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:
9 62 01 10
SUMMARY OF DOCTORIAT THESIS
Scientific supervisers:
1. Prof. Dr. Vu Manh Hai
2. Dr. Pham Huong Son
HA NOI - 2019
Thesis completed at:
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………………………………………
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INTRODUCTION
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, 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.
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 ornamental
flowers in Vietnam.
3. Scientific and pratical significance of the
thesis - Scientific significance
Valuable scientific data on the multiplication technology based on
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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.
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 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.
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 Vietnam
and outsides cited were involved in the document. Thesis content was
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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 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
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 4 - 6 cm long;
D. aphyllum
Subordinated living with thinh
and bended stems of
100 cm long. (Tran
1998).
about
Hop,
Long handled machete leaves
of 6 - 8 cm long, 1,5 - 2 cm
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 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).
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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
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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 diabete
treatment. 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 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
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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, 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 medium
with 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 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 2 g/l peptone medium was suitable for seed
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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; 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 6
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 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 μmol⋅m-2⋅s-1 and relative moisture at
85% – 95%.
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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 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 3 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 1 : 1 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 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
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 2
MATERIALS. CONTENTS AND METHODS OF STUDY
2.1. Materials
- Orchid accessions used in evaluation of morphological, micro operation
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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 seed
formation.
- 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 fibre
and “Don bang”…
- Nutritional sustances: Orchid Focus grow, Seaweed 95%, Striving buffalo
502, B1 Thai Lan, Growmore.
- Growth regulator: Superthrive.
2.2. Contents of study
- Study on biological characteristics of D. loddigesii and D. aphyllum
orchid species
- Study on in vitro propagation, synthetic seed creation techniques and in
vitro multiplication from preserved synthetic seeds of D. loddigesii and D.
aphyllum for its conservation
- 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
2.3.1 Research methods
* Research methods used in morphological evaluation of D. loddigesii and
D. aphyllum orchids
- Morphological description was based the method proposed by Nguyen
Nghia Thin (2007) concluding stem, leaf, flower characteriszation
- Accession analyse and specy nomination were based on morphological
comparision presented in Vietnamese orchids (Tran Hop, 1998) and
Vietnamese botany (Duong Duc Huyen, 2007).
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* 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 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
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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….
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+ 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 ± 2 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.
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CHAPTER 3. 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. 1 – 2 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 2 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 1 – 2 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 6 -7 liber vessels whereas 7 – 8 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 thanh 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 3 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 2 blocks of 4 pollen spaces each.
Flower of D. aphyllum orchid is single one or cluster of 2 – 3 flowers,
sprouted from non leaf existed stem with white colour and dark purple in the
centre. Three calyx petals have same dimension including 2 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 3 lobes in which 2 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 2 blocks of 4 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 và 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)
0
Contents of substances
D. loddigesii D. aphyllum
N
1
2
3
4
5
6
7
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 + 7 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 6 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 6 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 + 7 g/l agar +1 g/l
activated charcoal + 2 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 6 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 1 g/l Spirulina algae to medium
of VW + 20 g/l sucrose + 10% coconut water + 7 g/l agar + 1 g/l activated
charcoal + 2 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
1
16.82
17.10 6.14
6.22
30
2
14.50
14.60
5.00
5.10
30
3
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 + 7 g/l agar + 1 g/l activated charcoal
medium, resul was presented in tables 3.14 and 3.15.
For D. loddigesii orchid, supplying 1 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 6 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 6 weeks after cultured,
a little bit better than supplying with 1 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 CaCl 2.2H2O 100; 125
mM or sodium alginate 4% to CaCl2.2H2O 100 mM gave good effect of
synthetic seed formation of D. loddigesii and D. aphyllum orchids. It is also
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