linh et al, 2002) Currently, chrysanthemum is

grown throughout our country but mainly concentrated in the traditional areas of growing flowers, industrial parks, and resorts such as Ngoc Ha, Quang An, Nhat Tan (Hanoi), Dang Hai, Dang Lam (Hai Phong), Hoanh Bo, Ha Long (Quang Ninh), Trieu Son, Thanh Hoa city (Thanh Hoa) Go Vap, Hooc Mon (Ho Chi Minh City), Da Lat City (Lam Dong) with a total flower cultivation area about

2000 ha Particularly, Hanoi and Da Lat are ideal places for the growth and development of most of the imported chrysanthemum varieties (Dang Van Dong, 2000) In 2003, the whole country had 9430 ha of flowers and ornamental plants with a revenue of 482.6 billion VND, in which the chrysanthemum area is

1484 ha with a revenue of 129.49 billion VND In Hai Phong, the chrysanthemum is the second most important flower in the province’s production of fresh flowers after gladiolus Currently, there are some foreign

chrysanthemum This is a good sign for the development of the Vietnamese flower production industry in general, but also a concern for domestic flower producers In the southern provinces, Da Lat is the place with the largest chrysanthemum cultivation area Hasfarm is a foreign company investing in Da Lat, they provide 60% of the chrysanthemum demand of Ho Chi Minh City and some northern provinces

Currently in production, chrysanthemum can be grown all year round while in the past it can only be grown in the autumn-winter season, which is meeting the demand for chrysanthemum of consumers Chrysanthemum has a lower price than other flowers (400 - 800 VND per branch), so in both urban areas and rural, mountainous areas, chrysanthemum is consumed quite well (only after roses), especially on traditional Tet holidays and the full moon day Regarding the consumption market, Ho Chi Minh City has the largest consumption for cut flowers in Vietnam with the daily consumption demand

is from 40 thousand to 50 thousand branches per day, followed by Hanoi with the consumption demand from 25 thousand to 30 thousand branches per day Among all cut flowers for daily consumption, chrysanthemum accounts for

25 to 30% in quantity and 17 to 20% in value (Hoang Ngoc Thuan, 2003)

2.2.3 Chrysanthemum cultivation techniques

2.2.3.1 Chrysanthemum growth characteristics

The temperature range of Chrysanthemum is 10-35˚C, the suitable temperature for the plant to grow is 15-20˚C In Chrysanthemum, the seedling

period requires a higher temperature than other periods Besides, ensuring the optimal temperature during flowering period can achieve large, strong and

beautiful flowers The suitable soil humidity for Chrysanthemum is 60-70%,

while the air humidity requirement is 55-65%

Chrysanthemum is a short-day plant Seedling period requires little light,

while the branching period requires more light for photosynthesis The light affects deeply the yield and quality of flowers Flowering stage only requires 10-

11 hours of daylight, shorter than other growth stages

Chrysanthemum is suitable with soil that well-ventilated, rich in organic

matter and pH around 6.0-6.5 Chrysanthemum can survive for a few days in

drought condition but if it is prolonged it will affect plant growth, so watering is

important for Chrysanthemum

Chrysanthemum requires a quite large amount of fertilizer, so it needs to

be applied twice with NPK fertilizer (10-10-10) The amount of fertilizer for each time is 25 kg per 100m2 (Dao Thanh Van and Dang Thi To Nga, 2007)

2.2.3.2 Chrysanthemum cultivation techniques

In general, in Vietnam, there are some main seasons for growing chrysanthemum:

- Spring-Summer season: planting in March and April to harvest flowers

in June and July, used varieties are Vang he, Trang he, Tim he, etc

- Summer-Autumn season: planting in May and June to harvest flowers in September, October: used varieties are Vang he, Vang hoe, Tim he, etc

- Autumn-Winter season: planting August and September to harvest flowers in November and December, used varieties are Tim sen, Vang Dai Loan, Vang hoe, Vang nghe, Do nhung, Pha le, Trang hue, etc

- Winter-Spring season: Planting in October and November to harvest flowers in January and February, used varieties are Vang Dai Loan, Tim sen, Chi trang, Muong hong, Tia sao, Tho do, etc

 Cultivation techniques

Planting beds are 1.1-1.2 m wide with bed surface width is 80-90cm, bed height is 20-30cm depending on the season Bed preparation can be combined with the basal application

Plant density and distance depend on the number of flowers per plant The single flower varieties require a distance of 14 × 15cm or 15 × 15cm, the density is 40-45 plants/m2 The multiple flower varieties require a distance of 16

× 18cm or 18 × 18 cm, the density is 30-35 plants/m2

For newly planted plants, use a hose or watering can to water, the amount of water is small, enough to moist the soil, do not let the plants be soaked in water After planting 10-15 days or the weather is dry, furrow irrigation is necessary Let the water cover 2/3 of the furrows in 1 to 2 hours and then drain off the water

For 360 m2 of chrysanthemum, the requirement of fertilizer includes 1-2 tons of decomposed manure, 50 kg of superphosphate, 20 kg of potassium sulfate, and 20 kg of urea Manure and 30 kg of superphosphate are used in the basal application, the remaining amount of fertilizer is divided into 4 times to apply every 7 - 10 days

For the single flower varieties, it is necessary to prune the auxiliary branches and buds, remain only 1 bud on the main stem Pruning needs to be conducted as soon as the bud is young and small so that nutrition is not wasted For the multiple flower varieties, branches near the base of the plants should be pruned; the main bud also needs to be pruned for the even development of other buds

Light treatment to prevent early flowering: in Winter-Spring season, using 75W electric bulbs for extra 2 - 3 hours of light per day, continuously from planting until 30 days before flowering

When the plant height reaches 20 - 30cm, it is necessary to set stakes in the ground to help the plants growing upright without falling over As the plants grow higher, the net is also raised

The main pests on chrysanthemum are aphids, green worms, deep worms, etc Farmers can catch them by hand or use pesticides to prevent them Some pesticides used in chrysanthemum cultivation are Karate 2.5 EC, Supracide 40ND, Pegasus 500 SC, Supathion 40 EC, etc

The main diseases on chrysanthemum are leaf spots, powdery mildew, brown spots, and rust The diseases can be prevented by using plant protection products such as Topsin M-70 WP, Score 250ND, Anvil 5 SC, Roval WP, etc

7 - 10 days before harvest, farmers need to dilute potassium with water

to irrigate plants Flowers should be watered fully 1-2 days before cutting The flowers that bloom about 2/3 of the petals or bloom almost completely the outer petals can be harvested Cut the stem at the position of 10cm from the ground The flowers should be harvest in early morning or late afternoon, on dry days

Harvested flowers should be put in a cool house immediately for preliminary treatment, then soaked in 0.1% STS solution in 10 minutes, with the length of stem in the solution is 8 - 10cm

2.3 The scientific basis of tissue culture

2.3.1 Scientific basis of plant cell culture

The research on building regeneration system of plant cells is based on the totipotency of the cell, which means ‘the innate capacity (or a genetic potential) of a plant cell/tissue to regenerate into a whole new plant' Haberlandt (1902) was the first to propose that any cell of a multicellular organism has the ability to develop into a complete organism Modern biology has proven that every single cell carries the full genetic information of that organism When the right conditions are met, each cell can develop into a complete body

Theoretically, any differentiated plant cell (organized for a specific function: say leaves, roots, etc.) could be dedifferentiated (reversed to the embryo state) and redifferentiated (differentiated again to perform a specific

function) at will This basic ability is utilized in laboratories to mass-multiply plants for research or commercial purposes

The plant body consists of many different organs and organs, made up

of many different types of cells However, scientists have shown that these cells all originate from one original cell (zygote) In the first stage, this zygote cell divides to form many undifferentiated cells, which continue to divide into specialized cells for different organs and tissues This process is called cell differentiation Although the cells have specialized in separate tissues, they have not completely lost their ability to divide Under the right conditions, any part of the plant can return to the embryonic form and strongly divide (dedifferentiation), then forming a complete body (redifferentiation)

2.3.2 Plant cell culture techniques

Plant tissue culture is a common concept to all types of culturing microorganism-free plant material on artificial nutrient medium, in sterile conditions Tissue culture techniques include:

- Culturing young and mature plants

- Culturing organs: roots, stems, leaves, flowers, fruits, pollen, unfertilized ovules

- Culturing embryo: immature embryos and mature embryos

2.3.3 Culture medium

The culture medium is an important factor influencing plant cell culture Media composition varies depending on species, organ, and culture purpose Besides, it also changes according to the developmental stages and

culture purposes such as callus induction, shoot regeneration, root formation, etc Based on the composition and nutritional content, the culture medium can

be divided into four basic groups:

- Medium that is rich in nutrients: Murashige and Skoog (MS) media is representative of this group This is a group of media that is suitable for most cultured objects and developmental stages of cells

- Medium with sufficient nutrition: including N6, B5, SH This group is suitable for inducing callus formation and shoot differentiation

- Medium with poor nutrition: such as Nitsch media, suitable for anther culture for many types of plants, especially for Solanaceae plants

- Medium that is suitable for growing woody plants: WPM, DKW medium is representative of this group

Moreover, apart from the basic background media, depending on the purpose and the culture stage, sugar, vitamins, growth regulators, and organic compounds are added to create suitable conditions for the growth of cells

2.4 Research situation in the world and Vietnam

2.4.1 Research situation in the world

Nowadays, along with the development of modern science and technology, the biotechnology industry in the field of plant breeding has played a very important role Scientists have been focusing on the field of chrysanthemum breeding using many different methods: sexual hybridization, gene transfer into chrysanthemum cells, creating mutant chrysanthemum varieties, etc As a result, new varieties of chrysanthemum are created

Shibata et al (1998) successfully bred the "Moonlight" chrysanthemum, which was the result of a crossbreeding between C morifolium Ramat and C

pacifium Nakai, F1 hybrid was re-bred with C morifolium “Moonlight” variety

has single flower, flower diameter is 5 cm, has 25 greenish-yellow corolla

petals, the leaf is smaller and the petiole is longer than C morifolium, has a

chromosome set of 2n = 64

Mutation is one of the methods of selection that has been very

successful with chrysanthemum, creating new varieties with variations in flower

characteristics According to NBRI Newsletters (1989), when applying

gamma-ray treatment (1 - 3 krad) on 125 Dendranthema varieties, scientists obtained

mutated individuals in color and flower shape in M1 and M2 clones of 50 varieties, in which 36 are considered as new varieties, and they can conclude that the most suitable gamma ray concentration is 1.5 and 2.5 krad

Benetka and Pavingerova (1995) used the technique of transferring genes into the genome of the chrysanthemum variety to create a new variety According to Mitouchkina and colleagues in 2000, they have studied

rhizogenes to change the shape and structure of the chrysanthemum plant One

of the transgenic lines obtained exhibits a change in plant size, branching ability, flower shape as well as petal pattern, which is the source of materials to create a

new variety

Chrysanthemum can be propagated by many different methods: sowing, cutting branches, pruning buds, and tissue culture However, the cell culture method is the most researched by scientists in the world One of the factors that make up the success of the chrysanthemum production industry of some countries

in the world (the Netherlands, Japan, the USA, Thailand, etc.) is

using in vitro propagation technology to produce plants With in vitro propagation

technology, it is possible to produce a huge number of healthy, disease-free, genetically identical seedlings to meet production requirements

In 1990, when studying the effect of nutrient composition in culture media, Lunegent and Wardly (1990) concluded that the 1 - 2 cm high chrysanthemum stem segments grown in Benzyl Adenine culture media form 2 -

3 buds with no root, while in the media containing Indolebutyric acid 0.1 – 0.3 mg/l, they formed 1 - 2 buds and roots were formed

In 1990, Kenth and Toress successfully cultured tissue from the stem and leaf segments of purple chrysanthemum in MS medium Bud formation rate reached 100% and cultured plants flowered after 3 - 4 months on average

For the rapid multiplication of chrysanthemum in tissue culture, the use

of plant growth regulators is essential So scientists have focused on research effects of growth regulators on shoot regeneration, shoot multiplication, and root formation in tissue culture

Due to being popular in many countries around the world, chrysanthemum has become one of the first commercial targets in micropropagation, by using tissue culture techniques for large-scale production

(Levin et al 1988) Battacharya et al (1990) rapidly multiplied Chrysanthemum

morifolium from the callus achieved from stem and leaf cultures

Roest and Bokelmann (1973) studied and concluded that the combination of BA and IAA were suitable for the regeneration of

Chrysanthemum cinerariaefolium shoots in in vitro culture However, responses

to plant growth regulators are different in different species of chrysanthemum

Lazar and Cachita (1983) presented a research report showing that shoot was highest when culturing chrysanthemum in MS medium supplemented with IAA 0.1 mg/l + BA 1 mg/l

In 1994, Khan and colleagues studied the rapid multiplication of

Chrysanthemum morifolium Ramat, the results showed that the shoot Coefficient

of callus proliferation was high on the medium supplemented with BA 0.5 and 1.0 mg/l When BA 2.0 mg/l was added, the shoot Coefficient of callus proliferation was increased, but shoot development was inhibited (shoot height was lower in the treatment of BA 0.5 and 1.0 mg/l)

Gul's study in 2001 in tissue culture of chrysanthemum stem segments also showed that the number of newly formed shoots was highest when MS medium was supplemented with BA 0.5 mg/l An increase in the concentration

of BA added to the culture medium did increase the shoot multiplier, however, shoot growth was inhibited (Singh and Arora, 1995)

In 2002, Karim et al of Rajshahi University, Bangladesh conducted rapid propagation of Chrysanthemum morifolium by in vitro culture technique

He used IBA and Kinetin to stimulate shoot regeneration from nodal segments

of stem and meristem Results showed that the regeneration response was good

in MS medium supplemented with 1 mg/l (95% and 91% for nodal segments and meristem, respectively) Callus was formed on 1/2 MS medium with the combination of BA 0.5 mg/l + NAA 0.1 mg/l The asexual embryos formed on BA-only medium and developed like conventionally propagated seedlings

(Ihsan Ilahi et al., 2007) The average number of shoots obtained from meristem

culturing in MS medium containing IAA 0.1 mg/l was 3.9; IBA 1.0 mg/l was 4.1; similarly, the combination of the concentration of BA 1.0 - 2.0 mg/l with the IAA 0.1 - 0.2 mg/l gave a better result (6.9 - 7.0) compared with other treatments

Shatnawi et al (2010) studied the rapid shoot multiplication and rooting for the shoots of Chrysanthemum morifolium Ramat After 6 weeks, the research

results showed that MS medium supplemented with BA concentration of 0.3 mg/l was suitable for the rapid multiplication of shoots with the number of newly formed shoots of 4.35 shoots/sample In treatments with lower or higher

BA concentrations, the shoot Coefficient of callus proliferations were lower, besides, the higher concentration of BA inhibited shoot growth (lower shoot length) Compared with Kinetin, the addition of BA was shown to be more effective in rapid shoot multiplication (resulting in a greater number of shoots per sample) For the experiment studying the effects of growth regulators on the rooting of chrysanthemum shoots, his research results also showed that IBA, IAA, NAA supplemented with a concentration of 0.2 mg/l gave the highest rooting efficiency (18.75, 10.68 and 14.82 roots/shoot for IBA, IAA and NAA, respectively) Supplementation at higher concentrations inhibited root formation

as well as root length

2.4.2 Research situation in Vietnam

Scientists have determined that it is necessary to focus on the import, selection, creation and rapid multiplication of high-quality flower varieties, especially chrysanthemum, persimmon, gladiolus, etc Besides, technology transfer in cultivation also needs to be increased to enhance the value of

products, in which, the issue of varieties and farming techniques is of primary concern That is why the propagation has been interested in and researched by institutes, centers and universities

From 2003 to 2006, Dang Phuong Tram successfully carried out the research project "Application of biotechnology in propagation and high technology application to produce some imported flower varieties in Can Tho" which included chrysanthemum The chrysanthemum varieties propagated by tissue culture method, which were experimentally planted on the field with the cut flower method, gave good flowers and achieved commodity value in the winter-spring season The economic efficiency of chrysanthemum grown from the tissue culture method is also higher than those grown from the conventional method

Chrysanthemum can be propagated using many different methods, propagation methods have affected the quality of chrysanthemum To evaluate the effect of asexual propagation methods on the quality and efficiency of Vang Dai Loan variety production, Dang Van Dong (2005) studied four propagation methods: sprout separating, cutting branches from selected mother plants in the garden, tissue culture, cutting from tissue cultured mother plants The results are

as follows: the traditional methods (sprout separating and cutting) are simple and easy to conduct, but the infection rate is quite high from 9 to 15%, the quality indicators of plant are low such as the height of stem reached 77.4 to 82.2 cm Whereas the tissue culture method and stem cutting from tissue cultured mother plants had a low infection rate of 3 to 5%, the quality indicators

of plant were higher such as stem height reached 91.1 to 92.5 cm Using the cutting method from the mother plant cultured tissue cells gives high propagation coefficient, good seedling quality, commercial production efficiency is 1.7 times higher than traditional cutting method

Currently, the application of in vitro propagation techniques to produce

chrysanthemum has been successfully researched by many Vietnamese

scientists According to Nguyen Xuan Linh et al (1998), the regeneration and

propagation ability of chrysanthemum is very high: CN93 chrysanthemum with

a Coefficient of callus proliferation of 611/year, Vang Dai Loan is 510 610/year, Hong Dai Loan is 310 - 410/year and Do Ha Lan is 311/year The process of tissue culture propagation on chrysanthemum consists of 5 basic steps: creating clean materials, creating and rapidly multiplying shoots, creating

-a complete pl-ant, bringing the pl-ant to the nursery -and t-aking the pl-ant to the field

Study of Nguyen Thi Dieu Huong and Duong Tan Nhut (2004) on the

process of rapid multiplication of chrysanthemum varieties (Chrysanthemum

indicum L.) by using techniques of culturing meristem in 1/2 MS medium added

BA combining with NAA, IAA, IBA showed that the medium supplemented with NAA (0.2 – 0.5 mg/l), IBA (0.2-0.5 mg/l) had better rooting result than medium supplemented with IAA (0.2-0.5 mg/l)

Tran Thi Thu Hien et al (2007) studied the method of propagating

chrysanthemum variety CN97 by tissue culture and concluded that: in the rapid multiplication stage, MS medium with NAA 0.5 mg/l is the optimal rooting

medium for in vitro chrysanthemum buds

Lam Ngoc Phuong and Pham Le Tuan (2007) used leaves of chrysanthemum to create buds on MS culture medium with the combination of

BA and IAA After 4 weeks, the highest shoot formation rate was in the medium with high growth regulators concentration (IAA 2-4mg/l + BA 1-2mg/l) with the result of 52 - 64%, and the highest number of shoots is 4.8 - 6.1 The difference was statistically significant at 1% compared with the combinations at the lower concentration

PART III MATERIAL AND METHOD 3.1 Plant material

- Research subjects: 2 varieties of Chrysanthemum × koreanum: Striped

Dwarf and Yellow Dwarf

- Plant material: tissue-cultured seedlings

3.2 Time and location

- Location: Lab of Plant Biotechnological Applications, Faculty of Agronomy, Vietnam National University of Agriculture

- Time: September 2020 to February 2021

3.3 Researching contents

3.3.1 Evaluating the callus formation

3.3.1.1 Experiment 1: Effect of 6-BA on callus formation in Striped Dwarf

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.1 Treatment of experiment on the effect of 6-BA

on callus formation in Striped Dwarf variety

No Treatment 6-BA concentration (ml/l)

3.3.1.2 Experiment 2: Effect of 2,4-D on callus formation in Striped Dwarf

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.2 Treatment of experiment on the effect of 2,4-D

on callus formation in Striped Dwarf variety

No Treatment 2,4-D concentration (ml/l)

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.3 Treatment of experiment on the effect of 6-BA

on callus formation in Yellow Dwarf variety

No Treatment 6-BA concentration (ml/l)

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.4 Treatment of experiment on the effect of 2,4-D

on callus formation in Yellow Dwarf variety

No Treatment 2,4-D concentration (ml/l)

3.3.2 Evaluating the rapid multiplication

3.3.2.1 Experiment 5: Effect of 6-BA on rapid multiplication in Striped Dwarf

variety

- Base medium: 1/2 MS + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l +

Co 150 ml/l, pH = 5.7

- Materials: Callus formed in the previous stage, divided into small pieces

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.5 Treatment of experiment on the effect of 6-BA

on rapid multiplication in Striped Dwarf variety

No Treatment 6-BA concentration (ml/l)

- Materials: Callus formed in the previous stage, divided into small pieces

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.6 Treatment of experiment on the effect of 2,4-D

on rapid multiplication in Striped Dwarf variety

No Treatment 2,4-D concentration (ml/l)

- Materials: Callus formed in the previous stage, divided into small pieces

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.7 Treatment of experiment on the effect of 6-BA

on rapid multiplication in Yellow Dwarf variety

No Treatment 6-BA concentration (ml/l)

3.3.2.4 Experiment 8: Effect of 2,4-D on rapid multiplication in Yellow Dwarf

variety

- Base medium: 1/2 MS + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l +

Co 150 ml/l, pH = 5.7

- Materials: Callus formed in the previous stage, divided into small pieces

- Photoperiod: No light (in the dark)

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.8 Treatment of experiment on the effect of 2,4-D

on rapid multiplication in Yellow Dwarf variety

No Treatment 2,4-D concentration (ml/l)

3.3.3 Evaluating the shoot regeneration

3.3.3.1 Experiment 9: Effect of 6-BA on shoot regeneration in Striped Dwarf

variety

- Base medium: 1/2 MS + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l Co

150 ml/l, pH = 5.7

- Materials: Callus formed in the previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.9 Treatment of experiment on the effect of 6-BA

on shoot regeneration in Striped Dwarf variety

No Treatment 6-BA concentration (ml/l)

- Materials: Callus formed in the previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.10 Treatment of experiment on the effect of 6-BA

on shoot regeneration in Yellow Dwarf variety

No Treatment 6-BA concentration (ml/l)

3.3.4 Evaluating the shoot growth

3.3.4.1 Experiment 11: Effect of 6-BA on shoot growth in Striped Dwarf

variety

- Base medium: MS + NAA 0.5 ml/l + sucrose 30 g/l + agar 6 g/l, pH = 5.7

- Materials: Shoot formed in the previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.11 Treatment of experiment on the effect of 6-BA on shoot growth

in Striped Dwarf variety

No Treatment 6-BA concentration (ml/l)

- Materials: Shoot formed in the previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.12 Treatment of experiment on the effect of 6-BA on shoot growth

in Yellow Dwarf variety

No Treatment 6-BA concentration (ml/l)

3.3.5 Evaluating the root formation

3.3.5.1 Experiment 13: Effect of NAA on shoot formation in Striped Dwarf

variety

- Base medium: MS + 6-BA 1.0 ml/l + sucrose 30 g/l + agar 6 g/l + AC 0.2 g/l, pH = 5.7

- Materials: Shoot formed in previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.13 Treatment of experiment on the effect of NAA

on root formation in Striped Dwarf variety

No Treatment NAA concentration (ml/l)

3.3.5.2 Experiment 14: Effect of NAA on shoot formation in Yellow Dwarf

variety

- Base medium: MS + 6-BA 1.0 ml/l+ sucrose 30 g/l + agar 6 g/l + AC 0.2 g/l, pH = 5.7

- Materials: Shoot formed in previous stage

- Photoperiod: 10 hours of light and 14 hours of darkness

- Conditions of culture room: Stable room temperature of 25 - 26°C; Humidity is 60-70%

Table 3.14 Treatment of experiment on the effect of NAA

on root formation in Yellow Dwarf variety

No Treatment NAA concentration (ml/l)

- Callus weight (mg)

- Shoot height (cm): measure from shoot position on callus to the apical meristem

- Plant height (cm): measure from plant base to the highest leaf tip

- Root length (cm): measure from root base to root tip

- Number of leaves formed

- Number of roots formed

- Morphology of callus and shoots

3.4.4 Data processing

The data was measured, counted, collected, and then analyzed using Microsoft Excel and SPSS 22.0 statistical analysis software