69 A dvances in Environmental Biology, 3(1): 69-83, 2009 ISSN 1995-0756

69
A dvances in Environmental Biology, 3(1): 69-83, 2009
ISSN 1995-0756
© 2009, A merican-Euras ian Network for Scientific Information
T his is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Optimisation of Encapsulation-dehydration Protocol for the Orchid Hybrid Ascocenda
‘Princess Mikasa’
1, 2
Ranjetta Poobathy, 2 Helen Nair and 1Sreeramanan Subramaniam
1
School of Biological Sciences, Universiti Sains Malaysia (USM) , M i n d e n Heights, 11800, Penang,
Malaysia, 2 Department of Biotechnology, AIMST University, Batu 3½ , J a l a n B u k i t Air Nasi, Bedong
, 08100, Kedah, Malaysia,
Ranjetta Poobathy, Optimis ation of Encaps ulation-dehydration Protocol for t h e O rchid Hybrid
Ascocenda ‘Princes s M ikas a’, Am.-Eurasian J. Sustain. Agric., 3(1): 69-83, 2009
ABS TRACT
Ascocenda ‘P rin ces s M ikas a’ is a commercially important orchid hybrid. Long-term s torage of its
germplas m through cryos torage is a promis ing option of propagating clonal plants of this hybrid. In this s tudy,
protocorm like-bodies (PLBs ) of the orchid pretreated with s ucros e or s orbitol with an encaps ulationdehydration technique w e re c ryopres erved for 24 hours , prior to monitoring recovery through viability
obs ervations and the 2,3,5-triphenyltetrazo liu m c h lo rid e (TTC) as s ay. PLB s izes of 3 mm and 6 mm and
varying concentrations of s ucros e and s orbitol were tes ted to determine the bes t conditions for the method. The
viability of the PLBs after the cryopres ervation w e re d e t e rmined after a two-day dark and five-day light
recovery period in Part I of the experiment, and after a one-wee k d a rk re c overy period in Part II of the
experiment, conducted on defined culture media. It was fo u nd that the bes t PLB s ize for cryopres ervation was
6 mm, and th a t 0.50 M s ucros e or 0.25 M s orbitol pretreatments gave the highes t viabilities after the
cryopres ervation protocol. The bes t encaps ulated-dehydrated beads retained 40% of its water conten t d u ring
cryos torage. It appeared that either s ucros e or s orbitol, at appropriate concentrations , were e qually good as
pretreatments to ens ure viable PLBs were recovered after cryopres ervation.
Key words: Encaps ulation-dehydration . protocorm like-b odies (PLBs ). Ascocenda ‘Princes s M ikas a’ orchid .
2,3,5-triphenyltetrazolium chloride (TTC)
Introduction
The family Orchidaceae is clas s ified as one o f
the larges t and mos t divers e gro up of plants ,
containing almos t 20,000 to 25,000 s pec ies [46].
O v er 100,000 commercial hybrids are regis tere d
pres ently, mos t of th e m grown as cut flowers and
p otted plants [83]. A range of attractive hybrid s ,
varie ties or cultivars of s ympodial orchids , for
ins tance, the genus Oncidium or As cocenda, is
important in the cut-flower and potted-plant indus tries
[10]. Their popularit y is a t t rib uted to their
bewildering colour s chemes , s hapes and s izes , bloom
pers is tence, and their ability to travel long dis tances ,
hence their pos ition as one of the top 10 cut flowers
in the international market [46].
The orchid hybrid, Ascocenda ‘Princes s M ikas a’,
is a ma n -made epiphyte derive d fro m t h e
hybridization b etween the genera Ascocentrum and
Vanda (Fig. 1). This flower is highly des irable due
to the combination of traits expres s ed by the parents :
large flower s ize from Vanda, which is cons idered as
a promis ing progenitor for s y nthes izing a variety of
cut flower hybrids and the inte re s ting colour
combinations from Ascocentrum, a ls o cons idered as
an important parent in the production of miniature
Vanda hybrids . The hybrid plant pos s es s es upright
and narrow oviform le aves , with inflores cences
Corres ponding Author
Sreeramanan Subramaniam, School of Biological Sciences, Universiti Sains M alaysia (USM ),
M inden Heights, 11800, Penang, M alaysia.;Tel:604-6533528 Fax:604-6565125
E-mail:[email protected] / [email protected]
Adv. Environ. Biol., 3(1): 69-83, 2009
occuring twice o r t hrice a year. Jus t like the parents ,
this h y b rid is able to proliferate in warm, s unny
areas and in tropical climates , es pecially in T h a iland,
Philippines , India and M yanmar [36].
Germplas m pres ervation is important to s afeguard
biodivers ity and to s tore elite plants , the latter
important to dev e lo p and maintain new cultivars [7].
Important crops have been pres erved for long periods
through the es tablis hment of either field or in vitro
gene banks [17]. In vitro germination of orchids has
been long es t a b lis hed among orchid growers , and
mos t o rc h id s e e d s a re u s u a lly germinated
immediately a fter harves t from the parent plant, or
s tored for later propagation [83]. The s torage of
s eeds , a method of ex situ cons ervation, h a s always
been a favourit e among breeders , as this method is
cons idered as one of t h e bes t ways to pres erve
valuable genetic res ources [28], es pecially for s pecies
with limited reproductive capabilities . Orc h id s eeds
are us ually s tored at low temperatures for this
purpos e [28].
Thus , the develop me n t of a long-term
pres ervation method for embryos or protocorms is
important in the cons ervation o f t he orchid
germplas m, breeding programs , a n d the orchid
floricultural indus try [31]. Cryopres ervation has been
des cribed as the mos t valuable method us ed for longterm germplas m cons e rvation, as cryopres erved
materials re q u ire v e ry limit e d s p a c e , low
maintenance, and are protected from contamination
[66]. Cryopres ervation arres ts meta b o lic a n d
biochemical proces s es of cells and caus es energ y to
be un a vailable for kinetic or dynamic reactions ,
hence halting normal c e ll divis ion and growth
[77,21]. Thus , the explant c a n b e s tored without any
deterio ra tion or modification for unlimited periods
[1,38] as its gene t ic s tability and regeneration
potential is maintained [4,61,52]. Cry opres ervation
involves cryogenic (c ry o p ro t e c t a n t a n d low
temperature trea t ments ) and non-cryogenic (pre- and
pos t-s torage culture) protocols . The s ucces s of the
technique depends on germpla s m tolerance and
s ens itivity to the s tres s es incurred and accumu la t ed
at each s tage of the cryopres ervation procedure. The
bas ic cryopres ervation protoc o l in v o lv e s the
applic a tion of cryoprotectants and treatments prior
and s ubs equent to freezing, to protect and recover the
g e rmplas m material during and after s torage in liquid
nitrogen [85]. There mus t be minimized levels of
crys tallis able water within the plant material to
e n s u re high recovery percentages a ft e r t h e
cryos torage [85].
Three cryopres ervat ion methods for orchids are
a v a ila b le p re s e n tly: des iccation (a ir-d ry in g ),
vitrification, and encaps ulation-dehydration [28].
Encaps ulation techniques , e ither us ing alginate or
agar, may be u s e fu l for this purpos e as germplas m
immobilization may aid in regeneration and the
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s ubs equent orchid co ns ervation by protecting the
developing embryo or the dividing tis s ue mas s [42].
En c a p s u la tion of vegetative propa g u le s a n d
s ubs equent retrieval o f p lantlets have been reported
in s everal orchids [69,11,56,15,46] a s well, s howing
that enca p s ulation is an agreeable method of
cons erving the p la n t germplas m in vitro, as reported
in many end e mic and endangered orchids s uch as
Geodorum d e n s i fl o r u m [15,46]. Encaps ulationdehydration works throu gh the extens ive des iccation
o f p lant tis s ues prior to freezing, and relies heavily
on the bes t c ombination of techniques that mos t
effectively minimize ice formation during freezing
[17].
Encaps ulation-dehydration involves the placement
of explants in s odium-alginate beads , and the
s ubs equent progres s ive or non-progres s ive des s ication
in the pres ence of high s ucros e concentrations , which
beneficially affect the germplas m tolerance to the airdrying and ice crys tal g rowth during freezing [16].
The technique does not re q uire the addition of toxic
cryoprotectants s uch as dimethyls ulp hoxide (DM SO)
an d ethylene glycol (EG) [29], which are known to
kill c ryopres erved propagules during thawing.
A lginate encaps ulation protects the des ic c ated
germplas m from mec h anical and oxidative s tres s es
durin g s torage, analogous to an embryo s ac, and
allows eas ier handling of the plant materia l due to
their s mall s izes , hen c e often s uperior to naked buds
which are s us ceptible to fragility [58]. A lginate is
commonly us ed for encaps ulation purpos es due to the
polymer’s inertnes s , non-toxicity, cheapnes s , eas y
manipulability, and its availability in large quantities
[19].
Trials on the pres ervation and pro p a g ation of
plants through encaps ulation-dehy dration have been
promis ing. The ability to s tore encaps ulated PLBs for
long periods and at different temperatures will greatly
enhance the efficiency of micropropagation by this
s ys tem. The various parameters involved in the
encaps ulation of Dendrobium s onia, s uch as the s tage
of PLBs s uitable for encaps ulation, concentration of
gelling agents , and nutrient concentration in the
matrix has been s tandardized, wit h thes e conditions
impos ed on s tudies with three orc h id genera:
Dendrobium, Oncidium, and Cattleya [63]. There is
grea t p o t e n t ia l in inves tigating the us e of
encaps ulation-dehyd ra t io n a s a t e chnique of
cons erving endangered and commercially us eful
germplas m of orchids . M ore s tudies mu s t b e
conducted o n o p t imizing the techniques and
propagules u s ed in encaps ulation-dehydration to
p ro v id e the bes t pos s ible outcome in plan t
regeneration experiments , es pecially thos e of orchids .
A lthough c u rrently riddled with uncertainty and
s e t b a c ks , mo re s t u d ie s in t h is partic u la r
cryopres ervation method will beneficially affect how
commercially viable and e n d angered s pecies of
Adv. Environ. Biol., 3(1): 69-83, 2009
orchids are propagated in th e fu ture. The objectives
of this pres ent s tudy are:
to determine the optimal s ize of protocorm likebodies (PLBs ) that gives the bes t res ults with the
encaps ulation-dehydration me t h o d, to tes t the
s uitability of various concentrations of s ucros e and
s orbitol for p re c u lt u re in the encaps ulationdehydration of the PLBs , to obs erve the effe c t s of
encaps ulation-dehydration and cryopres ervation on the
water contents of the PLBs .
Materials and methods
Plant Materials
The in vitro-grown protocorm like-bodies (PLBs )
o f Ascocenda ‘Princes s M ikas a’ were us ed in this
s tudy. The entire res earch was conducted in two
parts : Part I, to obs erve and choos e the bes tperforming PLB s ize in cryopres ervation involving
both s ucros e a nd s orbitol pretreatments ; and Part II,
to run the entire e xp e riment us ing the chos en PLB
s ize, followed by viability tes t in g us ing the 2, 3, 5triphenyltetrazolium chloride as s ay.
71
s upplemented with s ucros e or s orbitol according t o
their predetermined pretreatment concentrations us ing
0 M (control), 0.25 M , 0.50 M , 0.75 M , a n d 1.0 M .
The excis ed PLBs w e re then pretreated by placing
the plates under continuous light (Os ram, 1600 ± 100
lux) for 18 hours .
Encapsulation of the pretreated PLBs
A fter the 18 h o u rs pretreatment period, the PLBs
were s us pended in univers a l bottles containing halfs trength liquid M S medium s upplemented with 3.0%
s odium alginate, but devoid o f hydrated calcium
c hloride (CaCl2 .2H2 O). The PLBs were pipetted w it h
150µL of t h e a lginate medium us ing a 1ml volume
micropipette fitted with a tip having a modified
diameter of s ix mm. The mixture was then dropped
into 0.1 M CaCl2 .2H2 O s olution, an d left to
polymerize for 30 minutes , with occas ional agitation.
The bea d s were then rins ed with liquid half-s trength
M S mediu m d e v oid of s ucros e, followed by a onehour incubation period in liquid half-s trength M S
medium s upplemented with 0.5 M s ucros e.
Dehydration and cryostorage of the beads
Preparation of culture media
A ll the M S [54] media and the neces s ary
s olutions that were required in the expe rime nts were
prepare d in advance. The media prepared included
half-s trength liquid M S medium for s ubcultures , halfs trength s olid M S media not s uppleme n ted with
s ucros e or s orbitol as control, and half-s trength s olid
M S media s upplemented with s ucros e or s o rb itol for
pretreat ment of the PLBs us ing the following
concentrations of 0.25 M , 0.50 M , 0.75 M , and 1.0
M . Half-s trength s o lid M S medium containing 0.30
M s ucros e was prepared for the growth recovery
s t ep, and half-s trength liquid M S medium containing
3.0% s odium alginate, but devoid of hydra t e d
calcium chloride (CaCl2 .2H2 O) for the encaps ulation
s tep. A s eparate s olution of 0.1 M CaCl2 .2H2 O was
prepared as the polymerizing agent for encaps ulation.
None of the media p repared contained hormones ,
except for the s ubculturing media, which was
required to encourage the divis ion and p roliferation
of the PLBs . This was a precautionary s tep to avoid
the treated PLBs from experiencing s evere s hock
form cryopres ervation. The pH values of all media
were adjus ted to 5.7–5.8 prior to autoclaving.
The incubated beads w e re placed upon s terile
filter papers in glas s Petri dis hes , and de h y d ra ted
under t h e s terile air flow of the laminar flow hood
for 100 minutes . The dehydrated beads were then
placed in s terile 2mL c ryovials (Nalgene Cryowares ).
The vials were firs t placed in a Dewar flas k for
2minutes , followed by s torage in LN 2 fo r 24 hours .
Thawing and growth recovery
The cryovials were retrieved from the LN 2 t ank
after 24 h o u rs a nd immediately thawed in a water
bath at 40°C for 90 s econds . T he thawed beads were
then placed on hormone-free half-s trength s o lid M S
media s upplemented with 0.3 M s ucros e, and
immediately incubated in th e d a rk a t ro o m
temperature (25°C). In Part I of the res e a rc h, the
beads were removed from the da rk a ft er 48 hours
and placed under a 16 hour/8 hour photoperiod fo r
5days . On the oth e r hand, the beads in Part II of the
res earch were placed in the dark continuous ly for a
week. Obs ervations were made every d a y on the
colour o f the PLBs encaps ulated within the beads ,
w it h t h e final obs ervation recorded prior to the 2, 3,
5-triphenyltetra zolium chloride (TTC) viability as s ay.
Excision and Pretreatment of the PLBs
The PLB clumps were as eptically teas ed apart and
meas ured into 3mm2 and 6mm2 s ingle PLBs us ing a
millimetre grid graph paper p laced under a s terile
g las s Petri plate. They were placed in plas tic Petri
pla t e s containing half-s trength M S s olid media
2,3, 5-Triphenyltetrazolium chloride (TTC) v iability
assay
In a TTC as s ay, c e ll s urvival is es timated by the
amount of formazan produced from the reduction of
T T C due to the action of dehydrogenas es in livin g
Adv. Environ. Biol., 3(1): 69-83, 2009
cells or tis s ue [72]. In this res earch, the TTC as s ay
was conducted according to the protocol des cribed by
Verleys en et al. [85]. A fter a week of incubation, t h e
beads w e re s liced open to remove the PLBs from
wit hin. Each PLB in a replicate were weighed as a
g ro u p , and then immers ed in 1.5 ml of the T T C
s olution cons is ting of 0.18 M TTC buffered by 0.05
M KH2 PO4 . The PLBs were incubated in the dark for
15 hours at room temperature (25°C). Next, the TTC
s olution was drained off, follo w ed by rins ing the
PLBs thrice with d is t illed water, and placing them in
7 ml of 95% e thanol in tes t tubes . The tes t tubes
were then boiled in a water bath at 100° C fo r 10
minutes . The extract obtained was cooled, a nd the
intens ity of the rednes s of the ext ra ct was meas ured
with a s pectrop h otometer at 530 nm, us ing 95%
ethanol as the blank.
Determination of the dry weight of the PLBs
A fter the TTC as s ay was conducted, the res idual
PLBs from the as s ay were rin s ed thrice with dis tilled
water and placed upon filt e r papers in glas s Petri
dis hes according to their re p licates . The uncovered
plates were placed in an oven for 24 hours at 80°C.
The dried PLBs were then cooled in a des ic c a t o r,
and weighed. The PLBs were continuous ly weighed
and replaced in the des iccator until a cons tant weight
was obs erved [84].
Experimental Design
Each replicate cons is ted of 5 PLBs , with three
replicates employed in Part I and 5replicates in Pa rt
II for e a c h pretreatment concentration. One s et of
control, cons is ting of three replicates tha t had not
been pretreated, was cryopres erved u s ing the exact
protocols employed for the other pretreated replicates
in Part I of the re s earch. In Part II, two s ets of
controls , each c o n s is ting of 5 replicates , were run
throughout the entire cryopres ervation protocol with
t h e re s t of the pretreated PLBs . 4 out of 5 PLBs
were randomly s elected for the TTC as s ay in Pa rt II,
with one PLB re ma ining in each replicate for
obs ervation. The data obtained were s ubjected to
a nalys is of variance us ing SPSS, at the 0.05
probability level. Variation among treatments was
analyzed us ing Tukey’s tes t.
Res ults and dis cus s ion
Part I Experiment Results
The viability obs ervations of the PLBs in Part I
of the res earch, bas e d on the colours obs erved after
growth reco v e ry, s howed that there was no
s ignificant difference between the 3mm and 6mm
PLBs in terms of the type of pretreatment and the
72
concentrations employed, although the 6mm P LBs
s eemed to have higher viabilities (Fig. 2). H o wever,
there was a difference in the abs orbance obtained for
both the s izes when each s ize w as compared as a
w h o le as the 6mm PLBs were s hown to have high e r
abs orbanc e w h en compared to the 3mm PLBs . No
s ignificant differences were obtained between the
s izes when they were compared a c c ording to their
pretreatment concentrations (Fig. 3). Bas ed on t h e s e
obs ervations , the 6mm PLBs were s elected to
continue Part II of the res earch. However, the firs t
part of the res earch was plagued with high variances
in the data obtain e d due to ins ufficient replicates , as
only 3 replicates were employed fo r each treatment.
Furthermore , it was evident that the abs orbance
obtained for the PLBs was a functio n of the s ize, the
s urface area a n d the amount of the tis s ue pres ent in
t h e s a mple, with 6mm PLBs giv in g h ig h e r
abs orbance when compared t o the minute 3mm
PLBs . Immediate brownin g (F ig. 4a) and bleaching
(Fig. 4b) of the PLBs w e re als o obs erved when the
PLBs were expos ed t o light (1600±100 lux), us ually
occurring 24 hours a ft er the expos ure. Hence, bas ed
on thes e irregularities , it was decided that 5
replicates cons is ting of 5 PLBs each were to be us ed
for each treatment in Part II of the res earch, and
abs orbance at 530 nm w a s to be recorded with
res pect to the dry weight of the tis s ue pre s ent in the
s ample. T he PLBs were s tored in the dark during the
growth recovery phas e in Part II of the res earch to
prevent bleaching and browning of the PLBs .
Part II Experiment Results
A mong all concentrations of s ucros e tes ted, the
highes t abs orbance per millig ram of PLBs was
recorded by 0.50 M s ucros e, followed clos ely by
0.25 M s uc ro s e (Fig. 5). The lowes t abs orbance
value was recorded by 1.00 M s ucros e. A s tatis tically
s ignificant difference was found was amo n g t he 5
s ucros e pretreatment concentrations . However, further
e xperiment s howed that there was no s ignificant
difference in us ing 0.25 M s ucros e and 0.50 M
s ucros e in pretreating the PLBs , s ugges ting that both
the concentrations yield s imilar viabilities in the
pretreatment of the PLBs . There was no s ignificant
difference between the controls and 0.75 M s ucros e
in their capability as pretreatment agents a s well,
althoug h being s ignificantly lower in their prowes s
compare d t o both 0.25 M and 0.50 M s ucros e.
P re t re a t me n t u s ing 1.0 M s ucros e y ie ld e d
s ignificantly lower res ults when compared to all the
other s ucros e concentrations (Fig. 4a).
A mong the various s orbitol pretreatment
concentrations , 0.25 M s orbitol recorded the highes t
abs orbance at 0.123, followed clos ely b y 0.50 M at
0.116 (Fig. 6). The lowes t v a lu e, 0.077, was obtained
from 1.00 M s orbitol. A s tatis tically s ignific ant
Adv. Environ. Biol., 3(1): 69-83, 2009
difference was found among the 5 s o rb it o l
p re t r e a t m e n t c o n c e n t ra t io n s . A ll s o rb it o l
concentrations us ed in the pretreatment recorded
s ignificantly different res ults from each other in their
abs orbanc e at 530 nm, except for the mean
differences between 0.25 M with 0.50 M , and 0.50
M with 0.75 M res pectively. Both groups of
pretreatment s yielded PLBs which remained green
when s tored continuous ly in the dark.
The meas urement of the dry weights of the PLBs
s howed that the PLBs were compos ed almos t entirely
of water, c o n s tituting a mean of 98.8% of the total
weight of each PLB. The fin a l water contents of the
PLBs were not s ignificantly different when compared
according to their pretreatment concentration s (Fig.
6), as both groups retained about 40% water, b u t
were different when compared according to t h e type
of pretreatment they unde rw e nt–either s ucros e or
s orb itol (Fig. 7). PLBs pretreated with s ucros e
generally los t more water than thos e treated with
s o rb itol. The res ults as a whole implied that both
s ucros e and s orbitol can be us ed as p re t re atment
prior to the encaps ulation and cryos torag e o f the
PLBs of Ascocenda ‘Princes s M ikas a’, and that they
b oth have their own mechanis ms that ens ured th e
effectivenes s of the pretreatment.
Discussion
Effects of the Sizes of PLBs on Viability
Synthetic s eed technology is currently cons idered
an effective alternative for propagating c o mmercially
important agronomic and horticultural crops , s uch as
s eedles s grape, s eedles s watermelon, s eedles s ja c k,
s eedles s cucumber, corn, cotton, s oybean, hybrid
tomato, hybrid cereals , forage legumes , pines , p otato
and banana. [62,63]. Synthetic s eeds of o rc hids are
frequently produced by encaps ulating protocorm likebo d ies (PLBs ) in an alginate matrix, s erving as a
lo w -c o s t , h ig h -v o lu me propagat io n s y s t e m.
A dvantages of s ynthetic s eeds over s omatic embryos
for propagation include eas e of h a n d ling during
s torage and trans portation, potential long-term s torage
w ithout los ing viability; and maintenance of t h e
clon a l nature of the res ulting plants [25,63].
However, the viability of the s elected explant or
tis s ue mus t be taken into account p rior to any
encaps ulation and cryos torage experime n t s , as the
s ucces s or failu re of the entire experiment depends
on the tis s ue. There is always t h e t hreat of
contamination and undes ira b le variations in explants
obtained in vivo. Hence, in many experiments ,
plantlets rais ed in vitro were the s o u rce of explants
for encaps ulation [44], as conducted in this res earch.
The PLB is the earlies t s tructure formed d uring
embryo development in orchid s eed germination, and
is unique to orchids [30,46]. Proliferations of
protocorms and p rotocorm like-bodies (PLB) are
73
us ually the only means of increas ing the number of
orchid s pecies that do not germinate well o r produce
fe w s eeds [55]. PLBs that have been s ubjected to
s ubcultures , trans fers , and cryos torage rarely dis play
the characteris tic green colour that indicate via b ility,
but ins tead generate three differen t a ppearances :
white or bleached, light ye llo w and brown [93].
How e ver, s hoots of the A frican Violet (Saintpaulia
ionantha W endl.) recovered from cryopre s erved
t is s ues of the plant were either pale green or y e llo w
[52], with greenis h appearance indic ating a quicker
regrowth a n d h ig h er viability. The yellowis h
appearance, as well as the bleaching and b ro w n ing
conditions , could be attribut e d to os motic s hock or
unfavorable regrowth conditions [74,52,93] reported
that the light yellow calli of the orchid Den drobium
candidum W a ll ex Lindl maintained their initial fas t
growth potential and bleached calli gradually turned
mois t while brown calli prolifera ted into lighter
coloured tis s ues followed by browning. Thes e tis s ues
needed hormone s upplementation s to continue
proliferation and development of h e a lthy green
tis s ues . A ll the cryos tored PLBs recovered fro m this
res earch were initially ligh t g re en and remained light
green when immediately recovered from the liquid
nitrogen and incubated in the dark, but underwent
either bleaching or browning within 24 hours of
expos ure to lig h t , as obs erved in Part I of the
res earch. No yellowing PLBs were obs e rv e d . On the
other hand, all the re c o v e re d PLBs in the s econd
p h a s e of the res earch maintained their light gre e n
colour when incubated in the dark continuous ly, with
the remaining beads not s ub je cted to the TTC as s ay
remaining green up to 6 weeks after the growth
recovery s tep. Hence, a higher degree of PLB
v ia b ility can be obtained if the PLBs are incubated
in the dark cont in u ous ly. This obs ervation has been
cited by Shatnawi and Johns on [67] and M oges et al.
[52] in the cryopres ervation of the s eeds of the
‘Chris tmas bus h’ (Ceratopetalum gummiferum) and
the s hoot tips of th e A frican violet (Saintpaulia
ionantha W endl.) res pectively, with both s tating that
this s t e p is es s ential to reduce s hock to the
cryopres erved plant tis s ue.
The s ize of tis s ues to be manip ulated als o play
an important role in the s urvivability of the tis s ues in
the s ubs equ ent s teps of an experiment. Throughout
the res earch, the 6mm PLBs ha d dis played better
viability compared to the 3mm PLBs , irres p e c tive of
the type of p retreatment chemical applied. This
implies that the 6mm P LBs are able to withs tand the
entire encaps ulation-dehydration protocol better than
th e 3mm PLBs , and hence were s elected to continue
with the s econd part of the res earch. Generally, PLBs
are s e le cted for tis s ue culture manipulations when
they are in the ra n g e of 3mm to 5mm, with
prot o c o rms of 3mm and 4mm s hown to be s uitable
for optimum convers ion of the encaps ulated PLBs o f
Cymbidium giganteum W a ll. PLBs s maller than this
s ize range dis played p o o r convers ion frequencies ,
Adv. Environ. Biol., 3(1): 69-83, 2009
74
Fig. 1: A s cocenda 'Princes s M ikas a' (W ikimedia, 2008)
Fig. 2:
Viability obs ervations ba s ed on the colour of the PLBs encaps ulated in the alginate matrix after being
placed on growth recovery medium. Only green and lig h t g re e n PLBs were deemed as viable, bas ed
on their ability to grow on hormon e -fre e me dia. Bleached, yellowing and brown PLBs were
cons idered as not viable.
Fig. 3:
Viability obs ervations b a s e d o n t h e abs orbance of the PLBs s ubjected to the TTC as s ay at 530 nm.
The data s howed large variances for PLBs with higher abs orbance and hence regarded as viable, while
PLBs with low abs orbance s howed lower variances .
Adv. Environ. Biol., 3(1): 69-83, 2009
75
Fig. 4:
(A ) The e n c a p s ulated PLBs browning after expos ure to light during the growth recovery s tep in Part
I ; (B) The encaps ulated PLBs bleaching after expos ure to light during the growth recovery s tep in
Part I.
Fig. 5:
The abs orbance value per milligram of PLB again s t the pretreatment concentration for both s ucros e
and s orbitol. No s ignificant difference was obs erved in the abs orbance of the PLBs in the TTC a s s ay
when both s ucros e and s orbitol were compared as a whole, hence s ugges ting that both the chemicals
may pos s es s s imilar efficacies in pres erving the PLBs of the orch id h y b rid A s cocenda 'Princes s
M ikas a'.
Fig. 6:
The final water content in the PLBs after the entire cryopres ervation protocol, prior to the TTC as s ay.
No s ignificant differences were found when the values were compared acro s s t h e c o n c entrations of
s ucros e or s orbitol employed, but a difference was obs erved when s ucros e was c o mpared to s orbitol
as a whole, with s ucros e pretreatment caus ing more water los s than s orbitol pretreatment.
Adv. Environ. Biol., 3(1): 69-83, 2009
Fig. 7:
76
The s everity of water los s e s c o mp a re d a gains t the type of pretreatment, des cribed as percentage of
w a ter weight. A lthough the graph implied that there was no difference in the amount of water lo s t
in both type of pretreatments , the analys is of variance indicated otherwis e.
pos s ibly as a res ult of tis s ue immatu rity, caus ing the
inability of the PLBs to withs t a n d encaps ulation or
lengthier emergence o f the PLBs from the alginate
caps ule [13,63]. The s ucces s of a cryopres ervation
experiment may rely on the various s tages of growth
and age of plant tis s ues . For ins tance, the torpedos tage embryos of Medicago sativa were s hown to be
s uitable for encaps ulation as the torpedo s tage
demons trated a rapid increa s e in embryo mas s and
the depos ition o f majority of the s torage res erves
[49,63]. Similarly, younger or older s omatic embryos
failed to produce h e a lthy plants before or after gel
encaps ulation in Geranium [25,63,]. s elected PLBs at
t h e leaf primordia s tage for s tudies on the
encaps ulation of Dendrobium s onia, Oncidium
‘Gower Rams ay’, and Cattleya leopoldii as they
dis covered that PLBs at the leaf primordia s tage gave
earlier leaf and primary root formation t han the promeris tematic s tage, a nd more complete germination
than the firs t leaf s tage PLBs , indicating that the
developmental s tage of PLBs us ed for encaps ulation
affected the germination percentage.
Effe c t s o f the
Pretreatment
Types
and
Concentrations
of
In this res earch, 0.25 M s ucros e, a nd both 0.25
M and 0.50 M s orbitol, the former produ c in g h igher
a b s orbance than the other, had been proven t o b e
effective as pretreatment in the e ncaps ulationdehydration of the PLBs of Ascocenda ‘Princes s
M ikas a’. However, bot h the s ugars might have been
effective for different reas ons entirely . Tokuhara and
M ii [76] in d icated that the morphogenetic res pons es
of the Doritaenopsis PLB c ells could be modified by
the concentrations and the type o f c a rbon s ources
applied in the media. Suc ro s e, when s upplemented
in t o a me d iu m, is c a t a b o lize d i n t o t h e
monos accharides glucos e and fructos e by extracellular
enzymes releas ed d uring the in vitro culture, hence
providing readily available nutrients for the explant
[24,76]. A s imila r phenomenon may have occurred in
t h e P LBs of the Ascocenda hybrid in this res earch
during th e p retreatment with s ucros e, hence the
optimal abs orbance at 0.50 M s ucros e, followed
clos ely b y 0.25 M s ucros e. On the other hand, Hilae
and Te-chato [27] dis covered that s o rbitol was a
s uitable o s moticum for s hoot and root induction in
oil palm as s hoots and roots were fo rme d
s imultaneous ly from s omatic emb ry os of oil palm.
T h is effect could be mimicry of the changes in
os molarity tha t occur in tis s ues s urrounding the
embryo within a real s eed [50]. Hence, th e imp a ct of
s orbitol in this s tud y could have been the direct
res ult of its os motic potential [35]. However, they
als o reported that increas ing concentrations of s ucros e
and s orbitol may heighten phenolic compound
formation within the s omatic embryos and promote
leaf b light s ymptoms , s imilar to the effects of water
s tres s . The oil pa lm plantlets died after being
cultured on such medium for two to three months .
This phenomenon may explain b oth the increas ed
browning obs erved in PLBs pretreated under high
concentrations of s ucros e, a n d the low abs orbance
values obtained for PLBs placed under hig h e r
concentrations of s ucros e and s orbitol pretreatments .
Sugar, when added in a culture medium,
functions both as a carbon s ource and as an os motic
regulator of water s tres s . It h a s been reported that
carbon s ources s uch as glucos e, fructos e, ma n n itol
and s orbitol play an important role in the germination
of s o matic embryos of as paragus [43] and cucumber
Adv. Environ. Biol., 3(1): 69-83, 2009
[41] further reported that a high co n centration of
s ucros e (0.25 M or 0.50 M ) could enhance
germinatio n of s omatic embryos in cucumber.
Os moticum increas ed the water s tres s in pa lm oil
s omatic embryos , inducing s hoots a nd/or root
forma t ion [27]. Os motic adjus tment is als o a
mechanis m involved in drought tolerance. Sucros e, a
dis accharide, is th e oretically thought to functions as
an os moprotectant, by s tabilis ing cellular membranes
and maintaining t u rgor [53,80]. Is lam and Ichihas hi
[32] had conc lu ded that s ucros e, a s ugar eas ily
me tabolized by cells , was s uitable for callu s
proliferatio n , while maltos e and s orbitol, both not
eas ily utilize d by cells , were s uitable for PLB
proliferation and PLB growth res pectively, b a s ed on
the effects of the three carbohydrate s ources on PLB
formatio n a nd callus growth in Phalaenopsis
embryogenic calli [76,33]. Higher con c e ntrations of
s u c ros e have als o been demons trated to be efficient
in the morphogenes is of underground organs in vitro
[73,65,68,57] and can be attributed to its nutritional
effect . However, this particular phenomenon may
als o be partly caus e d b y the low initial water
potential of the me dium, as the delay in the
develo p ment of Ipsea malabarica bulbs in media
co n t aining low concentrations of s ucros e was
attributed to the high initial water potential of t h e
media [47,91] theorized that high pretreatment
concentrations , in their cas e, 0.75 M s ucros e, allowed
viability retention of encaps ulated Dactylorhiza
fuchsii s eeds by reducing the bead drying rate, hence
s ugges ting that rapid dry in g rates may hinder the
s urvival of the orchid and its fungal s ymbiont.
Higher concentrations of os moticum w e re s aid to
protect the plant from des iccation injury.
The Final Effect of Pretreatment and Dehydration on
the Water Content of the PLBs
A lthough there were no s ignificant differences in
the final water content percentage of PLBs s ubjected
t o the various concentrations in the s ucros e an d
s orbitol pretreatments , a s ignificant difference was
obs erved between the types of pretreatment a p p lied
as a whole, with s ucros e-treated PLBs s howing a
greater water los s when compared to s orb itol-treated
PLBs , both at 61.7% and 56.0% res pec t iv ely.
J it s o p akul et al. [34] had reported that the regrowth
rate of non-cryopres erve d a n d c ry opres erved
protocorms of Vanda coerulea depended on the water
content of the precultured beads during dehydration,
conducted from between zero to 10 hours . The group
als o reported that highes t regrowth rate of the
cryopres erved orchid beads , at 40%, was achieved by
dehydrating the beads for eight hours , yielding a
final water content of 35%. This res ult was almos t
s imilar to the res ults obtained from the s ucros epretreated PLBs in this res earch (38.3%). The
77
optimal water content of alg inate beads is dependent
to a large extent on the plant s p e c ie s , for ins tance,
33% for apple [58], 19% for Eucalyptus [59] and
20% to 25% for Citrus [26]. For azalea, s hoots
encaps ulated in alginate beads with relatively h igh
water content (38.6%) recorded 40% s urvival after
cryopres ervation [84].
Both s ucro s e a nd s orbitol act as os moticum,
drawing water out of t he PLBs during pretreatment,
b u t both may pos s es s differing efficacies as a dire c t
re s ult of their chemical nature. The earlier mentione d
enzymatic degradation of s ucros e into glucos e and
fructos e in a culture medium is known to incre a s e
the os motic pres s ure in the medium, which in turn
lowers the water potential o f the medium, drawing
more water out of the cultu re d explants [76]. This
could be the reas on behin d the lower final water
contents of the s ucros e-pretreated PLBs when
compare d to the s orbitol-pretreated PLBs . Higher
concentrations of s ucros e may multiply t h is effect
and hence promote exces s iv e dehydration of the
PLBs , incurring toxicity and extens ive water s tres s
on the PLBs and overriding the nutrit io nal effects of
the s ugar [76]. Sorbitol, on the o ther hand, s imply
acts as an os motic des iccator [27].
The control of water content o f plant s amples
before freezing was the key facto r in developing
s ucces s ful cryoprotection protocols [92]. W hen PLBs
are not dehydrated s ufficiently, freezing-injury can
occur due to intrac e llu lar ice formation; on the other
hand, w h e n over-dehydrated, the os motic s tres s can
be damaging [5]. Hence, cells and tis s ues to be
cryopres erved mus t be s ufficiently des iccated in order
to be vitrified before imme rs ion into liquid nitrogen.
The vitrification (glas s formation) pro cedure for
cryopres ervation eliminates the controlled s low
freezing s tep and allows cells to be cryopres erved by
direct trans fer into liquid nitrogen [22,39,64,5].
demons trated that the application of exogenous A BA
or dehydration caus e d accumulation of s oluble
s ugars , followed by the accumulation o f heat-s table
proteins and dehydrin, a late-embryogenes is -abundant
(LEA ) protein in the PLBs of Dendrobium candidum,
with the latter occurring at relatively low water
c o ntents (1.0g water/g DW ). Soluble s ugars are s a id
to protect the cellular membrane through w a t er
replacement and to protect the cytoplas m by trans it
into a vitrified s tate [37]. The LEA proteins , a group
of heat-s table pro t e ins , are s aid to induce the ability
to tolerate des iccatio n [23,3,18,6,8]. The dehydrin
may achieve this by any of the three me t h o d s : the
s tabiliza t io n o f the membrane [14]; in vitro
cryoprotectant pro perties of the protein [40,90] or
through inhibition of the coagula t io n of a range of
macromolecules [12]. Th e re were als o theories that
interactions between s ugars and heat-s table proteins
might play a role in imp roving the dehydration
tolerance of plant cells . Oligos accharide s were found
Adv. Environ. Biol., 3(1): 69-83, 2009
to interact with LEA s to enhance the tole rance of
d eveloping s oybean s eeds [6]. Hence, with all t h e s e
pos s ibilities and evidence highlighted, there is a high
chance of dis covering s imila r mo lecules and
pathways being expres s ed in the PLBs of A s cocenda
‘Princes s M ika s a ’ that are s ubjected to the
pretreatment and dehydration s teps , hence pres enting
o p p o rt u n it ies for further res earch in t o t h is
encaps ulation-dehydration protocol.
The 2,3,5-Triphenyltetrazolium Chloride Assay
Succes s ful tis s ue cryopres ervation depends on the
technique and type of protection employed a g a ins t
damage fro m ultra-low temperature [21,51]. Tis s ue
regrowth in recovery medium, although very s ens itive
in as s es s ing cellular v ia bility, is time-cons uming.
Hence, various s taining methods s uch as the 2,3,5triphenylte t ra zolium chloride (TTC) tes t and vital
s taining with fluores cein d ia cetate (FDA ) [88] are
fre q u e n tly employed to determine the viability of
cells s ubjected to various s tres s factors s uch as cold,
s alinity and heat [86,31]. A s obs erved in this
res earch, as s es s ing s tres s parameters for s mall
explants us ed in cryopres ervatio n can be a difficult
affair. TTC s taining is a refe re nce method of the
International Seed Tes ting Organis ation (ISTA ) for
tes tin g s eed viability and can be us ed to tes t
biochemical activity of plant tis s ues aft er cold
tre a t ment [82,85]. The TTC as s ay, s uitable for large
cell a g g regates [31,71,86,87], is bas ed on the
enzymatic res piration of living plant c e lls , and
indicate re s piration levels in s amples tes ted [86].
A ctive dehydrogenas es in mitochondria reduce
colo rle s s TTC to red triphenylformazan [72,78].
Hence, living portions of tis s ue or s ingle cells s tain
red. A lthough widely u s ed as a fas t and inexpens ive
tes t, there have been problems as s ociated with its us e
as an indicator of pos t -t haw viability [60]. A bnormal
inorganic reactions may interfere and intens ify
formazan production from TTC [51,86] pointed out
that latent d a mage, which can des tabilize the cell
reaction, may be imperceptible s hortly after t hawing
and s ugges ted waiting for at leas t overnight after the
tis s ue is thawed prior to the tes t. In this res earch, the
thawed PLBs w e re allowed a recovery period of 48
hours under darknes s , followed by anoth e r 48 hours
of 16 hours /8 hours photoperiod in P art I and for a
week under total darknes s in Part II, to prevent s uch
damages from occurring in the encaps ulated PLBs .
Ve rleys en et al. [85] had demons trated that viable
tis s ues had relatively higher abs orbance values when
compared to non-viable tis s ues , when the abs orbance
of the TTC-treate d azalea nodal s egments was read
at 490 nm. They als o obs erved that the s tandard
errors recorded for both ty p e of tis s ues dis played
s imilar traits : viable tis s ues had higher s tandard error
valu es , while highly s tres s ed tis s ue had lower
78
s tandard error values . This exact obs ervation was
made in Part I of the re s e a rch: lower s ucros e and
s orbito l pretreatment concentrations dis playing high
abs orb a nce values had higher s tandard deviations ,
unlike higher pretreatment concentrations which
dis played lower s tandard de v ia tions . The group
forwarded two explanations for this phenomenon:
firs tly, reducing components pres ent in dead cells
c o uld reduce TTC. A s econd, more plaus ib le
explanation involves the metabolic a c t ivity of the
tis s ues . Viable tis s ues may pos s es s incons is tent
metabolic activities due to a number of fa c t o rs s uch
as the age and s tage of growth o f t he tis s ues , and
number of viable cells remaining in the tis s ue that
could contribute to the metabolic activity. Frozen or
non-viable tis s ues , which are les s metabolically
active, will reduce les s TTC, hence res ulting in a
lo w er s tandard error. Hence, the incons is te n t
abs orbance values obtained in Part I of the res earch,
involv ing the three mm and the s ix mm PLBs , could
have been attributed to the factors mentioned above.
Advantages a nd disadvantages of EncapsulationDehydration
The encaps ulation protocol e mployed in this
res earch, as previous ly conducted by our lab member
previous ly (unrecorded data) was fou n d to be
s atis fa c t o ry as the beads containing the PLBs could
be handled with eas e and s eemed to have s hown the
ability to protect the explants encaps ulated within. In
fact, s ome of the s ingle PLBs left behind in e a ch
replicate were o b s erved to have been proliferating,
eas ily breaking throug h the alginate s hell, three
weeks afte r growth recovery.
A n optimal ion
exchange between the s odiu m a n d calcium ions was
achieved us ing 3% s o d ium alginate s upplemented in
half-s trength M S med iu m and 0.1 M hydrated
calciu m c hloride as the complexing agent, producing
firm a n d c le a r is o d ia me tric beads . H ig h e r
concent rations of s odium alginate (4–5%) was found
to inhibit the convers ion of encaps ulated s hoot tips of
Phyllanthus amarus, a medicinally imp o rtant plant,
while lower co n c e ntrations (1–2%) caus ed the
formation of unmanage a b le and fragile beads by
prolongin g the polymerization period [70]. The M S
medium-s upplemented alginate matrix s erved as
artificial en d o s perm by providing nutrients to the
encaps ulated explants for plant regrowth [2,9]
discovered that th e a d dition of 1/2-M S nutrients in
the gelling matrix of Carica papaya, as conducted in
t h is res earch, enhanced its germin a t io n a n d
convers ion frequency [70]. The alginate coating
help e d in preventing the detrimental environmental
effects on the encaps ulated plant material [79,52,48].
demons trated that encaps ulated plant apices were able
t o w ithstand dras tic treatments s uch as preculture
with high s ucros e concentrations and des ic cation,
naturally harmful to nake d apices [52]. Formation of
Adv. Environ. Biol., 3(1): 69-83, 2009
intracellular ice crys tals during freezin g and/or
thawing was als o s hown to be de t rimental for
viabilit y, and was reduced by alginate encaps ulation
[81,52]. However, encaps ulation have b e en s aid to
inc re as e the lag period in the germination of the
Dactylorhiza fuchs ia s eed and the fungal s ymbiont.
One pos s ible explanation for this phenomenon is that
the beads impos ed a mechanical res is tance to growth,
with forces of up to 5 N cm-2 required t o rupture the
alginate b e a ds [91]. Thes e negative effects were not
obs erved in this res earch. In fact, the PLBs within
the beads s eemed to be able to s urv ive all the
treatment applied prior to and after cry opres ervation,
making the protocols applied in this res earch feas ible
for further deve lo pment and optimization for the
cryopres ervation of the orchid h y brid, Ascocenda
‘Princes s M ikas a’
79
2.
3.
4.
5.
Conclusions
T h is s tudy has s hown that an optima l
encaps ulation-d ehydration protocol for the orchid
hybrid Ascocenda ‘Princes s M ikas a’ can be achieved
us ing s ix mm P LB pretreated in either 0.50 M
s ucros e or 0.25 M s orbitol fo r 18 hours , followed by
encaps ulation in 3.0% s odium alginate s upplemented
in hormone-fre e half-s trength M S medium. The
PLBs , e n c a ps ulated and hardened in 0.1 M
CaCl2 .2H2 O, dehydrated for 100 minut e s u nder the
laminar flow hood and cryopres erved for 24 hours
before growth recovery, los t a mean 60.0% o f their
original water content regardles s o f the type of
pretreatment and concentrations applied. The s ize of
the PLB s elected, the t y p e and concentration of the
pretreatment applied in t h e protocol, as well as the
final water content in the PLBs after the end of the
entire treatment had proven to be great ly influential
in determining the s ucces s of the encaps ulationdehydration prot o c o l for this orchid hybrid. In order
to ens ure greater s ucces s o f this protocol, a more
accurate viability tes t can be applied at each s tage of
the protocol to as s es s the effect of the p a rt icular
s tage on the PLBs . Contro ls t hat do not undergo
cryopres ervation can b e e v aluated to meas ure the
effectivenes s of the protocols in pres erving the
v ia b ility of the PLBs . The recovery of the PLBs ca n
be as s es s ed u s in g different media or hormones .
Finally, molecular s tudies c an be performed to
dis cover what genes are trans crib e d in the orchid
during cryopres ervation, and to obs erve in teractions
b e t w e e n p ro t e in s e xp re s s e d d u r i n g t h e
cryopres ervation.
6.
7.
8.
9.
10.
11.
12.
13.
References
14.
1.
A s hmore, S.E, 1997. Curre n t i n
vitro
co n s ervation techniques . In: F. Engelmann (ed.),
Stat u s Re p o rt o n the Development and
A p p lication of in vitro Techniques for the
Cons ervation and U s e o f P la n t Genetic
Res ources , pp. 5–18. IPGRI. Rome, Italy.
Bapat, V.A . and P.S . Rao, 1992. Plantlet
re g eneration from encaps ulated a n d n o n e n c aps ulated des iccated s omatic embryos of
fores t tree: s andalwood (Santa l u m album L.).
J o u r n a l o f P l a n t B io c h e mis t ry a n d
Biotechnology, 1: 109–113.
Bartels , D ., M. Singh and F. Salamini, 1988.
On s e t o f d e s ic c a t io n t o le ra n c e during
development of the barley embry o . Planta, 175:
485–492.
Bens on, E.E., B. Reed, R.M . Bre n nan, K.A .
Clacher and D.A . Ros s , 1996. Us e o f thermal
analys is in the evaluation of cryopres ervat ion
protocols for Ribes nigrum L. germplas m.
Cryoletters , 17: 347–362.
Bian, H.W ., J.H. W ang, W .Q . Lin, N. Han and
M .Y. Zhu, 2002. A ccu mulation of s oluble
s u gars , heat-s table proteins and dehydrins in
cryopres ervation of protocorm-like bodies of
Dendrobium candidum by the air-drying method.
Journal of Plant Phys iology, 159: 1139-1145.
Blackman, S.A ., R.L. Obendorf, and A .C.
Leopold, 1992. M aturation proteins and s ugars in
des iccation tolerance of developing s oy b ean
s eeds . Phys iology Plant, 100: 225–230.
Bou ma n , H. and G.J. De Klerk, 1990.
Cryopres ervation of lily me ris t e ms . A c t a
Horticulturae, 266: 331–337.
Bradford, K.J. an d P.M . Chandler, 1992.
Expres s ion of dehydrin-like proteins in embryos
and s eedlings of Zizania palus tris and Oryza
s ativ a d uring dehydration. Phys iology Plant, 99:
488–494.
Cas tillo, B., M .A .L. Smith and U.L. Yadava,
1998. Plant regeneration from encaps ulated
s omatic embryos o f Carica papaya L. Plant Cell
Reports , 17: 172-176.
Chen, J.T. and W .C. Chang, 2000. Efficient
p la n t re g e n e r a t i o n t h r o u g h s o ma t ic
embryogenes is from callus cult ures of Oncidium
(Orchidaceae). Plan t Science, 160(2160): 87–93.
Chetia, S., P.C. Deka and J. Devi, 1998.
Germination of fres h and s tored enc a p s ulated
p ro t ocorms of orchids . Indian Journal o f
Experimental Biology, 136: 108–111.
Clos e, T.J, 1997. Dehydrins : a commonality in
the res pons e of plants t o d ehydration and low
temperature. Phys iology Plant, 100: 291–296.
Corrie, S. and P. Tandon, 1993. Propagation of
C y mbidium gigantium W all through h ig h
frequency convers ion of encaps ulated protocorms
under in vivo and in vitro conditions . Indian
Journal of Experimental Biology, 31: 61–64.
Danyluk, J., A . Perron, M . H o u d e , A . Limin, B.
Fowler, N. Benhamou and F. Sarhan, 1998.
A ccumulation of an acidic dehydrin in the
vicinity of the plas ma membrane during cold
acclimation of wheat. Plant Cell, 10: 623–638.
Adv. Environ. Biol., 3(1): 69-83, 2009
15. D a tta, K.B., B. Kanjilal and D. De Sarker, 1999.
A rtificial s e e d technology: development of a
protocol in Geodorum densiflorum (Lam) Schltr.
– an endangered orchid. Current Science, 76:
1142–1145.
16. Dumet, D ., F. Engelmann, N. Chabrillange, Y.
D u val, and J. Dereuddre, 1993. Importance of
s ucros e for the acquis it ion of tolerance to
des iccation and cryopres ervatio n o f oil palm
s omatic embryos . Cryoletters , 14: 243-/250.
17. Dumet, D., A . Grapin, C. Bailly and N. Dorion,
2002. Re v is it in g c ru c ia l s t e p s o f a n
encaps ulation/des iccation bas ed cryopres ervation
proces s : importance o f t hawing method in the
cas e of Pelargonium meris t e ms . Plant Science,
163: 1121-1127.
18. Dure, L.I., M . Crouch, J. Harada, T. David Ho,
J. M undy, R. Quatrano, T. Thoma s a n d Z.R.
Sung, Z , 1989. Common amino acid s equence
domains among the LEA proteins of higher
plants . Plant M olecular Biology, 12: 475–486.
19. End res s , R, 1994. Plant Cell Biotechnology.
Springer-Verlag, Berlin Heidelberg.
20. Engelmann, F, 1997. In vitro cons ervation
me t h o ds . In: J.A . Callow, B.V. Ford-Lloyd and
H.J. Newbury (Eds .), Biotechnology and Plant
Genetic Res ources , pp: 119–161.
21. En g e lma n n , F , 2000. Imp o rt a n c e
of
cryo p re s ervation for the cons ervation of plant
genetic res ources . In: En g elmann, F., Takagi, H.,
(eds .), Cry o p re s e rv a t ion of tropical plant
germplas m. Rome: International Plant Genetic
Res ources Ins titute, pp: 8–20.
22. F a h y, G.M ., D.R. M acfarlane, A .C. A ngell and
H.T. M e ry ma n , 1984. Vitrification as an
approach to cryopres ervation. Cryobiology, 21:
407-26.
23. Galau, G.A ., D.W . Hughes and L.I. Dure, 1986.
A bs cis ic acid in d u c t io n o f c lo n ed late
emb ryogenes is -abundent (LEA ) mRNA s . Plant
M olecular Biology, 7: 155–170.
24. Ge orge, E.F, 1993. Plant propagation by tis s u e
culture: component s of culture media. Exegetics
Ltd, London, pp: 313–336.
25. Gill, R., T. Senara t na and P.K. Saxena, 1994.
Thidiazuran ind uced s omatic embryogenes is
enhances viability of hydro gel-encaps ulated
s o matic embryos of Geranium. Journal of Plan t
Phys iology, 143: 726–729.
26. Gonzalez-A rna o , M .T., F. Engelmann, C.U.,
Villavicencio, M ., M orenza and A . Rios , 2000.
Cryopres ervation of citrus apices us ing the
e n c a p s u la t io n -dehydration t e c h n iq u e . In :
En g e lma n n , F . a n d T a ka g i, H . (eds .),
Cryopres ervation of Tropical Germplas m. Current
Res earch Progres s and A pplication. JIRCA S,
Rome, pp: 217–221.
80
27. Hilae, A . and S.Te-chato, 2005. Effects of
carbon s ources and s trength o f M S medium on
germination of s omatic embryos of oil palm
(Elaeis quineensis Jacq.). Songklanakarin Journal
of Science and Technolog, 27(3): 629-635.
28. Hirano, T., K. Is hikawa and M . M ii, 2006.
A dvances in Orchid Crypres ervation. Floriculture,
Ornamental and Plant Biot e c hnology, 2: 410-414.
29. H irata, K., S. Goda, M . Phunchindawan, D. Du,
M . Is hio, A . Sakai a n d K. M iyamoto, 1998.
Cryopres ervation of Hors eradis h Hairy Root
Cultures b y Encaps ulation-Dehydration. Journal
of Ferment a t ion and Bioengineering, 86(4): 418420.
30. Is hii, Y., T. Takamura, M . Goi and M . Tanaka,
1 9 9 8 . Ca llu s in d u c t io n a n d s o m a t i c
embryo g e n es is of Phalaenops is . Plant Cell
Reports , 17: 446–450.
31. Is hikawa, M ., A .J. Roberts on and L.V. Gus t a ,
1995. Comparis on of viability tes ts for a s s es s ing
cros s -adaptation to freezing, heat and s alt
s tres s es ind u c ed by abs cis ic acid in bromegras s
(Bromus inermis Leys s ) s us pens ion cultu red
cells . Plant Science, 107: 83-93.
32. Is lam, O.M . and S. Ichihas hi, 1999. Effect of
s u c ro s e, maltos e and s orbitol on callus growth
and plantlet regen e ra t ion in Phalaenops is ,
Doritaenopsis and Neofinetia. Jo urnal of the
Japanes e Society for Horticultural Science, 68:
1124–1131.
33. Is lam, O.M ., A .R.M .M . Rahman, S. M ats ui and
A .K.M .A . Prod h a n , 2003. Effects of Complex
Organic Extra c t s on Callus Growth and PLB
Regeneration through Embryoge n es is in the
Doritaenopsis Orchid. JA RQ, 37(4): 229-235.
34. J its opakul, N., K. Thammas iri and K. Is hikawa ,
2007. Cryopres ervation o f Va nda coerulea
protocorms by encaps ulation-dehydration method.
In: 33rd Congres s on Scie n c e and Technology of
Thailand.
35. K a ra mi, O. and G.K. Kordes tani, 2007.
Proliferation, s hoot organ o genes is and s omatic
embryogenes is in embry o g e n ic c a llu s of
carna t io n . Journal of Fruit and Ornamental Plant
Res earch, 15: 167-175.
36. Kis hor, R., P.S. Sh a Va lli Khan and G.J, 2006.
Hybridization and in vitro cultu re o f an orchid
h y b rid A s c o c e n d a ‘K a n g l a ’ . S c ie n t ia
Horticulturae, 108: 66–73.
37. Kos ter, K.L, 1991. Glas s formatio n and
d es iccation tolerance. Phys iology Plant, 96:
302–304.
38. Lambardi, A ., A . Fabbri and A . Caccavale, 2000.
Cryopres ervation of white poplar (P opulus alba
L.) by vitrification of in v itro-grown s hoot tips .
Plant Cell Reports , 19: 213–218.
Adv. Environ. Biol., 3(1): 69-83, 2009
39. Langis , R. and P.L. S t e p o n ku s , 1991.
Vitrification of is olat e d ry e p ro t o p la s t s :
protection agains t dehydration injury by ethylene
glycol. Cryoletters , 12: 107–112.
40. Lin, C. and M .F., 1992. DNA s equence analys is
of a comp lementary DNA for cold-regulated
Arabidopsis gene cor15 and cha racterization of
the COR15 polype p t ide. Phys iology Plant, 99:
519–525.
41. Lou, H. and S. K a ko , 1995. Role of high s ugar
concentration in inducing s omatic embryogenes is
f r o m c u c u mb e r c o t y le d o n s . S c i e n t i a
Horticulturae, 64: 11-20.
42. M allon, R., P. Barro s , A . Luzardo and M .L.
Gonzalez, 2007. Encaps ulation of mos s buds : an
efficient method for the in vitro cons ervation and
regeneration of the endangered mos s Splachnum
ampullaceum. Plant Cell, Tis s ue a n d Organ
Culture, 88: 41-49.
43. M amiy a , K. and Y. Sakamoto, 2000. Effects of
s ugar concentration and s trength of b a s al
medium on convers ion of s omatic embryos in
A s paragus officinalis L. Scientia Horticulturae,
84: 15-26.
44. M andal, J., S. Pattnaik and P.K. Chand, 2000.
A lg inate encaps ulation of Ocimum americanum
L. (Hoary Bas il), O. basilicum L. (Swee t Ba s il),
O . gratissimum L. (Shrubby Bas il) and O .
sanctum L. (Sacred Bas il). In Vitro Cellular
Developing Biologal Plant, 36: 287-292.
45. M a rt in , K .P ., 2003. Clonal propagation,
en c a p s u lation and reintroduction of Ipsea
m a l a b a r i c a (Re ichb. f.) J.D. Hook., a n
endangered orchid. In Vitro Cellular Developing
Biologal Plant, 39: 322-326.
46. M artin, K.P. a n d J. M adas s ery, 2006. Rapid in
vitro propagation of Dendrobium hybrids through
direct s hoot forma t io n from foliar explants , and
protocorm-like bodies . Scientia Horticulturae,
108: 95-99.
47. M artin, K.P. and A .K. Pradeep, 2003. Simp le
s trategy for the in vitro cons ervation of Ipsea
malabarica an endemic and endangered orchid of
the W es tern Ghats of Kerala , India. Plant Cell,
Tis s ue and Organ Culture, 74: 197-200.
48. M artinez, D., R. A rroy-Garcia and M . Revilla,
1999. Cryopres ervation of in vitro grown s hoottips of Olea europaea L. var. A rb equina.
Cryoletters , 20:29–36.
49. M cKers ie, B.D. and D.C.W . Brown, 1996.
S o matic embryogenes is and artificial s eeds in
fo rage legumes . Seed Science Res earch., 6:
109–126.
50. M erkle, S.A ., W .A . Parrott and B.S. Flinn, 1995.
M orphogenic as pects of s omatic embryogenes is .
In: Thorp e , T.A . (ed.), In Vitro Embryogenes is
in Plants , pp: 155-203.
81
51. M iku³a, A ., M . Niedziels ki and J.J. Rybczyns ki,
2006. The us e of TTC reduct io n as s ay for
a s s es s ment of Gentiana s pp. cell s us pens ion
v ia b ilit y a f t e r c ry o p re s e rv a t io n . A c t a
Phys iologiae Plantarum, 28(4): 315-324.
52. M oges , A .D., R.A . Shibli an d N.S. Karam, 2004.
Cryopres ervation of A frican Violet (Saintpaulia
ionantha W endl.) s hoot tips . In Vit ro Cellular
Developing Biologal Plant, 40: 389–395.
53. M undree, S.G., B. Baker, S. M o w la, S.Peters , S.
M arais , C.V. W ilingen, K ., Go v ender, A .
M aredza, S. M uyanga, J.M . Farrant and J.A .
Thoms on, 2002. Phys iological and molecular
ins ights into drought tolerance. A frican Journal
of Biotechnology, 1: 28–38.
54. M uras hige, T. and F. Skoog, 1962. A revis ed
medium for rapid gro w t h and bioas s ays with
tobacco tis s ue culture. Phys iologia Plantarum, 15:
473-497.
55. M urdad, R., S.K. Kuik, K.S. Choo, M .A . Latip,
Z.A . A ziz and R. Ripin, 2006. High frequency
multiplication of Phalaenopsis gigante a us ing
trimmed bas es protocorms tec hnique. Scientia
Horticulturae, 111: 73–79.
56. Nayak, N.R., S.P. Rath and S. Patna ik, 1998.
High frequency plant regeneration from alginate
e n c a ps u la t e d p ro t o c o rm-like b o d ie s o f
Spathoglottis plicata Bl., a terres trial orchid.
Phytomorphology, 48: 179–186.
57. Nayak, N.R., S. Sahoo, S. Patna ik a n d S.P. Rath,
2002. Es tablis hment of thin cros s s ection (T CS)
culture method for rapid microprop agation of
Cymbi d ium aloifolium (L.) Sw. and Dendrobium
nobile Lindl. (Orchidaceae). Science Horticulture,
94: 107.
58. Niino, T. and A . Sakai, 1992. Cryopres ervation
of alginate-coated in vitro-grown s hoot t ip s of
apple, pear and mulberry. Plant Science, 87:
199–206.
59. Paques , M ., M . Pois onnier, E. Dumas and V.
M onod, 1997. Cryopres ervation of dorman t and
n o n -d o rma n t b ro a d le a v e d t re e s . A c t a
Horticulturae, 447: 491–497.
60. Pelah, D., R.A . Kaus hik, A . Nerd and Y.
M izrahi, 2003. Validit y of in vitro viability tes ts
for predic t in g res pons e of different vine cacti in
the fie ld to high and low temperatures . Journal
of the Profes s ional A s s ociatio n for Cactus
Development, 5: 65-71.
61. Rajas ekaran, K., 1996. Regeneration o f plants
from cryopres erved embryogenic cell s us pens ion
and callus cultures of cotton (G ossypium
hirsutum L.). Plant Cell Reports , 15: 859–864.
62. Redenbaugh, K., P.R. Vis s , D. Slade an d J.A .
Fujii, 1987. Scale-u p : artificial s eeds . In: Green,
C.E., Some rs , D. A ., Haekett, W . P. and
Bies boer, D.D. (eds .), Plant tis s ue and cell
culture. Alan R. Lis s , New York, pp: 473–493.
Adv. Environ. Biol., 3(1): 69-83, 2009
63. Saipras ad, G.V. S. and R. P o lis etty, 2003.
Propagation of three orchid genera us ing
encaps ulated protocorm-like-bodies . In Vitro
Cellular Developing Biologal Plant, 39: 42–48.
64. S a kawa, Y., 1990. Orchids : other cons iderations .
In: P.V. A mmirato, D.R. Evans , W .R. Sharp. and
Y.P.S. Bajaj, (eds .), Handbook of plant cell
culture, 5. M cGraw-Hill, N e w Yo rk. p p :
638–653.
65. Santos , J ., I. Santos and R. Salema, 1998. In
v i t r o production of bulbs o f N a rc is s u s
bulbocodium flowering in the firs t s eas on of
growth. Science Horticulture, 76: 205–217.
66. S c o c c hi, A ., M . Faloci, R. M edina, S. Olmo s
and L. M rogins ki, 2004. Plant recovery of
cryopres e rved apical meris tem-tips of Melia
azedarach L. us ing encaps ulation/dehydration
and as s e s s ment of their genetic s tability.
Euphytica, 135: 29-38.
67. Shatnawi, M .A . and K.A . J o hns on, 2004.
Cryopres ervation by encaps ulation-dehydration of
‘Chris tmas Bus h’ (Ceratopetalum gummiferum)
s hoot tips . In Vitro Cellular Developing Biolo g a l
Plant, 40: 239–244.
68. Shirgurkar, M .V., C.K. John and R.S. Nadgauda,
2001. Factors affecting in vitro microrhizome
p roduction in turmeric. Plant Cell, Tis s ue and
Organ Culture, 64: 5–11.
69. Singh, F., 1992. M icropropagation of orchids –
Spathoglottis plicata and Epidendrum radicans.
In : Ba ja j, Y.P.S. (e d .), H ig h -t e c h a n d
mic ro p ro p agatio n IV. Bio t e c h n o lo g y in
agriculture and fores try, 20., Springer, New
York, Berlin, Heidelberg, pp: 223–243.
70. S ingh, A .K., M . Sharma, R. Vars hney, S.S.
A g a rw a l a n d K .C. Ba n s ali, 2006. Plant
regeneration from algiante-encaps ulated s hoot-tips
of Phylla nthus amarus Schum and Thonn, a
medicinally important plant s pecies . In Vitro
Cellular Developing Biologal Plant, 42: 109–113.
71. Steponkus , P .L., 1971. Cold acclimation of plant
tis s ue cultures . Cryobiology, 8: 386-387.
72. Steponkus , P.L. an d F.O. Lamphear, 1967.
Refinement of the triphenyltetrazolium chloride
method of d e termining cold injury. Phys iology
Plant, 42: 1423-1426.
73. Taeb, A .G. and P.G. A lders on, 1990. Effect of
low tempera t u re a n d s u c ro s e o n b ulb
development and on the carbohydrate s tatus of
bulbing s hoots o f t u lip in vitro. Journal of
Horticulture Science, 65: 193–197.
74. Tahtamouni, R.W . and R.A . Shibli, 1999.
P re s e rv a t io n a t low t e mp e ra t u re s a n d
cryopres ervation in wild pear (P y rus syriaca).
A dvance in Horticultural Science, 13: 156–160.
75. Tanaka , M , 1992. M icropropagation of
Phalaenops is s pp. In: Bajaj, Y.P .S. (Ed.), Hightech and Micropropagation. IV. Biotechnology in
Agriculture an d Forestry, 20. Springer-Verlag,
Berlin, Heidelberg, New York, pp: 246–266.
82
76
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
.Tokuhara, K. and M . M a s ahiro, 2003. Highlyefficient s oma t ic e mb ryogenes is from cell
s us p ens ion cultures of Phalaenopsis orchids by
adjus ting carbohydrate s ources . In Vitro Cellular
Developing Biologal Plant, 39: 635–639.
Towill, L.E, 1996. Vitrification as a method to
cryopres erve s hoot tips . In: Trig iano, R. S.,
Gray, D. J. (eds .), Plant tis s ue culture c o n cepts
and laboratory exercis es . CRC Pres s , Boca
Raton, FL., pp: 297–304.
Towill, L.E. and P. M azur, 1975. Studies o n the
reduction of 2,3,5-t riphenyltetrazolium chloride
as a viability as s ay for plant tis s ue cultures .
Canadian Journal of Botany, 53: 1097-1102.
Urag ami, A ., M .O. Lucas , J. Ralambos oa, M .
Renard and J. Dereuddre, 1993. Cryopres ervation
of micros pore e mbryos of oils eed rape (Brassica
napus L.) by dehydration in air with or without
alginate encaps ulation. Cryoletters , 14:83–90.
Valento v iè , P., M . Luxová, L. Kolaroviè and O.
Ga š paríková, 2006. Effect of os motic s tres s on
compatible s olut e s content, membrane s tability
and water relations in two maize cultivars . Plant
Soil and Environment, 52(4): 186–191.
Vandenbus s che, B. and M .P. De Proft, 1996.
Cryopres ervation of in vitro s ugar beet s hoot-tips
us ing the encaps ulation–dehydration technique:
development of a bas ic protocol. Cryoletters ,
17:137–140.
Van W aes , J.M . and P.C. Debergh, 1986. In
vitro germin a t io n of s ome W es t-European
orchids . Phys iology Plant, 67: 253–261.
Vendrame , W .A ., V.S. Carvalho, and J.M .M .
Dias , 2007. In vitro germination and s e e dling
development of cryopres erved Dend r o b ium
hybrid mature s eeds . Sc ie n tia Horticulturae, 114:
188-193.
Verleys en, H., E.V. Bocks taele and P. Debergh,
2005. A n encaps ulation–dehydration protocol for
c ry o p re s e rv a t io n o f the azalea c u lt iv a r
‘N o rd lic ht’ (Rhododendron simsii Planch.).
Scientia Horticulturae, 106: 402–414.
Verleys en, H., G. Samyn, E.Van Bo c ks taele. and
P. Debergh, 2004. Evaluation of analytical
t e c h n iq u e s t o p r e d i c t v ia b ilit y a ft e r
cryopres erv a tion. Plant Cell, Tis s ue and Organ
Culture, 77: 11-21.
W hiters , L.A , 1985a . Cryopres ervation of
cultured plant cells and protoplas ts . In: Vas il, J.
K. (ed.), Cell cu lt ure and s omatic cell genetics
o f p l a n t s . C e l l g r o w t h , n u t r it io n ,
cytodifferentiation, and cryopres ervation, vol. 2,
A cademic Pres s Inc., Orlando, Florida, pp: 253316.
W hiters , L.A ., 1985b. Cryopres ervation of
cultured plant cells and protopla s ts . In: Kartha,
K. K. (ed .), Cryopres ervation of Cultured Plant
Cells and Organs , CRC Pres s Inc., Boca Ra t on,
Florida, pp: 243-267.
Adv. Environ. Biol., 3(1): 69-83, 2009
88. W idholm, J.M ., 1972. T he us e of fluores cein
diacetate and ph e nos afranin for determining
viability o f c u lt u re d p la n t c e lls . Stain
Technology, 47: 189-194.
89. W ikimedia Foundation Inc., 2008. A s cocenda
[ i n t e r n e t ] .
A v a ila b le
a t
http://en.wikipedia.org/wiki/A s cocenda.
90. W is niews ki, M ., R. W ebb, R. Bals amo, T.J.
Clos e, X.M . Yu a n d M . Griffith, 1999.
Purification, immunolocalization, cryoprotective,
and antifreeze a ctivity of PCA 60: a dehydrin
from peach (P r u nus persica). Phys iology Plant,
105: 600–608.
91. W ood, B.C., H.W . Pritchard and A .P. M iller,
2000. Simulta n e o us pres ervation of orchid s eed
and it s fungal s ymbiont us ing encaps ulationdehydration is depen d e nt on mois ture content
and s torage temperature. Cryoletters , 21: 125136.
83
92. Zhang, Y.X ., J.H. W ang, H. Bian and M .Y. Zhu,
2001. Pregrow t h -d e s iccation: a s imple and
efficient procedure for the cryopres erv a t ion of
rice (Oryza sativa L.) embryogenic s us pens ion
cells . Cryoletters , 22: 221–228.
93. Zhao, P., F . W u, F.S. Feng and W .J. W ang,
2007. Protocorm-like body (PLB) fo rmation and
plan t regeneration from the callus culture of
Dendro b i u m candidum W all ex Lindl. In Vitro
Ce ll D e v e lo p mental Biology P la n t , d o i:
10.1007/s 11627-007-9101-2.