In vitro micropropagation of the orchid (dendrobium crystallinum var. alba)

The results showed that protocorms were formed from seeds culture on the ½ MS medium supplemented with 10% potatoes extract within 8 weeks. Protocorms which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar, 0.1 mg/l NAA and 1 mg/l BA were optimal for PLB formation (8.67 PLBs/sample) after 8 weeks culture. Protocorms converted into normal plants with well-developed shoots and roots on the ½ MS medium supplemented with 20 g/l sucrose, 10% potatoes extract, 0.5 g/l AC, and 7 g/l agar after about 90 days.

1

IN VITRO MICROPROPAGATION OF THE ORCHID (DENDROBIUM CRYSTALLINUM VAR ALBA)

Tran Thi Ngoc Lan1

1 South Central and Highland Institute of Science

Information:

Received: 07/08/2018

Accepted: 17/10/2018

Published: 11/2019

Keywords:

BA, Dendrobium crystallinum

var alba., NAA, PLB,

protocorm

ABSTRACT

Studies of micropropagation of Dendrobium crystallinum var alba were conducted in order to conserve and develop this precious orchid species The results showed that protocorms were formed from seeds culture on the ½ MS medium supplemented with 10% potatoes extract within 8 weeks Protocorms which were cultured on the ½ MS medium containing 30 g/l sucrose, 0.5 g/l AC,

7 g/l agar, 0.1 mg/l NAA and 1 mg/l BA were optimal for PLB formation (8.67 PLBs/sample) after 8 weeks culture Protocorms converted into normal plants with well-developed shoots and roots on the ½ MS medium supplemented with 20 g/l sucrose, 10% potatoes extract, 0.5 g/l AC, and 7 g/l agar after about 90 days PLBs converted into normal plants on the same medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, too No abnormal morphological changes were found

in these Dendrobium seedlings

1 INTRODUCTION

Vietnam has a tropical monsoon climate that is

appropriate for orchid growth such as the

Dendrobiums Dendrobiums are not a beautiful

species although they are highly valued in the

flower industry as potted plants and cut flowers

Even though these wild orchids are diverse, they

have been exploited almost to extinction The

Dendrobium crystallinum var alba is a very

graceful wild orchid of Vietnam and is a rare

species It is a epiphytic, sympodial orchid which

also grows in Hymalayan Myanmar, Thailand and

Vietnam at altitudes of 700 – 1700 m It has

beautiful, long-lasting flowers, noble aroma and

blooms in April- May every year It is classified as

a group of rare orchids and an endangered species

(EN, IUCN, Averyanov, 2005)

Micropropagation provided an important method for mass propagation of many orchid species There were perusals of Dendrobium micropropagation on the world and Vietnam Sunitibala & Rajkumar (2009) cultured seeds of Dendrobium transparens L on the ½ Murashige and Skoog (½ MS) suplemented with 1 mg/l NAA and 2 mg/l BA Nguyễn Văn Song (2011) cultured seeds of Dendrobium chrysotosum Lindl on MS medium Nguyễn Thị Sơn et al (2014) propagated Dendrobium officinale Kimura et Migo with sowing seeds on the Vacin Went and cultured nodal segment on the MS medium Vũ Kim Dung

et al (2016) propagated Dendrobium gratiosissimum Rchb.f Nguyễn Văn Việt (2017) cultured the Dendrobium lituiflorum Lindley seeds Rattana & Sangchanjirade (2017) propagated Dendrobium signatum Rchb.f However, there is no article refering to clonal

propagation of Dendrobium crystallinum var alba

This study reports micropropagation of

Dendrobium crystallinum var alba in order to

conserve and develop this precious orchid species

2 MATERIALS AND METHODS

2.1 Materials

Research subject: Den crystallinum var.alba plant with fruits were collected in the family garden Fruit was harvested 180 days after pollination in October, 2017 (fig 1)

Figure 1 Dendrobium crystallinum var.alba a Plants with their flowers b Plants with their fruits (arrow)

2.2 Experiment conditions

All tests were kept at 25±1οC under a photoperiod

of 16 h light/8 h dark, light intensity of 2000 lux

and 70% relative humidity

Basal cultured medium: MS, ½ MS, at pH 5.7, 121

οC, 1 atm, autoclaved for 20 minutes

2.3 Methods

2.3.1 Effect of different media and organic

supplements on germination of Den

crystallinum var.alba seeds

The mature capsules of Den crystallinum var.alba

were collected six months after pollination, soaked

in aqueous solution of commercial detergent

(Sunlight, Vietnam) for 10 min, then rinsed

thoroughly three times with sterile distilled water,

followed by dipping them in 70% ethanol for 20

sec Capsules were then surface sterilised by

dipping in 70% ethyl alcohol and flamed

immediately four to five times in laminar air flow

The capsules were then cut longitudinally in a

sterilised petri dish Seeds were scraped from the

capsules, mixed with 100 ml sterile distilled water

and pipetted into 1ml tubes and then cultured on

the surface of the medium Two different basal

consisting of MS, ½ MS, supplemented with different organic compound (15% coconut water (CW) – Thickness of coconut pulp = 3 – 4 mm; 10% potatoes extract – 100g potatoes were boiled, extract to 1 l solution; 5% mashed banana), 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar Seeds were cultured

in the bottles (volume = 500 ml with 70 ml medium/bottle) Each treatment had 5 bottles The percentage of orchid seed germination was obtained by estimating the surface area of seed germination in the tissue culture bottle with a diameter of 6.5 cm The total surface area of the tissue culture bottle was defined as 100% After cultivation for eight weeks, the percentage of seed germination was recorded Observations on the percentage germination of seeds, protocrom formation rate, shoot formation rate were recorded

8 weeks after culture

2.3.2 Effect of NAA and BA on protocorm proliferation of Den crystallinum var.alba

Protocorms from the above study were used as culture material (the average weight of 20 ± 5 mg) were proliferated on ½ MS supplemented with 30 g/l sucrose, 10% potatoes extract, NAA (0; 0.1; 0.2 mg/l), BA (0, 0.5, 1 mg/l), 0.5 g/l AC and 7 g/l agar

3

500 ml with 70 ml medium/bottle) with 20

explants/bottle Each treatment had 5 bottles

Observations on the protocorm-like body (PLB)

formation rate, number of PLB/explants, figure of

PLBs were recorded 8 weeks after culture

2.3.3 Effect of plant growth regulators (PGRs) on

development of protocorms and PLBs Den

crystallinum var.alba

Protocorms from the study (2.3.1) and PLBs from

the study (2.3.2) with being used as explants (the

average weight of 20 ± 5 mg) were cultured on the

½ MS supplemented with 20 g/l sucrose, 10%

potatoes extract, 0.5 g/l AC, 7 g/l agar, NAA (0;

0.5; 1 mg/l), BA (0; 0.5 mg/l) in examnination the

growth of these protocorms and PLBs They were

cultured in the bottles (volume = 500 ml with 70

ml medium/bottle) with 20 explants/bottle Each

treatment had 5 bottles Observations on figure of

seedlings, the PLB formation rate, the seedling

formation rate, height of shoot, number of leaves,

number of roots and length of root were recorded

90 days after culture Observation was conducted

on the developing stages of protocorms and PLBs

(4 weeks, 8 weeks and 90 days) after culture

2.3.4 Histological observation

Observation of the germination of orchid seeds and

their developing stages (4 weeks, 8 weeks and 90

days after culture) on the optical microscope (Olympus CX21, Japan) with degrees magnification of 40 - 100 times

2.3.5 Statistical analysis

All the experiments were set up in completely randomised design (CRD) Each treatment consisted of 3 replicates The difference among the treatment means was compared based on Duncan’s multiple range test (DMRT) analysis (with a level

of significance of P<0.05) Excel 2010 and the statistical package SPSS (Statistical Program Scientific System)16.0 was used for the analyses

3 RESULTS AND DISCUSSION

3.1 Effect of different media and organic supplements on germination of Den crystallinum var.alba seeds

Naturally, orchid seeds have a poor germination rate because of the small size of the seeds and the lack of cotyledons and endosperm Seeds can only germinate with isolated mycorrhizal fungi but seeds run the risk of being infected and die Consequently, the sample orchid seeds were propagated in asymbiotic seed germination The results of the study on germinated seeds of Den crystallinum var.alba on the different medium were observed at 8 weeks after culture (Table 1)

Table 1 Effect of different media and organic supplements on germination

of Den crystallinum var.alba seeds

rate of protocorms (%)

Survival and formation rate of shoots (%)

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05

It was observed that seeds had been green at 4

weeks after culture (Fig 2c), germinated and

formed green protocorms Protocorm had emerge

from capsules and come on growing at 8 weeks

after culture (Fig 2a, 2d) The most suitable media,

providing seed germination were ½ MS medium

supplemented with 15% CW and ½ MS medium

supplemented with 10% potatoes extract

(protocorm formation rate corresponding 98.67%

và 99.33%) The less protocorm formation rate

gained 26.33% và 37.33% of seeds which were

sowed on the MS medium and ½ MS medium

Combinations of organic supplements in the media

enhanced seed germination It show that the

importance of organic compounds such as coconut

water, potatoes extract… for orchid seeds

germination Den crystallinum var.alba is

epiphytic orchid, growth rate is low, so, the high

concentration of N, P, K compounds were not

suitable to culture orchid seeds However, the

minerals derived from organic compounds such as

coconut, potatoes extract were useful in culture

orchid seeds The reports on the world were

acknowledged this too (Arditti, 2008; Vũ Ngọc

Lan & Nguyễn Thị Lý Anh, 2013; Parthibhan và

cs., 2015, Nguyễn Văn Việt, 2017)

The best medium for seed germination in treatment

was 10% potatoes extract or 15% CW Potatoes

extract consists of carbohydrates, amino acids,

important vitamins (C, B1, B6…) and mineral

elements (potassium, iron, magnesium) (Molnár et

al., 2011; Sandoval et al., 2014, Parisa et al., 2014)

In the in vitro culture, potato had useful effects on

some orchid species such as Phalaenopsis and

Dendrobium (Rattana & Sangchanjirade, 2017)

This study found that the nutrients of CW and

potato were suitable for orchid seeds The

effciency of CW may depend on species, age and

thickness of coconut pulp At this study, CW was

undertaken of coconut fruits with the thickness of

coconut pulp = 3 – 4 mm was suitable for orchid

seeds germination However, CW was rather

expensive than potato Potato was easy to look for,

so, potatoes extract was selected in the following

(2009) for sowing seeds Den transparent L with using PGRs (1 mg/l NAA and 1mg/l BA), in this experiment only used MS medium and organic compounds for Den crystallinum var.alba seeds gemination

Beside protocorm formation, there was the low shoot formation rate from orchid seeds (0,33 – 7,67%) It show that orchid embryos can develop

to the different stages of seeds, accordance with reports on Dendrobium culture (Arditti, 2008; Vũ Ngọc Lan & Nguyen Thi Ly Anh, 2013, Vu Kim Dung và cs., 2016; Rattana & Sangchanjirade, 2017)

3.2 Effect of NAA và BA on protocorm proliferation of Den crystallinum var.alba

The results of the study on effect of NAA và BA

on protocorm proliferation of Den crystallinum var.alba were observed at 8 weeks after culture (Table 2)

The protocorm explants had showed the induction

of meristem, parenchyma with PGRs Protocorm is the germinated stage of orchid embryos, along with translation of PGRs sush as NAA, BA to differentiate to PLBs PLBs and protocorms are potential in embryo proliferation Subculture PLBs was conducted in micropropagation of orchid such

as Dendrobium transparent L.(Sunitibala & Rajkumar, 2009), Dendrobium aqueum Lindley (Parthibhan et al., 2015) or Dendrobium lituiflorum

Lindley (Nguyen Van Viet, 2017)

Adding to NAA, BA, potatoes extract was effective in forming secondary PLBs In fact, the composition of potatoes extract includes inorganic ions (e.g phosphorus, potassium), nitrogenous compounds, amino acids, vitamins…) All these suplements may be the reason for seed germination

in orchids (Arditti, 2008; Rattana & Sangchanjirade, 2017) Using BA individually had not much effective but the addition of BA in combination with NAA improved ability of cell division of explants The optimal concentrations

of NAA (0.1 mg/l ) and BA (1 mg/l) gave the highest PLB formation rate, the number of PLBs/explant (corresponding 100%, 8.67

5

PLBs/explants) The mean weight of PLBs was

higher the mean weight of protocorm ( 32.4 mg and

25.4 mg) and had deep green (Fig.2b) Compared

to PLB proliferation (multiplication factor: 4.2 in

protocorm proliferation of Dendrobium nobile

Lindl; Vũ Ngọc Lan & Nguyễn Thị Lý Anh, 2013),

the results of this study had higher multiplication

factor (8.67) The combined of auxin and

cytokinins was observed in present study is in accordance with report on Dendrobium transparens L (Sunitibala, 2009), Dendrobium signatum (Rattana K & Sangchanjirade, 2017)

Thus, the ½ MS medium suplemented with 0.1 mg/l NAA and 1 mg/l BA is optimal to form the secondary PLB

Table 2 Effect of NAA và BA on protocorm proliferation of Den crystallinum var.alba after 8 weeks culture

NAA

(mg/l)

BA (mg/l)

Formation rate PLB (%)

Number of PLBs/explant Figure of PLBs

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05

3.3 Effect of plant growth regulators on development of Den crystallinum var.alba protocorm and PLB after 90 days culture

Effect of plant growth regulators (NAA and BA) on development of Den crystallinum var.alba protocorm

and PLB after 90 days culture showed in table 3 and table 4

Table 3 Effect of NAA and BA on development of protocorm Den crystallinum var.alba after 90 days culture

Medium supplement

with

Formation rate PLB (%)

Formation seedling (%)

Average number

of roots

Average root length (cm)

Average

of shoot height (cm)

Average number

of leaves NAA(mg/l) BA(mg/l)

0 0 17.67c* 82.33b 2.67b 0.92c 3.18b 2.47b 0.5 0 37.33b 62.67c 2.74b 1.93b 3.15b 2.43b 0.5 0.5 7.67d 92.33a 4.13a 3.61a 4.36a 4.87a 1.0 0.5 66.33a 33.67d 1.13c 0.20d 0.52c 2.05b

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at P < 0.05

Table 4 Effect of NAA and BA on development of PLB, derived from protocorm Den crystallinum var.alba

after 90 days culture

Medium supplement

with

Formation rate PLB (%)

Formation seedling (%)

Average number

of roots

Average root length (cm)

Average

of shoot height (cm)

Average number

of leaves NAA(mg/l) BA(mg/l)

0 0 38.67b* 23.67c 1.57b 0.63c 0.76c 2.17c 0.5 0 39.33b 60.67b 1.79b 1.85b 2.07b 3.18b 0.5 0.5 9.67c 90.33a 4.22a 3.02a 4.19a 4.59a 1.0 0.5 69.33a 16.67d 0.53c 0.13d 0.55c 2.01c

*: Different alphabetical letters are significantly different according to Duncan’s multiple range tests at

P < 0.05

At treatment devoid of PGRs, there was 82.33% of

protocorms converted into seedlings (Table 3,

Fig.2h) but only 23.67% of PLBs converted into

seedlings (Table 4) Formation rate PLB in this

treament was also rather low (corresponding

17.67% and 38.67%) This show that the

embryogenesis nature of protocorms They can

invert into seedlings that no using PGRs However,

PLBs were not so PLBs need assisting of NAA

and BA to develop and convert into seedings (the

highest formation seedling at treatment

supplemented with 0.5 mg/l NAA and 0.5 mg/l

BA, 90.33%) Similar to protocorm, the seedling

formation rate was 92.33% in this result, too Shoot

height, number of roots, number of leaves, root

length of the seedlings in this treatment were

highest The combination of NAA and BA made

the cells loosen, elongate dimension, improved

ability of cell division, made the cell survive and

convert into seedling The combination of NAA

and BA in micropropagation is uasually applied in

orchid embryo proliferation or in shoot

proliferation with axenic pseudobulb segment

(Sunitibala & Rajkumar, 2009; Nguyễn Thị Sơn et

al., 2014; Parthibhan et al., 2015) or seedlings

formation of Dendrobium signatum Rchb.f

(Rattana & Sangchanjirade, 2017)

At treatment devoid of PGRs, PLBs still converted

into seedlings (23,67%) It show that PLBs can

develop to the different stages At treatment

medium suplemented with 1 mg/l NAA and 0,5

mg/l BA, PLB proliferation rate was high

(corresponding 66,33% and 69,33%) but formation

seedling rate was low (33,67% and 16,67%)

growth, lateral growth increases so it reduces height of shoot as well as root formation The combination of BA and auxin (NAA), the cells expand and differentiate, proliferate effectively protocorms In the medium concentraton of NAA and BA (0.5 mg/l), an increase cell division and shoot and root morphogenesis took place with organogenesis (Fig 2i, 2j.) Thus, the treatment with culture medium suplemented with 0.5 mg/l NAA and 0.5 mg/l BA was noted to differentiate protocorms and PLBs into seedlings

No morphological variations were observed in this study through micropropagation Den crystallium var alba with protocorms and PLBs (Fig 2i, 2j)

3.4 Histological observation

Protocorm is a earliest structure that is formed in embryogenesis of orchid seed and is united in all of Orchidaceae (Batygina et al., 2003; Arditti, 2008) Observating on optical microscope showed that, the orchid seeds with green embryos after 4 weeks culture (Fig 2c), formed protocorms after 8 weeks culture (Fig 2d), after that, formed shoots, roots, converted into seedlings after 90 days culture (Fig 2g) PLB derived from protocorm (Fig 2b, 2e) developed with the stages similar to protocorm (Stage 1: PLB formed meristem; Stage 2: PLB formed leaflets, vascular tissue; Stage 3: formed seedling with shoot and root) That was confirmed

in this study (Fig 2f, 2j) Thus, protocorms could directly convert into seedlings or proliferate, forming PLBs and PLBs could proliferate and convert into seedlings, too This is a effective orchid micropropagation

7

4 CONCLUSION AND PETITION

4.1 Conclusion

½ MS medium (suplemented with 10% potatoes

extract, 30 g/l sucrose, 0.5 g/l AC, 7 g/l agar) is the

most suitable for Dendrobium crystallinum var

alba seed germination (the protocorm formation

rate: 99.33%) PLBs were formed from protocorms

on the medium suplemented with 0.5 mg/l NAA

and 1 mg/l BA (number of PLBs/explant: 8.67)

Protocorms and PLBs converted into seedlings (corresponding 92.33% and 90.33%) on the ½ MS medium (supplemented with 10% potatoes extract,

20 g/l sucrose, 0.5 g/l AC, 0.5 mg/l NAA, 0.5 mg/l

BA and 7 g/l agar) after 90 days culture

4.2 Petition

The following studies are necessary to the growth and development of these seedlings in the ex vitro condittion for mass production this valuable species

Figure 2 Micropropagation Dendrobium crystallinum var.alba a Seeds germinated, formed protocorm after 8 weeks

culture; b PLBs formation from protocorms after 8 weeks culture; c The seeds with embryos after 4 weeks culture;

d Protocorms formation from seeds after 8 weeks culture; e PLB with shoot were formed from protocorms after 8 weeks culture; f Seedling formation from PLB, derived from protocorm after 90 days culture; g Seedlings formation from protocorm after 90 days culture; h Seedlings formation on no PGRs medium; i Seedlings formation derived from protocorm on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA; j Seedlings formation derived from PLB on the medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA Acknowledgements.

The authors would like to thank College of Economics and Technical Lam Dong for supporting this study

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