Effect of growth regulators on the tissue culture of turmeric (curcuma longa l)

Effect of TDZ and α-NAA on plant rapid propagation stage .... Effect of 2.4-D and α-NAA on plant rapid propagation .... In vitro culture, after regeneration, shoots were inoculated on MS

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF AGRONOMY

UNDERGRADUATE THESIS

TITLE: “EFFECT OF GROWTH REGULATORS ON

THE TISSUE CULTURE OF TURMERIC

GUARANTEE

I assure that the datas and research results in this thesis are real and objective Inaddition, they have never been used or protected in any scientific reports, articles or publications before

I asure that all supportation, helping and conduction to construct and complete this thesis has been thanked gratefully and all informations cited and references in this thesis has been acknowledged

Hanoi, March 01, 2022

Student

Ngo Minh Duy Bao

ACKNOWLEDGE

To complete this graduation thesis report, it is not only my own efforts, but also the dedication and enthusiasm of the teachers and the encouragement of family and friends

I would like to express my deep gratitude to the teachers who directly taught me and passed on the knowledge to make my own luggage in the future Especially the teachers in the Faculty of Agronomy - Vietnam Academy of Agriculture, teachers and aunts with enthusiastic hearts and love for their profession

I would like to sincerely thank Ph.D Pham Phu Long has enthusiastically guided, supported, encouraged and encouraged me throughout the process of writing this thesis

Finally, I would like to express my deepest gratitude to my family and friends, who have always supported me during my studies to complete this graduation thesis

Hanoi, March 01, 2022

Student

Ngo Minh Duy Bao

LIST OF CONTENTS

GUARANTEE i

ACKNOWLEDGE ii

LIST OF CONTENTS iii

LIST OF FIGURES vi

LIST OF CHART vii

LIST OF TABLES viii

LIST OF ACRONYMS ix

ABSTRACT x

PART 1: INTRODUCTION 1

1.1 Rationale 1

1.2 Aims and request of the study 3

1.2.1 Aims 3

1.2.2 Request 3

1.3 Objective and scope of study 4

1.3.1 Objective 4

1.3.2 Scope of study 4

PART II: LITERATURE VIEW 5

2.1 General introduction of Curcuma longa L 5

2.2 Origin and distribution 6

2.3 Classification 7

2.4 Botanical description 7

2.4.1 Shoot 7

2.4.2 Leaves 8

2.4.3 Reproductive organ 8

2.5 Living condition 9

2.5.1 Climate and soil 9

2.5.2 Light condition 10

2.5.3 Moisture condition 10

2.6 Pharmacological benefits 10

2.7 Production and consumption 11

2.8 Some scientific studies about Curcuma longa L in Vietnam 12

2.9 Some scientific studies about Curcuma longa L in the world 14

2.10 Propagation method 17

2.10.1 In vitro method 17

2.10.2 The scientific basis of the method 19

2.10.3 Advantages and disadvantages of in vitro method 19

2.10.4 Common problems in in vitro culture 20

2.10.5 Stages in propagation culture 21

2.11 Factors affecting the process of plant tissue culture 22

2.11.1 Explants 22

2.11.2 In vitro culture requirment 23

2.11.3 Stimulators 24

PART III: MATERIALS AND RESEARCHING METHOD 26

3.1 Research subjects 26

3.2 Materials 27

3.3 Location 27

3.4 Research time 27

3.5 researching method 27

3.5.1 Materials decomtamination and transplantation in In vitro medium 27

3.5.2 Evaluate plant measurment 27

3.6 Observation targets 31

3.7 Research method 32

3.7.1 Decontamination method 33

3.7.2 Rapid propagation method 33

3.7.3 Creatation complete plant method 33

3.7.4 Data analization method 33

PART IV: SOLUTION AND DISCUSSION 34

4.1 Solution in regeneration stage 34

4.1.1 Effect of 6-BAP on PLBs formation 34

4.1.2 Effect of 2.4-D on PLBs formation 34

4.1.3 Effect of 6-BAP on plant regeneration stage 35

4.1.2 Effect of 2.4-D on plant regeneration stage 39

4.2 Research method and solution in rapid propagation stage 42

4.2.1 Effect of 6-BAP and α-NAA on rapid propagation 43

4.2.2 Effect of TDZ and α-NAA on plant rapid propagation stage 48

4.1.5 Effect of 2.4-D and α-NAA on plant rapid propagation 54

CHAPTER V: CONCLUSION AND RECOMENDATION 62

5.1 Conclusion 62

5.2 Recommendation 63

REFERENCES 64

PICTURES IN RESEARCH 68

RESULTS OF THE ANALYSIS 69

LIST OF FIGURES

Figure 1.1 (a) Turmeric shoot system, (b).Turmeric flower, (c) Turmeric

rhizome, (d) Turmeric powder 9

Figure 1.2 Turmeric samples 28

Figure 4.1 Photos of explants of MS medium 38

Figure 4.2 Photos effect of 6-BAP on explants 39

Figure 4.3 Photos effect of 2.4-D on explants 42

Figure 4.4 Photo effect of 6-BAP and α-NAA on explants 48

Figure 4.5 Photos effect of TDZ on explants 53

Figure 4.6 Photos effect of 2.4-D on explants 58

LIST OF CHART

Chart 1.1 Effect of 6-BAP on plant measurementsError! Bookmark not defined

Chart 1.2 Effect of 2.4-D on plant measurements 59

Chart 1.3 Effect of 6-BAP and α-NAA on plant measurment 60

Chart 1.4 Effect of TDZ and α-NAA plant measurements 60

Chart 1.5 Effect of 2.4-D and α-NAA on plant measurements 61

LIST OF TABLES

Table 2.1 Formula of 6-BAP on bub regeneration stage 29

Table 2.2 Formula of 2.4-D on bub regeneration stage 30

Table 3.1 Effect of 6-BA and α-NAA on rapid proparation stage 31

Table 3.2 Effect of 2.4-D and α-NAA on rapid proparation stage 31

Table 3.3 Effect of TDZ and α-NAA on rapid proparation stage 31

Table 4.1 Effect of 6-BAP on PLBs formation 34

Table 4.2 Effect of 2.4-D on PLBs formation 34

Table 4.3 Effect of 6-BAP on plant measurements 35

Table 4.4 Effect of 6-BAP on number of bub and diameter 37

Table 4.5 Effect of 2.4-D on plant measurments 39

Table 4.6 Effect of 2.4-D on number of bub and diameter 41

Table 4.7 Effect of 6-BAP and α-NAA on plant measurment 43

Table 4.8 Effect of 6-BAP and α-NAA on number of bub and diameter 46

Table 4.9 Effect of 6-BAP and α-NAA on number of roots and root length 47

Table 4.10 Effect of TDZ and α-NAA plant measurements 48

Table 4.11 Effect of TDZ and α-NAA on number of bub and diameter 50

Table 4.12 Effect of TDZ and α-NAA on root length and number of root 51

Table 4.13 Effect of 2.4-D and α-NAA on plant measurements 54

Table 4.14 Effect of 2.4-D and α-NAA on number of bub and diameter 56 Table 4.15 Effect of 2.4-D and α-NAA on n number of roots and root length 57

LIST OF ACRONYMS

LSD Least Significant Difference

medium to achieve the best results of the Curcuma longa L The results of in vitro propagation researching of turmeric showed that the most suitable primary

materials for sampling are young shoot and shoot apical meristem with suitable time to decontaminate the samples is about 15- 20 minutes in HgCL2 0.1% In vitro culture, after regeneration, shoots were inoculated on MS medium

containing 6.5g/l agar, 30 g/l sucrose, 0.1 g/l activated carbon, 0.1 g/l inositol, supplemented with a combination of growth stimulants with the different concentrations were Cytokinin Benzylaminopurine (6-BAP), 2.4-D (2.4-Dicholorophenoxyacetic acid), Thidiazuron (TDZ) and Naphthlene acetic acid (α-NAA) Results were evaluated after 21-25 days of culture The culture medium was adjusted to pH = 5.8 and autoclaved at 1 atm pressure and 1210C temperature in 20 minutes Choose healthy turmeric shoots with large and 2-3

cm height Sample culture mode was performed with illumination intensity of

3000 - 4000 lux, lighting time 8 - 10 hours, temperature 250C ± 20C

PART 1: INTRODUCTION

1.1 Rationale

Turmeric contains a very valuable curcumin compound that is good for health care and disease free Curcumin is an anti-inflammatory and carcinogen tumor suppressor, then used for medicine preparation and dietary supplement to support the treatment of various diseases such as cancer, AIDS, diabetes, Alzheimer's, gastrointestinal ulcers, hepatitis B, C, antifungal, cholesterol removal, reduce blood fat, prevent obesity, anti-oxidant, scavenge free radicals,

make cosmetics (Hacher H et al., 2018) To produce turmeric products for

commercial and medicinal value Besides those successful applications in modern bioagriculture such as breeding and selecting new varieties, improving propagation techniques and post-harvest preservation of fresh and dried turmeric

tubers, one of the factors contributing to this achievement is in vitro propagation

technology to produce plant souces in large quantities and with a homogenous and disease-free genetic source

For a long time, people have known how to propagate plants by many methods From the way of sowing seeds to the way that people use branches or bubs to graft, called grafting branches With these great advantages, the propagation technology is widely applied, and the technology of tissue culture

using in vitro medium was created In vitro propagation is an effective tool to

rapidly produce new high-quality plants, evenly and in large quantities (Trieu Thi Thu Ha et al., 2014) Appllication in vitro method in study, using MS

medium and 1/2MS (Murashige and Skoog’s medium., 1962) Morever, propagation turmeric by In vitro method contributes to provide disease-free plants in large quantities in short time to reduce input costs, prevention costs, pests and diseases and sustainable development of turmeric production in Vietnam Inaddition, tissue culture has many advances about producing

pharmaceutical compounds have given rise to many pharmaceutical drugs such

as alkaloids, terpenoids, steroids, saponins, phenolics, flavanoids By transforming culture process, the yield of secondary compounds obtained can be

high (Plunkett et al., 2004)

The propagation of turmeric by cell culture method has given positive results in many countries such as Malaysia, Indonesia and India Here, the

experiments propagated by tissue culture from young bubs (Z spectabile) in

Murashige-Skoog’s medium (MS medium) supplemented with growth regulator IAA (indole-3-acetic acid), α-NAA (α-Naphthaleneacetic acid) and BAP (6-benzylaminopurine) have given high results and are considered an important

method important to recommend for turmeric growing regions (Chan L et al.,

2014) Beside, plant cell culture technique has many prospects and long-term applications in the production of natural compounds, especially those used in medicine This research will lead directly to stable quality and quantity products, less dependent on nature ( Nguyen HoangLoc, 2011)

Inaddition, some studies show that plant cell cultures are capable of producing greater secondary products than those extractation from wild plants Their advantage is that they can provide a single product continuously and reliably on the basis of:

- Plant cells can be grown under control conditions, so that, independent from natural condition, no need to transport and manage a large number of raw materials

- Product quality and performance can be controlled by removing

remove obstacles in the process of plant production

- Negation of biological effects on products are secondary compounds in natural (microorganisms and insects)

- Some metabolic products can be produced from culture fluid

suspension are higher quality than in the complete plant (Nguyen Hoang Loc, 2011)

Due to the advantages of plant tissue propagation and useful properties of turmeric, under the direct guidance of Dr Pham Phu Long, I have conducted research on the topic:

“Effect of growth regulators on the tissue culture of turmeric

(Curcuma longa L.)”

1.2 Aims and request of the study

1.2.1 Aims

Propagation rapidly of turmeric (Curcuma longa L.) by plant tissue

culture targeted to improve the propagation coefficient to keep the best advantages of the mother plants for the next generation, improve disease-free plant varieties by culture in antibiotic medium, and breed new varieties with higher yield by causing mutations to produce bigger organs and regenerate valuable genetic resources by reproducing shoots Depend on this object, I would like to provide more information, scientific data for the propagation of turmeric and selecting new varieties, contribute scientific resources for the production new plants, provide enough varieties with stable genetic quality, disease-free, for trading and conserving precious genetic resources

- Find out the best propagation medium, that is suitable to increase the quality of the plant's vegetative organs by creating healthy, green plants with developed vegetative organs, diseases-free, thereby creating quality plant source

- Determine the suitable medium for the rooting period to create completed plants that are eligible for transplanting in the nursery house

- Find out and limit the factors that interfere in the rapid propagation of turmeric, to ensure a healthy seed source that does not carry pathogens outside, thus helping to increase the quality of the plant

1.3 Objective and scope of study

Determine the suitable media for plant in regeneration stage

Determine the suitable media for plant in rapid propagation stage

PART II: LITERATURE VIEW

2.1 General introduction of Curcuma longa L

its analgesic and anti-inflammatory properties and its application in folk remedies for the treatment of visceral ulcers, detoxification and oncology (Do

Tat Loi, 2015) Morever, C longa L is a precious plant that is widely used as a

spice, medicinal plant, and has high value Red turmeric is an important crop in Vietnam and tropical and subtropical countries such as India, China, and Southeast Asian countries The rhizomes of red turmeric are used as an important incredient in medicine, cosmetic and spice (Bui Thi Thu Huong, 2020)

About 40 spices of genus Curcuma longa are indigenous to India, which point to its Indian origin (Velayuhan et al., 1999) Apart from Curcuma longa, several species of economic importance are available, such as Curcuma aromatica Salisd., Curcuma amada Roxb., Curcuma caesia Roxb., Curcuma aeruginosa Roxb., and Curcuma zanthorrizha Roxb., Curcuma caesia Roxb., Curcuma aeruginosa Roxb., and Curcuma zanthorrizha Roxb About 70-110

species of the genus have been reported throughout tropical Asia The species are seen as far away as China, Australia, and the South Pacific, while some other popular species are cultivated all over the tropics Turmeric, originating from India, reached the coast of China in AD 700 and reached East Africa 100 years later and West Africa 500 years later (Parry, 1969)

Inaddition, turmeric is a tropical plant, distributed mainly in India, Pakistan, Nepal, China, Japan, Bangladesh, Myanmar, Sri Lanka, Indonesia, Malaysia, Thailand, Taiwan, Iran, Vietnam, Argentina, Brazil, Cuba, Peru, Venezuela, Haiti, Liberia, Nigeria, Sudan, and the Maldives (Lahiri, 2014) Besides, Vietnam has rich source of turmeric, distributed in many provinces

such as Vinh Phuc, Hai Duong, Hung Yen, Nghe An, Quang Nam, Dong Nai, Binh Duong, Besides, turmeric is a medicinal plant, a traditional spice plant used often day in every Vietnamese family Turmeric is also an adaptable plant,

so it is cultivated by people on a variety of scales, from planting for self- subsistence to commercial cultivation (Ki Anh, 2008) Morever, turmeric is a light-loving plant, but it can grow and develop well in cold conditions in low light conditions from 30-50% That has been exploited for the intercropping of turmeric with many food crops, food crops and other industrial crops such as

corn, sugarcane, rubber, coffee to increase farming value for farmers (Dixit D et al., 2000) However, turmeric is a high land plant but it is also very sensitive to

soil moisture, thus the regions with an average rainfall of 1,500 - 3,000 mm/year, considered as a favorable condition for turmeric to increase yield and

development of (Kandiannan et al., 2002)

2.2 Origin and distribution

Turmeric is a tropical plant, distributed mainly in India, Pakistan, Nepal, China, Japan, Bangladesh, Myanmar, Sri Lanka, Indonesia, Malaysia, Thailand, Taiwan, Iran, Vietnam, Argentina, Brazil, Cuba, Peru, Venezuela, Haiti, Liberia, Nigeria, Sudan, and the Maldives (Lahiri, 2014) Besides, Vietnam has rich source of turmeric, distributed in many provinces such as Vinh Phuc, Hai Duong, Hung Yen, Nghe An, Quang Nam, Dong Nai, Binh Duong, (Phan Thi Hoang Anh, 2013) Turmeric is known as “Indian saffron” has been in used

dating back to 4000 BC It is mentioned in Ayurveda, the age - old Indian

system of medicine, and one encounter its name and use recorder in Dsankrit,

the ancient Indian language describing the ageless Vedas (ancient Indian scriptures), between 1700 and 800 BC during the period known as the Vedic age

(Nair and K P Prabhakaran, 2013)

In Malaysia, a paste of turmeric is spread on the mother’s abdomen and

on the ombilical cord after childbirth in the belief that it would ward off evil

spirits, and also would provide some medicine value, primarily antiseptic Both the East and the West have held turmeric in high esteem for its medicinals propertiers The Indus Valley Civilization dating back 3300 BC in western India was invole in a spice trade, of which turmeric was an important constituent The Creco- Roman, Egyptian, and Middle East regions were all familiar with turmeric meric (Raghavan, 2007) The crushed and powdered rhizome of turmeric was used extensively in Asian cookery, medicines, cosmetics, and fabric dying for more than 20.000 years (Ammon and Wahl, 1991) Early European explorers to the Asian continent introduced turmerict to the Western

world in the fourteenth century (Aggarwal et al., 2007)

2.3 Classification

- Scienctific name: Curma longa L

- Common name: Red turmeric, turmeric, Garden turmeric

2.4.2 Leaves

Leaves are oval-shapes, tapering at both ends, smooth on both sides, up to 45cm long and 18cm wide Sheathed petioles The leaves emerge from the branching rhizomes that lie just below the soil surface (Do Tat Loi, 2015)

2.4.3 Reproductive organ

Inflorescences composed grows from between leaves sheath, sparse cones shape, bracts are wide trough-shaped Corolla is plate -like, outer petals are pale yellow-green, divided into 3 lobes; upper lobe is larger, inner petals also divided into 3 lobes, 2 side lobes are vertical and flat, lower lobe is trough-shaped

During March and April, inflorescences begin to rise above the ground

On spots near inflorescences usually develop to sheaths and form new bubs In autumn, leaves begin to fade From November to December, secondary metabolites in rhizomes start to accumulate The fruits are 3 compartmental capsule; each seed has a coat (Do Tat Loi, 2004, 2015)

Red turmeric is a plant that has inflorescence but does not granulate seeds,

so that new crop must be plant by ramets or rhizomes This is a traditional propagation method in Vietnam, that cause large amount of plants initially, increasing the cost of the production process (Phung Tuan Giang, 2017)

Figure 1.1 (a) Turmeric shoot system, (b).Turmeric flower,

(c) Turmeric rhizome, (d) Turmeric powder

2.5 Living condition

2.5.1 Climate and soil

Turmeric is herbaceous perennial herb and having dormancy stage in stress condition such as cold wheather in temperated countries and the Northern

of Vietnam, which grows to three to five feet height and is cultivated extensively

in Asia, India, China, and other countries with a tropical climate It has the ability to adapt to wide range temperature conditions It can germinate in the temperature between 10 - 400C and normally growth in the range of 22 - 370C

(Ishimine et al., 2014) The optimal temperature for the development of leaves

and stems is about 25 - 300C (Le Kha Tuong, 2012)

Besides, turmeric can be grown in diverse tropical condition from sea level 1500 m above the sea level, at temperature range 20 - 350 C with an annual rainfall of 1500 mm or more, under rainfed or irrigation conditions Though it can be grown on different types of soils, it thrives best in well - drained sandy or clay loam soils with a pH range of 4.5 - 7.5 with good organic status (Jayashree

E et al., 2015)

2.5.2 Light condition

More-eve, turmeric is a light-loving plant, but it can grow and develop well in cold condition in low light conditions from 30-50%, turmeric is a light-loving plant, but it can grow and develop well in cold conditions in low light conditions That has been exploited for the intercropping of turmeric with many food crops, food crops and other industrial crops such as corn, sugarcane,

rubber, coffee to increase farming value for farmers (Dixit D et al., 2000)

C, antifungal, cholesterol removal, reduce blood fat, prevent obesity,

anti-oxidant, scavenge free radicals, make cosmetics (Hacher H et al., 2018)

The group of curcuminoid pigments includes curcumin and its derivations as demethoxycurcumin and bisdemethoxycurcumin are the main

groups, responsible for the important biological activities of turmeric (Ki Anh, 2008) Recent studies show that curcuminoids, especially curcumin, have a wide range of pharmacological effects broad as anti-inflammatory, antibacterial, antiviral, strong antioxidant, anti- UV rays, tumor growth inhibition and neuroprotection (β-amyloid resistance) Curcuminoids are currently used as pharmaceutical substances in clinical research for patients with lung cancer, cancer rectum, rheumatoid arthritis, Alzheimer's disease, psoriasis, (Truong Thi Phuong Lan, 2019)

Especially, with new technology, active ingredients curcumin and nano curcumin has been successfully extracted and safe for human and great benefits

in the prevention and treatment of a number of common diseases (Phan Thi Hoang Anh, 2013) Morever, anticancer activity of curcuminoids obtained from the ethanol extract of turmeric The result showed that they have cytotoxic

activity against ovarian cancer cells OVCAR-3 in humans (Syu et al, 1998) The studies of Jiang et al., (1996) and Hanif et al., (1997) also showed that

curcuminoids isolated from black turmeric have the ability to inhibit tumor growth and cytotoxicity to colon and cancerous cell lines human liver

epithelium (Hanif R et al., 1997), (Jiang M.C., 1996)

2.7 Production and consumption

Red turmeric has been present in almost every Vietnamese farmer family from the region from sea to plains, midlands and mountains throughout our country However, in recent years, due to increasing consumption demand, red turmeric has grown in many localities in Vietnam, especially in the provinces of the Northern-Central area and Red River Delta with an estimated scale of over 5,000 ha, yield fluctuates from 15-20 tons/ha, output is about 100,000 tons/year Production amount of red turmeric in our country is consumed in both domestic and export forms, in that domestic consumption accounts for about 30%, extant

is exported The area of lower hills and mountains in Thanh Hoa and Bac Giang

is over 10,000 ha, the delta area along the rivers in Hung Yen and other provinces is over 12,000 hectares India is the largest producer, consumer and exporter of turmeric in the world The quality of Indian turmeric is also considered the most attractive in the world because of its high content high curcumin The world's annual turmeric output is produced approximately 80% in India, the remaining production belongs to China, Burma, Nigeria, Bangladesh and some other countries Consequently, producing and expansion turmeric are promoted in production zones as well as contributed the heat for domestic markets and exported market (Le Cong Hung, 2018)

However, in recent years with extraction technology, turmeric products have been widely consumed on a global scale Therefore, consumption of turmeric products tends to increase in Europe and America and Australia with an

output of millions of tons/year (June et al., 2015)

2.8 Some scientific studies about Curcuma longa L in Vietnam

Bui Thi Thu Huong et al., (2020) have researched and built an in vitro process of red turmeric (Curcuma longa L.) with high multiplication coefficient,

good quality, which can be used for production Research results show that using a combination of alcohol 700 for 30 seconds and HgCl2 0.1% for 15-20 minutes is the most suitable for disinfection, with disinfected explant ratio 75%, the survival ratio 86.67% The most suitable medium is MS medium, that is supplemented with 30g/l sucrose, 6.5 g/l agar, 100ml coconut water, 0.5 mg/l

IBA, with 100% rooting ratio Tran Viet Ha et al., (2018) have studied successfully of application of in vitro tissue culture of Zingiber rezumbet The

results showed that, disinfecting explants with alcohol 700, rinsed in HgCl2

0.1% for 9 minutes, and cultured on MS medium, supplemented with 1.2 mg/l BAP; 0.5 mg/l Kinetin; 0.2 mg/l NAA and 30 g/l sucrose reaches 100% budding ratio, with 4.08 multiple times, after 5 weeks Rooting ratio reaches 100%, with

average length 5.5 cm, average number of roots 5.7 roots/plant, in MS medium, supplemented with 0.2 mg/l NAA and 30 mg/l sucrose, after 5 weeks

Truong Thi Phuong Lan (2019) has shown that the addition of 1.5 mg/L AgNO3 to the medium (including in vitro plant formation, culturing callus) both

increased the efficiency of the culture process compared to other studies previously Successfully isolated 4 genes involved in synthesis curcuminoids In which the DCS gene has the largest role in the genes involved in the synthesis of curcumin in black turmeric The effect of some elicitors (yeast extract and salicylic acid) on the ability to accumulate curcumin and the expression level of related genes Le Cong Hung (2018) selected the N8 turmeric variety with higher yield than other varieties > 20%, from 29.0 - 48.2 tons/ha depending on farming conditions, content Curcumin is high from 6.0 - 6.5% N8 turmeric variety is approved by the Ministry of Agriculture and Rural Development, accepts to product trially in the Northern Midland and Mountainous Provinces and the North Central Coast in 2017 Morever, develop a process of propagation and cultivation to achieve production and efficiency High economy has been recognized, according to the N8 turmeric variety was propagate the following

methods: (i) in vitro method and (ii) propagated by rhizomes, harvesting after 18

months gives the highest yield and increases more than 50% compared to 12 months

Le Kha Tuong (2012) collected genetic resources of ginger and turmeric innprovinces of Quang Ninh, Lai Chau, Hai Duong, Nghe An, Quang Nam, Lam Dong, Dong Thap with 121 ginger varieties and 66 turmeric varieties and information about ginger and turmeric in these above provinces The study carried out propagation, description and evaluation of 121 ginger varieties and

66 turmeric varieties in Tan Vinh and Nhan Trach communes, Luong Son district, Hoa Binh province in 2008 and 2009 As the results of description and evaluation of the collection The project has selected 2 sets of excellent varieties

including 10 varieties of ginger and 10 varieties of turmeric, which is a source of materials for comparison and selection of promising varieties for production

Phan Thi Hoang Anh (2013) investigated the curcuminoid extraction process in combination with the extraction of essential oils from fresh turmeric and dried turmeric (10-12%), the obtained curcuminoid had high purity (> 95%) and high yield, high extraction (7.8% on absolute dry weight) In addition, 30 derivatives of curcuminoid, 22 curcumin derivatives, 1 demethoxycurcumin derivative and 7 bisdemethoxycurcumin derivatives were synthesized, of which

10 are completely new derivatives that have not been published at home and abroad Especially the methyl pyrazolrcurcumincarboxilate derivative (derivative 19) has great potential for further research and development into medicine for prostate cancer

2.9 Some scientific studies about Curcuma longa L in the world

Acording to Chan, L.K and Thong, W.H (2014), Zingiber officinale buds from the rhizomes were used to produce in vitro shoots These explants

produced the largest number of multiple shoots, 9.8 shoots per explant, when were cultured on MS (Murashige and Skoog, 1962) medium supplemented with 2.0 mg/L benzyladenine (BA) and 2.0 mg/L indole butyric acid (IBA) This

medium was also found to be suitable for in vitro propagation of other Zingiberaceae species: Alpinia conchigera, Alpinia galanga, Curcuma domestica, C zedoaria and Kaempferia galanga Both C domestica and C zedoaria produced more multiple shoots when were cultured in the liquid

proliferation medium, MS medium containing 2.0 mg/l BAP and 2.0 mg/l IBA

To maintain the in vitro plantlets of Zingiberaceae species, they were required

to subculture every four weeks After executing proper acclimatization protocol,

in vitro plantlets of Alpinia galanga, A conchigera, Curcuma domestica, C zedoaria, Kaempferia galanga and Zingiber officinale could be successfully

planted in the field with high percentage of survival

Balachandran S M et al (2013) showed that Rhizome buds, excised from three Curcuma spp., and ginger, inoculated aseptically on MS medium

with varying levels of BAP and kinetin, produced multiple shoots For shoot multiplication, a concentration of 3.0 ppm BAP was found to be optimum for all

the species In vitro plants were successfully established in the field and were

morphologically uniform A simple method to extend the subculture interval was used and its relevance to germplasm conservation is discussed Dixit D and Srivastava N.K (2000) showed that the changes in essential oil, CO2 exchange rate and distribution of photosynthetically fixed 14 CO2 into curcumin, essential oil, amino acids, organic acid and sugars were determined in developing leaves, rhizome and roots of turmeric Of the total 14 CO2 assimilated by plants, first, second, third and fourth leaves fix 31, 23, 21 and 9%, roots 4%, rhizome 6%, oil 0.01% and curcumin 4.6% of gm fresh weight rhizome Leaf area, its fresh and dry weight and CO2 exchange rate increase up to third leaf The incorporation of

14 CO2 into sugars was maximal followed by organic acid, amino acid and essential oil at all stages of leaf development Assimilates translocated to roots and rhizome showed similar trend of incorporation in fractions as in leaves

Hatcher H et al (2008), showed that Curcumin has a surprisingly wide

range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive and chemotherapeutic activity The pleiotropic activities of curcumin derive from its complex chemistry as well as its ability to influence multiple signaling pathways, including survival pathways such as those regulated by NF-KB, Akt, and growth factors; cytoprotective pathways dependent on Nrf2; and metastatic and angiogenic pathways Curcumin is a free radical scavenger and hydrogen donor, and exhibits both pro- and antioxidant activity Curcumin is remarkably non-toxic and exhibits limited bioavailability Curcumin exhibits great promise as a therapeutic agent, and is currently in human clinical trials for a variety of conditions, including multiple myeloma,

pancreatic cancer, myelodysplastic syndromes, colon cancer, psoriasis and

Alzheimer’s disease Hanif R et al (1997) showed that Curcumin, the active ingredient of the rhizome of the plant turmeric (Curcuma longa L.), a commonly

used spice, prevents cancer in animal tumor models Its mechanism of action is unknown; curcumin may act by inhibiting arachidonic acid metabolism To explore the mechanism of curcumin's chemopreventive effect, we studied its role in proliferation and apoptosis in the HT-29 and HCT-15 human colon cancer cell lines

Plunkett G.M et al (2004) showed that as currently circumscribed,

Polyscias is the second largest genus in Araliaceae, with 150 species, and found throughout much of the Old World tropics Recent studies have shown that the genus is paraphyletic, including as many as eight additional genera in a broad

“Polyscias sl” clade Syu W J., (1998) showed that bioassay-directed

fractionation of an EtOH extract of Curcuma zedoaria led to isolation of an

active curcuminoid, which was identified as demethoxycurcumin (2) by comparison of its 1H and 13C NMR spectra with literature data and by direct comparison with synthetic material Curcumin (1) and bisdemethoxycurcumin (3) were also obtained Curcuminoids (1−3) were synthesized and demonstrated

to be cytotoxic against human ovarian cancer OVCAR-3 cells The observed

CD50 values of 1, 2, and 3 were 4.4, 3.8, and 3.1μg/ml, respectively Three additional novel compounds, 3,7-dimethylindan-5-carboxylic acid (4), curcolonol (5), and guaidiol (6), were also isolated from the EtOH extract The structures and relative stereochemistry of 4−6 were determined by spectroscopic methods and X-ray crystallographic analysis

According to Jiang M.S et al., (1996) showed that curcumin, which is a

widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis Morever, report that curcumin induces cell shrinkage, chromatin condensation, and DNA

fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene‐transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT‐29, human kidney cancer cell 293, and human hepatocellular carcinoma Hep G2 cells, but not in primary culture of mouse embryonic fibroblast C3H10TI2, rat embryonic fibroblast, and human foreskin fibroblast cells in a concentration‐ and time‐dependent manner Many cellular and biochemical effects of curcumin in mouse fibroblast cells have been reported, such as inhibition of protein kinase C (PKC) activity induced by phorbol 12‐myristate 13‐acetate treatment, inhibition of tyrosine protein kinase activity, and inhibition of arachidonic acid (AA) metabolism Treatment of NIH 3T3 cells with the PKC inhibitor staurosporine, the tyrosine kinase inhibitor herbimycin A, and the AA metabolism inhibitor quinacrine induces apoptotic cell death

2.10 Propagation method

2.10.1 In vitro method

Plant tissue culture is a combination of techniques used to maintain and grow plant cells, tissues or organs under sterile conditions on nutrient-rich culture media with ingredients identified

Various techniques in plant tissue culture can provide certain advantages over traditional propagation methods, including:

Producing exactly the number of clones helps to produce high-quality flowers, fruits, or other desirable traits

- Producing mature plants quickly in a period of time

- Producing high numbers of plants without seed or pollination to produce seeds in short time

- Regeneration completely plants by genetically modified plant tissues

- Creating plants in sterile conditions, so that they can be transported without minimizing the possibility of spreading diseases, pests or pathogens

- Producing plants from seeds, that often have low germination rate or low growth ability, e.g orchids,

- Clean up plants infected with certain viruses or other infectious agents and rapidly multiply these plants as a source of clean raw materials for fields and agriculture

Plant cell culture is based on the fact that many plant cells have the ability

to regenerate into completed plants (also called totipotency - the ability of single cells to differentiate into specialized cells with Unlimited quantities) Single cells, plant cells without a cell wall (protoplasts), leaf, root or stem fragments, can often be used to generate to new cells on cultures supplemented with substances nutrients and hormones

Modern plant tissue culture is carried out under sterile conditions, HEPA filtered air, and incubator Sources of materials: living plants taken from the environment are often contaminated with microorganisms, so the surface sterilization of the primary material (cultivars) in a chemical solution (usually using alcohol, sodium, calcium hypochlorite or mercury clorua) is needed Subsequent cultures are usually placed on top of a solid culture medium, but are also sometimes placed directly into the liquid medium, especially when resting cell cultures are used Liquid and solid culture media usually contain inorganic salts and some organic nutrients, vitamins and phytohormones The solid medium is prepared from the liquid medium by the addition of gelling agents

The composition of the medium, especially the plant hormones and the nitrogen source (nitrate, ammonium or amino acid salts) have a profound influence on the morphology of the tissues grown from the primary source For example, an enhancement of auxin stimulates root growth, while an enhancement of cytokinin stimulates shoot growth The balance of both auxin and cytokinin will produce callus, but the morphology of the product will depend on the individual plant species as well as on the environmental

composition When tissue cultured plants grow, Parts of plant are often cut and transferred to a new medium (subcultured) to allow plants continue to grow

2.10.2 The scientific basis of the method

2.10.2.1 Totipotent of plant cell

+ The cell contains the genome that determines the genotype of the species, carrying all the information of the species Can reproduce asexually when cultured in a suitable medium for complete plants

+ Plant cells are totipotential That means, all cells will have the same genome and be able to reproduce asexually Therefore, they can be cultured in a favorable environment to produce new individuals with uniform traits, genotypes and phenotypes

2.10.2.2 Cell differentiation and dedifferentiation

+ Differentiation: the process that regulates the transformation of embryonic cells into specialized cells for different tissues and organs to perform different functions

+ Cell dedifferentiation: is the converting process of cells to a certain function back to their original stage and trongly dividing

2.10.2.3 Medium

+ MS medium includes: carbon source, nitrogen source, macronutrients Stimulators

+ Auxin, Cytokinin, Giberillin, Ethylen, Abscisic axit, Inositol

2.10.3 Advantages and disadvantages of in vitro method

2.10.3.1 Advantages

In vitro methods improve many of the problems that other methods faced

such as grafting, matching Creating homogenous plants, disease-free and characteristic can be produced by materials selection, with good traits inherited

well-from the mother plants next generation evenly 100% In vitro propagation

method does not waste much area, not be affected by weather as well as external

conditions Capable of producing precious and high-value plant varieties all year such as orchids, ginseng, etc The production process is taken place actively without dependence on the time and season of the year The propagation

coefficient of in vitro method is very higher than other methods, from a small

part of a plant, it is possible to multiply hundreds or thousands of new plants, including the method of propagation from seeds within 1- Two years can produce millions of plants, which does not able to compare to the speed and

multiplier of in vitro propagation The time to mass production of plants in in vitro propagation is also faster than other methods

In breeding, the quality and quantity of varieties are the most important target Base on the propagation method from the shoot apical meristem, people not only create the high number of new plants but also reach the quality of free-virus plants Virus density gradually decreased near the growth apex area, but the growth apex was completely free of virus (Morel and Martin, 1952)

2.10.3.2 Disadvantage

The main disadvantage of in vitro propagation method is that it requires

expensive equipments and higher techniques than other methods, so the cost is

not competitive Not all plants can be propagated in vitro The ability to generate

mutations is high and plants can be infected Despite the large multiplication coefficient, the new plants are produce in small sizes and sometimes undesirable plants appeare The organic organic substances synthesization ability is poor

because in vitro plants are provided with an artificial source of carbohydrates

In vitro seedlings are often grown in glass or plastic bottles, so the

humidity is often saturated, so that when transferring plants to autotrophic environment, the plants often lose moisture balance, causing wilting and death

2.10.4 Common problems in in vitro culture

Infection of microoganism and fungus is the biggest problem when conducting experiments and influence the growth and nutrient absorption

2.10.5 Stages in propagation culture

Stage 1: Selection and Preparation of samples

This is the first stage of in vitro propagation The possibility of explants

contamination depends on how they are collected and sterilize under aseptic conditions Each sample has suitable temperature and humidity requirement when sterilizing The mother plant must be disease-free, especially viral The samples must be medium or young shoots, clean and fresh

Stage 2: Sterilization and transferation

This is the important stage before the sample is cultured This stage should ensure the following requirements: the samples are sterilized cleanly, the space in the carbinet have to keep cleanly, after sterilization, the sample should

be quickly transferred into the medium

Before decontamination, use alcohol 700to wash off the dirt, then wash it with distilled water 2 to 3 times, finally use 0.1% HgCl2 for 15-20 minutes, then wash again with distilled water for the second time

Stage 3: PLBs formation

In this stage, materials are transplanted in media have simulators to observe the bub creation ability and the number of buds in each material The materials will be inoculated on MS medium supplemented with stimulators such

as 6-BAP and α - NAA, 30 g/l sucrose, 7 g/l agar and 0.1 g/l myo- inositol in standard condition

Stage 4: Regeneration and propagation

After transferring the plants to the siutable medium, the plants begin to regenerate and produce shoots after about 7 days, and then begin to separate the plants to other media for rapid propagation This is the period of rapid regenaration

After 21-25 days, begin transferring the plants from the rapid multiplication medium to the best growth medium to create roots, stems and leaves At this stage, the indicators on stems and leaves will develop and are most suitable for monitoring

Stage 5: Trasplanting plants to nursary house

When the explants reach a certain size before they can be transferred to the nursery house, a small amount of auxin (α-NAA) is added to stimulate rooting However, the plants were still able to produce roots even from the medium that does not contain the rooting regulator

At this stage, plants move from the artificial medium to the outside, the nursery, so it is necessary to ensure some of requirements belove:

+ Plants have to ensure the standard of morphology: number of leaves, number of roots and height

+ Suitable material: clean, porous and drainage

+ Actively adjust the humidity and light of the nursery to suit the plants

2.11 Factors affecting the process of plant tissue culture

2.11.1 Explants

Murashige T (1974) persuade that the importance of appropriate explant selection and shows that most organs can be used for tissue culture It is important to note that several factors in sample selection include genotype, suitable organ, physiological age, season, growth stage, sample health, and sample source

Genotype profoundly affects the culture process With tobacco being used

as a explant, Cheng and Smith (1973) reported genomic differences in core

tissue growth cultures Furthermore et al., (1990) reported that the genotype

affects the number and diameter of callus through the pollen culture of

Lycopersycon esculentum Mill tomato

Murashige T (1974) suggested that almost all types of organs and tissues

are capable of using in vitro culture He suggested that cultures differ in

different species, such as in Petunia using apical buds for culture, according to Doerschung and Miller (1976) who suggested that sprouts are suitable as cultures in plants germinated from seeds

The real age and the seasonal age of the explants showed an important influence on cell differentiation and physiological age According to Pierik (1970) who noted that roots arise on young leaves and do not arise on old leaves

In recent years, many research results have shown that in vitro samples

have higher regenerative ability than samples taken from mother plants in the field or in nurseries such as Azalea (Economou and Read, 1986) However, many other studies have shown that the mother plants have a very important

influence on in vitro culture Beside, Morel and Martin (1952, 1955) reported

that culture apical meristem to remove the virus to produces disease-free plants and emphazise that care must be taken in the selection of cultures especially for diseased plants a large number of diseased plants were propagated

2.11.2 In vitro culture requirment

a Temperature

The suitable temperature for tissue culture is 250C ± 20C, which has a

profound effect on plant growth and development in vitro through physiological

processes such as respiration or formation of cells and organs

2002) Light quality Directly affects plants in vitro, because too strong or too

weak light condition, also affects physiological process, metabolism and nutrient absorption

d Gas condition

The gas composition in the cultural glass bottle that affects plant growth, for example, O2, CO2 and ethylene are the most investigated gaseous components in the culture medium Humidity is also effect due to its influence

on obsortion and transpiration

2.11.3 Stimulators

Growth stimulators or growth hormones are organic compounds (including natural products from plants and artical compounds) They have the effect of regulating the processes of plant growth and development However, stimulators only increase metabolism without directly participating in metabolism It cannot replace nutrients Stimulators effects strongly in very small amounts on cellular metabolism, at high concentrations they can act as inhibitors In the composition of the culture medium, the stimulants work as a key to open and close the gene activity, control the morphogenesis and the synthesis of active ingredients The effect of stimulants is related to the inhibition and induction of enzyme synthesis in plant cells, which activate parts

of the DNA molecule Each stimulator substance has its own function, but in the plants, they have function to control the cell division activities of plants, they are often involved not one but many substances Depending on each stage of culture and development of plants, the combination of these substances is different There are two main groups: auxin and cytokinin, in addition to gibberellin and ethylene

Auxin

Auxins activate macromolecular compounds (proteins, cellulose, pectin) and prevent their decompose process Auxins are considered to be the most important plant hormones because they play a very fundamental role in the

coordination of cell growth and differentiation necessary for the normal development of plants Auxin, along with a number of other regulators, ensures the mass formation of dividing cells into a complete plant body In tissue culture, substances such as: Indol acetic acid (IAA), α-Naphthyl acetic acid (α-NAA), 2,4-Dichlorophenol acetic acid (2,4-D), Indol butyric acid (IBA) are applied normaly

In plants, cytokinins have a great effect on enhancing DNA and protein synthesis as well astimulating metabolism

Gibberellin

Gibberellin research has its origins in Japan in the 19th century, when a disease of rice was shown to be due to a fungal infection The symptoms of the disease including overgrowth of the seedling and sterility were later shown to be due to secretions of the fungus Gibberella fujikuroi (now reclassified as Fusarium fujikuroi), from which the name gibberellin was derived for the active component The profound effect of gibberellins on plant growth and development, particularly growth recovery in dwarf mutants and induction of

bolting and flowering in some rosette species, prompted speculation that these fungal metabolites were endogenous plant growth regulators and this was confirmed by chemical characterisation in the late 1950s ( Hedden and Peter, 2020) Gibberellins are now known to be regulators of many phases of higher plant development, including seed germination, stem growth, induction of

flowering, pollen development and fruit growth (Sponsel et al., 2010)

Mineral element amount

MS medium (Murashige and Skoog, 1962) is the most commonly used in

in vitro culture But different species require different culture medium The

toxicity of MS medium has also been observed in some species (Sommer, 1962; Vieitez, 1963) due to its high content of macro elements, which can be toxic to cells, especially protoplasts The simplest method is to reduce the concentration

by the environment during the culture process

PART III: MATERIALS AND RESEARCHING METHOD

Turmeric (Curcuma longa L.) was grown and colected at the Faculty of

Agronomy, Vietnam National Academy of Agriculture

3.4 Research time

The study is began from the end of September 2021 to March 2022

3.5 researching method

3.5.1 Materials decomtamination and transplantation in In vitro medium

Selecting the materials, are young rhizomes reach 2 to 4 cm length will be cut After removing all of dirt and soil, they will be decontaminated with 700

ethanol for 30 seconds, then thoroughly wash carefully with sterilized distilled water for 30 seconds Next, they are decontaminated in HgCl2 0.1 % in 15-20 minutes and wash again in distilled water for 30 seconds The removing decontaminated materials in media, which contained stimulators such as 6 - BAP, α- NAA, TDZ and 2.4-D Affter 21 to 25 days, the measurement is implemented about observation targets such as the plant height, leaves length, root length, leaves width, number of leave, number of roots, number of bub and diameter

3.5.2 Evaluate plant measurment

3.5.2.1 Experiment 1: Effect of stimulation in survival ability of PLBs

formation

- Base medium: ½MS medium with growth regulator, added sucrose 30 g/l + agar 6 g/l +0.1gAC, pH = 5.8

- Materials: The shoot of turmeric plants The turmeric samples are collected from Trau Quy - Gia Lam - Ha Noi

- Method: In this experiment, the part of shoot, after decontaminated through alcohol 700 in 1 minute and HgCl2 0.1%, in 15-20 minutes, move all the material into MS medium, to observe the survival ratio, the total number of shoots are 50 samples, with 3 time replication The experiment is observed after 21-25 days

Figure 1.2 Turmeric samples

Table 1.2 Effect of 6-BAP in survival ability of PLBs formation

Table 1.3 Effect of 2.4-D in survival ability of PLBs formation

3.5.2.2 Experiment 2: Effect of growth regulators in bub regeneration stage

In this experiment, the materials from young apical meristems are

transplanted in in vitro medium to observe the effect of growth There are a few

observation targets such as the plant height, the number of bubs and the diameter Subsequently, the explants will be inoculated on MS medium supplemented with stimulators, 30 g/l sucrose, 7 g/l agar and 0.1 g/l myo- inositol Explants are propagated at a temperature of 250C ± 20C and humidity about 40%

Table 2.1 Formula of 6-BAP on bub regeneration stage