0 MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND 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 ORCHIDS Speciality: Crop science Code: 62 01 10 SUMMARY OF DOCTORIAT THESIS Scientific supervisers: Prof Dr Vu Manh Hai Dr Pham Huong Son HA NOI - 2019 Thesis completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Scientific supervisors: Prof Dr Vu Manh Hai 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: National Library Library of Vietnam Academy of Agricultural Sciences Library of……………………………………… INTRODUCTION 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, food poisoning 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 as well resulting in strongly germplasm erosion In addition, the germination ability of these orchids in natural condition is very low whereas its distribution is quite limited, its availability, is therefore, met a lot of difficultities in terms of areas existed and individual quantity as well Because of that, the study on multiplication technology targeted firstly for preservation 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 paid great 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 medicine resources, 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 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 ornamental flowers in Vietnam 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 suitable reservation, exploitation for its development in Vietnam condition will be accordingly 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 and significant materials used in universities, colleagues and concerned training programs 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 from various 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 medicinal and cosmetic production for highly valued domestic consumption and oriented exportation as well 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 have prolonged the conservation duration with low cost, high percentage of its germination (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 scale propagation shoulde be established 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 Vietnam and outsides cited were involved in the document Thesis content was devided into diiferent parts : introduction (3 pages); Chapter 1: Over all documentation 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 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 Characters Stems Leaves Flowers D loddigesii Subordinated living, small shrubs with smooth and bended stems of 10 - 20 cm long and long internodes (Tran Hop, 1998) Oblong leaves of - cm long; D aphyllum Subordinated living with thinh and bended stems of 100 cm long (Tran 1998) about Hop, Long handled machete leaves of - cm long, 1,5 - cm - cm wide and easily droped wide (Vietnam red book (Tran Hop, 1998) 2007) Clusters of 1-2 flowers of dark Clusters of – flowers of pink color with dark pots light purple color generated located in leaf sides Petals are from leafless stems Petals round with smooth hairs in its with three veins are light edges and yellow colored yellow color Flowering centre 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, food poisoning 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 seriously exploited in recent years resulting in very high risk of germplasm erosion in Vietnam 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 the main concern in global markets that is estimated of about 10% in comparision with total flower quantity consumed resulting in 438 mil USD annually in 2007 – 2012 period (Jayarama Reddy, 2016) Netherland ranks the first country over the world in orchid production and 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 limited accounting approximately 10% of total flower cultivated area Present production of orchid flowers in Vietnam is implemented in two orientations: The industrial production is applied for newly released orchid varieties or introduced ones whereas utilization and exploitation of indeginous orchids is prioritized for available cultivars mainly taken from the forests It is also obvious that orchid production in Vietnam does not meet the demand of domestic consumtion, only 30 - 40% orchid quantity required is produced 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 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 (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 diabete treatment In addition, polysaccharides and phenol included sustances such as moscatin, moscatilin and tricetin ', 4', 5'-trimetyl ete 7-O-βglucopyranoside 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 are helpful 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 botanical lassification 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 Duy Duong (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 world 1.5.2.1 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 had been regenerated after protocorm like bodies were transferred onto the same basal medium with 1.0 mg/l αNAA The plantlets, - 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 medium with 15% coconut water and 0.1 mg/l TDZ within weeks Mohammad Musharof Hossain et al., (2013) studied on multiplication of D aphyllum orchid and found that Phytamax (Sigma) + mg/l BA medium was helpful in seed germination whereas Phytamax + mg/l BA + mg/l αNAA 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 was found to be suitable for nodal segments and VW medium was found to be suitable 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 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 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; Nhut et 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 months 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) in D densiflorum (60 days preserved seeds) 1.5.2.4 Study on cultivation of Dendrobium Mohammad Musharof Hossain et al., (2013) stated that mixture of brick pieces, charcoal pieces, coco peat and vermiculite at : : 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 months and mixture of wood charcoal and C rumphii bulk at 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 μmol⋅m-2⋅s-1 and relative moisture at 85% – 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 : ratio covered with moss and polyethylene bags Abdul Aziz Mirani 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 at mg/l and then changed to mixture of cocopeat: 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 : ratio and slowly diluted N, P, K fertilizer compound at 20 : 20 : 20 ratio combined with 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 – 10 days intervals should be effectively applied when in vitro planlets were transferred to nursery from laboratory tubes for 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 foaming slag : seaweed at 2:2:1 ratio was considered favourable for plant 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 and reccommended CHAPTER MATERIALS CONTENTS AND METHODS OF STUDY 2.1 Materials - Orchid accessions used in evaluation of morphological, micro operation 11 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 nursery period - Experiment 23: Study on the determination of suitable media for plants in production gardens - Experiment 24: Study on nutritional regime for plants in production gardens - Experiment 25: Study on supplying Superthrive for plants in production gardens Criteria observed: + 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… 12 + Criteria observed in in vitro culture and synthetic seedcreation Infected explant ratio (%), survived plant ratio (%), number of PLBs/tTCL (PLBs) formed, ratio of PLBs formed (%), ratio of protocorm formed explant (%), multiplication coefficience of protocorm, shoot height (cm); number of leaf/shoot, germinated seed ratio (%), number of shoot/seed, shoot length (mm), rooted seed ratio (%), number of root/shoot + Growth and development criteria (nursery period and production garden) - Survived plant ratio (%), plant height (cm), plant stem diametter (cm), number of leaf/plant, leaf length (cm), leaf width (cm), number of twig/plant, average number of root/plant 2.4 Experimental conditions Sites and time duration of study - Studies on morphological characteristics of Dendrobium were carried out at the Centre for Experimental Biology, The Institute of Applied Technology in 2013 – 2014 period - Study on micro structure of Dendrobium was implemented at the laboratory of Biological Subject – Agronomy Faculty – Vietnam National University of Agriculture in 2014 – 2015 period - Fundamental bio-chemical composition of D loddigesii and D aphyllum orchids was analysed at the Laboratory of Standard Chemistry Faculty – The institute of Pharmacy – Minystry of Health in 2013 – 2014 period - Studies on propagation, synthetic seed creation and cultivating technology were carried out at the Plant tissue culture Laboratory and in green houses of Centre for Experimental Biology, National Center for Technological Progress – Ministry of Science and Technology in 2013 – 2017 period Conditions for tissue culture - Temperature at laboratory 25 ± 20C, relative humidity 60 - 70%, light duration 16 h/day, light intensity 1.500 - 2.300 lux Conditions for plant management - Young plants were planted in net houses covered by black colored net with > 70% protected light, temperature 25 ± 0C, atmosphere humidity 70 - 80%, wartering twice a day - Pest control: spray Antracol 70WP at 0,1% concentration at 10 days intervals 2.5 Data treatment method Biological statistics were used for data treatment with Excel and IRRISTAT 5.0 soft wears 13 CHAPTER RESULTS AND DISCUSSIONS 3.1 Biological characteristics of D loddigesii and D aphyllum 3.1.1 Morphological characteristics of D loddigesii and D aphyllum orchids Both D loddigesii and D aphyllum orchids have thin, filamentous and aerial roots with cylendar form, blue colour and turn brown with time Root system of these species when grown in pots develop closely surrounding media or bended downwards Its have good capacity or regeneration, water and nutrition exploitation from soil D loddigesii and D aphyllum have lots of long stems of many internodes that consist one sleep bud each Stems of D loddigesii orchid are thin, cylinder formed, bended with 40.2 cm long, 0.5 cm diametter whereas oblong, cylinder formed and bended stems of 70.4 cm long and 0.8 cm diameter were observed in D aphyllum orchid Leaves of D loddigesii orchid are wedge shaped and tounge formed with sharp peak, dark blue colour, 5.0 cm long, 1.5 cm width 17 leaves available in a stem was also observed D aphyllum orchid has sharp peak, look like leather with smooth edges Its leaves are light blue coloured, soft and thin, 7.62 cm long, 3.10 cm width with 32 leaves on a stem Flowers of D loddigesii orchid are single, purple whitenish with yellow colour in the centre – flowers was found on a leaf existed stem Flower stalks are light blue, 0.4 – 0.8 cm long Flowers of 5.2 cm diameter are covered with thin and light purple coloured slides Calyx petal are oblong with shrpless peak and side petals of 2.5 cm long and 0.7 cm width Corola petals are elip formed, 1.7 cm long and 1.0 cm width Petal curved edges are surrounded with white lines, yellow colour in the centre and white in the base, 1,9 cm long and 3.1 cm width Flowers of D loddigesii are fragrant, occurred in April to August period and existed for 30 – 35 days Flowere of D aphyllum orchid were formed along non leaf available stem in clusters of – flowers each Flower stalks are light purple coloured, 0.2 – 0.6 cm long Flowers are white, 4.8 cm diametter with purple coloured strips and covered with thin slides of light white colour Three calyx petals are the same in dimenssion in which two side ones are 3.6 cm long and 0.9 cm width Corola petals are oblong with almost sharp peak, 3.4 cm long and 3.6 cm width D aphyllum orchid flower in April to June period and flower can be available from 27 to 30 days 3.1.2 Micro structures of D loddigesii and D aphyllum orchid species Generally, dimensions of root and its organs such as bark, epidermis, incorrect endosperm … between D loddigesii and D aphyllum are not much 14 different The most significant difference in in the number of liber vessel Root of D loddigesii orchid consists of -7 liber vessels whereas – ones was found in case of D aphyllum This may be related to water and mineral absorbability from soil of plant, thus, D aphyllum orchid is considered better than D loddigesii in draught tolerance It is also mentioned that wartering to keep moisture constant was significantly recommended for D loddigesii cultivation The stem dimension of D aphyllum is 1.12 times higher with liber vessel is 1.5 times higher D loddigesii In D aphyllum liber vessels are located closely to epidermal layers whereas libers are concentrated in the middle of stem No much difference in big liber dimension between D loddigesii and D aphyllum orchids was found The abve mentioned characters proves that the resistance to unfavourable conditions of D aphyllum is a little bit better than D loddigesii orchid And, because of that, it is necessary to have good pergular for D loddigesii when grown in the gardens and tieing its shoots closely to strong supporters.… Leaves of D aphyllum orchid are thin and soft with large leaf blades whereas D loddigesii has hard and thick leaves, 2.5 times thicker than D aphyllum but the width of D loddigesii leaf blade is much lower than D aphyllum It is also found that the thickness of assimilative tissues of Nghe tam is 2.78 times thiker than D aphyllum whereas the same dimension of liber vessels located in main veins was also recorded Flowers of D loddigesii orchid are single ones, purplish white and yellow in the centre, sprouted on leaf existed stems covered by light purple coloured slides Calyx petals are oblong with sharpless peaks, side petals of 2.2 – 2.5 cm long and o.7 cm width are bigger than opposite ones (1.6 – 1.9 cm long; 0.7 – 0.8 cm width) Corola petals are oval, nearly round peaks, 1.6 – 1.7 cm long, 0.8 – 1.0 cm width) whereas lip petals have white colour in curved edges yellow in th ecentre, 1.7 – 1.9 cm long and 2.1 – 3.1 width diveded into lobes of yellow colour with smooth hairs in the centre Lip petals are closely placed with stamen at the bases State axil is short and semi cylinder formed, 0.7 – 0.8 long whereas stigma with three spaces is light green, 3.1 – 3.5 cm long Pollen with sharp cover is located at the peak and devided into blocks of pollen spaces each Flower of D aphyllum orchid is single one or cluster of – flowers, sprouted from non leaf existed stem with white colour and dark purple in the centre Three calyx petals have same dimension including side petals of 2.7 – 3.6 cm long; 0.7 – 0.9 width and opposite petal of 2.5 – 3.1 cm long; 0.6 – 0.7 width Corola petal is oblong with almost sharp peak, 2.7 – 3.1 cm long and 1.0 – 1.1 width whereas lip petal was observed as round shaped one 15 with long hairs in the edges, 3.1 – 3.7 cm long; 3.0 – 3.6 cm width and devided into lobes in which side lobes are purple and the middle one is whitenish covered with smooth hairs and white veins located in the centre Lip petals are closely placed with stamen axis at the base Stamen axis is short and semi cylinder shaped, 0.9 – 1.3 cm long whereas ovary of three spaces is light purple, 2.4 – 3.1 cm long Pollen of blocks of pollen spaces each with sharp cover is located at flower peaks 3.1.3 Fundamental bio-chemical compositions of D loddigesii and D aphyllum orchids Data presented in table 3.6 showed that: total alkaloids content of two species was quite same that accounted for 0.35% Total flavonoids contents were quite low, 0.92% in D loddigesii and 0.56% in D aphyllum whereas higher values were reported in total polysaccharides content: 1.42% in D loddigesii and 4.2% in D aphyllum, much higher than the contents of flavonoids and alkaloids The contents of protein, Ca, Mg Fe of these orchids are not much different 6%, 12030 mg/kg, 1726 mg/kg, 168 mg/kg respectively for D loddigesii and 6,6%, 14821 mg/kg, 2829 mg/kg, 134 mg/kg for D aphyllum orchid Table 3.6 Main bio-chemical compositions of D loddigesii and D aphyllum (The Institute of Pharmacy, 2013 - 2014) Contents of substances D loddigesii D aphyllum N Total Alkaloid (%) Total Flavonoid (%) Total Polysaccharid (%) Protein (%) Ca(mg/kg) Mg(mg/kg) Fe(mg/kg) 0.35 0.92 1.42 6.0 12030 1726 168 0.35 0.56 4.2 6.6 14821 2829 134 3.2 Study on in vitro propagation, synthetic seeds creation techniques and in vitro multiplication from preserved synthetic seeds of D loddigesii and D aphyllum for its conservation 3.2.1 Determination of sterilizers and sterilized duration of explants cultured Utilization of NaOCl at 3% concentration for 15 minutes and 1‰ cefotaxime gave good effect, highest ratios of survived samples were observed (79.9% for D loddigesii and 86.0% for D aphyllum) 16 3.2.2 Study on protocorm like bodies (PLBs) creation from traverse thin cell layer (tTCL) 3.2.2.1 Effect of BA to formation ability of PLBs from (tTCL) VW + 20 g/l sucrose + 10% coconut water + g/l agar medium supplemented 1,5 mg/l BA gave the best result in terms of number of PLBs created and percentage of tTCL that produced PLBs (30.12 PLBs/tTCL; 52.03% tTCL produced PLBs for D loddigesii and 28.71 PLBs/tTCL, 49.22% tTCL produced PLBs for D aphyllum at weeks after cultured 3.2.2.2 Effect of BA + αNAA compound to the formation of PLBs from tTCL Table 3.10 Effect of BA + αNAA compound to the formation of PLBs from tTCL (6 weeks after cultured) Phytohormore PLBs produced Number of concentration (mg/l) samples (%) PLBs/tTCL BA αNAA Dl Da Dl Da 1.5 0(Đ/C) 52 03 30.40 30.12 17.83 1.5 0.5 38.0 49.22 21.00 28.71 1.5 1.0 31.0 38.30 19.00 20.70 1.5 1.5 21.9 19.96 12.80 12.30 1.5 2.0 16.0 14.80 7.00 7.45 CV (%) 3.1 4.3 3.3 3.6 LSD0,05 7.6 1.93 0.4 0.19 Data in table 3.10 showed that: incomparision with VW supplemented BA medium, αNAA and BA compound spplied to medium used for D loddigesii had no effect to the formation of PLBs presented by unchangeable numbers of PLBs created and percentage of tTCL produced PLBsinh PLBs For D aphyllum orchid, the supply of 1,5 mg/l BA combined with 0,5 mg/l αNAA gave good effect, 49,22% samples produced PLBs and PLBs number regenerated from tTCL was 28,71, PLBs were healthy and green 3.2.3 Regeneration of shoots from protocorm like bodies (PLBs) 3.2.3.1 Effect of combination (BA + IBA) on regeneration of shoots from PLBs BA+ IBA compound supplied to medium promoted the formation of shoot produced from PLBs at weeks after cultured in which the compound of 1,5 mg/l BA + 0,5 mg/l IBA was regarded the best in terms of number of shoot and its height (6.8 shoots/a explant, 2.86 cm high for D loddigesii and 6.9 shoots/a explant, 2.90 cm high for D aphyllum) 3.2.3.2 Effect of mashed pumpkin on regeneration of shoots from PLBs 17 Supply of mashed pumpkin juice to medium promoted significantly shoot regeneration presented by increasing the number of shoot created In the medium of VW + 20 g/l sucrose + 10% coconut water + g/l agar +1 g/l activated charcoal + g/l peptone + 1,5 mg/l BA + 0,5 mg/l IBA added with 30 g/l mashed pumpkin, 10.6 shoots regenerated/a sample of 4.20 cm high (for D loddigesii) and 11.6 shoots regenerated/a sample of 4.43 cm high (for D aphyllum) were observed at weeks after cultured It is, however, reported that number of shoot created was oriented to decrease if 30 g/l up concentration was supplied 3.2.3.3 Effect of Spirulina algae powder on regeneration of shoots from PLBs Data in table 3.13 showed that: adding g/l Spirulina algae to medium of VW + 20 g/l sucrose + 10% coconut water + g/l agar + g/l activated charcoal + g/l peptone + 1,5 mg/l BA + 0,5 mg/l IBA + 30 g/l mashed pumpkin with pH of 5.5 gave the best effect to regeneration of shoot from PLBs Table 3.13 Effect of Spirulina algae powder on regeneration of shoots from PLBs (8 weeks after cultured) Mashed Spirulina No shoots/ Shoot explant height (cm) pumpkin algae Shoot situation (g/l) (g/l) Dl Da Dl Da 30 0,0 (Đ/C) 10.59 11.60 4.20 4.34 30 16.82 17.10 6.14 6.22 30 14.50 14.60 5.00 5.10 30 11.24 11.30 4.50 4.46 3.6 2.16 3.0 1.02 2.9 1.42 3.3 0.41 CV (%) LSD0,05 Big and healthy with specific green colour Big and healthy, with dark green colour Big and healthy with specific green colour Small shoot, small and light green colour 3.2.4 Study on production of complete in vitro plants In this study, IBA and PAA at various concentrations were added to VW + 20 g/l sucrose + 10% coconut water + g/l agar + g/l activated charcoal medium, resul was presented in tables 3.14 and 3.15 For D loddigesii orchid, supplying mg/l IBA significantly promoted 18 the growth of young plants, plants of 6.5 cm high with 5.3 leaves and 7.3 roots each, long and chubby roots with 2.87 cm long were recorded at weeks after cultured (Table 3.14) In case of D aphyllum (Table 3.15), supplying 0,5 mg/l PAA gave the best result, presented by plant height (6.96 cm), leaf and root number (5.7 leaves and 7.4 roots/a plant) root length (3.02 cm) at weeks after cultured, a little bit better than supplying with mg/l IBA Table 3.14 Effect of IBA to root formation of orchid in vitro shoots (6 weeks after cultured) IBA concentrations (mg/l) 0,0 (Đ/C) 0.5 1.0 1.5 2.0 CV (%) LSD0,05 Plant height (cm) Dl Da 3.50 3.52 5.30 5.38 6.50 6.80 5.00 5.72 4.60 5.35 3.3 3.0 0.93 0.39 Leaf Root Root length number number (cm) Dl Da Dl Da Dl Da 3.00 4.20 5.30 4.08 4.00 4.1 0.6 3.00 4.50 5.40 4.10 3.90 3.0 0.38 2.50 6.20 7.30 4.90 3.70 3.7 0.32 2.50 6.00 7.00 5.10 4.10 3.8 0.53 1.12 2.13 2.87 1.92 1.26 3.9 0.74 1.14 2.24 2.80 1.86 1.24 3.8 0.16 Table 3.15 Effect of PAA to root formation of orchid in vitro shoots (6 weeks after cultured) PAA Plant height concentrations (mg/l) (cm) 0,0 (Đ/C) 0.5 1.0 1.5 2.0 CV (%) LSD0,05 Dl 3.53 6.16 5.42 5.10 4.20 3.3 0.79 Da 3.52 6.96 5.50 5.22 4.60 3.3 0.31 Dl 3.00 5.18 5.46 5.08 5.02 4.5 0.35 Leaf Root number number Da 3.00 5.7 5.1 4.8 4.2 3.1 0.42 Dl 2.50 6.50 5.40 4.50 3.60 4.0 0.34 Da 2.50 7.40 5.90 5.20 4.50 3.2 0.46 Root length (cm) Dl 1.10 2.46 2.00 1.30 0.92 2.7 0.91 Da 1.15 3.02 2.20 1.60 1.03 3.1 0.19 3.2.5 Study on technology of synthetic seed formation 3.2.5.1 Effect of sodium alginate (%) and CaCl2.2H2O (mM) at various concentration to synthetic seed formation and germination Supply of sodium alginate 3% existed PLBs to CaCl2.2H2O 100; 125 mM or sodium alginate 4% to CaCl 2.2H2O 100 mM gave good effect of synthetic seed formation of D loddigesii and D aphyllum orchids It is also 19 mentioned that, only sodium alginate 3% promoted significantly synthetic seed germination (92.0% for D loddigesii and 95.2% for D aphyllum) 3.2.5.2 Effect of contacting duration with 100 mM CaCl 2.2H2O solution to synthetic seed formation, germination and growth Germination highest ratio of synthetic seed was obtained when sodium alginate 3% solution contacted with CaCl2.2H2O 100 mM one for 30 minuits for both D loddigesii and D aphyllum orchids 3.2.5.3 Effect of BA + IB compound to the germination and growth of synthetic seeds A compound of mg/l BA + 0,5 mg/l IBA gave the best effect for synthetic seed in terms of germination, rooting, root and shoot number and bud growth (96% seeds germinated, 88% seeds rooted, 4,1 roots/a seed, 7.6 shoots/a seed, 13 cm high for D loddigesii and 98%, 88%, roots, shoots and 14.2 cm respectively for D aphyllum) 3.2.6 Study on reservation of synthetic seeds 3.2.6.1 Study on the effect of temperature to stored duration of synthetic seeds The best result of synthetic seed germination was recorded when stored at 4°C (68.0% and 71.2% germinated seeds for D loddigesii and D aphyllum respectively) The growth of shoots sprouted from these seeds of D loddigesii and D aphyllum were also healthy stored by shoot height (4,15 cm and 4.0 cm), rooted shoot percentage (68.2% and 70.0%), root number/a seed (2.6 and 3.0) respectively at 16 weeks after stored 3.2.6.2 Effect of ABA to to stored duration of synthetic seeds Supply of ABA 20 mg/l gave the best effect to synthetic seed germination (51.1% for D loddigesii and 50.02% for D aphyllum at 24 weeks after preserved) After 24 weeks of preservation, germination of synthetic seed was decreased with time 3.2.6.3 Effect of Natri benzoat, topsin-M and carbendazim to survival of preserved synthetic seeds Of three preservative substances mixed with artificial films, carbendazim at 3000 mg/l concentration gave the best effect, better than natri benzoat and topsin-M, low infected seed ratio and high germinated one (68.2% for D loddigesii and 70.2% for D aphyllum at 24 weeks after preserved) were observed It is, however, obvious that at too low (0 200mg/l) or too high (4,000 mg/l up) concentrations, high ratio of infected seeds and low ratio of germinated seeds were also recorded 20 3.2.7 Study on in vitro propagation of preserved synthetic seeds 3.2.7.1 Effect of BA + PAA compound to rapid multiplication of protocorm and shoots from preverved synthetic seeds VW medium supplemented with 1,5 mg/l BA + 0,5 mg/l PAA was considered the most appropriate for propagation of protocorm and preserved synthetic seeds presented by number of protocorm produced (8.5 for D loddigesii and 8.3 for D aphyllum), number of shoots created (7.5 for D loddigesii and 7.3 for D aphyllum) and shoot height (4.3 cm for D loddigesii and 4.3 for D aphyllum) And, what is more, shoots produced in this media looked more healthy compaired to other treatments 3.2.7.2 Effect of mashed tomato to rapid multiplication of protocorm and shoots from preverved synthetic seeds Results available in table 3.26 showed that: 1,5 mg/l BA + 0,5 mg/l PAA regulator compound added with mashed tomato of 10 g/l to 100 g/l concentrations in medium of VW + 20 g/l sucrose + g/l agar + 10% coconut water + 1g/l activated charcoal at pH of 5,5 positively affected to protocorm and preserved shoots multiplication in which mashed tomato added at 70 g/l concentration gave the best result for both D loddigesii and D aphyllum in terms of number and health of protocorm and shoots produced Table 3.26 Effect of tomato juice to rapid multiplication of protocorm and shoots from preverved synthetic seeds (8 weeks after cultured) Content Mult ratio of Mult ratio Height of Qual of mashed protocorm of shoot shoot (cm) of tomato Dl Da Dl Da Dl Da shoot (g/l) 0,0 (Đ/C) 8.50 8.40 7.40 7.20 4.40 4.30 +++ 10 9.50 9.80 7.90 9.40 4.70 4.60 +++ 30 10.20 10.10 8.10 10.10 4.90 4.80 +++ 50 10.40 11.20 10.90 10.50 5.20 5.30 ++++ 70 12.80 16.60 12.30 12.90 5.40 5.60 ++++ 100 9.70 10.60 11.10 12.00 3.20 3.30 +++ CV (%) 3.8 3.0 3.7 3.0 3.0 3.0 1.04 1.00 0.34 0.33 0.67 1.16 LSD0,05 Notes: Medium used:(VW + 20 g/l sucrose + g/l agar + 10% coconut water + g/l activated charcoal) + + + + Chubby, hard shoots with dark green colour 21 + Chubby shoots with specific green colour + Small shoots and leaves with light green colour +Small shoots and leaves with light yellow colourt 3.2.8 Study on in vitro complete plant production from preserved synthetic seeds Adding 0,5 mg/l PAA to VW + 20 g/l sucrose + g/l agar + 10% coconut water + g/l activated charcoal medium at pH of 5,5 gave the best effect to rooting process of young plants before moving to nursery 3.3 Study on management of in vitro plants of D loddigesii and D aphyllum in nursery period 3.3.1 Growth comparision of in vitro plantlets multiplicated from shoot and from preserved synthetic seeds in nursery period Height of plants multiplicated from synthetic seeds was significantly at 5% level lower than that of plants traditionally propagated by in vitro culture method (6.63 cm; 6.58 cm in the first method and 7.10 cm; 7.00 cm in the later at 12 weeks after grown for D loddigesii and D aphyllum respectively) No difference of numbers of newly produced of leaves and roots was reported It is also mentioned that young plants produced by these methods were quite familiar in morphological characteristics in terms of colours and shapes of stem, leaf and root etc… 3.4.2 Study on determination of suitable time for moving in vitro plants to nursery Table 3.29 Effect of plant moved seasons to survival and growth of in vitro plants in nursery period (12 weeks after planted) Survived Plant height Number of New roots/a plant ratio Seasons (cm) leaves plant (%) Dl Da Dl Da Dl Da Dl Da 78.00 83.00 7.15 7.06 6.30 6.80 3.50 3.46 Autumn (18/8/2016) 62.00 60.00 5.60 5.48 5.60 5.53 2.20 2.10 Winter (18/11/2016) 72.0 74.0 6.42 6.54 5.80 6.10 2.73 2.50 Spring (18/02/2017) 76.00 75.0 6.86 6.70 6.03 6.23 3.00 2.80 Summer (18/5/2017) 4.4 3.9 4.8 4.6 3.7 3.8 CV (%) 0.24 0.22 0.15 0.19 0.24 0.24 LSD0,05 22 Results (Table 3.29) indicated that: Autumn was considered as the best season for moving plants to nursery proved by high percentage of plants survived (78% for D loddigesii and 83% for D aphyllum) and healthy growth (7.15 cm high, 6.3 new leaves, 3.5 new roots a plant for D loddigesii and 7.06 cm; 6,8 leaves, 3.46 roots for D aphyllum) 3.3.3 Study on determination of suitable media for in vitro plants cultinated in nursery Medium of Sphagnum moss + Pumice Stone (50:50) was considered suitable for D loddigesii in vitro cultivated in nursery presented by highest ratio of survival (94%), healthy growth (7.8 cm heigh, 7.20 leaves and 3.9 new roots a plant) whereas Sphagnum moss + coconut fibre (70 : 30) medium gave the best effect (92% survived plants of 7.88 cm heigh, 7.48 leaves and 4.5 new roots/a plant) 3.3.4 Study on nutrional adding for in vitro plants in nursery period Utilization of B1 – Thailand product sprayed a week intervals at ml/l dose promote effectively the growth of D loddigesii and D aphyllum in nursery peiod presented by healthy plants with long and chubby roots (Table 3.31) Table 3.31 Effect of nutrional substances to the growth of in vitro plants in nursery period (12 weeks after cultivated) Plant height Number of Number of newly Fertilisers (cm) leaves produce roots Dl Da Dl Da Dl Da Water (Control) 6.00 6.50 5,79 5.51 1.93 1.83 B1- Thailand 9.10 8.76 7,93 8.13 4.60 5.30 Growmore 8.30 8.20 7,48 7.82 4.10 4.80 (30:10:10) Ðầu trâu 502 7.30 7.24 6,62 6.79 3.40 3.33 (30:12:10) CV (%) 4.6 4.7 4,5 4.6 4.4 4.6 LSD0,05 0.23 0.29 0,33 0.42 0.29 0.26 Notes: Medium of Sphagnum moss + Pumice Stone (50:50) was used for D loddigesii (Dl) Medium of Sphagnum moss + coconut fibre (70:30) was used for D aphyllum (Da) 3.4 Study on orchid cultivating techniques of D loddigesii and D aphyllum in production gardens 3.4.1 Study on determination of suitable media for orchid in production gardens 23 For D loddigesii orchid, medium of pine bark + Pumice Stone + Sphagnum moss (40 : 40 : 20) characterized by light, well spongy and ventiolated was recorded suitable for plant growth 913.35 cm high; 0.37 cm diameter, 1,2 twigs; 9.6 leaves of 5.05 cm long and 1.29 cm width) For D aphyllum orchid, “Don bang” medium gave the best effect to plant growth presented by healthy plants (13.46 cm high; 0.41 cm in diametter), twig produced (1.1); lots of healthy leaves (9.7 leaves of 4.98 cm long and 1.35 cm width) and good look 3.4.2 Study on nutrional regime for orchid in production gardens On the basic medium mentiond in part 3.4.1, adding B1 Thailand + sea weed 95% promoted significantly the growth of orchid plan, better than other compounds such as B1 Thailand + Grow more or B1 Thailand + Orchid focus Grow The healthiest plants were also recorded in this treatment 3.4.3 Study on supplying Superthrive for orchid plants in production gardens Adding Superthrive with 0.2 ml/l dose impacted positively to orchid plant growth of both species, plant height (18.4 cm for D loddigesii; 23.7 cm for D aphyllum at weeks after planted) and stem diametter (1.06 cm for D loddigesii; 1.21 cm for D aphyllum at weeks after planted) were much higher than control (no adding) and applied with 0.1 ml/l Superthrive In addition, more twigs and leaves produced, healthier leaves presented by leaf dimensions were also reported in this treatment CONCLUSIONS AND RECOMMENDATIONS Conclusions D.loddigesii and D aphyllum orchids have racemiform roots and bended stems D loddigesii flowers are single, purple whitenish and yellow in the centre with long stalk whereas D aphyllum flowers are white with purple strips and dark purple in the centre, located closely as clusters with small stalk Micro structure of these orchids suit to subordinate life with good draught tolerant because of few stoma existed Apart from decorational function, D loddigesii and D aphyllum can be used in pharmacy because some useful pharmaceuticl substances such as alkaloids (0.35%), flavonoids (0.92% in D loddigesii; 0.56% in D aphyllum); polysaccharides (1.42% in D loddigesii; 4.20% in D aphyllum) were included Chemicals and media suitably used in in vitro culture of D loddigesii and D aphyllum are: sterilizers: 3% NaOCl diluted with 1‰ cefotaxime for 15 Media for PLBs production from tTCL: VW + 20 g/l sucrose + 10% 24 coconut water + g/l agar + g/l activated charcoal + 1,5 mg/l BA for D loddigesii and VW + 20 g/l sucrose + 10% coconut water + g/l agar + g/l activated charcoal + 1,5 mg/l BA + 0,5 mg/l αNAA for D aphyllum; Media for shoot regeneration from PLBs: g/l activated charcoal + g/l peptone + 1,5 mg/l BA + 0,5 mg/l IBA + 30 g/l mashed pumpkin + g/l Spirulina algae Medium for In vitro rooting of shoots were VW medium supplemented with g/ activated charcoal + mg/l IBA for D loddigesii and g/l activated charcoal + 0,5 mg/l PAA for D aphyllum Media for synthetic seed formation: MS + 3% sodium alginate + mg/l BA + 0,5 mg/l IBA + 20 g/l sucrose + g/l activated charcoal + 20 mg/l ABA + 3000 mg/l carbendazim contacting with CaCl2.2H2O 100 mM for 30 min; Synthetic seeds was stored well in dark at 4°C; synthetic seed high germinated (68.2% for D loddigesii and 70.2% for D aphyllum at 24 weeks after preserved); Media for protocorm and shoots sprouted from preserved synthetic seed: 1,5 mg/l BA + 0,5 mg/l PAA + 70 g/l mashed tomato Medium for In vitro rooting of shoots from synthetic seed after storage: 1g/l activated charcoal + 0,5 mg/l PAA, pH of 5,5, (number of protocorm (12,8 for D loddigesii and 16,6 for D aphyllum), number of shoots (12,3 for D loddigesii and 12,9 for D aphyllum) Suitable season for moving in vitro plants to nursery was Autumn Sphagnum moss and Pumice Stone (50:50) mixture increased survived plant ratio for D loddigesii (94%) whereas Sphagnum moss + coconut fibre (70:30) was suitable for D aphyllum (92% survived plants) Adding B1 Thailand every week at ml/l dose improved plant health (8.76 cm – 9.10 cm high; 7.9 – 8.1 leaves and 4.6 – 5.3 newly formed roots each) Suitable media for plant cultivated in production period were pine bark + Pumice Stone + Sphagnum moss (40 : 40 : 20) for D loddigesii and “Don bang” for D aphyllum In addition, nutrional foliar of ml/l B1 + 0,5 g/l sea weed 95% + 0,2 ml/l Superthrive gave good effect to plant growth (18.4 cm high, 1.06 cm in diameter, 2.7 twigs and 12.8 leaves produced for D loddigesii and 23.7 cm 1.21 cm, 2,4 and 12.0 respectively for D aphyllum at weeks after planting Recommendation Multiplcation and cultivation techniques of D loddigesii and D aphyllum resulted from the study should be accordingly recommended in to production Further studies on flowering and pharmaceutical evaluation of D loddigesii and D aphyllum orchids propagated from synthetic seeds should be continued 25 THESIS RELATED SCIENTIFIC WORKS PUBLISHED Nguyen Thi Lai, Pham Huong Son, Nguyen Huu Cuong (2016), “Structural study of Dendrobium aphyllum (Roxb.) Fisher and Dendrobium loddigesii Rolfe”, Journal of Vietnam Agricutural Sciences and Technologies, No 10(71), pp 27-31 Nguyen Thi Lai, Pham Huong Son (2017), “Study on the production of synthetic seed for Dendrobium aphyllum (Roxb.) Fisher”, Journal of Vietnam Agricutural Sciences and Technologies, No, 1(74), pp 84 - 90 Nguyen Thi Lai, Pham Huong Son, Vu Manh Hai, Tong Xuan Trung (2018), “Micropropagation of Dendrobium loddigesii Rolfe by thin cell layers culturing’, Journal of Vietnam Sciences and Technologies, No 60(5), pp 60 - 64 Nguyen Thi Lai, Vu Manh Hai, Pham Huong Son, Pham Minh Duy, Bui Thi Thanh Phuong, (2018), "A study on cultivation of (Dendrobium loddigesii Rolf) in nursery”, Journal of Vietnam Sciences and Technologies, No 60(12), pp 36 - 39 ... Reddy, 201 6) Netherland ranks the first country over the world in orchid production and exportation (39.7 %), followed by Thailand (28.4 %); Taiwan (10 %), Singapore (10 %) and New Zealand (6 %) Main... Survived plant ratio ( %), plant height (cm), plant stem diametter (cm), number of leaf/plant, leaf length (cm), leaf width (cm), number of twig/plant, average number of root/plant 2.4 Experimental... Stone (50:5 0) mixture increased survived plant ratio for D loddigesii (94 %) whereas Sphagnum moss + coconut fibre (70:3 0) was suitable for D aphyllum (92% survived plants) Adding B1 Thailand every