Nghiên cứu đặc điểm sinh học và kỹ thuật nhân giống phục vụ bảo tồn hai loài lan nghệ tâm (dendrobium loddigesii rolfe), hạc vỹ (dendrobium aphyllum (roxb ) fisher) của việt nam tt tiếng anh

AND TRAINING RURAL DEVELOPMENTVIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN THI LAI STUDY ON BIOLOGICAL CHARACTERISTICS AND PROPAGATION TECHNOLOGY USED FOR PRESEVATION OF Dendrobium lo

AND TRAINING RURAL DEVELOPMENT

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

NGUYEN THI LAI

STUDY ON BIOLOGICAL CHARACTERISTICS AND

PROPAGATION TECHNOLOGY USED FOR

PRESEVATION OF (Dendrobium loddigesii Rolfe, Dendrobium aphyllum (Roxb.) Fisher) VIETNAM

1 Prof Dr Vu Manh Hai

2 Dr Pham Huong Son

HA NOI - 2019

VIETNAM ACADEMY OF AGRICULTURAL SCIENCES

Scientific supervisors:

1 Prof Dr Vu Manh Hai

2 Dr Pham Huong Son Advocate 1:

Advocate 2:

Advocate 3:

The thesis will be defended before Council for thesis assessment at Institutional level held at Vietnam Academy of Agricultural Sciences at

…… hour ……minute, day …… month…….year

This Thesis can be referred to at:

1 National Library

2 Library of Vietnam Academy of Agricultural Sciences

3 Library of………

1 Necesity of the thesis to be studied

Dendrobium loddigesii Rolfe and Dendrobium aphyllum (Roxb Fisher belonging Dendrobium genus are considered as pretty forest orchid species

with high value of medicine and commerce As mentioned in the documents

of traditional Chinese medicine, D loddigesii Rolfe can be used as a blood

anti-freezer and in protection of some cancer diseaseas orcured in human

organs like stomatch, lung, (Tsai et al., 2010); prospate, oesophagus…and in treatment of diabetes (Veronika Cakova et al., 2017); skin color whitening (Ho Kyung Jung et al., 2015) D.aphyllum (Roxb.) Fisher takes an important

role in treatment of some diseases like cough, throatsore, flame burning, foodpoisoning etc….(Vietnam red book, 2007)

As a matter of fact, D loddigesii and D.aphyllum orchids have been

seriously exploited in recent years for ornamental decoration and medicine aswell resulting in strongly germplasm erosion In addition, the germinationability of these orchids in natural condition is very low whereas itsdistribution is quite limited, its availability, is therefore, met a lot ofdifficultities in terms of areas existed and individual quantity as well.Because of that, the study on multiplication technology targeted firstly forpreservation and development of these orchid species takes an very

important role in which in vitro propagation and synthetic seed create

techniques aimed to prolong the seed conservation duration should be paidgreat attention to This direction is regarded as an effective way not only for

D loddigesii and D.aphyllum orchids but also for other rarely precious ones

as well With the aim of diversification of Vietnam valuable medicineresources, partly improvement of vital environment and orchid producers

living standard, a sicientific thesis entitled “Study on biological characteristics and propagation technology used for preservation

(Dendrobium loddigesii Rolfe, Dendrobium aphyllum (Roxb.) Fisher)

Vietnam orchids” was proposed for implementation.

2 Objectives of the thesis

To evaluate the fundamental biological characteristics of D loddigesii and D aphyllum orchid species and in vitro propagation techniques aimed to

its reservation for development of highly valuable medicinal and ornamentalflowers in Vietnam

3 Scientific and pratical significance of the

thesis - Scientific significance

Valuable scientific data on the multiplication technology based on

biological characteristics of D loddigesii and D aphyllum orchids should

be obtained from the results of the thesis and from which suitablereservation, exploitation for its development in Vietnam condition will beaccordingly proposed

Results conducted from the systematized study on agro-biological and

medicinal chracteristics and synthetic seed production techniques of D loddigesii and D aphyllum orchids will be of valuable information

considered as the fundamental basic for further studies related andsignificant materials used in universities, colleagues and concerned trainingprograms as well

- Practical significance

Results obtained from the study actively contributed in the reservation

of rarely precious orchid species named D loddigesii and D aphyllum

presented by abundant germplasm with over all information collected fromvarious locations in Vietnam

With the in vitro multiplication techniques resulted from the study, a big quantity of disease - free, healthy plantlets and synthetic seeds of D loddigesii and D aphyllum orchids have been produced with high ratio of

propagation to be supplied to large scale of production aimed to medicinaland cosmetic production for highly valued domestic consumption andoriented exportation as well

4 Newly proposed contribution of the thesis

-Thesis is a synthesized scientic study on morphologycal characteristics,micro strucrure and bio chemistrical ingradients of valuable medicinal and

ornamental orchid precies named D loddigesii and D aphyllum.

- In vitro propagation technical procedures of precious D loddigesii and

D aphyllum orchids have been established that can be easily and effectively

applied in the condition of Vietnam because of material availablity

- Techinical procedures of synthetic seed production and in vitro post

preserved synthetic seed multiplication of these orchid species haveprolonged the conservation duration with low cost, high percentage of itsgermination (68% to 70%) without variety character change

- Fundamental techniques used in in vitro and in nursery propagation of

D loddigesii and D aphyllum orchids have been sucessfuly dertermined,

from which completely technological procedure used for large scalepropagation shoulde be established

5 Structure of the thesis

The thesis consists of 130 pages uncounted the references and appendix

36 tables, 36 pictures and figures, 169 scientific works published in Vietnamand outsides cited were involved in the document Thesis content was

devided into diiferent parts : introduction (3 pages); Chapter 1: Over alldocumentation concerned and scientific basic of the thesis (34 pages);Chapter 2: Material, content and methods of the study (14 pages); Chapter 3:Results and discussion (72 pages); Concusions and proposals (2 pages);References (16 pages) and appendix (25 pages).

CHAPTER 1 OVER ALL DOCUMENTATION

1.1 Origin and distribution of D loddigesii and D aphyllum orchid

species in Vietnam

Dendrobium genus was nominated in 1799, Dendrobium word was understood as on trees growned orchid and was called “Dendrobium” by

Vietnamese (Tran Duy Quy, 2005)

In Vietnam, Dendrobium genus is very diversiform with specific distribution D loddigesii orchid was available in Thai Nguyen, Ha Giang, Lai Chau and Nghe An provinces (Averyanov et al., 2005) whereas D aphyllum specy has been found in the provinces of Lam Dong, Khanh Hoa, Lao Cai, Bac Kan, Ninh Thuan (Averyanov et al., 2005; Vietnam red book,

2007)

1.2 Main botanical characteristics of D loddigesii and D aphyllum

orchids

Bảng 1.1 Main botanical characteristics of the orchid species studied

Subordinated living, small Subordinated living with thinhshrubs with smooth and bended

Stems stems of 10 - 20 cm long and and bended stems of about

100 cm long (Tran Hop,long internodes (Tran Hop, 1998).

1998)

Oblong leaves of 4 - 6 cm long; Long handled machete leaves

of 6 - 8 cm long, 1,5 - 2 cmLeaves 1 - 2 cm wide and easily droped wide (Vietnam red book

(Tran Hop, 1998) 2007).

Clusters of 1-2 flowers of dark Clusters of 1 – 3 flowers ofpink color with dark pots light purple color generatedlocated in leaf sides Petals are from leafless stems PetalsFlowers round with smooth hairs in its with three veins are light

edges and yellow colored yellow color Floweringcentre Flowering season: April season: April - May (Tran

- August (Tran Hop, 1998; Zhu Hop, 1998; Vietnam red book,

Guanghua et al., 2009). 2007)

1.3 Utilised value and exploitation status of D loddigesii and D aphyllum orchids in Vietnam

As reported by Chinese traditional medicine, D loddigesii can be used

as a blood anti-freezer and in protection of some cancer diseaseas orcured in

human organs like stomatch, lung, (Tsai et al., 2010); prospate, oesophagus…and in treatment of diabetes (Veronika Cakova et al., 2017); skin color whitening (Ho Kyung Jung et al., 2015) D aphyllum takes an important role in degest improvement (Zhao Yong Ling et al., 1994) and

treatment of some diseases like cough, throatsore, flame burning, foodpoisoning etc….(Vietnam red book, 2007)

Since D loddigesii and D aphyllum orchids can be used as ornamental

and medicinal crops with highly economic value, they have been seriouslyexploited in recent years resulting in very high risk of germplasm erosion inVietnam

1.4 Production and consumption situation of Dendrobium in Vietnam

and over the world

For orchid generally, pot and cut flowers have been considered as themain concern in global markets that is estimated of about 10% incomparision with total flower quantity consumed resulting in 438 mil USDannually in 2007 – 2012 period (Jayarama Reddy, 2016)

Netherland ranks the first country over the world in orchid productionand exportation (39.7%), followed by Thailand (28.4%); Taiwan (10%),Singapore (10%) and New Zealand (6%) Main import countries are Japan(30%), United Kingdom (12%), Italy (10%), France (7%) and United States

of American (6%) Of cut flower orchids exchanged in the world markets,

Dendrobium (85%) and Phalaenopsis, Cymbidium ( 15%) are considered to

be principal ones (Cheamuangphan et al, 2013; De et al., 2014) In Netherland, the production of pot Dendrobium has increased significantly a

quantity of about 40 to 50 mil of orchid individuals produced annually was

reported (De et al., 2014).

In Vietnam, the area under orchid cultivation has been limitedaccounting approximately 10% of total flower cultivated area Presentproduction of orchid flowers in Vietnam is implemented in two orientations:The industrial production is applied for newly released orchid varieties orintroduced ones whereas utilization and exploitation of indeginous orchids isprioritized for available cultivars mainly taken from the forests

It is also obvious that orchid production in Vietnam does not meet thedemand of domestic consumtion, only 30 - 40% orchid quantity required isproduced in country and the rest has to be imported from outsides

1.5 Study on biology and multiplication situation of Dendrobium in

Vietnam and over the world

1.5.1 Study on biology of Dendrobium

Study on leaf and pseudobulbs morphological and physiological

characteristics of 4 orchid species D chrysotoxum, D officinale D crystallinum and D crystallinum in the relation with maintaining water balance was reported by Shi-Jian Yang et al., (2016) whereas Metusala et al., (2017) focused on micro morphological structure of stem and leaf of D capra and D Arcuatum in different environments related to its adaptability

in draught condition

As reported by Chinese traditional medicine, D loddigesii can be used

as a blood anti-freezer and in protection of some cancer diseaseas orcured in

human organs like stomatch, lung (Tsai et al., 2010), treatment of diabete type 2 (Zhang et al., 2011) Results conducted from bio-chemical studies showed that some useful substances were availabe in D loddigesii orchid

such as shihunine, 9,10 dihydrophenanthrene 2,4,7 triol, moscatin,

loddigesiinols C, moscatilin, gigantol and tristin (Li Chunyan et al., 2013).

Ho Kyung Jung et al., (2015) found that chemicals extracted from D loddigesii orchid inhibited the activity of melanin and tyrosinase but

promoted the development of dendrite resuting in skin whitening

Veronika Cakova et al., (2017) reported that moscatilin available in D loddigesii can be used for prevention the development of some cancer

diseases orcured in human organs like stomatch, lung, prospate,oesophagus…while Loddigessinol G-J, Crepidatuol B are useful in diabetetreatment In addition, polysaccharides and phenol included sustances such

as moscatin, moscatilin and tricetin 3 ', 4', 5'-trimetyl ete

7-O--glucopyranoside in stem of D aphyllum orchid can inhibit the production of

NO, that is of good help for immne system

In the other hands, anti oxidized substances extracted from D aphyllum

orchid such as superoxidedismutase, catalase and glutathione peroxidase arehelpful for human health whereas amino acids like Ala, Val, Ile, Leu, Tyr,Phe, Try, Pro, Met and Cys are also considered as highly anti oxydisers with

good nutrions (Huifan Liu et al., 2017).

In Vietnam, almost scientific works recently have focussed on the

morphologies of Dendrobium orchid botanical organs, viz, stem, leaf, root

system etc…(Tran Hop, 1998; Vietnam red book, 2007; Duong Duc Huyen,2007), whereas the studies on biological micro structure and botanicallassification based on morphological markers were still limited

Cao Phi Bang et al., (2017) implemented a study on the growth, anatomy and transpiration characteristics of micropropagated D anosmum

Lindl during ex vitro acclimatization process Nguyen Thi My Duyen et al., (2012) studied on morphological based classification of Dendrobium and from which three new species namely D pulchellum, D gatton Sunray, D moschatum was nominated These species were closely relative with genetic

coefficiences of 96.5% and 95.0% respectively In a study done by Tran DuyDuong (2015), the morphological index was combined with molecular

markers in polymorphic evaluation of 32 Vietnam indigenous Dendrobium accessions including higly valued medicinal species such as D jenkinsii, D lindleyi, D aphyllum…

It seems, however, no study on bio-chemical composition of

Dendrobium orchid has been reported in Vietnam up to now.

1.5.2 Study on propagation of Dendrobium over the world1.5.2.1 Study on in vitro propagation

Various scientific works on in vitro propagation have been done over the world Dake Zhao et al., (2013) had a research into multiplication and in tube flower formation of D wangliangii Xin Qian et al., (2014)

implemented a study on in vitro flowering and fruiting in culture of D officinale Calluses were induced from shoot-tip explants of D officinale on

MS medium with 0.2 mg/l BA and 0.05 mg/l αNAA Multiple shoots hadNAA Multiple shoots had

been regenerated after protocorm like bodies were transferred onto the same

basal medium with 1.0 mg/l αNAA Multiple shoots hadNAA The plantlets, 2 - 4 cm high, maintained

in vitro were induced to flower, and the highest rates of inflorescence

(83.2%) and normal flowers (73.6%) were produced on the MS mediumwith 15% coconut water and 0.1 mg/l TDZ within 9 weeks

Mohammad Musharof Hossain et al., (2013) studied on multiplication

of D aphyllum orchid and found that Phytamax (Sigma) + 1 mg/l BA

medium was helpful in seed germination whereas Phytamax + 1 mg/l BA +

1 mg/l αNAA Multiple shoots hadNAA was considered to be useful for protocorm multiplication In case of D jerdonianum Wight, Sagaya Mary et al., (2016) mentioned that

MS medium was suitable for flower stalk culture while KC medium wasfound to be suitable for nodal segments and VW medium was found to besuitable for nodal segments

Tapash Kumar Bhowmik et al., (2017) stated: Phytamax (PM) medium

supplemented with 20 g/l sucrose gave good effect for seed germination of

D palpebra, better than PM medium supplemented with glucose or fructose Thejaswini et al., (2017) reported that mashed tomato and coconut water 15% were regarded the best for in vitro seed germination of D ovatum (Willd.) whereas Edy Setiti WidaUtami et al., (2017) concluded that VW

medium supplemented with 2 g/l peptone medium was suitable for seed

germination, shoot formation and protocorm development.

In addition, the studies on orchid propagation related to thin cell layer(TCL) had been also implemented and applied in some orchid species such

as D chrysotoxum (t-TCL of 2 mm thick, done by Saranjeet Kaur, (2017); Paromik Bhattacharyya et al., (2018); D aphyllum (t-TCL done by; Parthibhan et al., (2018) and D aqueum Lindley (t-TCL originated somatic

embryo regeneration done by some researchers)

In Vietnam, some studies on orchid propagation have been recently

concerned, eg a study on orchid multiplication from t-TCL of in vitro stem

of D aduncum implemented by Nguyen Thanh Tung et al., (2010), studies

on orchid multiplication from in vitro seed of D nobile Lindl (Vu Ngoc Lan

et al., 2013), D officinale (Nguyen Thi Son et al., 2014), D lituiflorum Lindley (Nguyen Van Viet, 2017), D transparens (La Viet Hong et al., 2017), D officinale (Le Thi Diem and Vo Thi Bach Mai, 2017),…

1.5.2.2 Study on synthetic seed formation of orchids

Up to now, protocorm and PLBs have been mainly used in the studies

on synthetic seed formation of orchids (Bunnag et al., 2010; Zhang et al.,

2011; Mohanty et al., 2013; Bustam Suryanti et al., 2013; Sukhumpinij et al., 2015; Nhutet al., 2005; 2007; Tran Thi Ngoc Lan, 2013).

1.5.2.3 Study on the growth and development of orchid plants generated from preserved or non preserved synthetic seeds.

Using RAPD technique for polymorphic evaluation of Cineraria maritana, Srivastava et al., (2009) reported that there was no genetic

difference between plantlets generated from non preserved seeds and 6months preserved ones The same result was also reported by Mishra et al.,

(2011) in Picrorhiza kurrooa (3 months preserved seeds) and Mohanty et al., (2013) inD densiflorum(60 days preserved seeds)

1.5.2.4 Study on cultivation of Dendrobium

Mohammad Musharof Hossain et al., (2013) stated that mixture of brickpieces, charcoal pieces, coco peat and vermiculite at 1 : 1 : 0.5 : 0.5 ratio

was considered suitable for plantlet cultivation of D aphyllum while Winarto et al., (2015) recommended Cycas numphii used in the first 2 months and mixture

of wood charcoal and C rumphii bulk at 1: 1 ratio used in the next duration were favourable for D ‘Zahra FR 62’ and D ‘Gradita 31’ It was also mentioned that

plantlets could be grow better (90 – 100% plant survived) by 50% net covering

to keep light intensity down to 100-150 μmolmol⋅m-2⋅s-1 and relative moisture at85% – 95%

Selvaraju Parthibhan et al., (2015) showed that high ratio of survived

plants of D aqueum was obtained when grown in brick pieces and charcoal

medium at 1 : 1 ratio covered with moss and polyethylene bags Abdul AzizMirani et al., (2017) studied on D nobile, and concluded that 100% planlets

would be survival at 120 days after growing when grown in MS rooting medium involved αNAA Multiple shoots hadNAA at 3 mg/l and then changed to mixture of coco- peat: desert sand: perlite at 1,0 : 1.0 : 0.2 ratio

Apart from fertilizers, studies on the utilization of biological substances

and growth regulators to promote the growth of Dendrobium were also carried out For D cv sonia -17 grown in green house, Bhatt et al., (2012)

reported that GA3 sprayed at 15 mg/l produced significantly maximum number of shoot, shoot length and number of flowers per plant BA at 15mg/l was found beneficial for number of leaves per plant in both first and second month after.

As a matter of fact, the study on cultivating technology of Dendrobium in Vietnam was still limited Pham Thi Lien et al., (2010) proposed a procedure

of cultivation used for Dendrobium grown in the North of Vietnam in which

the utilization of mixture of charcoal and coconut fibre at 1 : 1 ratio andslowly diluted N, P, K fertilizer compound at 20 : 20 : 20 ratio combinedwith Growmore foliar was paid great attention to Vu Ngoc Lan (2012)reported that powder of coconut fiber medium was suitable for the growth of

D.nobile and D.chrysanthum and spraying Komix 2ml/l at 7 – 10 days intervals should be effectively applied when in vitro planlets were

transferred to nursery from laboratory tubes for 1 month on Hoang Xuan

Lam (2014) recommended for D cherry red, April was suitable time to transplant in vitro plants to nursery while mixture of charcoal : volcano

growth; It was also recommended that the utilization of foliar Growmore(N:P:K = 30:10:10) and growth regulator Atonik 1,8 SL (0,1%) should be

effectively applied Nguyen The Cuong et al., (2018) stated that in oder to promote the growth of D.officinal the sensity of 44 plant per square metter

and foliar Growmore application should be paid great attention andreccommended

CHAPTER 2 MATERIALS CONTENTS AND METHODS OF STUDY 2.1 Materials

- Orchid accessions used in evaluation of morphological, micro operation

and bio-chemical analyse were D loddigesii orchid collected and screened in Dinh Hoa – Thai Nguyen province and D aphyllum ones in

Hon Ba – Khanh Hoa province

- Materials used in tissue culture were taken from bud tips of D loddigesii and D aphyllum orchids with 7 - 10 cm high.

- Materials used in somatic embtyo creation were PLBs (protocorm like bodies) formed from growth peak culture At 6 weeks after cultured,

unique PLBs were cut into pieces of 3 – 4 mm long for synthetic seedformation

- In nursery period: in vitro plantlets multiplied from synthetic seeds of 5 –

6 cm high with 4 – 6 leaves and 3 – 5 roots

- In production gardens: Plants of 6 – 7 cm high with 6 -7 leaves and 3 – 4 newly formed roots

- Organic Juices extracted from tomato, pumpkin…

- Materials used in in vitro rapid multiplication: saccaroza, charcoal

- Media materials: Sphagnum moss, Pumice Stone, pine bark, coconut fibreand “Don bang”…

- Nutritional sustances: Orchid Focus grow, Seaweed 95%, Striving buffalo

502, B1 Thai Lan, Growmore

- Growth regulator: Superthrive

- Study on cultivating technology for in vitro plants of D loddigesii and D aphyllum orchids in nursery period.

- Study on cultivating technology for young plants of D loddigesii and D aphyllum orchids in production gardens

2.3 Research Methodology and experimental design

* Methods used in micro structure evaluation of D loddigesii and D aphyllum orchids

- Study on micro structural characteristics of root, stem and leaf was based

on improved botanical evaluation proposed by Nguyen Nghia Thin (2007)

* Metods used in fundamental bio-chemical analyse of D loddigesii and

D aphyllum orchids

- Total alkaloid content determination was based on acid - base method presented in (Vietnam Pharmacy IV, 2009)

- Total flavonoid content determination was based on UV-VIS method

(Mudasir Sultana et al., 2012).

- Total polysaccharids content determination was based on UV-VIS method (Chinese Pharmacy, 2010)

- Mineral (Ca, Mg, Fe) content determination was based on atomic absorb spectrum (AAS)

2.3.2 Experimental design

- Experiment 1: Study on the effect of NaOCl and sterilized duration to

accession situation

- Experiment 2: Study on the effect of disinfectant cefotaxime to survival

ratio of explants cultured

- Experiment 3: Study on the effect of BA to PLBs formed from traverse

thin cell layers (tTCL)

- Experiment 4: Study on the effect of BA and αNAA Multiple shoots hadNAA compound to PLBs

formed from traverse thin cell layers (tTCL)

- Experiment 5: Study on the effect of combination (BA + IBA) on

regeneration of shoots from PLBs

- Experiment 6: Study on the effect of mashed pumpkin on regeneration

of shoots from PLBs

- Experiment 7: Study on the effect of Spirulina algae on regeneration of

shoots from PLBs

- Experiment 8: Study on the effect of IBA to root formation of the shoots

- Experiment 9: Study on the effect of PAA to root formation of the

shoots

- Experiment 10: Study on the effect of sodium alginate concentration (%)

and CaCl2.2H2O (mM) to the formation and germination of synthetic seeds

- Experiment 11: Study on the effect of contacting duration with

CaCl2.2H2O solution to synthetic seed germination

- Experiment 12: Study on the effect of BA and BA + IBA compound to

the germination and the growth of synthetic seeds

- Experiment 13: Study on the effect of temperature to stored duration of

synthetic seeds

- Experiment 14: Study on the effect of ABA to stored duration of

synthetic seeds

- Experiment 15: Study on the effect of natri benzoat, carbendazim and

topsin - M to the storage of synthetic seeds

- Experiment 16: Study on the effect of BA + PAA compound to rapid

multiplication of protocorm and shoots

- Experiment 17: Study on the effect of mashed tomato to rapid

multiplication of protocorm and shoots

- Experiment 18: Study on the effect of PAA to complete in vitro plant

formation after storage time

- Experiment 19: Growth comparision of in vitro plantlets multiplicated

from bud and from preserved synthetic seeds in nursery period

- Experiment 20: Study on the determination of suitable time for moving

in vitro plantlets to nursery

- Experiment 21: Study on the determination of suitable media for in vitro

plantlets in nursery period

- Experiment 22: Study on nutritional supply for in vitro planlets in

+ Root related criteria:

- Root number/quantity (a plant), root length (cm)

- Dissect criteria: bark dimension, zylem dimension, number and

dimension of vessel…

- Dimension of different tissues…

+ Leaf related criteria:

- Leaf shapes: oblong shape, tonge formed shape; sharp peak shape leaf lenth (cm), leaf width (cm)

- Micro dissect criteria: dimension of different tissues, numer and

dimension of vessel

+ Flower related criteria:

- Flower diametter (cm), length of flower shoot (cm), flower petal dimension (cm): dimensions of different flower organs…

- Micro dissect criteria: pollen, stage…