ORIGINAL ARTICLE

Storability, post-storage conversion and genetic stabilityassessment of alginate-encapsulated shoot tips of monopodialorchid hybrid Aranda Wan Chark Kuan ‘Blue’ 3 Vanda coeruleaGrifft. ex. Lindl.

Saikat Gantait • Uma Rani Sinniah

Received: 14 March 2012 / Accepted: 19 August 2012 / Published online: 9 September 2012

� Korean Society for Plant Biotechnology and Springer 2012

Abstract An efficient short-term storage system of syn-

thetic seeds, produced using in vitro shoot tips of the

monopodial orchid hybrid Aranda Wan Chark Kuan

‘Blue’ 9 Vanda coerulea Grifft. ex. Lindl. (AV), was

developed. In vitro shoot tips (3–4 mm) were successfully

encapsulated, resulting in uniform spherical beads (cap-

sules), using 3 % sodium alginate with 75 mM

CaCl2�2H2O. Maximum (*100 %) conversion (into

plantlets with shoot and root) of capsules (or synthetic

seeds) was achieved on quarter-strength Murashige and

Skoog regrowth medium, while full-strength MS medium

was required for effective conversion of non-encapsulated

shoot tips. The capsules showed distinct difference in their

response to temperature during storage. The conversion

efficiency declined upon storage duration at both 4 and

25 �C, with those stored at 25 �C being more tolerant to

storage. Capsules stored at 4 �C had rapid deterioration and

faced complete death within 160 days while those stored

for 200 days at 25 �C showed relatively high conversion

(71.6 %). An inter-simple sequence repeats fingerprinting

approach, employed on indiscriminately chosen plantlets

from converted capsules (following 4 and 25 �C of stor-

age), ensured the post-storage genetic stability.

Keywords Capsules � Conservation � Conversion �Encapsulation � Germplasm exchange � ISSR � Sodium

alginate � Synthetic seeds

Abbreviations

AV Aranda Wan Chark Kuan ‘Blue’ 9

Vanda coerulea Grifft. ex. Lindl

ISSR Inter-simple sequence repeats

MS Murashige and Skoog (1962)

Introduction

Alginate-encapsulation of plant organs have been used to

produce synthetic seeds or artificial seeds which can serve

as an efficient tool for storage, germplasm conservation,

and direct distribution of planting materials for propagation

(Germana et al. 2011). The technology utilizes artificial

gelling matrix to encapsulate propagules such as somatic

embryos, shoot tips, nodal segments, or protocorms which

can be sown under in vitro or direct ex vitro conditions

(Martin 2003). High efficiency of propagation, limited

requirement of space, easy-to-handle during transport, and

potential for storage are some of the key features of

encapsulation technology which make it attractive to

researchers for germplasm conservation and plant material

exchange (Nyende et al. 2003).

The exploitation of the encapsulation technology has

proven to be quite fruitful specifically for a number of

Orchidaceae species, for example seeds and protocorms of

Spathoglottis plicata (Khor et al. 1998), protocorms in

Cymbidium gianteum (Corrie and Tandon 1993), Vanda

coerulea (Devi et al. 2000), Dendrobium, Oncidium, and

Cattleya (Saiprasad and Polisetty 2003), and bulbs in Ipsea

malabarica (Martin 2003). In orchids, the use of proto-

corms (bipolar structure) as plant material for the produc-

tion of synthetic seed appears to be quite common.

S. Gantait � U. R. Sinniah (&)

Department of Crop Science, Faculty of Agriculture,

Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

e-mail: umarani@agri.upm.edu.my

123

Plant Biotechnol Rep (2013) 7:257–266

DOI 10.1007/s11816-012-0257-9

However, very few reports on the use of unipolar explants

such as shoot tips for encapsulation are available in

Orchidaceae (Vanilla; Divakaran et al. 2006). Reports on

success of shoot tip encapsulation on other plant species

(Danso and Ford-Lloyd 2003; Rai et al. 2009; Hung and

Trueman 2011; Faisal et al. 2012; Mehrotra et al. 2012)

have indicated that the juvenility of shoot tips with high

mitotic activity may facilitate the revival process more

rapidly.

Despite wide utilization of encapsulation technology in

micropropagation and storage of orchids, the genetic

fidelity of synthetic seed-derived propagules have been

completely unassessed. The escalating exploitation of

synthetic seed technology for propagation and germplasm

preservation obliges genetic stability assessment of prop-

agules subsequent to their conservation (Dehmer 2005). To

date, methodical germplasm sampling and their molecular

status assessment via DNA marker technology intervention

has turned into and efficient practice (Mishra et al. 2011).

Among the various available DNA uniformity/polymor-

phism detection systems, inter-simple sequence repeats

(ISSR) has been proven to be simple, efficient and repro-

ducible to detect clonal fidelity (Gantait et al. 2010).

Aranda Wan Chark Kuan ‘Blue’ 9 Vanda coerulea

Grifft. ex. Lindl. (AV) is one of the admired hybrids in the

orchid industries of the tropics. AV is renowned for its

exemplary blooms and has gained exceptional commercial

importance as a cut-flower (Lee et al. 1996). The most effi-

cient way to propagate this hybrid is via in vitro culture, as the

multiplication rate is low through conventional propagation

(Gantait and Sinniah 2012). To facilitate the sustainable in

vitro propagation and distribute this economically viable

hybrid plant material, alginate-encapsulated artificial seed

development and their storage will be a promising approach.

Storage of seeds is greatly influenced by the temperature.

However, the response of synthetic seeds to storage temper-

ature appears to be species specific, responding to either 4 �C

(Saiprasad and Polisetty 2003; Lisek and Olikowska 2004;

Singh et al. 2010; Sharma and Shahzad 2012) or room tem-

perature (Devi et al. 2000; Mohanraj et al. 2009; Hung and

Trueman 2011; Gantait et al. 2012). Interestingly, there are

scanty reports existing on the superiority of temperatures

inbetween the two mentioned.

In view of the importance of the AV orchid, the

advantages of the encapsulation technology and genetic

integrity of regenerants are a major concern, and the

objective of the present study was to develop an encapsu-

lation technique for synthetic seed production in AV using

in vitro shoot tips to ensure a steady supply of uniform

quality propagules. The study assessed the effect of Mu-

rashige and Skoog (1962) (MS) media in different strengths

on the regrowth of encapsulated shoot tips (capsules).

Efforts were also made to test their ability to retain

viability following storage at room as well as low tem-

perature (4 �C) for different durations. Post-storage genetic

uniformity of plantlets from converted (into plantlets with

shoot and root) capsules was analyzed using ISSR markers.

Materials and methods

Plant material

In vitro shoot tips measuring 3–4 mm in length were iso-

lated from 25-day-old in vitro cultures (Fig. 1a) of AV

(Gantait and Sinniah 2012), when these were loosely

connected with each other as well as with the multiple

shoot clusters (Fig. 1b). The shoots were washed with

sterile water and blotted dry on a sterilized filter paper

before subjecting them to encapsulation.

Alginate-encapsulation

Shoot tips (3–4 mm) were suspended in autoclaved low-

viscosity sodium alginate (alginic acid sodium salt from

brown algae) (Sigma, St. Louis, MO, USA) gelling matrix

with concentrations of 1, 2, 3, 4, or 5 % (w/v) containing

half-strength liquid MS medium with 3 % sucrose (w/v),

for 10 min. The variable concentrations of sodium alginate

were tested to check for the optimum concentration

resulting in uniform and steady capsules. To obtain cal-

cium-alginate beads, aliquots of the alginate solution

(c. 0.2 ml), each containing one shoot tip, were aseptically

taken into a 5-mm-diameter Pasteur pipette and gently

dropped individually into 100 ml of 75 mM (w/v) sterile

calcium chloride (CaCl2�2H2O) (Sigma) solution, used as

the complexing agent in a 250-ml beaker. The droplets

containing AV shoot tips were kept in calcium chloride

solution for 30 min with continuous shaking to allow

complete polymerization of sodium alginate. The calcium

chloride solution was then decanted and the alginate-

encapsulated shoot tips (i.e. capsules) (5–6 mm diameter)

were retrieved by washing thrice with sterile-distilled

water. The entire process was carried out in aseptic con-

dition under a laminar air flow chamber.

Evaluation of media for regrowth

The capsules along with non-encapsulated in vitro-derived

shoot tips were transferred into 50-ml culture vials con-

taining 20 ml of one of four culture media for shoot

regrowth. The media treatment consisted of semi-solid full-

strength MS, �MS, MS or �MS, all containing 3 %

(w/v) sucrose. The pH of the medium was adjusted to 5.8

using 0.1 N NaOH and 0.1 N HCl prior to adding 2.5 %

gelrite (Merck). The medium was sterilized at 121 �C at

258 Plant Biotechnol Rep (2013) 7:257–266

123

1 kg cm-2 for 20 min in an autoclave (Hirayama, Japan).

The capsules were incubated at 25 ± 1 �C under a 14-h

photoperiod, which was provided by cool, white fluores-

cent light (Phillips Life Max, Thailand) with an irradiance

of 60 lmol m-2 s-1 photosynthetic photon flux density

(measured with LI-COR, Lincoln, USA). The days taken

for new shoot and root initiation (i.e. conversion), and

conversion percentage were recorded for capsules as well

as non-encapsulated shoot tips, based on daily observa-

tions. An evaluation was made based on these two

parameters to optimize the most favorable media for con-

version of both capsules and non-encapsulated shoot tips.

Storage of capsules

Encapsulated shoot tips were transferred into sterilized

miniature screw-capped polypropylene vials (measuring

5.5 9 1.5 cm), each vial containing five capsules. To

assess the after-storage conversion efficiency of capsules,

they were stored at cold (4 �C) or room temperature

(25 �C) for 40, 80, 120, 160, or 200 days without

illumination.

Assessment on viability of stored capsules

To determine the longevity of stored capsules, samples

were removed from vials sequentially after each storage

period as mentioned earlier and cultured on �MS medium

(optimized conversion medium as determined from first

experiment). Days taken for conversion of capsules and

percent of capsules showing conversion were determined.

Converted plantlets were subsequently transferred for ex

vitro acclimatization in a mixture of perlite and sand (2:1;

v/v) (Gantait and Sinniah 2012).

Fig. 1 Alginate-encapsulation of in vitro-derived Aranda Wan Chark

Kuan ‘Blue’ 9 Vanda coerulea Grifft. ex. Lindl. (AV) shoots.

a Proliferating multiple shoots at 25 days of culture (bar 2 mm),

b separated shoot tips (3 mm) from multiple shoot clusters for

encapsulation (bar 5 mm), c calcium-alginate bead with a AV shoot

tip inside (capsule) (bar 3 mm), d bulk of capsules after immersed in

sterile water (bar 8 mm)

Plant Biotechnol Rep (2013) 7:257–266 259

123

Isolation and quantification of DNA

Genomic DNA isolation was carried out homogenizing

50 mg of tender leaves of each of the ten (five from 4 �C

and five from 25 �C) randomly chosen 10-month-old

ramets (plantlets from converted capsule) and ortet (control),

using DNeasy plant mini kit (Qiagen) and re-suspended

in 50 ll elution buffer. The concentration of DNA was

determined by a UV–vis spectrophotometer (Perkin Elmer,

Germany) and quality of genomic DNA was checked fol-

lowing electrophoresis on 0.8 % agarose gel.

PCR amplification

The 25 ll of optimized polymerase chain reaction (PCR)

mixture contained 40 ng template DNA, 5 ll 19 Taq

polymerase assay buffer, 2 mM MgCl2, 200 lM of each

dNTP, 0.1 lM of each primer and 0.5 U Taq DNA poly-

merase in HPLC grade sterile water. The PCR amplifica-

tion comprised of an initial denaturation at 94 �C for 5 min

followed by 35 cycles of 60 s at 94 �C, 60 s at annealing

temperature and 60 s at 72 �C, and final extension at

72 �C for 7 min, carried out using Eppendorf PCR system

(Eppendorf Mastercycler Gradient, Germany). Overall,

nine ISSR primers (Gantait et al. 2010) were employed

(Table 1). Aliquots of 5.0 ll amplified PCR products,

along with DNA ladder, were resolved by electrophoresis

on 2 % agarose gel in 19 TAE buffer, stained with ethi-

dium bromide (10 lg l-1 TAE buffer).

Scoring of amplified fragments

Gels were scanned with the Bio Imaging System (Syngene,

UK) and analyzed using GeneSnap software. The sizes of

the PCR products were compared to the molecular size

standard 100 bp DNA ladder (Fermentas, ThermoFisher

Scientific, Lithuania). The well-separated and consistently

reproducible, amplified DNA fragments (bands) were

scored as being present or absent for ISSR markers. To

detect the genetic uniformity, the resulting banding pat-

terns were compared between DNA samples for each ISSR

primer. Data were scored as 1 for presence and 0 for

absence of a DNA band in each ramet and oret.

Data analysis

Each experiment was repeated five times with 25 samples

per replication. Completely randomized design (CRD) was

followed for all the experiments. Each shoot tip or capsule

was treated as a single experimental unit. The collected

data were analyzed statistically using one-way analysis of

variance (ANOVA). Treatment data (mean ± SE) were

compared based on Duncan’s multiple range test (DMRT)

(Duncan 1955) at a P value = 0.05 using SAS�v.6.12

(SAS Institute 1999) software package. Percentage values

were transformed using arcsine before ANOVA was car-

ried out and converted back to the original scale (Compton

1994).

Results and discussion

The present study is the first report of synthetic seed pro-

duction using alginate-encapsulation of in vitro AV shoot

tips, and their short-term storage with genetic uniformity

for germplasm swap between laboratories and sharing to

nurseries. Shoot tips are highly meristematic tissues, which

often develop directly into plantlets; hence, the use of shoot

tips as synthetic seeds can be highly advantageous for rapid

plantlet establishment upon short-term storage.

Encapsulation of in vitro shoot tips

Based on different levels of sodium alginate used in the

present study, the capsules exhibited divergence in mor-

phology (clearness, form, and consistency). A gelling

matrix of 3 % sodium alginate with 75 mM CaCl2 solution

provided the best and most uniform capsules. This

Table 1 Inter-simple sequence

repeats primers, used for post-

storage genetic stability

assessment of encapsulated

shoots of Aranda Wan Chark

Kuan ‘Blue’ 9 Vanda coerulea

Grifft. ex. Lindl., their

sequences with number and size

range of amplified fragments

Primer(s) Sequence (50–30) Tm (�C) Number of

scorable

bands/primer

Total number

of bands

Size range (bp)

IS-6 (GA)8C 52 7 77 270–750

IS-7 (GT)8A 50 4 44 530–950

IS-8 (AG)8C 52 4 44 180–1,350

IS-9 (TG)7T 46 7 77 220–1,200

IS-11 (CA)8G 52 7 77 340–1,550

IS-12 (GT)8C 52 5 55 430–1,330

IS-61 (GA)8T 50 8 88 280–1,640

IS-63 (AG)8C 52 5 55 150–1,450

IS-65 (AG)8T 50 4 44 230–1,700

260 Plant Biotechnol Rep (2013) 7:257–266

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formulation permitted us to attain more or less sphere-

shaped beads (Fig. 1c) disseminated like a monodisperse

population. The average size of beads achieved as a result

of this practice was 5 mm in diameter (Fig. 1d). Applica-

tion of calcium alginate encapsulation matrix for storage of

in vitro shoot-derived synthetic seeds was described earlier

by Pattnaik and Chand (2000) in the Spathoglottis plicata

orchid. Most recently, Faisal et al. (2012) and Mehrotra

et al. (2012) also employed alginate matrix to encapsulate

Rauvolfia serpentina and Glycyrrhiza glabra shoot tips for

temporary storage. The use of 3 % solution of sodium

alginate and 75 mM CaCl2 in this study facilitated opti-

mum ion exchange amid Ca2? and Na?, producing isodi-

ametric, clear and compact capsules. Lower levels (1–2 %)

of sodium alginate were not suitable since the capsules

were of irregular shape and extremely squashy to grip,

whereas the capsules were stiff, resulting in extensive

setback in shoot emergence (data not presented) at higher

concentrations (4 and 5 %) of sodium alginate. Similar

results were also reported by Lata et al. (2009) in Cannabis

sativa L. and Singh et al. (2009) in Spilanthes acmella (L.)

Murr. during their assessment on influential role of sodium

alginate concentrations on capsule-matrix quality and

ensuing conversion. As reported by Nagesh et al. (2009) in

Curculigo orchioides Gaertn., lower levels of sodium

alginate have the potential to result in CaCl2 toxicity (due

to the large quantity of absorption for an extended period)

and abridged resistance of capsules. In contrast, the higher

resistance capsules formed eventually absorb a lesser

quantity of CaCl2 owing to higher levels of sodium alginate

(3 %). Hence, CaCl2 toxicity could be less, resulting in

higher frequency recovery.

Influence of MS media strength on conversion

of capsule

The MS media strength gave different responses in relation

to conversion of encapsulated and non-encapsulated shoot

tips. Irrespective of MS media strength, conversion of

capsules as well as non-encapsulated shoots was observed

within 1 week of culture (Fig. 2), but the percentage of

conversion differed. For non-encapsulated shoot tips, full-

strength MS was proven to be the best, whereas reduction

of MS strength resulted in reduced plantlet conversion

percentage. But, during conversion of capsules, an opposite

trend was observed. The maximum conversion (99.6 %) of

capsules was achieved using �-strength MS medium, with

complete conversion into plantlets taking place following

4 weeks of culture, whereas the lowest conversion

(86.6 %) was recorded in full-strength MS. This result

clearly shows the importance of full-strength MS on con-

version of shoot tips. According to Ara et al. (2000), the

existence of nutrients (MS medium) in the gel matrix

actually works like a nutrient bed around the shoots, hence

influencing conversion. Bajaj (1995) also stated that the

nonflexible configuration and outsized pores of these

water-insoluble gels make them competent for encapsula-

tion of live plant cells, as they permit the swap of sub-

stances to and from the adjoining medium. Based on this,

shoot tips embedded in high MS gelling matrix probably

received a double dose of MS strength as both the gelling

matrix as well as the regrowth media had full-strength MS,

while the superlative performance observed in �MS media

in comparison to the three additional higher strengths is

seemingly attributable to the optimal total concentration of

nutrients in the alginate gel matrix (prepared in �MS

medium) as well as regrowth substrate (�MS medium).

The additional three media might be disadvantageous

owing to an overdose of the nutrient element content

resulting in nutrient toxicity.

0

1

2

3

4

5

6

7

8

9

Full MS ½ MS MS ¼ MS

Day

s to

con

vers

ion

Media strength

Encapsulated shoot tips Non-encapsulated shoot tips

a

b b

ba a

aa

a

75

80

85

90

95

100

105

Full MS ½ MS MS ¼ MS

Con

vers

ion

(%)

Media strength

Encapsulated shoot tips Non-encapsulated shoot tips

d

a

c c

b

d

a

d

b

Fig. 2 Influence of MS media strength on a days to conversion and

b conversion % of encapsulated (capsules) as well as non-encapsu-

lated shoot tips of Aranda Wan Chark Kuan ‘Blue’ 9 Vanda

coerulea Grifft. ex. Lindl. Data represent mean ± standard error of

25 replicate explants per treatment in five repeated experiments. Data

expressed as percentages were transformed using arcsine prior to

ANOVA and converted back to the original scale for demonstration in

the table (Compton 1994). Data for each column followed by different

alphabets are significantly different according to Duncan’s multiple

range test (Duncan 1955) at P = 0.05

Plant Biotechnol Rep (2013) 7:257–266 261

123

The accessibility to nutrients could be the chief limiting

factor for importunate growth of the encapsulated plant

tissue into complete plantlets. Therefore, assembling a

nutrient pool, either endogenously or exogenously, for the

encapsulated plant tissue is indispensable to facilitate the

advanced regrowth frequency (Gantait et al. 2012). In our

study, �MS and �MS media served the nutrient require-

ment for both the inside and exterior parts of capsules quite

effectively.

Influence of storage temperature and duration

on conversion of capsule

The present study has revealed that storage at room tem-

perature (25 �C) proved to have potential for conversion of

capsules. Figure 3 compares the conversion proficiency of

capsules stored at 4 and 25 �C for 40, 80, 120, 160, and

200 days of storage. The percentage of capsules converting

into plantlets declined progressively with the rise in the

duration of storage, both at 4 and 25 �C, with capsules

stored at 25 �C being significantly more (P = 0.05) resis-

tant compared to those stored in 4 �C (Figs. 3b, 4a). With

the progression of storage period up to 120 days at 4 �C, a

mere 23.4 % conversion (Fig 3a), initiated at *24 days of

transfer (Fig. 3b), was achieved, with most of the capsules

turning necrotic, shrunken and brown (Fig. 4b i), resulting

in complete death within 160–200 days of storage. Con-

trastingly, capsules stored at 25 �C were observed to be

green, with regrowth potential and undamaged bead con-

sistency (Fig. 4b ii). These capsules recorded 71.6 %

conversion even at 200 days of storage. Nevertheless, a

delay in the time taken for conversion to occur with

increased storage time was the only setback (for example,

at 200 days of storage, conversion occurred at 15.6 days in

comparison to 7.3 days after 40 days of storage) (Fig. 3a).

For capsules stored at 25 �C for 40 days, the first plantlet

conversion was observed at 1 week (7.3 days) of inocula-

tion in regrowth media (i.e. �MS), while capsules stored at

4 �C for the same time period took 11.3 days to germinate.

The merit of 25 �C storage over 4 �C for encapsulated

shoot tips in the present study corresponds to the earlier

report of Hung and Trueman (2011) on Khaya senegalensis

shoot tips, where they also highlighted that the ease of

storage at 25 �C is highly encouraging for germplasm

exchange, because capsules can be maintained under lab-

oratory conditions and then dispatched and processed

conveniently. In compliance with our study, other

researchers have also reported that explants take time to

recover from cold storage stress after returning to prolif-

erating culture conditions (Ballester et al. 1997). The

increased time required for regeneration might be related to

the restricted oxygen supply in the air-tight vials with

inadequate air space (Fig. 4a) during extended storage.

This opinion corresponds to the former study of Gantait

et al. (2012) who reported that the decline in conversion

into plantlets of the stored encapsulated embryos could

presumably be allied to deficiency of oxygen in the gel

bead.

In the current study, the failure of extended storage at

4 �C corresponds to the previous reports, where, for low

temperature (4 �C) storage, the storage life of encapsulated

AV was relatively short (Gantait et al. 2012), likewise the

conversion rate of encapsulated embryos of Asparagus

cooperi (Ghosh and Sen 1994) was reported to be low.

Similarly, the regrowth of encapsulated nodal segments of

Punica granatum L. also showed prominent decline fol-

lowing storage at low temperature (Naik and Chand 2006).

In recent years, Singh et al. (2010) and Sharma and

Shahzad (2012) reported merely 60 days storage (at 4 �C)

0

5

10

15

20

25

30

35

40

40 80 120 160 200

Day

s to

con

vers

ion

Storage duration (days)After 4°C storage After 25°C storage

e

g

c

f

b

e

a

dcd

na

a

0

10

20

30

40

50

60

70

80

90

100

40 80 120 160 200

Con

vers

ion

(%)

Storage duration (days)After 4°C storage After 25°C storage

g

a

h

b

i

c

j

de

na

b

Fig. 3 Influence of storage temperature and duration on a days to

conversion and b conversion % of encapsulated shoot tips (capsules)

of Aranda Wan Chark Kuan ‘Blue’ 9 Vanda coerulea Grifft. ex.

Lindl. (growth period of 35 days). Data represent mean ± standard

error of 25 replicate explants per treatment in five repeated

experiments. Data expressed as percentages were transformed using

arcsine prior to ANOVA and converted back to the original scale for

demonstration in the table (Compton 1994). Data for each column

followed by different alphabets are significantly different according

to Duncan’s multiple range test (Duncan 1955) at P = 0.05. na not

applicable due to non-viable capsule

262 Plant Biotechnol Rep (2013) 7:257–266

123

of encapsulated nodal segments in Eclipta alba (L.) and

Decalepis hamiltonii Wight and Arn, respectively. Storage

at room temperature (25 �C) implemented in this study was

effective for short-term storage, and handling without

refrigerated containers, and even storage up to 200 days

gave considerable conversion (71.6 %) in AV. The con-

version of capsule into complete plantlets commence with

breakage of the capsule wall by emerging of shoot pri-

mordia (Fig. 4c) followed by root development. Eventu-

ally, AV plantlets having more than three shoots and roots,

developed from a single capsule (Fig. 4d) within 32 days

of capsule inoculation in regrowth medium. Randomly

selected plantlets, relocated for ex vitro acclimatization,

were readily acclimatized and established in perlite plus

sand (2:1; v/v) in 45 days, showing 88 % success (data not

presented). As a matter of fact, the resulting shoots were

vigorous, green, and devoid of any morphological disorder.

Another vital consequence of this investigation was the

consistent emergence of uniform shoots of more or less

similar length from the capsules during the process of

conversion. From the success of the study at hand, it

appears that the storage of capsules in air-tight polypro-

pylene vials (though having a restricted supply of oxygen

for respiration) proved to be suitable to protect loss of

humidity which appears to be hugely essential for retention

of viability of the encapsulated shoots, since sodium algi-

nate has been reported to succumb to rapid dehydration,

and their high porosity can also result in marked cell and

nutrient leakage which may further result in non-viable

capsules.

Fig. 4 Short-term storage and conversion of in vitro-derived encap-

sulated shoot tips (capsules) of Aranda Wan Chark Kuan ‘Blue’ 9

Vanda coerulea Grifft. ex. Lindl. a Storage of capsules (at 4 and

25 �C) within polypropylene vials, each containing five capsules and

rest empty space (bar 10 mm), b comparison of capsules after

120 days of storage; i capsule stored in 4 �C showing necrosis, ii

capsule stored in 25 �C showing fresh green morphology (bar 7 mm),

c capsules on �MS medium showing initiation of new shoot

primordia from capsule after 3 days of transfer (bar 5 mm),

d conversion of capsule into plantlets with multiple shoots (Sht)

and roots (Rt) after 32 days of transfer (bar 15 mm)

Plant Biotechnol Rep (2013) 7:257–266 263

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According to Danso and Ford-Lloyd (2003), the decline

in conversion as the result of prolonged storage (e.g.,

200 days in the present study) could be due to inhibited

respiration of tissues. Deterioration at the time of storage is

a process which cannot be evaded, but can effectively be

slowed down if the storage environment is conducive. The

hindrance behind the process of delayed conversion

observed in this study is related to a certain degree of

deterioration that may have occurred.

For the storage and on-demand supply of plant materials

for propagation or germplasm exchange, encapsulation of

AV shoots appears to be a promising tool. Previously, in

many other endemic and endangered orchids, like Renan-

thera imschootiana (Chetia et al. 1998), Geodorum densi-

florum (Datta et al. 1999), and Ipsea malabarica (Martin

2003), the similar usage of the encapsulation technique for

storage has also been reported. According to Rai et al.

(2008), a significant feature of the encapsulated vegetative

propagules is their ability to retain viability after storage

for a sufficient amount of time required for the exchange of

germplasm.

Genetic uniformity of the germinated capsules

Nine ISSR primers were successfully employed to assess

the trueness where the ramets showed no discernible dif-

ferences among them and as compared to the oret in the

ISSR analysis. A total number of 51 reproducible mono-

morphic bands were scored per clone using nine primers.

Finally, 561 bands from all the ramets and orets under

assessment revealed no variation. Each primer amplified

these distinct bands with molecular size, ranging between

150 bp in IS-63 to 1,700 bp in IS-65 (Table 1) with an

average of *5.5 bands per primer. As no ISSR polymor-

phism was observed among oret and the plantlets regen-

erated from synthetic seeds (ramets) after 200 days of

storage at 4 and 25 �C storage conditions (Fig. 5), it was

proven that the uniformity of the regenerated capsules was

maintained, indicating high genetic stability among the

clones. This study is of particularly great significance as

these clones are uniform. We suggest that this high-value

economically important orchid hybrid can be conserved up

to 200 days without the risk of genetic instability.

Fig. 5 Agarose gel

electrophoresis of amplified

ISSR fragments of post-storage

capsule-regenerated clones

(ramets) (1–5 at 4 �C and 6–10

at 25 �C) of Aranda Wan Chark

Kuan ‘Blue’ 9 Vanda coerulea

Grifft. ex. Lindl. and the control

(oret) (lane P) showing

monomorphic bands generated

by a primer IS-6 and b primer

IS-11. Lane M 100-bp ladder

264 Plant Biotechnol Rep (2013) 7:257–266

123

The observed normality and homogeneity of the plants

generated in this study, strongly suggest that the encapsu-

lation of shoot tips (using as synthetic seed) is a reliable

approach for storage of AV. Our results substantiate reports

of genetic stability of encapsulated shoot tip-regenerated

(medicinal) plants of Eclipta alba (Ray and Bhattacharya

2010), Picrorhiza kurrooa (Mishra et al. 2011), Bacopa

monnieri (L.) (Ramesh et al. 2011), Cannabis sativa (Lata

et al. 2011), and Glycyrrhiza glabra (Mehrotra et al. 2012)

after short-term storage periods. However, it is well doc-

umented that direct regeneration from organized tissue like

the meristem reduces the chances of genetic integrity loss.

In vitro culture represents a physiological stress that is

characterized by disruption of normal developmental con-

trols (Cassels and Curry 2001), which can ultimately lead

to various kinds of aberrations at the nucleotide sequence

level. However, in vitro storage of explants, specifically

shoot tips, presumably did not affect the genetic integrity at

this stage (e.g., up to 200 days in the present study); nev-

ertheless, it needs further confirmation. Very few scientists

have assessed genetic stability of, in particular, capsule-

derived plantlets, whereas there has so far been no such

report on orchids until our present study.

Conclusion

This report provides information on the successful use of

alginate encapsulation and synthetic seed production of AV

in vitro shoots and their storage. The synthetic seed

development protocol illustrated here offers a substitute

scheme for mass propagation and germplasm distribution

of this important orchid hybrid to laboratories and exten-

sion centers in distant places. Exultant plant recovery from

encapsulated shoot tips following room temperature stor-

age for more than 200 days indicates that the technique

described in this report can be potentially exploited for

short-term storage with the retention of genetic uniformity.

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