Study on the technical measures for the cultivation of ngoc linh ginseng (panax vietnamensis) in vitro seedling in nursery

VIENAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY UNDERGRADUATE THESIS TOPIC: STUDY ON THE TECHNICAL MEASURES FOR THE CULTIVATION OF NGOC LINH GINSENG PANAX VIETNAMENSIS IN

VIENAM NATIONAL UNIVERSITY OF AGRICULTURE

FACULTY OF AGRONOMY

UNDERGRADUATE THESIS TOPIC: STUDY ON THE TECHNICAL MEASURES

FOR THE CULTIVATION OF NGOC LINH

GINSENG (PANAX VIETNAMENSIS) IN VITRO

SEEDLING IN NURSERY

Student code : 611646 Class : K61-KHCTT Supervisor : Dr Thieu Thi Phong Thu

Assoc.Prof.Dr Le Hung Linh Department : Crop Science

HA NOI -2021

ACKNOWLEDGE

There is an undeniable fact that, beside the own effort, there are no successes that are not associated with support or assistance whether more or less, directly or indirectly by others This thesis would not have been possible if

I did not have support of many individuals and organizations Therefore, I would like to extend my sincere gratitude to all of them

First of all, I would like to express my thankful to the lecturer in Faculty

of Agronomy of Vietnam National University of Agriculture with their knowledge and dedication to convey valuable knowledge to me during the time

of studying at university

Secondly, I am using this opportunity to express my deep and sincere gratitude to Dr Thieu Thi Phong Thu and Associate prof Dr Le Hung Linh who in site of being extraordinarily busy with her duties, took time out to hear, guide and keep me on the correct path It was a great privilege and honor to work and study under their guidance

I am also grateful to Department of Molecular Biology - Institute of Agricultural Genetics, Experimental station in Van Giang - Hung Yen and technical support in the implementation I would not forget to express my special thanks to Dr Khuat Thi Mai Luong and all personnel of Institute Without Dr Khuat Thi Mai Luong and others superior knowledge and experience, this undergraduate thesis would not like in quality of outcomes, and thus their guidance and support have been essential

However, due to limiting my professional knowledge and lacking in practical experience, the content of the topic can not avoid the deficiencies I look forward to receiving recomments from lecturers and all staffs at the

TABLE OF CONTENTS

ACKNOWLEDGE i

TABLE OF CONTENTS ii

LIST OF TABLE iv

LIST OF GRAPH v

LIST OF FIGURE vi

LIST OF FIGURE vi

ABSTRACT vii

PART 1: INTRODUCTION 1

1.1 Background 1

1.2 Objectives and requirements 2

1.2.1 Objectives 2

1.2.2 Requirements 2

PART 2: LITERATURE RVIEW 3

2.1 Introduce about Ginseng 3

2.2 General introduction to ginseng species of the Panax genus in Vietnam 7

2.3 Introduce about Ngoc Linh ginseng 11

2.3.1 The original of Ngoc Linh ginseng 11

2.3.2 Botanical characteristics, growth and development of Ngoc Linh ginseng, external requirements of Ngoc Linh ginseng 13

2.3.3 Natural conditions in the distribution area of Ngoc Linh ginseng Climate condition 16

2.3.4 The value of Ngoc Linh ginseng 17

2.4 The state of Ngoc Linh ginseng research and development 18

2.4.1 Researches on propagation from seeds 19

2.4.2 Researches on in vitro clonal propagation 19

2.5 Researches optimize the growth and development process of Ngoc Linh ginseng 20

2.5.1 The condition of planting and caring for Ngoc Linh ginseng 20

2.5.2 Research on growing medium Ngoc Linh ginseng 21

PART 3 CONTENT AND METHOD OF RESEARCH 25

3.1 Research materials 25

3.2 Place and time of the research 27

3.3 Research content 27

3.4 Experimental parameters 29

3.5 Data processing methods 29

PART 4 RESULTS AND DISCUSSION 30

4.1 Research on standardization of in vitro seedlings Ngoc Linh ginseng 30

4.2 Effect of the substrate on the viability and growth of Ngoc Linh ginseng in vitro seedlings 34

4.2.1 Experiment in laboratory conditions at Van Giang Experimental Station 34

Figure 4 5: Ngoc Linh ginseng in vitro seedling used in the experiment 35

4.2.2 Experiment in net house conditions at the Experimental station in Sa Pa 37

4.2.3 Experiment in nursery conditions at Sa Pa experimental station 39

4.3 Effect of plant age on in vitro seedling adaptability in vitro in nursery 42

4.4 Effect of spraying time of AT Chibozin on the growth of in vitro seedlings in the nursery 46

4.5 Assessment of pests and diseases of Ngoc Linh ginseng in Sa Pa 49

PART 5 CONCLUSIONS AND SUGGESTIONS 53

5.1 Conclutions 53

5.2 Suggestion 53

REFFERENCE 54

ACTIVE IMAGE 56

LIST OF TABLE

Table 2.1: Species of ginseng were registered 3 Table 2.2: Panax species in the world 6 Table 4.2: Effects of substrate on in vitro seedling adaptability in the net house conditions 38

Table 4.3: Table 4 3 Effects of substrate on in vitro seedling adaptability in nursery

conditions 40

Table 4.4: Effect of plant age on in vitro seedling adaptability in nursery 44 Table 4.5: Effect of spraying time of AT Chibozin on the growth of in vitro seedlings

in the nursery 48

LIST OF GRAPH

Graph 4.1: Describe the survival rate of Ngoc Linh ginseng in vitro seedling planting

in three location 42 Graph 4.2: Effect of plant age on in vitro seedling adaptability in nursery 45

LIST OF FIGURE

Figure 2.1: The most commonly of ginseng 4

Figure 2.2: Distribution of Ginseng species in the world 5

Figure 2.3: Ngoc Linh ginseng 15

Figure 3.1: Ngoc Linh ginseng in vitro seedling 25

Figure 3.2: Perlite rock and Vermiculite of Namix company 26

Figure 4.1: Ngoc Linh ginseng tubers have no morphogenetic differentiation 30

Figure 4.2: The ginseng tubers have not split clearly between buds and node 32

Figure 4.3: Ngoc Linh ginseng tuber is morphogenetic differentiation 32

Figure 4.4: Ngoc Linh ginseng tuber have morphogenetic differentiation clearly 33

Figure 4.1: Ngoc Linh ginseng in vitro seedling used in the experiment 35

Figure 4.6: Ngoc Linh ginseng in vitro seedlings in the net house 37

Figure 4.7: Ngoc Linh ginseng in vitro seedlings in Sapa 39

Figure 4.8: Ngoc Linh ginseng in vitro seedlings growth in Sapa 45

Figure 4.9: Experiment spraying AT Chibozin after 20 days planting 47

Figure 4.10: Photos of bare slugs harming Ngoc Linh ginseng seedling in Sa Pa 51

Figure 4.11: Image of diseases of Ngoc Linh ginseng seedlings in Sa Pa 52

ABSTRACT

Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) is proven to have

high medicinal value, and is classified as the most precious ginseng in the world Nowadays, Ngoc Linh ginseng have been overexploited so seed propagation must be faced many difficulties This study aimed to choose the suitable technique to raise the survival rate, the adaptability in the natural environment of

Ngoc Linh in vitro seedling In this study, Ngoc Linh in vitro seedlings were

used the initial materials to survey the effect of other substrates (humus, perlite rock, vermiculite mineral organic fertilizers and peat moss), the effect of age plant ( 1 year old, 1.5 years old and 2 years old) and the effect of time spray

biological products (AT Chibozin) on the adaptive capacity of in vitro seedlings

in the nursery The result showed that the mixture substrate with forest humus, vermiculite and perlite rock in the ratio of 2: 3: 1 is suitable for a high survival rate of over 80% Seedlings have a high survival rate, strong plants and good growth and development The age of plants are appropriate to take the seedlings

to the nursery which ensures good growth and development are plants from over 1.5 years old Research results on the effect of spraying time of AT Chibozin on

the growth of seedlings in vitro in the nursery, the seedlings have the best

growth and development when spraying 35 days after planting Throughout the survey in nursery, Ngoc Linh ginseng grown in Sapa has identified a number of pest such leaf worms, black cutworms, armyworm, grasshoppers, brown crickets, bare slugs and maggots in bulb and root; and some diseases as disease

are caused by fungus: Rhizoctonia Solani , ring spot Alternaria alternata or rust Puccinia sp Based on this systematic review, this is the basis for Ngoc

Linh ginseng plant can be acclimatized to the region with similar ecological

PART 1: INTRODUCTION 1.1 Background

Ngoc Linh ginseng is an endemic species in Viet Nam and the world It

has been known to the scientific name Panax vietnamensis Ha et Grushv Ngoc

Linh ginseng has been identified as valuable medicinal plants, unique value to use as well as the value of genetic resources According to Phung Tuan Giang from Tho Xuan Duong Oriental Medicine Pharmacy in Hanoi, the stem and root

of Ngoc Linh ginseng contain 52 saponin, which has many new compounds as

majonoside-R2 (type saponin only found in P vietnamensis), ginsenoside-Rh5 (20-O-Me-G-Rh1), vina-ginsenosid R-25, etc (Korean ginseng (P ginseng) has

about 25 types of saponins) Due to many good uses for human health, high economic value, Ngoc Linh ginseng has been exploited at risk of extinction, losing rare genetic resources

In recent years, Ngoc Linh ginseng has been entered in the list of development and conservation priority species But, in fact, the production of propagating Ngoc Linh ginseng is still facing many difficulties as: (1) Did not take interest in the adequate value and potential of it The production is still fragmented, the indiscriminate exploitation of Ngoc Linh ginseng has occurred, resulting in the exhaustion of variety sources (2) While the demand for the Ngoc Linh ginseng variety in our country is huge, it does not have the stable supply of seedlings in both quantity and quality The production of seedlings following traditional methods with effective seed is not high The reason is that

it takes time to develop into mature plants and flower seeding takes 4-5 years, the germination rate of seeds is low 50-60% This is the limiting factor which affected the successful development of Ngoc Linh ginseng production in Viet Nam

To respond the pressing needs, the rapid multiplication method using embryonic cloning technique is already giving high efficiency This method allows for the shortest possible time, the large-scale rapid multiplication, high

uniformity plants, disease-free and keep the genetic characteristics of the

parents However, Ngoc Linh ginseng seedlings in vitro need to be trained to

enhance the adaptability in the natural environment with the ability growth and development well Thus the theme: "Study on the technical measures for the

cultivation of Ngoc Linh ginseng (Panax vietnamensis) in vitro seedling in

nursery" is necessary and practical significance

1.2 Objectives and requirements

1.2.1 Objectives

Studying the technical measures for cultivation of Ngoc Linh ginseng

(Panax vietnamensis) in vitro seedling in nursery to choose the suitable technic

to raise the survival rate, the adaptability in the natural environment of seedling

in vitro at the nursery stage

- Evaluating the effects of nutritional probiotics and disease probiotics on

the survival rate, the growth and disease rates of Ngoc Linh ginseng seedlings in vitro at the nursery stage

PART 2: LITERATURE RVIEW 2.1 Introduce about Ginseng

Ginseng belongs to the Araliacaea family, carrots, celery and radishes also belong to this family Araliacaea consists of about 700 species of plants, divided

into 70 genus, one of which is the Panax genus The Panax genus has 5-13

species (Persons và Davis, 2007), but among it only 5 species have been

officially registered (Table 2.1) Panax japonicum has been found naturally

growing in the mountains of Japan since ancient times The Japanese have

grown Panax ginseng and exported to China and Hong Kong (Persons, 1994) Panax notoginseng has pharmacological characteristics similar to those of the two most valuable species (P quinquefolium and P ginseng) and it is becoming increasingly popular as a herb Panax trifolium is called dwarf ginseng or

ground nut, are founded in American (Persons and Davis, 2007)

Table 2 1: Species of ginseng were registered

China

nut

North America

Canada (Persons and Davis, 2007)

Ginseng (Panax ginseng C A Meyer) is a perennial herbaceous plant

cultivated for its highly valued root in Korea, northeastern China, the northern United States, and Canada (But et al 1995) It is traditionally considered one of

the most potent medicinal plants Ginseng plants are known by various names depending on their origin, cultivation, the food processing method used, and so

on Many ginseng cultivars are currently valuable commercially Three major

species are included in ginseng; one is Panax ginseng C A Meyer, referred to

as ‘‘oriental ginseng,’’ which is distributed primarily in Asia and East Siberia;

another is P quinquefolium L., commonly called ‘‘American ginseng;’’ the other is P notoginseng (Burkill) F.H Chen (Sanchi), which is called ‘‘Chinese ginseng’’ (Chang and But 1986) The species P ginseng includes several

accessions and cultivars: Chunpoong, Yunpoong, Jakyung, Gopoong, Kumpoong, Sunpoong, Hwangsook, and Sukju Among these, three cultivars—Chunpoong, Gopoong, and Kumpoong—are suitable for generating ‘‘red ginseng’’ (steamed ginseng roots) because they have good root shapes

Figure 2 1: The most commonly of ginseng

The Panax genus includes 16-18 species (Sharma and Pandit 2009) The species composition of the Panax genus (Panax L.) has many changes

notoginseng Chen; American ginseng (Panax quinquefolius L.); Japanese Ginseng (Panax japonicus C A May); Narrow-leaf ginseng (Panax wangianus

S C Sun); Panax bipinnatifidus Seem; Panax bipinnatifolius var angustifolius (Burk) Wen); Panax stipuleanatus Tsai et Feng; Panax zingiberensis Wu et Feng; Panax trifoliatus L.; and Vietnamese Ginseng (Panax vietnamensis Ha et

Grushv) (Thuong Dong Nguyen et al., 2007) Based on its geographical or

commercial origin, ginseng is divided into two main things: Panax ginseng Asian and Panax quinquefolium American Most of the genus is distributed in

Asia, from East to North Asia to the near Himalayas and there are only three species in North America (Figure 2.1)

Figure 2 2: Distribution of Ginseng species in the world

Table 2 2: Panax species in the world

Angustifolius

Narrow leaf pearlginseng

(Source: http://www.theplantlist.org/browse/A/Araliaceae/Panax)

In particular, the composition species of Panax genus are valuable as

medicines, some species of this genus are currently grown on a large scale in many countries, not only within the scope of the Oriental traditional medicine

but worldwide as Panax ginseng; Pseudo-ginseng (P pseudo ginseng); American Ginseng (P quiquefolius) and P notoginseng, following by

Lee(1992), Li(1995), Nguyen(1998) (Tap Nguyen, 2005)

The species of the ginseng have been known and used very early in

oriental medicine Nowadays, with the knowledge about the medicinal

properties of species of the genus Panax, ginseng is increasingly widely used in

medicine and cosmetic chemistry

2.2 General introduction to ginseng species of the Panax genus in Vietnam

Vietnam is a country with a tropical monsoon climate, hot and humid through year-round, with many sub-terrain and diverse climates, so Viet Nam has a diversity of species and distribution of precious medicinal plants Among

the species of the naturally growing Panax genus are Vu Diep Ginseng (P bipinnatifidus), P stipuleanatus, Lai Chau ginseng (P vietnamensis var fuscidiscus), and Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) (Le Thanh Son and Nguyen Tap, 2006) Vu Diep ginseng and P stipuleanatus are

widely distributed on Hoang Lien Son mountain in 2 provinces: Lao Cai and Lai Chau Ngoc Linh Ginseng is distributed on Ngoc Linh mountain in Kon Tum and Quang Nam provinces and Lai Chau ginseng is endemic of Lai Chau province All of them are appreciated not only in terms of economic value but also for their therapeutic and health-promoting effects

Lai Chau Ginseng is a new medicinal plant, little-known in Vietnam It is

a perennial herb, distributed in Pu Si Lung mountain and the neighborhood of mountain(Mường Tè andin the western of Sìn Hồ nearly border China) and Pu Sam Cap mountain located between Sin Ho districts and Tam Duong last to Lai Chau City Lai Chau Ginseng habitat is under the primeval forest, dense forests, which adjacent between the low mountain belt and the medium mountain belt,

on the A1 floor of the soil Humic Acrisols (ACu) which is weathered of shale and silicate rock, rich humus and nutrients, under the tropical monsoon climate

in the mountains, summer rainny, there is no apparent dry period, the cold weather have at least 5 months (Phan Ke Long et al 2013) Lai Chau Ginseng has a solitary inflorescences sprouting in the top of the stem, the flower has a white stamen and the stigma splits into half to the base of the lower pot The

plants usually flowers in July to September, fruits are flattened, the shape is disproportionate, self-pollinated In addition, the plant are pollinated by butterfly bees through the process of absorbing honey, thereby increasing genetic diversity for Ginseng However, the method of breeding with Lai Chau Ginseng seeds faces many difficulties such as pollination capacity, low grain rate, scattered ripe seeds so it is difficult to harvest, especially, Lai Chau Ginseng is

in flowers and set fruit after 3-4 years of planting, the percentage of seeds germinated is low

Lai Chau ginseng has the same main morphological characteristics as P Vietnamensis var fuscidiscus K Komatsu, S Zhu & S.Q Cai variety was

recently described in a standard sample collected in southern Yunnan, China

Lai Chau ginseng differs from both types of Vu diep ginseng Panax japonicas var bipinnatifidus and Panax stipuleanatus are caused by single leaflets rather than split feathers On the contrary, it is the most similar to Panax vietnamensis

which is discovered and described in Ngoc Linh in its major morphological features Although the medicinal content is inferior to Ngoc Linh ginseng and Lai Chau Ginseng, all parts of Vu Diep ginseng are also used as medicines

Rhizomes of P bipinnatifidum Seem are used to as a tonic which helps to

increase sexual health, hemostasis, anti-stress, improve memory, reduce blood sugar levels with patients’s diabetes (Bich, 2004), activation of the metabolism, anti-inflammatory, antioxidant, prevent cancer, and cardiovascular benefits The uses are made up pharmacological activity called saponin triterpene

Vu Diep ginseng is a herb that lives for many years with 30-100 cm high Its rhizome is fat, branched, horizontal and usually above ground with 1.5-3.5

cm in diameter The leaf-bearing stem consists of 1-3, depending on the number

tops; flower stalks is 5-10 cm, carrying 20-90 flowers Flower stalks are thin, 1.5 cm long It has yellow-green flowers, 5 small sepals, 5 petals and 5 stamens Its tubers include 2 cells with split nozzle into two Its ruits are in shape of sphere or flattened sphere with 0.6-1.2 cm in diameter and red when ripe It has

1-2 seeds; however, if there is only 1 seed, it is because the other is flat Seeds are nearly spherical or near pea-like with white gray, hard shell and hilum seeds Its flowers blossom from April to May but fruits from May to September or October Vu Diep ginseng can be propagated naturally by seeds However, the method of propagation with its seeds has many difficulties because of low pollination ability and seeding rate, scattered ripe seeds as a result of difficultly harvesting and especially Vu Diep ginseng after 3-4 years bear fruit Ripe fruit is usually eaten without seeds, falling seeds are eaten by a small brown squirrel The rhizome is broken or exploited with old parts, the tips of the rhizome (with sleeping buds) remains able to regenerate The entire stem bearing leaves fades

in winter, at the beginning of spring next year, from the tips of the rhizome, new

shoots will sprout According to the latest statement by Nguyen Huu Tung et al (2011),the root of Vu Diep ginseng P bipinnatifidum Seem contains 10 types of

oleanolic saponins In which, there are 3 new types of saponins: unknown bifinoside

A, bifinoside B and bifinoside C and 7 groups of compounds have been announced, including: narcissiflorinemethylester, chikusetsusaponin Iva, pseudoginsenoside RP1 methylester, pseudoginsenoside RT1 methylester, stipuleanoside R2 methyl ester stipuleanoside R1, and momordin Iie These compounds inhibit the development of

some cancer cell lines (Nguyen et al., (2011))

Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv) is a type of grass,

perennials Ngoc Linh ginseng distinguishes from the closest species naturally growing in Vietnam such as Vu Diep ginseng by the shape of the leaflets (the leaflets are oblong, the two pointed ends with the leaflets are deeply lobed and

in shape of feather) The length of the stalk is from 15-30 cm to 5-10 cm, its

flower color is vory-white or light green with lemon yellow, its fruit color when ripe is red and black spots at the top with red Ngoc Linh ginseng distinguishes

P Stipuleanatus by stalk length It has stalk length of 15-30 cm Its stalks are

beyond the canopy with 5-10 cm, as high as canopy) Its flower color is ivory white or light green with yellow green Its fruit color when ripe is red with black dots at the top and red with no black dots at the top Ngoc Linh ginseng is of high economic value, but it is now absent from the natural environment Ngoc Linh ginseng has a very narrow distribution area, requiring a very strict living environment with slow growth It is overexploited for medicinal purposes In the past two decades, due to people’s intense hunting, from having reserves of several tens of tons in the wild, so far, Ngoc Linh ginseng is facing extinction in the Vietnam Red Book and becoming a In many subjects, priority is given to conservation and development

The last of the four genus Panax species is P Stipuleanatus Basically, its

shape is very similar to Ngoc Linh ginseng Herbaceous plants live for many years with 30-50cm tall It has propeller compound leaves, and grows round 3-4 each Its common petiole is 3-6cm long, bearing 3-7 long lanceolate leaflets, having notched edges, having hard bristles in the tendons on both sides The

leaflets are 0.6-1.2cm long P Stipuleanatus has single canopy flower cluster at

the top of the stem with pale yellow flowers with 5 sepals, 5 petals, 5 stamens and 2 pots below It has flat spherical berries and white, spherical particles and it’s red when ripe This kind of plant flower in May - July, fruits ripe in August - October People choose good seeds from plants that have grown for 3-4 years, putting seeds into soil in October - November, and in February - March plants grow However, it takes a year in January or February to plant seedlings After

prolin, histidine, lysine, cysteine; inorganic substances like Fe, Ca P Stipuleanatus also contains ginseng-like compounds Plant parts such as roots,

leaves, and flowers contain saponosid compounds of the dammaran group In addition, there are also other valuable ingredients such as amino acids,

polyacetylen and panaxytriol and so on The tubers of P Stipuleanatus have a sweet, slightly bitter taste and temperament P Stipuleanatus has very good

energy-boosting effect which is similar to that of ginseng such as shortening clotting time, losing blood stasis and increasing blood flow in the coronary arteries of laboratory animals Moreover, it helps to increase myocardial contractility at low doses, have aphrodisiac effects on female sexual endocrine function, which expresses in estrogenic and gonadotropic activities It also helps peripheral vasodilation not to affect blood pressure and central nervous system, harmonizes immunomodulatory and has the effects of mental stimulation, anti-depression

2.3 Introduce about Ngoc Linh ginseng

2.3.1 The original of Ngoc Linh ginseng

Ngoc Linh ginseng was first discovered in Central Vietnam by the ethnic minorities Xe Dang The ethnic in here often use Ngoc Linh ginseng as a kind of forest root that they often call "wormwood root” or concealed medicinal plant

Classification:

- Scientific name: Panax vietnamensis

- Name of two parts: Panax articulatus KL Dao (1973) ex Ha et Gruskv

- Sub family: Aralioideae

- Genus: Panax

- Species: P Vietnamensis

Distribution: Vietnam is one of the few countries on the world's ginseng

distribution map Specifically, some species of ginseng belong to Araliaceae

family have been found in high mountain areas, such as Vu Diep ginseng (P bipinnatifidus) and P stipuleanatus in Lao Cai and Lai Chao; Lai Chau Ginseng (P vietnamensis var fuscidiscus) is endemic ginseng of Lai Chau province and Ngoc Linh ginseng (P vietnamensis) in Kon Tum and Quang Nam So far, Ngoc

Linh ginseng has only been discovered in the Ngoc Linh mountain area of Quang Nam and Kon Tum provinces This is also the southernmost limit (at 150

north latitude) of the distribution map of the genus Panax L in the world Ngoc

Linh is the second highest mountain range in Vietnam, with geographical coordinates from 107º50' - 108º7' east longitude and from 15000' - 150010 'north latitude This mountain range has the highest peak Ngoc Linh at 2.598 m In the past, Ngoc Linh ginseng grew naturally from an altitude of about 1.500 m to 2.200 m, mainly concentrated in 1.800-2.000 m Ngoc Linh ginseng grows in mountainous districts of Ngoc Linh in Dak To district, Kon Tum province or in Nam Tra My district, Quang Nam province In addition, Ngoc Linh ginseng is also distributed at the mountain Ngoc Lum Heo in Phuoc Loc commune, Phuoc Son district and possibly even at the top of Ngoc Am in Quang Nam province, according to the latest survey results At the altitude of 1.500 to 2.600m, Ngoc Linh ginseng grows thickly under the forest canopy along the moist streams on humus soil The limit and distribution of this species in Ngoc Linh mountain now have had many changes

2.3.2 Botanical characteristics, growth and development of Ngoc Linh ginseng, external requirements of Ngoc Linh ginseng.

Ngoc Linh ginseng is a perennial herbaceous plant, the rhizome-stem has scars and the vertebrae due to the pneumatophores die gradually with the height

of about 40-100 cm high This species of ginseng begins to appear at an altitude

of 1.500 m or more above sea level From 1.700 – 2.000 m, this species grows in populations along the stream banks, with the moisture content over 80% The soil is rich in organic humus, under the mixed forest canopy between broad leaves and conifers In terms of rhizome, Ngoc Linh ginseng has lean rhizome with 1-3.5 cm in diameter, the length of rhizome depends on the number of years of growth, light yellow or earthy yellow, many internodes and scars left by stalks every year–each is equivalent to 1 year old The rhizome has many rootlets and longitudinal wrinkles, mild fragrance and bitter but slightly sweet taste Tuberous roots of Ngoc Linh ginseng are located at the end of rhizomes, spindle-shaped, cylindrical, sometimes humanoid, light yellow, bearing many rootlets and horizontal veins Planted ginseng has more developed tuberous roots and usually comes in three forms: carrot-type, spindle - shaped and most commonly tuber-form The stems of Ngoc Linh ginseng erect, green or purpleish, with 5-8 mm in diameter, usually blighted every year after growing season Rhizomes can branch many times and form a ginseng bush, but very rarely Ngoc Linh ginseng has propeller-shaped compound leaves, growing at the tip of the stem The leaf stalk is 2-12 cm long, each compound leaf usually has 5 leaflets that are inverted, lanceolate or oval, serrated edge, pointed leaf tip and wedge-shaped leaf base Leaf veins are feather-shaped, usually have 10 pairs, and veins are networked Blades are green, thin, easy to tear, with many hard bristles from 1-2 mm long which is less in lower surface Flowers in clusters usually appear in plants with 3 or more compound leaves Each cluster has 50-120 flowers, stalks are 1- 1.5 cm long Flowers are light greenish yellow,

3-4 mm in diameter, including 5 sepals forming a bell shape, 5 petals, 5 white stamens Anther has oval shape, it is attached back The gourd is 1-1.5 mm tall, has 2 carpels, but usually only one develops Flowering season varies by region but usually starts from April to June The fruit is slightly spherical or spherical slightly flattened back-abdomen, concentrated in the center of the foliage, with 0.8-1 cm long and approximately 0.5-0.6 cm wide After two months it starts to turn from green to dark green, berry-yellow, when ripe to orange - red with an irregular black dot at the top of the fruit Each fruit includes one seed, some contain 2 seeds and the average number of fruit is about 10 to 30 fruits Seeds are mainly in shape of kidney, a few flat spherical fruits contain 2 seeds The average weight of one fruit is 275 mg and one seed is 75 mg The fruit season is from July to September On the eastern slopes of Ngoc Linh in Quang Nam province, the fruitful season may be 1 month slower than the western slopes of Kon Tum province At the beginning of January every year, new buds appear

At the end of October, the pneumatophores die gradually, leaves fall, leaving a scar on the head of ginseng and the plant begins to hibernate through December When harvested, only ginseng from 3 years old or preferably over 5 years old should be collected Winter is also the season to harvest the best rhizomes of ginseng

Figure 2 3: Ngoc Linh ginseng Growth cycle of Ginseng Ngoc Linh:

- From January to March, ginsengs appear and develop aerial stem, leaves and flower canopy

- From April to June, plants flower, flowers bloom and fruiting

- From July to September, the fruits are ripe and fully ripe, lasting until October

- From November to the end of December, stems are largely destroyed, propagules and other organ which take shape on the ground are formed

2.3.4 Natural conditions in the distribution area of Ngoc Linh ginseng

Vegetation

Ngoc Linh ginseng grows under moist forests, humus, broadleaf trees in the lowland and medium ridges contiguous areas (cloud forests) at an altitude of approximately 1500 to 2000 meters above sea level, on shale and silicate rocks The places where Ngoc Linh ginseng grows naturally are usually under the forest canopy that has never been affected It is where there are often many old trees that are dead, collapsed, rotting, creating a thick rotting vegetation layer andmulch, porous, usually moist soil layer Canopy cover is about 60-90% Very

rarely, Ngoc Linh ginseng regenerates randomly on the edge or under the forest canopy which has been strongly destroyed, has a great slope Sometimes it is washed away strongly in the middle of mountains or rocks where there is a lot of light Although the weather in the Central Vietnam is the end of the dry season and the beginning of the rainy season, Sam Ngoc Linh grows strongly in Spring

- Summer, due to its distribution at an altitude of over 1,500 m and the original vegetation creating a humid environment, in the Ngoc Linh mountain area

Climate condition

Ngoc Linh is one of the high mountain range so this is the intersection between two air currents, the northeast monsoon and the southwestern air current, along with the characteristics of high coverage density, height, etc All

of these create a tropical climate characteristic of Ngoc Linh mountain Therefore, this is the ideal place to meet ecological requirements for the growth

of Ngoc Linh ginseng Ngoc Linh ginseng is suitable for daytime temperatures from 20°C-25°C or 15°C-18°C at night They can live for a long time, even over

100 years and grow quite slowly Another very different feature of this region is the relatively large annual rainfall, high humidity, low evaporation, low temperature, etc Although the weather in the Central Vietnam is the end of the dry season and the beginning of the rainy season, Sam Ngoc Linh grows strongly in Spring - Summer, due to its distribution at an altitude of over 1.200

m and the original vegetation creating a humid environment, in the Ngoc Linh mountain area

Soil characteristics

According to the analysis of numbers and data, the topsoil of areas where Ngoc Linh ginseng grows is mainly formed by plant remains Ngoc Linh

According to statistics on soil nutrients, physical and chemical properties, especially the standards of the content of the surface layer are very high The thickness of the surface layer is mainly mollusks, ensuring the thick for the ginseng root to develop favorably Growth conditions of Ngoc Linh ginseng are usually certain, not easily changed Therefore, in order to propagate and preserve Ngoc Linh ginseng, it is necessary to have a specific plan

2.3.5 The value of Ngoc Linh ginseng

Ngoc Linh ginseng is a special ginseng species, ranked top 4 best ginseng species in the world, scientifically and economically valuable, with saponin content, amino acid content, trace minerals in roots, leaves and tuber than other ginseng species The important point that makes the high value of Ngoc Linh ginseng is that in its composition, there are 52 different saponins In which, there are 26 types of ginseng with chemical structures commonly found in other ginsengs such as Korean ginseng, Japanese ginseng and Western ginseng and there are 26 types of saponins with new structures, especially in Ngoc Linh ginseng In addition to their pharmacological effects, Ngoc Linh ginseng also helps reduce stress, depression, oxidation, etc Due to limiting the distribution area and overexploitation, Ngoc Linh ginseng becomes scarce and are introduced IUCN Red List (2003), as well as the list of species restricted to exploitation and commercial use Facing the danger of extinction of precious ginseng varieties, the Government of Vietnam has decided to establish a national restricted area where ginseng grows concentrated in two provinces of Kon Tum and Quang Nam, and put Ngoc Linh ginseng on the list of types of trees banned from illegal exploitation and sale

Ngoc Linh ginseng as well as most medicinal herbs of high economic value which develop under the forest canopy are non-timber forest products Regarding Ngoc Linh ginseng, it is estimated that with an initial cost of 3 billion per hectares After 5 years, it can be earned 30 billion VND It is a dream figure

for people living off the forest Many localities are looking for different ways to plant Ngoc Linh ginseng varieties However, Ngoc Linh ginseng which is currently absent in the natural environment is an endemic plant, growing in certain places with a very narrow distribution area (only growing under the canopy

of old forest around the foot of Ngoc Linh mountain) It requires very strict living environment (planted at 1.200-2.000m high), and soil conditions as well as coverage, climatic conditions It has slow growth butis usually overexploited for medicinal purposes In the past two decades, due to the intense hunting of people, from having reserves of several tens of tons in the wild, so far, Ngoc Linh ginseng

is facing extinction and recorded in the Vietnam Red Book as a result of one plant with priority given to conservation and development

Currently, Ngoc Linh ginseng is grown in a number of gardens in the two protected areas of Quang Nam and Kon Tum provinces such as Chot Sam (Mang Ri commune, Tu Mo Rong district, Kon Tum province) and Tra Linh Lieu Medicinal Station (Tra commune) Linh, Nam Tra My district, Quang Nam province) with a total planting area of about 10 hectares Ngoc Linh ginseng is grown scatteredly in the gardens of some households on Ngoc Linh mountain Recently, Ngoc Linh Ginseng Company in Kon Tum province has started planting more than 100 hectares in Te Xang commune, Tu Mo Rong district, Kon Tum province In addition, there are many studies to preserve and develop Ginseng Ngoc Linh into an advantageous commodity plant in Vietnam Now, Kon Tum province has 9 communes certified for geographical indication of Ngoc Linh ginseng in 2 districts Tu Mo Rong and Dak Glei with an area of about 400 hectares

2.4 The state of Ngoc Linh ginseng research and development

Highlands Scientific Research Institute, Center of Ginseng and Medicines City

Ho Chi Minh City, Institute of Tropical Biology, Center of Biotechnology City

Ho Chi Minh City, Military Medical Academy, Department of Science and Technology of Kon Tum province, Department of Science and Technology of Quang Nam province

2.4.1 Researches on propagation from seeds

In ancient times, ginseng seeding was done with two main forms: propagation from seeds and from the tip (underground rhizomes) But the results achieved are still limited as the number of ginseng seeds produced is low, which means that the quantity is small, the quality is not good and it is easily lost due

to natural disasters and epidemics and it is difficult to meet the increasing demand of consumers Within the framework of the project "Complete research

on seed production process, planting techniques and development planning for

Ngoc Linh ginseng in Kon Tum (Panax vietnamensis Ha et Grushv.), Scientists

from the Institute of Medicine and The Department of Science and Technology

of Kon Tum province have conducted researches on the procedure of propagating Ngoc Linh ginseng from seeds (Nguyen Ba Hoat, 2004) Ngoc Linh ginseng as well as most other plants are propagated from seeds, however, the researchers believe that propagation from this seed has some specific characteristics In which, the process of harvesting ripe fruit is the main factor determining the germination rate (the maximum germination rate is 94%) This seed production process is very simple, but soil preparation for the nursery must

be focused because it requires a high natural humus content As a result, it is possible to shorten the sleeping time of seeds, especially seedlings in nurseries

as well as in gardens will develop earlier and more favorably

2.4.2 Researches on in vitro clonal propagation

Ginseng clonal embryos were successfully induced on the direct

environment from the roots (Asaka et al., 1993); (Chang and Hsing, 1980),

leaves (Tirajoh et al., 1998), cotyledons (Choi and Soh, 1994), zygotes (Choi

and Soh, 1996) Research on ginseng embryogenesis in liquid medium was also

performed by Claire et al., (2000) Gorpenchenko et al., (2006) studied the

transfer of rolC gene by Agrobacterium rhizogenes bacteria to obtain callus types which are capable of developing asexual embryos Various methods to improve the incidence of Ginseng clonal embryos have also been studied such as

growing in liquid medium (Claire et al., 2000), using polyamine (Claire et al.,

2002) or osmotic treatment (Choi and Soh, 1997)

In Vietnam, Nguyen Ngoc Dung is one of the first researchers studying clonal propagation in Ngoc Linh ginseng by biological methods through

regeneration from callus (Nguyen Ngoc Dung, 1995) Duong Tan Nhut et al

(2010) also conducted the asexual propagation of Ngoc Linh ginseng through

the organ generation method In this study, procedures for in vitro propagation

and experimental planting at the nursery stage were presented After 6 months,

1000 in vitro ginseng planted outside the nursery area showed faster growth rate

than that of Ngoc Linh ginseng grown with seeds with the survival rate of 87%

In the research framework of the project "Research on building a molecular indicator for inspection, exploitation and development of Ginseng

Ngoc Linh (Panax vietnamensis)", the research team was completed the in vitro

propagation process for Ngoc Linh ginseng from the stage of entering the callus model to a complete seedling (Le Hung Linh, 2019)

2.5 Researches optimize the growth and development process of Ngoc Linh ginseng

2.5.1 The condition of planting and caring for Ngoc Linh ginseng

Ginseng is a very slow-growing, perennial and shade-loving plant In our

canopy of deciduous plants and deeply underground roots such as birch or oak The best soil condition for growing ginseng that has highly humidity, has been the ability to well-drain water Ginseng usually thrives alongside woody herbaceous plants such as ferns Unless the ginseng grow, the woody herbaceous plants will not thrive in over levels Therefore marshy soils or clay soils are not suitable because another basic condition is that soils must be well-drained Ginseng seeds can be sown from late summer or in spring at a depth about 1-3

cm and a part of 15-30 cm About 70 - 90 kg of seed is required per hectare and this area should be covered with a layer mulch 5 - 8 cm of leaf humus Using seed has many advantages such as cheaper, less risk as pests and diseases, the development of plants will have the typical characteristics of wild ginseng, higher prices However, seeds can be sown only under ideal moist soil conditions, low germination rate and plants may germinate after 2 years, so it can take up 6-10 years to get commercial products In nature, ginseng is less affected by pests and diseases, but growing ginseng stimulated pests and

diseases Ginseng suffers from major diseases: fungi (mycorrhiza fungus and foliar fungus are common) The best way to prevent pests and diseases is to

improve health plants through a careful selection of soil areas, irrigation and ventilation However, some plant protection drugs are still recommended to the appropriate levels in each case of necessity In general, in terms of food and pharmaceutical safety, ginseng cultivation has no harmful effects on the environment, ginseng products ensure food safety and hygiene

2.5.2 Research on growing medium Ngoc Linh ginseng

In vitro seedlings are plants that are cultured and propagated completely

in artificial media that are adjusted to the optimal living condition for plant growth and development Seedlings need to be trained to increase survival rate and adaptability in the natural environment before taking out the nursery Therefore, the roles of substrate is very important The substrate is a material

that helps create a favorable environment for the plant to grow The substrate is usually mixed from other materials such as compost (bark, green waste), coir, wood fiber, moss, peat or mineral ingredients such as vermiculite, clay, pumice and perlite to achieve equilibrium air and water holding capacity for plants

Humus is the last remaining product of the natural decomposition of organic matter The role of humus is so important which has affected the growth and development of plants, helping to increase the fertility of the soil, and improving the soil environment is more quality Besides, humus works to improve soil texture help the soil retain water, keep nutrients better Humus helps provide nutritional elements N, P, K, Ca, Mg, and trace elements for plants

to use Soil rich in humus, plants still grow and develop well without the use of chemical fertilizers Humus has slow mineralization, but often forms inorganic substances in the form of usable plants, so it still ensures plant nutrition In humus-rich soil, plants is still growing and developing well without the use of chemical fertilizers Humus has slow mineralization, but it often forms inorganic substances in the form of usable plants, so it ensures nutritious for plants

Perlite is a unique volcanic mineral When expanded, each granular, snow white particle Perlite is sterile with a neutral pH and contains many tiny, closed cells or bubbles The surface of each particle is covered with tiny cavities, which provide an extremely large surface area These surfaces hold moisture and nutrients and make them available to plant roots In addition, because of the physical shape of each particle, air passages are formed which provide optimum aeration and drainage In addition, Perlite stone helps keep nutrients for plants, just enough moisture for plants Based on mixing Perlite stone with the soil that have been ability to loosen and nourish the soil, not compacted, and prevent the

Vermiculite is a natural mineral that is used in many different fields from construction to horticulture They are mainly made up of silicate iron - aluminum - magnesium Vermiculite rock have many sizes after the soil and impurities have been removed Vermiculite rocks help improve soil structure through soil aeration efficiency Air can be easily to circulate in the soil, increase the ability of plants to retain water and nutrients

Peat moss is a dead-fiber material that is formed when moss and other living materials decompose in swamps or peat mines This peat was discovered

in the 1900s, and is used in tree planting Its main component is moss Peat moss has the ability to keep water and nutrients effectively In addition, it improves soil texture and consistency

Organic fertilizers are fertilizers that contain multi, medium, and micro nutrients in the form of organic compounds, used in agricultural production Its fertilizers have origin, having formed from manure, waste cattle, poultry, stem residues, leaves, agricultural by-products, peat or organic substances from domestic waste, kitchen, aquatic and seafood factories, etc When using fertilizing, organic fertilizers help improve the soil, increase fertility and loosen the soil through supplying and applying humus, OM, microorganisms to the soil and plants

Following author Northen (1974) said that the substrate consists of pureed pine bark, sand (or 8 parts pureed asminda), peat with ratio 3:1:1 or 3:8:1 which

is suitable for transplanting orchid seedlings in vitro This substrate gives a high survival rate of orchid seedlings in vitro and plants have good growth and

development

Author Masstalerz (1997) said that in the United States often use the treatment with components including: clay humus, clay loam and catcos that followed mixed ratio (calculated by volume) 1: 2: 2, 1 : 1 : 1 or 1: 2: 0, used as a

pot for seedlings They have high survival rates and good growth and development

Ha Thi Thuy et al (2005) has experimented with growing lily tubers in vitro on different types of substrates This substrate includes sand, harmonious

humus at the ratio of 1: 1 and Pumice stone with rice husks at the ratio of 2: 1 The authors concluded that Pumice stone with rice husk at the ratio of 2: 1 is the best substrate On this substrate, the tubers germinate evenly, strongly, have good quality and high survival rate

In general, the study of planting medium is not popular, it is only used mainly for ornamental plants and Bonsai trees There have been a number of Institutes conducting research on suitable substrates for growing orchids, chrysanthemums, and carnations, but it has just stopped at the experimental stage that has not been widely put into production The specific reason is that this is a new field, the second is due to the complex composition of the substrates, unable to create the substrate

PART 3 CONTENT AND METHOD OF RESEARCH

3.1 Research materials

- Seedling: Ngoc Linh ginseng in vitro seedling from 1 to 2 years old has

1-3 leaves Seedling is tubers and pyramid-shaped with main shoots and nodes around the stem (or tubers have many buds and nodes, with silk roots 1-2 cm long) (Figure 3.1)

Figure 3 1: Ngoc Linh ginseng in vitro seedling

- Preparation of substrate: humus, perlite rock, vermiculite mineral organic fertilizers and peat moss

Humus comes in many varieties from many different origins In this study, the humus used is one of humus which is formed under the forest canopy including mulch and coarse humus in Sapa The humus has a near neutral pH,

the main composition of which are humic acids, fulvic acids and unfinished

organic compounds In the humus, there are many residues of decomposing plants, so humus has high ventilation and good drainage ability

In this experiment, perlite rock and vermiculite minerals were used in the products of Namix company Perlite is a unique volcanic mineral and rich silic for using in gardens Perlite has a very good aeration effect for the soil, good drainage ability for plants Besides, Vermiculite is a group of minerals (iron-magnesium-aluminum) which are mined from rock Vermiculite helps to improve soil structure through efficient soil aeration and the development of roots quickly, which increases the ability of plants to retain water, nutrients and germination rate

Figure 3 2: Perlite rock and Vermiculite of Namix company

There are many different types of organic fertilizers and are suitable for each plant In the experiment, the organic fertilizer are used to vermi-compost

and impact as well as their ability to retain water and make the soil loose and retain moisture for a long time In vermi-compost, it has a pH = 7, so it makes the root zone active in a neutral environment, helping to increase the plant's nutrient absorption Vermi-compost contains Humid Acid that absorbs in the state easily, helps to stimulate the growth of plants as well as beneficial microorganisms in the soil Besides, vermi-nutri vermi-compost reduces the carbon acid content in the soil and increases the nitrogen concentration that helpls plants absorbs easily and effective

- Biological products: AT Chibozin provides organic nutrition through foliar to ginseng plants which provides nutrients to help plants development, increase the growth of stems, branches, leaves and tubers Beside,

AT Chibozin not only helps to increase resistance but also helps plants withstand extreme weather

3.2 Place and time of the research

Research period: from August 1, 2020 to January 31, 2021

Location: Department of Molecular Biology - Institute of Agricultural Genetics; Experimental station in Van Giang - Hung Yen; Experimental station

Content 2: Research on the effect of the substrate on the adaptive capacity of

in vitro seedlings in the nursery

CT1: 100% of forest humus

CT2: Forest humus and perlite rock mixed in the ratio 1: 3

CT3: Forest humus,perlite rock and vermiculite mineral mixed in the ratio

of 2: 1: 3;

CT4: Forest humus, organic fertilizer (vermi-compost), and peat moss mixed in the ratio of 2: 1: 1

Methods of optimizing the substrate: Place 1: In laboratory conditions

with temperature 200C, humidity 65%, light intensity 30% (15 µmol.m -2 .s -1) Place 2: In net house condition in Experimental station in Sa Pa with temperature 18-200C, humidity 60-70%, coverage 70% Place 3: In nursery conditions with temperature 16 - 180C, humidity 65 – 75%, coverage 70%

Healthy seedlings about 1.5 years old with micro tubers and silk roots, uniform plant height of about 5 cm are grown in pots containing different nutrient media Total number of experimental plants: 1place x 10 plants / CT: 3 places x 4 treatments x 3 replicates = 360 plants Monitoring and evaluation viability and growth indicators (height plant) after 60 days of planting

Content 3: Research on the effect of plant age on the adaptive capacity of in vitro seedlings in the nursery

CT1: 1 year old in vitro seedling

CT2: 1,5 years old in vitro seedling

CT3: 2 years old in vitro seedling

Methods: Seedlings studied in the experiment were taken from previous

studies with the age from 1-2 years old Experimental plants with good vitality, uniform height of plants of age were planted in pots containing nutrient substrates (the substrate was a mixture of forest humus soil, vermiculite mineral and perlite rock mixed in the ratio of 2: 3: 1) and place planting on beds in

evaluation viability and growth indicators (height plant ) after 60 days of

planting

Content 4: Research on the effect of time to spray AT Chibozin nutritional product on the growth of seedlings in vitro of the nursery

CT1: Spraying at 20 days after planting

CT2: Spraying at 35 days after planting

CT3: Spraying at 50 days after planting

Methods: The seedlings studied in the experiment are from 1.5 years old

Experimental plants with good vitality, uniform height of plants of age were planted in pots containing nutrient substrates (the substrate was a mixture of forest humus soil, vermiculite mineral and perlite rock mixed in the ratio of 2: 3: 1) and place planting on beds in nurseries with the same environmental conditions Total number of experimental plants: 10 plants / CT: 3 treatments x

3 replicates = 90 plants Monitoring and evaluation of viability and growth indicators (plant height) 40 days after spraying with nutritional products

3.4 Experimental parameters

- Survival rate (% unit)

- Growth characteristics of in vitro seedlings: height plants (cm)

- Parameters of the diseases: Survey pests and diseases (aphids, death of seedlings, root rot, rust, etc.) in the nursery

- Parameter the occurrence and development of pests and diseases to have timely control plans

3.5 Data processing methods

The data was measured, counted, collected and then analyzed using Excel

and IRRISTAT 4.0 biological statistical analysis software

PART 4 RESULTS AND DISCUSSION

4.1 Research on standardization of in vitro seedlings Ngoc Linh ginseng

In order to standardize the traits of a good seedling, the morphology of seedling is an important issue because it allows us to find the morphological characteristics of a seedling that increases the viability and growth of a seedling

Ngoc Linh ginseng in vitro has many different morphologies, through monitoring, four basic morphologies of the tissue culture of Ngoc Linh ginseng stem have been recorded The characteristics and abilities survival and development of each morphology of the tuber are varied

Morphology 1: Ngoc Linh ginseng tubers are in a friability, have not split

on the tubers: tubers are characterized by connective tissues but they are not closely linked together Besides, it forms "fake bulbs" that are in a soft and spongy state