Pak. J. Bot., 48(2): 693-697, 2016.

A STUDY ON THE ISOLATION OF PROTOPLASTS FROM MESOPHYLL

CELLS OF DENDROBIUM QUEEN PINK
RAMSHA AQEEL1,2, MARIUM ZEHRA1,2, SYEDA KAHKASHAN KAZMI2, SAIFULLAH KHAN2,3*,
HAMMAD AFZAL KAYANI2,4 AND AMEER AHMED MIRBAHAR2
1
Department of Biotechnology, University of Karachi, Karachi, Pakistan
2
Biotechnology Wing, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi, Pakistan
International Center for Chemical and Biological Sciences (ICCBS), University of Karachi-75270, Pakistan
3
Department of Agriculture and Agribusiness Management, University of Karachi, Karachi, Pakistan
4
Shaheed Zulfiqar Ali Bhutto Institute of Science and Technology, Pakistan
*Corresponding author’s e-mail: drsaif65@gmail.com.

Abstract

Protoplasts were successfully isolated from one month old In vitro grown plantlets of Dendrobium cultivar Queen pink.
The enzyme solution used was composed of 1% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.1% Pectinase, 0.3 M
mannitol, 10 mM CaCl2.2H2O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Protoplast highest yield with
15.7x104 protoplasts per 1.5 gm freshly chopped leaves were obtained when digested in enzyme solution for 4 hrs on a rotary
shaker with an agitation speed of 45 rpm in dark conditions. Protoplasts were filtered with 45µm nylon sieve and washed with
0.3 M mannitol solution supplemented with 10 mM CaCl2.2H2O and 10 mM MES, and purified with 0.3 M sucrose solution
gradient. Purification of protoplasts on a sucrose mannitol gradient yielded clean protoplasts that were free from debris.

Key words: Protoplast isolation protocol, Dendrobium Queen Pink, Protoplast yield, Protoplast viability.

Introduction Protoplast isolation and somatic hybridization can

help in plants genetic improvement, to develop new
Orchidaceae family has more than 1000 genera and flower colors, pathogen resistance, somaclonal variation,
approximately 22,000 species (Dodson, 2016). Orchids functional genomics, genetic transformation, conservation
occur throughout the world, they are abundant in tropical biotechnology and pharmaceutical products. Protoplast
regions of America, Africa and Asia, as these are the main technology is also used to produce top quality flowering
regions where orchids are cultivated on large scale. ornamental plants (Musharof et al., 2013). In the family
Orchids are most remarkable due to their diversity and Orchidaceae, studies on protoplast started in 1978 and
floral structure. Most of orchid varieties are epiphytic, first isolation of protoplasts was reported in Renentanda
they use rocks or static objects for support and derive from protocorms (Teo & Neumann, 1978a), from leaves
their nutrients and water from the atmosphere and debris,
of Cattleya and Phalaenopsis (Sajise & Sagawa, 1991).
and however some species grow in the ground, under
Seedlings of Dendrobium and Paphiopedilum were used
forest or grassland areas (Robert & Calaway, 1960).
Orchidaceae has gained some special significance for protoplast isolation by (Teo & Neumann 1978 b; Price
among other cut flower families in recent times from & Earle, 1984) and in Cymibidium (Oshiro & Steinhart,
horticultural point of view. Dendrobium cultivars are 1991) protoplast of Aranda was isolated by (Loh & Rao,
available in all seasons throughout the year especially 1985; Koh et al., 1988; Kanchanapoom & Tongseedam,
autumn because it takes relatively lesser time for its growth. 1994). However, successful colony formation of
Dendrobiums has great economic value and are protoplast was reported in Dendrobium cultivars by
commercially profitable flowers in the world which is (Kuehnle & Nan, 1990). Successful protoplast isolation
suitable for making bouquets and flower arrangements was also achieved in other ornamental plants like from
because of their vibrant colors like yellow, purple, white and callus of Lilium longiflorum overig by (Yousuf et al.,
pink due to its long vase life (Yuphin et al., 2006). Orchids 2015). Protoplast technology has been growing over time
are not only ornamental plants but also serve as a source of and carries great significance and attention, it has
traditional medicines. Boiled bulbs of Bletia purpurea and its achieved great progress. Genetic manipulation through
liquid helps to treat the poisoning caused by fish. Similarly, protoplast technology like somatic hybridization,
Spiranthes diuretica is thought to be a strong diuretic in cybridization or direct gene transfer can be exploited for
Chile (Tzi et al., 2012; Bulpitt et al., 2007). Dendrobium plant improvement if a reliable and efficient plant
formosum has an amazing ability to fight tumors’ (Prasad & regeneration system for isolated protoplast can be
Koch, 2014). Orchids not only serve as medicines but also developed (Papadakis & Roublakis, 2002).
provide food or used as food supplements, like Dendrobium Many outstanding Dendrobium intrageneric and
salaccense leaves has been eaten with rice. Tubers of some interspecific hybrid cultivars had developed from
species of Gastonia are used like a potato in some regions of conventional breeding methods and through somaclonal
Asia and orchids also provide an alternate use of glue from variations resulting from tissue culture techniques.
pseudo bulbs (Dodson, 2016). Among various cultivars of Somatic hybridization through protoplast fusion allows
Dendrobium, Queen pink cultivar has widely been used for the hybridization of different sexually incompatible
ornamental purpose due to its bright pink color of ever genera and species (Davey et al., 2005). The regenerative
blooming flowers. plantlets brought to existence with protoplast culture
694 RAMSHA AQEEL ET AL.,

through an efficient method and have great potential as a protoplast fraction was formed at interphase of washing
biotechnological tool for orchid advancement. The aim of solution and sucrose solution, then gently removed with a
this research was to optimize an efficient protocol for pasture pipette. The purified protoplasts were then washed
isolation of viable protoplasts from leaves of In vitro twice with washing solution and separated by
grown plantlets of Queen pink cultivar of Dendrobium. centrifugation at 750 rpm for 4mins and resuspended in
1.5ml washing solution (Fig. 2D).
Materials and Methods
Determination of the Yield of protoplasts: The yield of
Source of plant material: Dendrobium Queen pink pods the protoplast was counted using haemocytometer under a
were grown in the greenhouse of H.E.J. Biotechnology compound microscope (Nikon ECLIPSE TE 300, Japan).
wing through artificial pollination. These pods were surface The protoplasts were viewed at 40× magnification and the
sterilized with flame and inside seeds were exposed number of protoplasts observed was recorded. Total
through longitudinal cutting, and placed on MS medium. protoplast yield was calculated using the equation below:
Orchid seeds were successfully germinated in growth room
Total cell count × Total vol. of cell suspension
under 25±2oC with a 16/8 hrs photoperiod. Leaves of these Protoplast yield =
4 × Weight of fresh tissues (g)
In vitro grown orchid plantlets were used as a source of
explants for protoplast isolation and culture (Fig. 2A). Viability test: The protoplasts viability was examined by
adding 50ul protoplasts suspended in washing solution
Explants preparation prior to enzyme exposure: In and then mixing with 25ul FDA (Fluorescein diacetate)
vitro raised leaves of Dendrobium Queen pink were stock solution prepared per (Nadel, 1989). Viable cells
excised and chopped transversely into fine pieces with were observed under a fluorescence microscope (Nikon
sharp scalpel for protoplast isolation (Fig. 2B). All TE 2000 E, Japan).
chopped leaves were weighed in sterilized Erlenmeyer
flask under laminar flow hood for fresh weight Protoplast culture for regeneration: Each sample of
determination. Four different measurements i.e., (0.5gm, purified protoplasts was washed thrice with modified
1.0 gm, 1.5 gm and 2.0 gm) were used to treat them with K&M media (Kao & Michayluk, 1975) excluding nucleic
the same volume and concentration of enzyme. acids and amino acids except glutamine with pH adjusted
to 5.7. About 2.6ml of fresh K&M medium supplemented
Effect of Incubation time on enzyme solution: with growth regulators 0.5 mg/l NAA, 0.4mg/l BAP and
Appropriate time needed to isolate more viable 0.5mg/l zeatin (Kunasakdakul & Smitamana, 2003) were
protoplasts from chopped leaves of Dendrobium Queen added into each test tube. Protoplasts were taken from test
pink was very important. Viable protoplast with high yield tubes and transferred onto 6 cm Petri dishes. Petri plates
was determined with 0 hrs, 2 hrs, 4 hrs and 6 hrs were sealed with parafilm and incubated in dark at 25°C.
incubation period. The percentage of dividing protoplasts was monitored
after 1 week of culture.
Protoplast isolation: The protoplast isolation procedure
was performed under aseptic conditions inside the laminar Results
air flow cabinet. Protoplast isolation solution consists of
cell wall digesting enzymes (1% Cellulase Onozuka R-10 Effect of amount of explants on yield: The chopped
(Phyto technology laboratories), 0.5% Macerozyme leaves of Dendrobium Queen pink gave high yield as well
Onozuka R-10 (Serva) and 0.1% Pectinase Onozuka R-10 as good strength of viable protoplasts at the amount of
(Sigma Aldrich) along with the osmotic or washing 1.5gm leaves (Fig. 3C&G). In 1.5gm of leaves, protoplast
solution with (0.3 M mannitol, 10 mM CaCl2.2H2O and yield was 15.7 × 104 protoplasts/g FW and showed high
10 mM MES, pH 5.8) which were dissolved in distilled viability (Fig. 1A). On the other hand leaves with 0.5gm,
water. The protoplast isolation solution 8ml was added to 1.0gm and 2.0gm in amount resulted in low yield and
fresh weight samples of explants, then enzyme-leaves poor viability as compared with 1.5gm of chopped leaves
as mentioned in (Fig. 3A, B&D) and (Fig. 3E, F&H).
mixture was incubated on rotary shaker at 45 rpm in dark
at 25ºC for 4 hours to facilitate the liberation of
Effect of incubation time on yield: The best incubation
protoplasts (Fig. 2C).
time for protoplast isolation from Dendrobium cultivar
Queen pink in vitro leaves was 4 hrs, at this incubation
Protoplast purification: After incubation, suspension of
time more viable protoplasts with high yield were
digested tissues in the isolation mixture was filtered
obtained (Fig. 4C&G). Protoplast yield in 4 hrs enzyme
through a 45µm nylon mesh sieve and the filtrate was incubation time was 15.7×104 protoplasts/g FW as shown
centrifuged at 750rpm for 5min in a swing bucket in (Fig. 1B). In 6 hrs incubation time protoplast yield was
centrifuge (Anke, China). The supernatant was removed 3.6×104 protoplasts/g FW, large size protoplast with more
using pasture pipette. The pellet of protoplasts was debris were obtained (Fig. 4D&H). This was due to high
washed twice with washing solution through enzymatic treatment time as more digestion of cells
centrifugation at 750 rpm for 4mins to remove all traces occurred so more debris were isolated with less viable and
of enzyme solution. Finally, the protoplast pellet was low yield protoplasts (Fig. 4D). After 2 hrs of incubation,
transferred to the 10ml screw capped centrifuge tube very less viable cells with very low yield were obtained
containing 4ml of 0.3M Sucrose solution and then i.e. 1.0×104 protoplasts/g FW (Fig. 4B&F).
centrifuged at 300 rpm for 4 mins. The floating viable
PROTOPLASTS ISOLATION PROTOCOL OF D.QEEN PINK 695

Fig. 1A. Effects of different amount of explants on protoplast Fig. 1B. Effect of incubation time on protoplast isolation of
isolation of Dendrobium Queen pink. Dendrobium Queen pink.

A B C D
Fig. 2. A. In vitro grown plantlets of Dendrobium cultivar Queen pink. B. Chopped leaves. C. Incubated chopped leaves in enzyme
solution. D. Isolated protoplasts in a test tube.

A B C D

E F G H

Fig. 3. A. Protoplast viability in 0.5gms of leaves. B. Protoplast viability in 1.0gms of leaves. C. Protoplast viability in 1.5gms of
leaves. D. Protoplast viability in 2.0gms of leaves. E. Protoplast yield in 0.5gms leaves under bright field. F. Protoplast yield in
1.0gms of leaves under bright field. G. Protoplast yield in 1.5gms of leaves under bright field. H. Protoplast yield in 2gms of leaves
under bright field.
696 RAMSHA AQEEL ET AL.,

A B C D

E F G H

Fig. 4. A. 0hrs yield of viable protoplasts. B. 2hrs yield of viable protoplasts. C. 4hrs yield of viable protoplasts. D. 6hrs yield of
viable protoplasts. E. 0hrs yield of protoplast under bright field. F. 2hrs yield of protoplasts under bright field. G. 4hrs yield of
protoplasts under bright field. H.6hrs yield of protoplasts under bright field.

Discussion yield and viability. Similarly 0.3g leaves of Dendrobium

pompadour was incubated in 5ml enzyme solution (1%
(a) Effect of amount of explants on protoplast yield cellulase, 0.5% driselase, 1% macerozyme) provided best
and viability: The successful isolation of protoplasts yield of viable protoplasts reported by (Kanchanapoom et
depends on many factors; it is also related to the source al., 2001). Also highest viable protoplast yield was
plant and the conditions of the applied methods. The achieved when 0.5g leaves of Dendrobium crumenatum
isolation conditions are extremely important for the were used (Chong et al., 2010). (Yuphin et al., 2006)
release of protoplasts, without losing cell viability and to reported that 1.0g leaves of Dendrobium sonia gave
get maximum yield. The result of this study on highest viability as well as yield.
Dendrobium cultivar Queen pink demonstrated that the
protoplast yield and viability also influenced by the (b) Effects of incubation time on protoplast yield and
amount of explants i.e., leaves used. According to (Pindel, viability: To determine the suitable duration required for
2007) mesophyll tissues of cymbidium are good source obtaining the highest yield of protoplasts, the explants
material for protoplast isolation and culture. were incubated with enzymes for different incubation
We used different amount of explants i.e., 0.5gm, periods like 0, 2, 4 and 6 hrs (Fig. 4). The low yield of
1.0gm, 1.5gm and 2.0gm leaves of In vitro grown viable protoplasts was achieved after 0 hr and 2 hrs of
Dendrobium cultivar Queen pink plantlets (Fig. 2). incubation; protoplasts were smaller in size and oval in
Among these, the highest yield of 15.7×104 protoplasts/g shape (Fig. 4A&B). In 0 hrs and 2 hrs incubation time cell
FW with greater viability was achieved when leaves of wall was removed, it might be due to osmotic stress
1.5g were used as shown in (Fig. 3C&G). because this was insufficient time for enzymes to release
(Prasertsongskun, 2004) found that a low protoplast yield large number of protoplasts (Fig. 4E&F).
was obtained in the absence of pectinase. This showed The yield of protoplasts was increased when the
that the presence of pectinase was essential to increase incubation time extends to 4 hrs which is 15.7×104
protoplast yield. protoplasts/g FW (Fig. 1B). Protoplasts appeared in proper
The freshly isolated protoplasts were of spherical shape and size and the organelles had seen to be aggregated
shapes with different sizes and appeared yellow green in at a point within the membrane (Fig. 4G). When incubation
FDA staining. In addition, protoplasts derived from time was increased up to 6 hrs, yield and viability of
Dendrobium mesophyll cells were stable after staining protoplasts decreased and protoplasts were observed large
(Fig. 3). According to (Pindel, 2007) 0.3g of Cymbidium in size with more debris, this is because 6 hrs was more
was incubated in 10 ml enzyme solution (1.2% than enough time for enzymes to digest whole cell wall
macerozyme and 0.5% pectinase) they had given highest (Fig. 4H), it caused protoplasts to burst.
PROTOPLASTS ISOLATION PROTOCOL OF D.QEEN PINK 697

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(Received for publication 24 February 2015)