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AUTHENTICITY
I declare that this thesis is my own work, except for those sections explicitly
acknowledged, and to my knowledge the main content of the thesis has not been
previously submitted for a degree at any other university.
Rajnesh R. Prasad Sant
DATE: 27/09/2001
ABSTRACT
From this study it was discovered that the hormone-free clonal propagation system developed
by Thinh (1997) for Colocasia esculenta var. antiquorum, based on enhanced axillary
branching (multiple shoot formation) through liquid TDZ medium shake culture and
subsequent carry over effect on hormone-free medium, did not work with the tropical taro
variety, Tausala ni Samoa, of Colocasia esculenta var. esculenta type. After two four-week
cycles on liquid TDZ shake medium cultures, no enhancement in proliferation rates was noted
and no carry over effect was observed when these plants were transferred to hormone-free
media.
The vitrification method of cryopreservation was experimented with cultivars of the tropical
taro (Colocasia esculenta var. esculenta) and the technique was shown to have potential for
the cryopreservation of taro from Pacific Island countries. Out of the eight taro cultivars
experimented with, three, namely E399, CPUK and TNS, were successfully cryopreserved
with average recovery rates of 20, 29 and 29%, respectively.
The optimum vitrification protocol for the cultivars E399 and CPUK was; using shoot-tip
donor plants cultured on solid MS in large jars for three months as sources of shoot-tips,
which consisted of the apical dome surrounded by two leaf primodia; preculturing these shoot-
tips overnight (16hr) on 0.3 M sucrose medium; loading with liquid MS supplemented with 2
M glyceroi + 0.4 M sucrose for 20 min at 25°C, dehydrating with PVS2 for 12 min at 25°C
followed by rapid immersion in LN. Thawing was done by shaking the shoot-tips rapidly for
90 sec in waterbath at 40°C, followed by rehydration in liquid MS medium supplemented with
1.2 M sucrose for 15 min. The shoot-tips were then plated on a layer of filter paper on MS
medium supplemented with 0.3 M sucrose and left overnight in the dark. Next day, they were
transferred onto MS medium supplemented with 0.1 M sucrose and maintained in the dark for
three days, then transferred to dim light (10 u.molm"V) for one week before exposure to
normal culture conditions. Piantlets were produced after about two months.
For cultivar TNS, the optimum vitrification protocol was preconditioning explants on solid
MS supplemented with 90 g/1 sucrose for seven weeks prior to dissecting and cryopreserving
the shoot-tips without any preculture, The vitrification procedure was same as described
above.
During this study, it was found that the vitrification protocol has to be optimized for each
individual taro cultivar.
DEDICATION
This thesis is dedicated to my parents, Ram Prasad and Jai Wati, who had the vision
and courage to rise above their experiences.
AKNOWLEDGEMENTS
I would like to acknowledge my supervisors, Dr. Mary Taylor, the Tissue Culture Specialist of
Regional Germplasm Center at the Secretariat of the Pacific Community and Professor Anand
Tyagi, Head of the Department of Biology at The University of the South Pacific, whose
efforts have made this project a success. Dr. Taylor's relentless and enterprising efforts as a
supervisor in the still infant field of cryopreservation of tropical species deserve a special
mention.
The help and support given to me by the RGC personnel, Samila, Eliki, Raghani, Rohini and
Kiran was invaluable. My special thanks to Samila for inaugurating me in the 'art and craft' of
plant tissue culture.
I wish to extend my gratitude to staff of SPC and technical staff of USP, whose names 1 do not
mention out of the fear of inadvertently missing out some, for their help and support.
I am indebted to two people, without the advice, suggestions and guidance of whom this
project may not have succeeded. Firstly, Dr. Barbara Reed of National Clonal Germplasm
Repository, Oregon, USA, through whom I got my introduction to the world of
cryopreservation. She had been my mentor throughout my research. Secondly, Dr. Nguyen
Tien Thinh of Nuclear Research Institute, Dalat City, Vietnam, who showed me the finer
details of the vitrification method he developed for taro. It was only after his expert insight
and suggestions that I achieved positive results.
My wife, Nischal Lata, deserves special recognition for her understanding and courage for the
trials she endured as a newly wed wife of a research student and her help with the
proofreading of my thesis.
Last, but not the least, I would like to thank my sponsors, AusAID, who under the TaroGen
project provided the funds with which this work was carried out.
LIST OF APPENDICES
Appendix 1: Recent successful cryopreservation of shoot apices by vitrification 89
Appendix 2: Bacteriological 523 Medium (Viss) 91
CHAPTER 1
1.0 INTRODUCTION
1.1 Description of Taro (Colocasia esculenta (L.) Schott Aracea)
Taro is a common name of a species of the Colocasia genus that belongs to the Araceae
family. It is a herbaceous plant one or two metres tall with peltate shaped leaves attached to
one metre stout petioles that clasp around the base. The underground cylindrical corms are
30cm in length and 15cm in diameter with short internodes and a superficial and fibrous root
system (Purseglove, 1972; Strauss, 1983). The yellow unisexual flowers are borne on a stout
peduncle that is shorter than the petiole with about 20cm spathe covering the spadix. The
spadix has male flowers on the upper portion and a mixture of female and sterile flowers on
the lower part (Purseglove, 1972; Wilson, 1990).
Only some taro cultivars flower naturally. However, flowering can be achieved through
artificial induction (Katsura et al, 1986; Miyazaki et at, 1986; Wilson, 1990). The nutritive
composition of corms is as follows: water 63% - 85%, carbohydrates 13% - 29%, protein 1.4%
- 3.0%, fat 0.2% - 0.4%, fiber 0.6% - 1.2% and ash 0.6% - 1.3%. Vitamins B and C are present
in appreciable amounts in the corm. The leaves contain 87.2% water, 6.0% carbohydrates,
3.0% protein, 0.8% fat 1.4% fiber, 1.6% ash, and they are excellent source of vitamin C
(Purseglove, 1972).
1.2 Taxonomy
Taro (Colocasia esculenta (L.) Schott) belongs to the monocotyledonous Araceae along with
two other important root crops, Alocasia and Xanthosoma. There are confusions in the
taxonomy of different Colocasia cultivars with edible tubers. The general agreement now, as
first suggested by Purseglove (1972) and Pluckett (1983), is that there is one species,
Colocasia esculenta (L.) Schott, with two botanical varieties. The first variety is Colocasia
esculenta var. esculenta, commonly known as taro, dasheen or cocoyam. The second variety is
Colocasia esculenta var. antiquorum, commonly known as taro or eddoe.
The important differences between the two cultivars include; antiquorum has small central
corm with many side cormels or tubers while esculenta has a large central corm. In
antiquorum, the sterile spadix is longer than male section and is three times or more than that
of esculenta. Antiquorum is hardier of the two - withstands lower rainfalls, colder climates and
lighter poorer soils, and has longer storage periods (Purseglove, 1972). Throughout this study,
taro will be used to refer to.Colocasia esculenta var. esculenta.
1.3 Distribution and Use
Taro is commercially cultivated throughout the humid and semi-humid tropics, as well as
some warm regions of temperate areas with irrigation or rainy conditions. Worldwide, taro
ranks fourteenth among staple vegetable crops with about 9.2 million tonnes produced
globally from 1.8 million hectares with an average yield of 5.1 t/ha (FAO, 1996). Regions
with active taro cultivation include South-East Asia, Pacific Islands (Papua New Guinea
[PNG] inclusive) the South and Central China, Japan, India, West Indies and West and North
Africa.
Almost all parts of taro plants are useful, either palatable or otherwise. However, as previously
stated, the tuber is the most nutritive and considered most delicious. The conns are roasted,
baked or boiled. The young leaves, including laminas and petioles, are used as vegetables.
Blanched young shoots can also be eaten like asparagus. Some common dishes include the
Hawaiian 'Poi', 'Che Mon Sap' of Viet Nam, and 'Callaloo' soup in Trinidad (Purseglove,
1972; Thinh, 1997). Taro corn) puree is used as a low allergic and easily digested baby food.
Taro corm confections and leaves are also commercially processed in some countries (Nip,
1989; Tuia, 1997). It is also used as a traditional medicine. Root extracts are used to treat
rheumatism and acne, while leaf extracts help clot blood, neutralize snake poison and act as a
purgative (Winarno, 1990). Corms from wild plants and inferior cultivars as well as cooked or
fermented silage from waste leaves and corms are used as animal feeds.
(
1.4 Importance of Taro in Pacific
Taro cultivation has been practiced in the Pacific Island countries (PICs) for centuries. It is
thought to have reached the Polynesian islands about two thousand years ago from the Indo-
Malayan region. Lebot et al. (2000) have suggested that there are two distinct gene pools in
South Asia and Melanesia and these probably reflect natural differentiation of the species on
each side of the Wallace line. Over generations, taro has found its way into the rituals and
fabric of local custom and culture in the Pacific and forms an important part of traditional
ceremonies and auspicious occasions. It is also a staple food in the Pacific region and almost
all parts, including the corm, leaves, stem and flowers are prepared as various delicacies. Taro
is of great economic importance in the Pacific region. It is an important, and in some cases the
major, export of a number of Pacific Island countries (Jackson, 1994; Taylor, 2001).
There are believed to be about two thousand taro cultivars in the Pacific region (Hunter et al.,
1998). A report from an Australian Center for International Agricultural Research (ACIAR)
funded project presented at the taro genetic conservation strategy workshop in Suva, Fiji (5"1-
7th September 2001) revealed the significant taro diversity in the region, especially in PNG.
Concerted efforts are required to conserve this diversity as significant genetic erosion has
occurred due to agricultural intensification, pests, diseases, natural disasters, and civil unrests.
Taro diversity is important for long-term food security as highlighted in the case of taro leaf
blight (TLB) disease outbreak in Samoa in 1993. The introduction of exotic TLB tolerant and
resistant varieties from Palau and the Philippines was used to combat the disease.
1.5 Conservation of Taro Germplasm
The options for taro germplasm conservation can be categorized in two main areas, in situ, and
ex situ conservation.
1.5.1 In situ conservation
In situ conservation is the conservation of ecosystems and natural habitats and the
maintenance and recovery of viable populations of species in their natural surroundings and, in
the case of domesticated or cultivated species, in the surroundings where they have developed
their distinctive properties. It encompasses genetic reserves, on-farm and home garden
conservation (Maxted et al, 1997; Engelmann, 2000). In situ conservation of cultivated
species is primarily concerned with the on-farm maintenance of traditional crop varieties (or
land races) and with forage and agroforestry species (Taylor, 2001).
Farmers have been practicing on-farm conservation for centuries, through the selection and
maintenance of those varieties best suited to the local ecological conditions and social and
cultural requirements. However, it is a strategy about which very little is known. It is only in
relatively recent years that studies have been conducted in attempts to determine the scientific
basis of on-farm conservation. In addition to this, crops grown in the field situation are
vulnerable to pest and/or disease attack, and to climatic extremes.
It is also a system that is at the mercy of farmers' desire and ability to maintain the various
cultivars (Taylor, 2001). A preliminary study was carried out within the Taro Genetic
Resources Conservation and Utilization Project (TaroGen) that showed the impact of market
pressure on the varieties chosen by the farmers for cultivation. Economically good varieties
attracted more interest from fanners' than traditional ones, leading to abandoning of
traditional varieties. This influence of new varieties on the impact of traditional varieties
highlights the need to generate more knowledge and information about on-farm conservation.
f
1.5.2 Ex situ conservation
Ex situ conservation can be in the form of biosphere reserves, botanical gardens, field
genebanks, seeds and in vitro storage which includes slow growth and cryopreservation
(Maxted et al., 1997; Engelmann, 2000; Taylor, 2001). The first two forms are not suitable for
taro germplasm conservation.
1.5.2.1 Seeds
Most taro plants do not flower naturally hence storage of taro germplasm as seeds has not been
a feasible option. However, the technology to make taro flower through gibberellic acid
induction (Miyazaki et al., 1986; Katsura et al, 1986; Wilson, 1990) is now available and the
possibility to store taro germplasm as seed is being investigated under the TaroGen project.
Although seeds could be used to conserve genes, they are not an option for maintaining
genetically true-to-type germplasm.
1,5,2.2 Field Genebanks
According to a Consultative Group on International Agricultural Research (CGIAR) report
(1989), field genebanks are more aptly called field collections, as they tend to be working
collections. As a short to medium term activity, field collections are effective as working
collections. They can be a useful system for conserving farmer-preferred varieties. In addition
to this, they also facilitate screening, evaluation, breeding and distribution.
Field genebanks face setbacks in that maintenance is labour-intensive and expensive due to
demands for heavy machinery such as tractors and harvesters, vehicles for transport of
material to and from field, labour, fuel, repairs, fertilizers and chemicals, buildings for storage,
insurance and so on (Jerret and Florkwoski, 1990). The cost and maintenance of field
genebanks as opposed to in vitro storage (slow growth) is being investigated under the
TaroGen project.
Other issues that are particularly pertinent to the Pacific situation include climatic extremes
such as hurricanes, flooding and drought, limited resources, high staff turnover, and land
ownership disputes. Another drawback is the loss of plants through insects, pests, pathogens,
and diseases as in the Solomons in 1975 and the annihilating outbreak of TLB in Samoa in
1993 (Jackson, 1994). Individual plants may also be lost through human errors.
The regional countries do not have the economy to facilitate collection and genetic
conservation of taro in field genebanks. Losses of taro genetic resources have already occurred
in some countries due to natural disasters (flooding and cyclones in Vanuatu), diseases (TLB
in Samoa) and ethnic conflicts (Solomon Islands).
1.5.2.3 In vitro storage
In vitro storage of taro involves micropropagating shoot tips and maintaining them under slow
growth conditions in tissue culture laboratories. The conditions that can be used to induce
slow growth include: lowered incubation temperatures, raised osmolarity of the culture
medium and decreased oxygen concentration in the culture containers. Some studies have
showed that taro can be stored under slow growth conditions (high osmolarity and low
temperature) for over a year (Staritsky et at, 1986; Zandvoort, 1987; Bessembinder et al.,
1993; Taylor, 1998).
(
In vitro storage offers the following advantages:
• explants free from pathogens and safe from natural disasters
• relatively smaller space required to house cultures
• better (visual) monitoring of cultures
• high propagation potential of cultures
• slow growth conservation in in vitro is apt for short to medium term storage for accessions
that are being distributed on regular basis
The drawbacks can be:
• high establishment costs of a tissue culture lab and recurrent funding needed for
maintenance with very precise storage conditions, frequent monitoring, costly chemicals,
and specialized staff. Labour needs and costs for the running of an in vitro active genebank
can also be high, especially if operating on a large scale. Jerret and Horkowski (1990)
have estimated that two to three technical staff are necessary for initiating, maintaining,
and subculturing a collection of 1000 accessions
• cultures can be lost through bacterial and/or fungal contaminations, human errors and
power failures in the absence of backup facilities
• genetic instability could compromise genetic conservation. Genetic instability due to
somaclonal variation may have genotypic and phenotypic consequences such as changes in
chromosome number and structure, loss of secondary product production, and
characteristical changes such as disease resistance or plant height (Withers, 1987; Taylor,
1988; Ashmore, 1997; Thinh, 1997).
To date there have been no studies to assess the genetic stability of taro under slow growth
storage. For the PICs, the lack of technical staff and the high costs of establishing and
maintaining tissue culture labs make in vitro conservation not a viable option.
1.5.2.4 Crvopreservation
In light of the various challenges and shortcomings experienced with both genetic reserves and
field genebanks, cryopreservation is the safest and most cost-effective option for the long-term
conservation of Plant Genetic Resources (PGR) at present (Withers and Engels, 1990; Maxted
et al., 1997; Engelmann, 2000). Cryopreservation has the following advantages over other
PGR conservation methods:
• large number of accessions can be stored in a small space (for example: a 25L Dewar
could store germplasm for 8000 individual plants) for unlimited time
• the collection is housed in the safe and secure confines of a laboratory and this obliterates
the external threats which other ex situ and in situ methods are susceptible to
• ensures protection against loses through culture contaminations
• maintains genetic fidelity of the germplasm collection.
However, the following requirements have to be met in setting up cryopreservation facilities
for any crop:
• well developed tissue culture technology for the crop
• round-the-clock electricity supply
• a reliable supply of liquid nitrogen
• modern, well-maintained laboratory facilities
• appropriate training for staff (university courses, regional workshops, sponsorship for
study and travel)
(Ashmore, 2001).
('
1.6 TaroGen Project
As mentioned before, an outbreak of TLB (Phytophthora colocasiae) in the Samoan Islands in
1993 had a devastating impact on the taro industry as well as subsistence fanners. Farmers
were forced to abandon the crop, which had severe economic implications. It also resulted in
the loss of genetic resources. Collaborated effort of concerned authorities, interested parties
and international donors to address the problem resulted in the TaroGen project in 1997.
The project is funded by Australian Aid for International Development (AusAID) and
implemented by the Secretariat of the Pacific Community (SPC). It aims to collect, improve
and conserve the regional taro varieties. According to the project design document of TaroGen
(1997), the aim of the conservation component of the project is to overcome the difficulties
that all Pacific Island countries face in maintaining field collections of taro which are costly
and subject to loss from natural disasters, pest attack and neglect due to fluctuating financial
support. To facilitate the regional countries' conservation efforts, a Regional Germplasm
Centre (RGC) has been established at SPC, Suva within the project. The RGC was established
in response to the realization that increased collaboration among the countries of the region is
crucial in PGR conservation in the Pacific (Sajise, 2001).
As part of the TaroGen project, taro conservation strategies were to be investigated. These
included on-farm conservation, in vitro slow growth storage and cryopreservation.
Cryopreservation was considered necessary, as one of the outputs of the project would be a
collection of approximately two thousand taro accessions collected from PICs. This is
valuable material accumulated because of its genetic diversity. The only safe and effective
method for conserving this taro germplasm on a long-term basis would be cryopreservation.
The purpose of this study is to investigate the applicability of cryopreservation for storage of
regional taro germplasm. The vitrification protocol developed by Thinh (1997) on Japanese
taro would be assessed for its competency with taro from PICs.
10
CHAPTER 2: LITERATURE REVIEW
2.0 CRYOPRESERVATION
2.1 Introduction
Cryopreservation is storage at ultra low temperatures such as that of liquid nitrogen (-196°C)
where all cellular divisions and metabolic processes cease and plant material has unlimited
span of storage without alteration or modification. In 1956, Sakai carried out the first
successful experiment on storage of plant tissue by freezing with liquid nitrogen on mulberry
twigs (Sakai, 1995). In 1968, Quatrano showed cultured cells of flax pretreated with
dimethylsulphoxide (DMSO) to withstand freezing to -50°C. Several new cryopreservation
techniques have been developed in. recent years for application to a larger range of tissues and
organs, in myriad infrastructural situations (Engelmann, 1997a; Takagi, 2000).
2.2 Freezing Implications
The principal challenge faced during cryopreservation is avoiding the irreversible damage
caused, to tissue by the crystallisation of water contained by all organic matter (cells, callus,
shoot tips, embryos). During freezing, tee can form either outside the cells (extracellular
freezing) or inside (intracellular freezing). Cryopreservation involves dehydrating plant
material prior to liquid nitrogen (LN) cooling to avoid water crystallisation damage.
However, plant cells can survive cooling only to a minimum critical temperature, which is
species dependant, and can recover only after a 40-50% critical minimum volume reduction
(Merymann et at, 1977; Sakai, 1995). Excess shrinkage and excessive dehydration can cause
cell injuries in various ways. Endocytotic vesicles may form on cell plasmolysis through
membrane infolding and fusion. These vesicles could cause cells to burst on rehydration.
Other forms of injury could be a dysfunctional cell membrane, coagulated, precipitated and
subsequently toxic cell contents, and protoplasmic pH alterations. Cellular proteins can also
get denatured at low temperatures (Levitt, 1980; Gordon-Kamn and Steponkus, 1984a,b,c;
Singh and Miller 1985; Steponkus and Lynch, 1989; Steponkus et at, 1993; Pearce, 2001). In
light of above, the dehydration process should be optimised to dehydrate plant material
enough to cryopreserve, whilst avoiding excess shrinkage and excessive dehydration injuries.
2.3 Dehydration and Vitrification
Cryopreservation employs two mechanisms for the treatment of intracellular water. The first
mechanism exploits freeze-induced dehydration, where slow cooling causes cell exterior and
medium to cool and form ice. The supercooled, but still liquid interior, responds by shedding
water to equilibrate built up internal aqueous vapour pressure (Engelmann, 1997a; Ishikawa et
at, 2000). The second mechanism, termed vitrification, involves the transition of water
directly from the liquid phase into an amorphous phase or glass, whilst avoiding the formation
of intraprotoplasmic ice crystals (IIC). Rapid thawing prevents IIC formation due to
devitrification.
Vitrification (glass formation) occurs readily in highly concentrated and viscous solutions that
become solid without forming crystals upon rapid cooling (Burke et at, 1976; Fahy et at,
1984; Steponkus, 1985; Sakai et at, 1990; Engelmann, 1997a). Effective vitrification
solutions, called cryoprotectants, consist of high molecular weight compounds or osmoticums
such as 5-10% w/v or v/v levels of DMSO, sucrose, glycerol, abscisic acid (ABA) or proline
(Kartha, 1985; Benson, 1999). Treating plant material (cells in suspension, calluses, embryos
12
and apices) firstly with cryoprotectants and then rapidly cooling causes the vitrification of the
cells. This avoids mechanical damage caused by the formation of HC.
The glass formed has lower vapour pressure than extracellular ice and therefore, further
dehydration of cells and excessive cell shrinkage is avoided. The glass also prevents the build-
up of cell solutes and thus prevents solute toxicity and pH alterations. The highly viscous glass
stops all chemical reactions requiring molecular diffusion. These factors render the plant
material dormant and stable for unlimited length of time (Fahy el al., 1984; Sakai et ah, 1990;
Fujikawa & Jitsuyama, 2000). However, very high concentrations of cryoprotectants (CP) are
toxic to plant materials. Hence the concentration, combination and length of exposure to CP
has to be optimized for different species and type of plant material to be stored.
2.2 Cryopreservation Strategics '
According to Sakai (1993), all the cryopreservation protocols developed over the years fall
into four general categories based on the dehydration treatments before LN immersion. All
strategies aim to overcome cell damage resulting from intracellular crystallization and freeze
dehydration. This is achieved either by glass formation intracellularly (classical methods) or
both intra and extracellularly (complete vitrification method).
The four general categories are:
2.2.1 Conventional slow prefreezing method
This method relies on dehydration of cells by the formation of extracellular ice on slow
cooling so that the cytosol gets concentrated enough to vitrify on LN cooling. The explants are
treated with cryoprotectants (at 0°C for 2-3 hrs) then subjected to slow cooling at rates ranging
13
from 0.1"C /min to 10°C /min depending on species and type of explant using a programmable
freezer. An ice inoculation step at temperatures of -7°C to -10°C facilitates extracellular
crystallization.
The explants are plunged in LN after further slow freezing to -35°C to -40°C, where any
unfrozen extracellular solution and cytosol are sufficiently concentrated to vitrify. Although an
important and efficient protocol, its drawbacks are in the expensive equipment and long
procedural time required (Thinh, 1997). Furthermore, the equipment used in this method
requires high maintenance and therefore is not really viable for low-tech laboratories.
2.2.2 Simple freezing method
This method involves exposing material to concentrated cryoprotectants for extracellular
dehydration instead of slow freezing. The explants are then subjected to prefreezing
temperatures (-30 to -40°C) in an ordinary freezer for a length of time dependant on species
(e.g. 1 hr) before rapid cooling in LN. Although this method eliminates the use of expensive
freezing equipment and ice inoculation step, it is not suitable for use with multicellular
structures such as apical meristems or shoot tips (Sakai et al., 1991; Nishizawa et at, 1992).
2.23 Complete vitrification method '•>
Complete vitrification is when, both, extracellular and intracellular glasses form upon cooling
in LN. This is achieved by sufficiently dehydrating cells, tissues and organs with concentrated
vitrification solutions (PVS) at 0°C or 25°C prior to plunging in LN. PVS not only dehydrates
cells, but also penetrates into cellular interspaces inducing freeze tolerance. However, the
osmotic and chemical toxicity of PVS to plant tissues necessitates optimizing exposure. This
can be achieved either by gradually increasing the concentration of the additive cryoprotectant
14
solutions (i.e. 10-»20->40-»60->80-M00%) or loading the plant material with less
concentrated cryoprotective solutions prior to PVS exposure. The latter is referred to as two-
step vitrification method (Takagi, 2000).
The steps for complete vitrification method can be generalized as follows:
1. preparation and selection of appropriate samples
2. preculturing dissected shoot-tips with osmoticum (e.g. 0.3-0.6M sucrose)
3. treatment with loading solutions
4. dehydration by exposure to PVS at 0°C or 25°C
5. rapid immersion in LN
6. rapid rewarming (40°C)
7. rehydration with unloading solution (e.g. 1.2M sucrose)
8. conditioning apices under favourable conditions for recovery
(Takagi, 2000).
The complete vitrification method has been successfully applied to a range of explant types.
These include protoplasts (Langis and Steponkus, 1991), cell suspensions (Huang et al,
1995), nucellar cells (Sakai et al, 1990), somatic embryos (Uragami, 1989), shoot tips and
meristems (Yamada et al, 1991; Niino et al, 1992b,c; Reed, 1992; Matsumoto et al., 1994;
Matsumoto et al, 1998; Towill and Jarret> 1996), axillary buds (Takagi et al, 1994) and bud
clusters (Kohmura et al, 1992). This method has the advantages of being simple, quick and
easy to carry out and no expensive or sophisticated equipment is required.
2.2.4 Air drying method
It involves concentrating cellular liquids to vitrifiable levels through evaporation with sterile
air from a laminar airflow cabinet (LAF) or silicagel. Optimal water content for successful
15
vitrification is 20-30%. This could be achieved by drying periods of two to ten hours,
depending on the plant species. Plants are conditioned to withstand such levels of dehydration
by low temperature hardening or preculturing with ABA or high sucrose concentrations
(Dereuddre et al, 1990; Uragami et al., 1990; Plessis et al, 1991; Dumet et al, 1993a; Sakai,
1993). Air drying of explants may be direct or after being first encapsulated in alginate beads
as in the recent technique of encapsulation-dehydration (ED) (Takagi, 2000).
The steps for encapsulation-dehydration method can be generalized as follows:
1. preparation and selection of appropriate samples
2. encapsulation of dissected shoot apices in alginate beads
3. preculturing beads with osmoticum (e.g. sucrose, sorbitol, glycerol)
4. desiccation in (LAF) or with silica gel
5. rapid immersion in LN I
6. rapid rewarming (40°C)
7. conditioning apices under favourable conditions for recovery
(Takagi, 2000).
Apart from the incipient success of Fabre and Dereuddre (1990) with ED of Solatium shoot
tips, other successes with cryopreservation are cell suspensions (Bachiri et al., 1995), somatic
embryos (Dereuddre et al., 1991; Hatanaka et al., 1994) and shoot tips (Plessis et al, 1991;
Niino and Sakai, 1992a; Suzuki et al, 1994). Air drying and the modified ED method also
have the advantages of being devoid of expensive appliances and easy to conduct. However,
the disadvantages are that they can be time consuming and explants exhibit a slower post LN
regrowth (Thinh et al., 2000).
16
2.3 Technical Issues for Cryopreservation of Shoot Apices
2.3.1 Introduction
Shoot apices, meristems or shoot tips, are plant organs with an ordered structure and less
differentiated cells. This enables vigorous recovery into plantlets without callus formation or
genetic artifacts (Ashwood-Smith, 1985; Kartha, 1985; Sakai, 1993; Thinh, 1997). Kartha et
al. (1980) and Bajaj (1983) found plants regenerated from cryopreserved apices of strawberry
and cassava to be normal. Oil palms regenerated from cryopreserved somatic embryos also
had normal vegetative and floral development (Engelmann, 1991).
A comparative study on the effects of slow growth and cryogenic storage on the stability of
plants revealed that while cryopreserved Panax ginseng and Catharanthus roseus maintained
normal secondary metabolite production, slow growth reduced it markedly (Mannonen et al.,
1990). A study on potato plants by Harding (1991) showed plants regenerated from
cryopreserved apices to be normal, whereas those stored on mannitol supplemented medium
for six months under slow growth conditions depicted modifications in the Restriction
Fragment Length Polymorphism (RFLP) pattern.
Other studies on evaluation of plants rejuvenated from cryopreserved material also show that
plant material can be safely stored under LN without change. Cote et al. (2000) studied the in-
field behaviour bananas {Musa AA sp.) obtained after regeneration of cryopreserved
embryogenic cell suspension and found no difference at the agronomic level between plants
produced from cryopreserved embryogenic cell suspensions and control plants. Genetic
examination of Picea glauca engelmanni complex for somaclonal variation resulting from
cryopreservation by Cyr et al. (1994) detected no genetic variation. Similarly, randomly
amplified polymorphic DNA (RAPD) fingerprint evaluation of white spruce [Picea glauca
17
(Moench) Voss.] trees regenerated from embryogenic clones cryopreserved for 3 and 4 years,
suggested primary genetic stability (DeVerno et al., 1999).
The new vitrification methods have been optimized to successfully cryopreserve shoot apices
with good recovery. To date a number of shoot apices of vegetatively propagated tropical
monocots (VPTM) have been successfully cryopreserved (Appendix 1). Factors pertaining to
successful cryopreservation of shoot apices include:
2.3.2 Efficient in vitro culture system
The most common source of apices for cryopreservation is in vitro maintained shoot cultures.
The advantages of in vitro cultures are that duplication of adequate numbers of plant material
can be achieved quickly, the material is pests and pathogen free, and the right conditions can
be maintained to induce the optimum physiological conditions essential for cryo survival. An
adequate tissue culture system is also necessary for effective preculturing of dissected apices
and vigorous recovery of cryopreserved shoot apices without intermediate callus formation. In
vitro culture system may be needed to be optimised for cryo success as what may be effective
for conventional micropropagation may not be best for cryo success. Culture conditions such
as light, solid or liquid media have been found to play crucial roles for cryo success (Thinh,
2001). -._
2.3.3 Selection of size and development stage of shoot apices
Shoot tips, comprised of the apical dome with a few leaf primodia, are generally considered to
be the best propagules for cryopreservation of clones (Towill, 1996; Takagi, 2000). The
optimum type and shape of shoot tip to be used is species dependent. The best type of shoot
tips for the VPTM, banana, Cymbidium, Cymbopogon, pineapple and taro, were found to be
18
partly covered ones 0.5 - 1 mm in size (Thinh, 1997; Thinh et al, 2000). These ST consisted
of the apical dome partly covered by the second leaf primodia. Escobar et al. (1997) found that
cryopreserving smaller cassava shoot tips ( 1 - 2 mm and apical dome partly covered by 2-3
leaf primodia) markedly increased the recovery rate.
Another essential factor for cryopreservation success is the optimal developmental stage at
which the explants are used for freezing. Explants from rapidly growing cultures are
recommended as actively dividing cells have characteristics, such as under developed vacuolar
system and dense cytoplasm, which render them more cryopreservation tolerance (Kartha and
Engelmann, 1994; Withers, 1985; Engelmann, 1997a). It has also been found that increasing
the duration for which mother plants are maintained on standard medium without subculture
also increases the recovery rate after cryopreservation (Thinh, 1997; Yongjie et al, 1999).
f
2.3.4 Factors affecting post-thaw survival of shoot apices in the vitrification method
Thinh et al. (2000) categorised the various factors affecting VPTM, namely banana,
Cymbidium, Cymbopogon, pineapple, and taro cryopreservation success as follows; in
descending order of importance:
2.3.4.1 Explant structure
The best explants for cryopreservation are meristems with partially covered apical domes.
Such explants are also referred to as shoot-tips (ST). Taro shoot structure, like most
vegetatively propagated monocots, consists of apical meristems very well covered by the outer
leaf petioles, which are tubular shaped and have interfolded, thick leaf bases. Two consecutive
leaf petioles of an apex are buffered by air spaces. Thinh (1997) postulated that such apex
organization could prevent vitrification solutions from penetrating to apical dome cells. Hence
19
the best shoot-tip structure was found to consist of the apical dome covered by two leaf
primordia.
2.3.4.2 Loading treatment
Cryoprotectants (section 2.3) can be a source of cellular injury. However, their toxic effects
can be minimized by pretreatment (referred to as loading treatment in this study) with sugars,
sugar alcohols, and amino acids introduced via solid or liquid medium (Nag and Street, 1975;
Reid and Walker-Simmons, 1990; Luo and Reed, 1997). Pretreatment chemicals benefit plant
cells and tissue by rendering reduced cell size and the cytoplasm to vacuole ratio, enhanced
ability to take up cryoprotectants, resistance to dehydration injury through cell wall and/or cell
membrane modifications, stabilized membrane bilayers and enzymes and prevention of toxic
levels of compounds accumulating in membranes during dehydration and freezing by acting in
a colligative manner (Heber et at, 1971; Volger and Heber 1975; Withers and King 1979;
Steponkus, 1984; Crowe et al, 1990; Dumet et at, 1993b; Luo and Reed, 1997).
Thinh et al. (2000) found that loading treatment enhanced markedly, both tolerance to PVS2
dehydration (see below) and post-thaw survival of meristems of all the species tested. This is
corroborated by other studies of different species such as rye protoplasts (Langis and
Steponkus, 1991), meristems of wasabi and lily (Matsumoto et al, 1994,1995a) and meristems
and callus of currant (Luo and Reed, 1997). Thinh (1997) found that exposing taro ST to a
loading solution consisting of 2M glycerol + 0.4M sucrose in liquid Murashige and Skoog
(1962) (MS) for 20 minutes gave the highest recovery rates.
20
2.3.4.3 PVS2 dehydration
Only sufficiently dehydrated plant material can survive LN cooling. One of the most effective
and universally used solutions for dehydrating plant material is Plant Vitrification Solution 2
(PVS2) developed by Sakai et al, 1990. It consists of 30% glycerol + 15% ethylene glycol +
15% dimethylsulphoxide + 0.4M sucrose in liquid MS. However, PVS2 can be harmful to
plant material due to high osmotic pressure and chemical toxicity. Hence, exposure time to
PVS2 has to be optimized for different species, and in some cases cultivars (Thinh, 2001). It
has been found that PVS2 sensitive plants can be adequately dehydrated by incubating at 0°C
instead of the normal 25°C. Thinh (1997) optimized taro ST exposure to PVS2 as lOmin at
25°C.
2.3.4.4 Preculture with high sucrose concentrations (
In the studies by Thinh et al. (2000) previously stated, it was found that preculturing the
dissected ST on MS medium supplemented with 0.3M sucrose enhanced the post-thaw
survival of ST of all the plant species, with banana as the only exception. Several theories
have been put forward to explain the positive effects of preculturing. One suggestion is that
the high osmotic pressure resulting from the high level of sucrose in a culture medium can
trigger certain responses in plant cells, such as the accumulation of ABA and/or proline. The
presence of these substances can protect against freezing damage. Mohapatra et al. (1998)
report that ABA induces the synthesis of new proteins that confer cold tolerance to plants.
Studies have found that sucrose and proline stabilize membrane biiayers and enzymes during
desiccation and freezing (Steponkus, 1984; Crowe et al., 1990; Dumet et al, 1993b) The
preculture medium also facilitates in holding dissected ST awaiting cryopreservation.
However, the preculturing stage can be eliminated when using preconditioned plants (section
2.3.4.5).
21
2.3.4.5 Sucrose preconditioning
While cold-hardening (4-5°C) of ST donor plants has enhanced the cryopreservation success
of temperate species, it is not an alternative with temperate species (Bajaj, 1985; Thinh, 1997;
Thinh et at., 2000). However, preconditioning the shoot-tip donor plants on medium
supplemented with high sucrose concentrations has enhanced cryopreservation success of
tropical species. Such plants undergo physical and physiological changes that assist in
withstanding cryo treatments. The plants have stunted growth, with thicker leaf blades, shorter
petioles and more rigid tissues. This change enables better dissection of the tiny ST with little
damage. The ST become morphologically uniform and have reduced water content with a
build up of stress-responsive solutes (soluble sugars and free proline). This mediates more
efficient vitrification of cytosol on immersion in LN (Thinh, 1997;Thinh et ah, 2000).
22
CHAPTER 3
3.0 STUDY ON IN VITRO CULTURE OF TARO USING SHAKE
CULTURE WITH LIQUID 0.5 TDZ MEDIUM
3.1 Introduction
In vitro plants are the preferred sources of explants for cryogenic work. They have advantages
over field and screen house materials in being less loaded with pathogens (bacteria and
fungus), more physiologically homogenous and are already adapted to in vitro culture
conditions. Taro has been micropropagated via callus or protocorm-like bodies induced by
combinations of cytokinins (kinetin or BAP) and auxins (2,4-D or NAA or IAA) or even
incorporations of taro extracts in the culture media (Mapes and Cable, 1972; Jackson et al.,
1977; Irawati and Webb, 1983; Oosawa et al., 1984; Tim et al., 1990; Sabapathy and Nair,
1992,1995). These systems could compromise the genetic stability of the germplasm
collection through artifacts such as somaclonal variations (Scowcroft, 1984). Nagata (1995),
Clemente et al. (1994), Thinh (1997) developed systems for multiplication through
enhancement of axillary branching or reproduction from axillary buds.
In addition to the multiplication systems mentioned above, a three-stage cycle (0.5 tng/1 TDZ -
0.8 mg/1 - BAP - 0.005 mg/1 TDZ) system was developed in the laboratory at The University
of the South Pacific, Samoa by the Pacific Regional Agriculture Programme (PRAP) tissue
culture project (1997). This system was used to bulk up the required number of taro plants in
this study (section 4.1.1). This system has the drawbacks of being lengthy, costly due to high
prices of TDZ and BAP and the use of such relatively high concentrations of growth hormones
could compromise cryo success (Reed, 2001:pers. comm.; Thinh, 2001;pers. comtn.)
23
Thinh (1997) established a non-hormone system for clonal production of taro plants
(Colocasia esculenta var. antiquorum). Shoot-tips maintained on liquid shake culture
supplemented with 0.1- 0.5 mg/1 TDZ had an average production rate of 4.3 offshoots after
five subcultures. The offshoots continued to proliferate at a similar rate when transferred to
hormone free medium. Cormel slices from these plants also produced three to four offshoots
when placed on solid MS medium. The applicability of this protocol to a tropical taro
{Colocasia esculenta var. esculenta) cultivar was investigated.
3.2 Materials and Method
3.2.1 Taro plant material
In vitro plants of the Fijian cultivar, Tausala ni Samoa, maintained on hormone free solid MS
were obtained from the RGC. Two centimetre explants including the corm base and an
adjoining length of stem were excised from these plants and used for the experiment.
5.2.2 Cycle One
Twenty explants were inoculated on 0.5 mg/1 TDZ in liquid MS medium in McCartney bottles
. (hereafter referred to as tubes). Ten tubes were placed on the shaker at 80 rpm while the other
ten were left standing on the shelf under normal culture conditions (section 4.2). Observations
were recorded on weekly basis.
3.2.3 Cycle Two
After the fourth week, the tubes for both treatments in cycle two were divided into three
categories depending on the number of offshoots per explant:
24
3.4 Discussion
The hormone-free clortal propagation system developed by Thinh (1997) for Colocasia
esculenta var. antiquorum, based on enhanced axillary branching (multiple shoot formation)
through liquid TDZ medium shake culture and subsequent carry over effect on hormone-free
medium, did not work with the tropical taro variety Tausala ni Samoa of Colocasia esculenta
var. esculenta type. After two four-week cycles on liquid TDZ shake medium cultures, no
enhancement in proliferation rates was noted and no carry over effect was observed when
these plants were transferred to hormone-free media.
In contrast, the three-stage system being used in this study produced an average of four to six
offshoots from the variety (Tausala ni Samoa) during routine bulking up. Furthermore, when
these plants were transferred to hormone free medium, some carry over effect of TDZ was
noted as these plants produced offshoots at similar rates in the first subculture on hormone-
free medium. However, these effects diminished on subsequent subcultures on hormone-free
medium.
The different response of this variety of taro (Tausala ni Samoa of esculenta type) to liquid
TDZ shake medium could be a genotypic response as the study carried out by Thinh (1997),
• used antiquorum type of taro cultivated in Japan. In tissue culture studies with these two types
of taro, var. antiquorum has always been shown to be more responsive in tissue culture
(Jackson et ah, 1977; Arditti and Strauss, 1979; Irawati and Webb, 1983; Tim et al., 1990). As
var. antiquorum has a small central conn with pronounced lateral branching, it is possible that
little manipulation of the tissue culture system is required to encourage further branching,
compared to the var. esculenta, which produces a central corm with very few lateral branches.
A comparative investigation of responses of different cultivars of var. esculenta to shake and
non-shake cultures would be an interesting study.
27
Finally, it was interesting to note that there was no difference in proliferation whether cultures
in liquid medium were shaken or left to stand. This implies that aeration is not crucial with
cultures of taro in liquid medium.
28
CHAPTER 4
4.0 METHODOLOGY
4.1 Materials and Method
4.1,1 Plant material
In vitro stock plants of taro (Colocasia esculenta var. esculentd) were obtained from the
Secretariat of the Pacific Community (SPC) Regional Germplasm Centre (RGC), Suva. The
regional taro collection is comprised of taro varieties from several different PICs. A sample of
varieties was selected to be representative of the diversity in the region by choosing varieties
from all countries contributing to the regional collection. The plants were exposed to the
following micropropagation cycle to bulk up sufficient material for experimentation: four
weeks on MS solid medium + 0.5 thidiazuron (TDZ), followed by three weeks on solid MS +
0.8 6-benzylaminopurine (BAP), then for three weeks on solid MS + 0.005 TDZ. When
sufficient material had been generated, the accessions were cultured in full strength liquid MS
for one month to lessen any carry-over effects of the plant growth hormones. From these
plants, 2 cm explants, including some basal corm and an adjoining length of leaf cluster bases,
were excised and conditioned on the vaiious media stated in section 4.4.1.1.
Initially, the following cultivars were bulked up for experimentation:
29
4.2 In vitro Culture Conditions
The basic medium used to micropropagate material for this study was that developed by
Murashige and Skoog (1962) for tobacco tissues and always contained 30 g/1 sucrose, unless
stated otherwise. The pH of the culture medium was adjusted to 5.8 ± 0.1 with 0.1 M KOH
and/or HC1 solutions. The gelling agent used for solid medium was 7.5 g/1 Agar Type A
(Sigma Chemical Co., Germany). The medium was sterilized by autoclaving at 121°C at 103
kPa for 15 min. Two types of micropropagation culture vessels were used: McCartney bottles
containing 10 ml medium, referred to as Small Jars (SJ), and 100 ml Mayonnaise bottles
containing 15 ml medium, referred to as Large Jars (LJ). SJ were initially used for
micropropagating all in vitro plants. Later, LJ were used for cultures in an attempt to improve
the quality of ST donor mother plants. Culture vessels Were incubated at 25°C under light
intensity of 50 umolm'V1 (cool white fluorescent lamps) and a photoperiod of 16 hr. All
tissue culture consumables were obtained from Biolab Scientific Ltd. - New Zealand.
4.3 Procedure for Bacterial Screening
The in vitro plants were inoculated on half strength liquid MS at pH 6.9 for one month to
encourage bacterial and fungal growth (Reed et at, 1995; Tanprasert and Reed, 1997a,b). All
the explants that failed to produce contaminants in that medium were tested on bacteriological
523 medium (Appendix 2). The bases of the explants were streaked on the 523 medium plates
and the explants were cultured on solid MS. The plates were monitored for bacterial growth
for up to a month (Viss et ah, 1991; Reed and Buckley, 1999). The explants depicting
bacterial growth were discarded.
31
4.4 Vitrification of VPTM
The steps for the vitrification procedure are outlined in general below. During this study the
techniques used were modified for many of the steps in the vitrification procedure. The initial
method was modified to optimize results by reducing handling damage, and to ensure that the
times for each treatment were accurate in their duration. Both methods are described below
under Initial Method and Modified Method.
4.4.1 General procedures
4.4.1.1 Conditioning
The explants are conditioned with different sucrose concentrations before being
cryopreserved. The conditioning consists of either preculture or preconditioning:
Preculture
In vitro plants are cultured on solid MS. Shoot-tips (ST) are dissected from these plants and
precultyred on solid MS medium supplemented with 0.3 M sucrose in 100 x 15 mm Petri
dishes and left overnight (16 hi") in the dark at 25 ± 0.5°C before being cryopreserved. [This
treatment will be referred to as overnight preculture (ONP) treatment throughout this study].
Preconditioning
Preconditioning involves culture of the mother plants on high sucrose medium. ST were
dissected from these plants and directly cryopreserved without preculture.
32
4.4.1.2 Shoot-tip dissection
ST dissected from in vitro plants conditioned in the different ways described above are 0.8 -
1.0 mm in size and consist of the apical dome surround by two leaf primodia. ST dissection is
done under the binocular-dissecting microscope placed in the LAF using scalpel blades.
4.4.1.3 Loading treatment
This involves loading the ST for 20 min at 25°C with a loading solution (LS) that consists of 2
M glycerol + 0.4 M sucrose prepared in liquid MS at pH 5.8.
4.4.1.4 Dehydration with PVS2I*
This involves dehydrating the ST with the plant vitrification solution 2 (PVS2) (Sakai et al.,
1990) which consists of 30% w/v glycerol + 15% w/v ethylene glycol (EG) + 15% w/v DMSO
+ 0.4 M sucrose prepared in liquid MS at pH 5.8.
4.4.1.5 Immersion in liquid nitrogen
The ST are immersed very quickly in LN.
4.4.1.6 Thawing
The ST are removed very quickly from the LN and warmed at 40°C in a water bath.
33
4.4.1.7 Unloading
This involves rehydrating the ST by incubating for 15 min at 25°C in liquid MS + 1.2 M
sucrose solution, referred to as unloading solution (ULS).
4.4.1.8 Recovery
The ST are inoculated on recovery medium and incubated under favourable conditions for
regrowth into plantlets.
4.4.2 Initial Method
4.4.2.1 Conditioning f
Same as in section 4.4.1.
4.4.2.2 Shoot-tip dissection
ST dissection was done under the binocular dissecting microscope (Olympus) placed in the
LAF cabinet (BTR Environmental Pty Ltd.), using size 12 scalpel blades (Paragon, England).
4.4.2.3 Loading treatment
A 1 ml polyvinyl cryovial (Nalgene) was placed in the LAF cabinet and filled with 0.8 ml of
LS. Seven ST were collected at the tip of a scalpel blade with a needle and dipped into the
cryovial of LS. After a few minutes the sides of the cryovial was tapped lightly to sink any
floating ST. The ST were immersed in the LS for 20 min at 25°C.
34
4.4.2.4 Dehydration with PVS2
After 20 min, the LS was sucked out of the cryovial with a sterile Pasteur pipette. The ST were
then washed three times with PVS2 solution. PVS2 solution was dispensed into the cryovials
using the Pasteur pipette with enough force to swirl the ST. The ST were left in the last wash
of PVS2 for 10, 12, 15 and 30 min.
4.4.2.5 Immersion in liquid nitrogen
PVS2 was sucked out of the cryovial with the sterile Pasteur pipette and a fresh sample was
filled in. The cryovial was attached to a cryocane, which was put in a cryocan and plunged
quickly into LN contained in the Dewar flask. The ST were left overnight in LN.
The cryocane with the cryovial containing the ST was removed from the LN and plunged
quickly into waterbath at 40°C and kept in for 1 min. Then it was transferred to 25°C water for
1.5 min. The cryovial was then taken inside the LAF cabinet for unloading the ST.
PVS2 was sucked out of the cryovial with the sterile Pasteur pipette and the ST were washed
three times with ULS. The ULS was dispensed into the cryovials using the Pasteur pipette
with enough force to swirl the ST. The ST were left in the last wash of ULS for 15 min.
35
4.4.2,8 Recovery
After 15 inin, the ST were sucked up into the Pasteur pipette and dropped onto a sterile filter
paper placed in a Petri dish. Using a needle, the ST were transferred onto solid MS
supplemented with 0.1 M sucrose. The ST were maintained in dim light for a week then
transferred to normal culture conditions as stated in section 4.2. If ST were green after twenty
days, they were recorded as surviving the cryopreservatioii process. A recovery scoring was
only recorded if the ST were still green and had started to grow into plantlets after four to six
weeks.
4.4.3 Modified Mfithod
4.4.3.1 Conditioning (
Same as in section 4.4.1.
4.4.3.2 Shoot-tip dissection
Same as in section 4.4.2.2.
4.4.3.3 Loading treatment
Seven ST were wrapped in a tissue paper prior to loading. The wrapping procedure was as
follows: (Figure 4.1)
a sterile 2x2 cm tissue paper (TP) was spread in a Petri dish and wetted with a few drops
ofLS
36
using scalpel blade, and looking under the microscope, the ST were carefully put onto the
TP. All ST were placed in the center of the TP.
1st Fold: two sharp pointed forceps, held in each hand, were used to hold two corners of
the TP and fold the TP in half - trapping the ST in the middle.
2nd Fold: the same two corners of the TP, together with the corners of the layer underneath
it, were held again and folded back.
3rd Fold: another two corners, at right angles one to the ones held previously, of the folded
TP were held and folded perpendicular to the first two folds but reaching only half way
across.
4lh Fold: the opposite two corners were then folded over on the remaining unfolded half.
The TP with the wrapped ST was immersed in 15 ml of LS contained in a 40 mm sterile glass
Petri dish for 20 min at 25°C.
('Proline pretreatment (PPT)
As an alternative investigation, trials were carried out where the ST of cultivar E399 were
soaked in 5% proline solution for 2 hours prior to loading with LS. PVS2 exposure times of
10, 12, 15, 30 and 40 min at 25°C were investigated with PPT.
4.4.3.4 Dehydration with PVS2
The TP was lifted from the LS with a pair of sterile forceps, and blotted dry on two layers of
sterile filter paper in a Petri dish. The TP was then immersed in 5 ml of PVS2 contained in a
15 ml Petri dish for the times stated in section 4.4.2.4.
37
4.4.3.5 Immersion in liquid nitrogen
When 50 sec of PVS2 exposure time was left, the TP with the ST was transferred to a 1 ml
polyvinyl cryotube containing 0.8 ml fresh PVS2. The cryotube was attached to a cryocane
and plunged quickly into LN contained in a wide mouth flash exactly at the end of the PVS2
exposure time. The ST were held in the LN for at least an hour.
4.4.3.6 Thawing
The cryocane with the cryovial containing the ST was transferred very quickly from the LN to
a waterbath at 40°C and shaken vigorously for 90 sec. The cryovial was transferred to the LAF
cabinet for unloading the ST.
The TP was removed from the cryovial with a pair of sterile forceps, blotted dry on two layers
of sterile filter paper in a Petri dish and immersed in 5 ml of PVS2 contained in a 15 ml Petri
dish. After 10 min, the TP was unwrapped so that the ST could float in the ULS for the next 5
At the end of 15 min, using the scalpel blade the ST were lifted from the ULS onto a sterile
filter paper placed on solid MS supplemented with 0.3 M sucrose. The plated ST were left
overnight in the dark at 25°C. The following day, they were inoculated on solid MS + 0.1 M
sucrose plates. The cultures were maintained in the dark for three days and then transferred to
38
dim light for one week before exposure to normal culture conditions as stated in section 4.2.
Survival and recovery were scored as in section 4.4.2.8.
4.5 Experimental Procedures
Using the above procedures, the following variables were investigated:
• PVS2 dehydration times of 12, 15, 30 min at 25°C and prolonged PVS2 exposure at 0°C
(on ice) for cultivar E399 at 40, 60 and 90 min
• PPT trials for cultivar E399 at PVS2 exposure times of 10, 12, 15, 30 and 40 min at 25°C
• Overnight preculture on 0.3 M sucrose medium
• Preconditioning on 60, 90 and 120 g/1 sucrose medium for 4 and 7 weeks (NOTE: 60, 90
and 120 g/1 sucrose media are referred to as 60S, 90S and 120S, respectively, throughout
this study) (
• Age of ST donor plants; 1, 2, 3 and 4 months for ONP trials and 4 and 7 weeks for high
sucrose preconditioning trials
• Size of culture containers; post-LN recoveries of ST dissected from mother plants grown
in small jars and large jars were evaluated
39
5.2.2 Post-LN recovery rates for all trials conducted for all culture conditions at PVS2
exposure time of 12 tnin
As presented in section 5.1 above, the best results were attained with PVS2 exposure time of
12 min. Other results for this treatment were 17% average recovery for two months old
explants of cultivar CPUK with 60S preconditioning. The recovery rate for an individual trial
under these conditions was 50% with the same cultivar (Table 5.6). Using 90S
preconditioning, 7% average recovery was obtained for cultivar CPUK using seven weeks old
explants. No success was achieved with cultivar E399 for 60S preconditioning with 12 min
PVS2 exposure time. However, 7% average recovery for seven weeks old explants with 90S
preconditioning was obtained with this cultivar. 12% and 7% average recoveries for three and
four months old plants respectively, was achieved with cultivar TNS for ONP treatment.
Under the same conditions, the recovery rate for individual trials were 71% for three months
old plants and 14 and 29% for two months old plants. For TNS cultivar, average recoveries of
10% each for one and two months old explants, was obtained with 60S preconditioning.
Under the same conditions, the recovery rates for individual trials were 29% for one month
old explants and 14 and 43% for two months old explants.
5.2.3 Post-LN recovery rates for all trials conducted for all culture conditions at PVS2
exposure times of 15 and 30 min v
As shown by the results presented in Tables 5.4 and 5.5, PVS2 exposure times of 15 and 30
min are not favourable for post-LN recovery with the taro cultivars investigated. The only
success achieved for both times under all conditions was 4% average recovery for two
months old plants of cultivar E399 with ONP treatment. However, high survival rates for
ONP trials for these two cultivars (20 and 30% for two of the three E399 trials and 20, 30 and
40% for the three TNS trials) were obtained with PVS2 exposure time of 30 min. Ten percent
43
survival was also obtained for two of the three 120S trials for cultivar TNS with PVS2
exposure time of 30 min (Table 5.5). In all of these survival cases, one outer leaf of the shoot-
tip stayed green and had some expansion but turned brown and died after 4 to 6 weeks.
5.2.4 Effects of prolonged PVS2 on post-LN recovery rates
All trials conducted for prolonged PVS2 exposure at 0°C (on ice) for cultivar E399 at 40, 60
and 90 min were unsuccessful (section 4.5).
5.3 Effects of Prolonged PVS2 on Post-LN Recovery Rates
All proline pretreatment trials conducted for cultivar E399 were unsuccessful (sectionf
4.4.3.3).
As shown in Table 5.6, it appears that eultivar E399 is the most tolerant to PVS2 as it had
recoveries for all 10, 12 and 15 min exposure times.
5.4 Effects of Different Preconditioning on Post-LN Recovery Rates
5.4.1 Effects of overnight preculture on 0.3 M sucrose medium on post-LN recovery rates
No post-LN recoveries were obtained for cultivars CIMG, CIRA, N13, N14 and P3 using
ONP treatment. As shown in Tables 5.3 and 5.6, cultivars CPUK, E399 and TNS could be
successfully cryopreserved through overnight preculture on 0.3 M sucrose medium. For
cultivars CPUK and E399, the best recovery rates were obtained with this treatment (section
5.1). As shown in Table 5.2, low average recovery rates of 12% for three months old plants
and 7% four months old plants were obtained for cultivar TNS with ONP treatment.
44
However, a high recovery rate of 71% for an individual trial was obtained for this cultivar
(Table 5.6). ONP treatment trials for ST of cultivar E399 dissected from plants
preconditioned using 60S conditions were unsuccessful (Table 5.2).
5.4,2 Effects of preconditioning on high sucrose concentrations on post-LN recovery rates
5.4.2.1 Effects of preconditioning on 60 g/l sucrose medium on post-LN recoverv rates
The only success obtained for cultivar E399 with preconditioning treatments was a low
recovery rate of 10% for 60S preconditioning with PVS2 exposure time of 10 min (Table
5.1). No successful recoveries were obtained for cultivar CPUK for 60S preconditioning with
PVS2 exposure time of 10 min (Table 5.1). For this cultivar, an average recovery rate of 17%
for PVS2 exposure time of 12 min was achieved with this treatment (Table 5.2). The
recovery rate for an individual trial under these conditions was 50% (Table 5.6). The data in
Tables 5.1, 5.2 and 5.6 show that cultivar TNS achieved the highest recoveries of all cultivars
with 60S preconditioning treatment. An average recovery rate of 14% was obtained with
PVS2 exposure time of 10 min (Table 5.1) whereas an individual trial under the same
conditions had a recovery rate of 43% (Table 5.6). As shown in Table 5.2, a low average
recovery rate of 10% with PVS2 exposure time of 12 min was obtained for cultivar TNS. The
recovery rates for individual trials werfr 14, 29 and 43%. Generally, it can be said that
preconditioning on 60 g/l sucrose medium was not very effective in enhancing post-LN
recovery rates.
5.4.2.2 Effects of preconditioning on 90 g/l sucrose medium on post-LN recoverv rates
As can be seen from Table 5.2, no post-LN recovery was achieved for cultivar CIMG using
90S preconditioning. This was also true for cultivar CPUK using 90S preconditioning with
45
PVS2 exposure time of 10 min (Table 5.1). However, a low 7% average recovery rate with 12
min PVS2 exposure using 90S preconditioning was obtained for this cultivar (Table 5.2).
Using 90S preconditioning, the trials for cultivar E399 were unsuccessful with 10 min PVS2
exposure time (Table 5.1), while low average recovery rates of 6% were obtained for both 12
min and 15 min PVS2 exposure times (Tables 5.2 and 5.4). While no success was obtained
for cultivar TNS with 90S preconditioning for 10 min and 15 min PVS2 exposure times
(Tables 5.2 and 5.4), the optimum average recovery rate (29%) for all 90S preconditioning
trials was obtained with 12 min PVS2 exposure. The recovery rates for individual trials were
17, 17, 29 and 100% (Table 5.6). All successes for all cultivars stated above using 90S
preconditioning were for explants preconditioned for seven weeks.
An interesting observation was that three cryopreserved shoot-tips had one axillary bud each
appearing from their bases. One of such shoot-tips was from a CPUK trial while other two
were from two separate TNS trials. The new buds were alive and growing for two weeks for
the CPUK and one of the TNS trials, then turned brown and died. The new bud in the other
TNS trial survived to grow into a plantlet.
5.4.2.3 Effects of preconditioning on 120 g/1 sucrose medium on post-LN recovery rates
For cultivar E389, an average recovery rate of 3% was achieved using 120S preconditioning
and a PVS2 exposure time of 10 min (Table 5.1). However, this cultivar had endogenous
bacterial contamination, hence this result may not represent its true success potential for
cryopreservation. A low average recovery rate of 3% was also obtained for cultivar TNS with
120S preconditioning for PVS2 exposure time of 10 min (Table 5.1).
46
5.5 Effects of Age of ST Donor Mother Plants on Post-LN Recovery Rates
From the evaluation of the post-LN recovery rates of ST dissected from mother plants grown
for different lengths of time without subculture, it was discovered that using older mother
plants as sources of ST is more conducive to post-LN viability. As stated in section 5.1, the
optimum age of ST donor mother plants for cultivars CPUK and E399 was three months with
ONP treatment. However, leaving the mother plants too long without subculture conferred a
decline in post-LN recoveries as no recoveries were achieved for either of cultivars CPUK and
E399 with four months old plants (Table 5.2). For cultivar TNS, seven weeks old plants were
best with 90S preconditioning treatment.
5.6 General Observations/Results '
• All plants grown in large jars for one to four months on hormone free solid MS were on
average 17 to 20 cm tall, had 2 to 4 leaves that were 2.5 to 5 cm long and 1.5 to 3.5 cm
wide and produced 12 to 25 roots that ranged from 11 to 25 cm in length (Figure 5.1).
These plants produced very few offshoots (0 to 4). In contrast, plants grown in small jars
were of roughly half the size and dimensions of those from large jars (Figure 5.1). These
characteristics correlated with the quality of ST dissected from plants grown in different
jars. The ST dissected from plants grown in large jars were larger, less succulent, more
rigid, more homogenous and easier to dissect than their counterparts grown in small jars.
• Different growth was observed for different cultivars when subjected to medium with high
sucrose concentrations. While cultivar TNS did not grow at all on 90S medium and had
retarded growth on 60S medium, the cultivars CPUK and E399 grew almost normally on
47
60S medium and had retarded growth on 90S medium (Figure 5.2). In addition to this, all
the cultivars listed in Table 4.1 had no growth on 120S medium.
For the cultivar TNS, the successes from 90S culture conditions (Table 5.6) came from
explants that had no growth at all. The outer two to three leaves of the 2 cm explants
cultured on the 90S medium turned brown and the explant had a dead appearance.
However, on dissecting the inner ST was healthy, turgid and very easy to dissect.
ONP and 120S trials were conducted on the taro cultivars listed in Table 4.1. All trials
were unsuccessful except a 120S trial for cultivar E389 (Table 5.1). While the ST
cryopreserved from these plants died on the recovery medium, the bacteria flourished.
All trials conducted for ST donor plants grown in small jars and/or liquid MS were
unsuccessful, ,except the cultivar E389 success stated above.
During the post-LN recovery process, the surviving shoots-tips could be identified two to
four days after thawing from LN. The survivors would swell and start turning green. Four
to six weeks later they would form the first leaves and begin rooting. About two months
after thawing, they would form small plantlets (Figure 5.3).
CHAPTER 6
6.0 DISCUSSION
6.1 Effects of Quality of the ST on Post-LN Recovery Rates
The quality of the apices (shoot-tips or meristems) is considered the most important factor for
cryopreservation success (Thinh, 1997; Takagi, 2000; Thinh et at, 2000). The various ways to
acquire optimum shoot-tip quality for high post-LN recovery rates are discussed below.
6.1.1 Shoot-tip structure and dissection technique
Good dissection technique is essential for obtaining good quality shoot-tips that can survive
the stresses of cryoprotectant toxicity and LN cooling. This requires a good understanding of
the shoot-tip structure and sufficient shoot-tip dissection practice. As emphasized by Thinh
(1997) and Thinh (2001:pers. comm.), the optimum shoot-tip should consist of the apical
dome surrounded by two leaf primodia. As shown in Figure 6.1, taro meristems are
surrounded by a cluster of leaf petioles that are tubular in shape and interfolded at the bases
(Thinh, 1997). Adjacent petiole bases are buffered by air spaces. Hence, PVS2 can only reach
and dehydrate the dome area when the apical dome is partly covered. This is possible when
two outer leaf primodia surround the apical dome. The shoot-tips should be dissected without
any damage to these leaf primodia. Any damage to the shoot-tips will result in wounding,
which can increase the phytotoxic effect of the cryoprotectant solution. This will prove fatal
when the plant tissues are under the stress of LN cooling. In addition, wounding increases the
possibility of callus formation, which is to be avoided in the conservation of clonal material.
56
Only the shoot-tips with the right structure and size have any chances of post-LN recovery.
This held true for the successful trials conducted under this study.
6.1.2 Quality of shoot-tip donor plants
Shoot-tip quality can be improved by enhancing the quality of shoot-tip donor plants
(Pennycooke and Towill, 2000; Thinh et al., 2000; Reed, 2001:pers. comm.; Thinh, 2001:pers.
comm.). In this study it was observed that plants grown in small jars were smaller in height,
had fewer and smaller leaves, and produced less extensive roots than those grown in large jars.
The shoot-tips dissected from such plants were watery, small, transparent, fragile and hard to
dissect. These shoot-tips did not survive LN cooling. In contrast, shoot-tips from plants grown
in large jars were larger, robust, more homogenous and could be dissected more easily and
without damage. In this study, all the shoot-tips that survived LN cooling were those dissected
from plants grown in large jars.
Secondly, combining high levels of cytokinins with a liquid culture medium can induce
hyperhydricity of the shoot-tips (Palupe, 1997). This can result in the shoot-tips being more
susceptible to physical and cryopreservation damage. The shoot-tips dissected from plants
micropropagated under these conditions were similar to those described for small jar shoot-
tips above and did not survive LN cooling. To rectify this problem, the stock plants bulked up
on the described 0.5TDZ-0.8BAP-0.005TDZ cycle were cultured for one month on liquid MS
to reduce the effect of the hormones, then grown on hormone free solid MS before shoot-tips
were dissected from them. The successes for ONP trials presented in section 5.4.1 were for
such shoot-tips. However, cultivars CIMG, CIRA, N13, N14 and P3 received the same
treatments but had no success for any of the ONP preculture trials (section 5.4.1). This is
likely to be due to different sucrose sensitivities of these cultivars as discussed below.
57
Thirdly, an optimal physiological state necessary to inculcate shoot-tip tolerance to
dehydration and cryogenic procedures can be induced through preculture or preconditioning.
Subjecting explants to high sucrose medium can reduce the water content of cells, thus making
them more suitable for cryopreservation. It also reduces physiological heterogeneity of apices
by synchronizing the metabolism of all apical cells (Lambardi, et al., 2000; Takagi, 2000).
Engelmann (1997b) elucidated that post dissection preculture of apices reactivated metabolism
through starch accumulation in all cells, resulting in high survival for coffee, date palm and
sugarcane. Chemical analysis of taro plants preconditioned on medium with 120 g/1 sucrose
indicated higher levels of soluble sugars, proline and starch than those grown on medium with
30 g/1 sucrose (Thinh 1997).
Hitmi et al. (1999) found that increasing the concentration of sucrose as well as incubation
time on medium conferred freezing tolerance in Chrysanthemum cinerariaefolium. They
discovered that these conditions increased the levels of intracellular sucrose, fructose, glucose,
ABA and proline in the cells. High cell content of these chemicals is conducive to better post-
thaw viability. Bachiri et al. (2000) found that pretreatment of Arbidopsis thaliana suspension
cells with high sucrose concentrations increased the cell content of soluble sugars, essentially
sucrose or trehalose, and monosaccharides as well. High cell content of substances like
sucrose and ABA may also trigger genes synthesizing new proteins that may posses
cryoprotective characteristics (Shriver and Mundy, 1990; Chandler and Robertson, 1994;
Gosti et at., 1995; Koch, 1996; Zhu et al, 1997; Pahis, 1998; Reinhoud et al., 2000).
As stated in section 5.2, for cultivars CPUK and E399, overnight preculture on 0.3 M (73 g/1)
sucrose medium is more favourable for obtaining higher post-LN recovery rates than
preconditioning ST donor plants on medium with high sucrose concentrations (60 or 90 g/1
sucrose). However, for TNS, preconditioning on high sucrose concentration of 90 g/1 seems to
be the better option for enhancing post-LN recovery rates. These differences could be due to
58
the different sensitivities of the cultivars to varying sucrose concentrations (section 5.6). In
light of the observation that the successes of 90 g/1 sucrose preconditioning for cultivar TNS
were from explants with no growth at all, it would be interesting to investigate the effects that
preconditioning on higher sucrose concentrations such as 120 g/1 sucrose, have on the other
cultivars listed in Table 4.2. Unfortunately, no conclusions could be drawn on the effects that
preconditioning on high sucrose medium has on the post-LN recovery rates for the cultivars
listed in Table 4.1 as all perished due to endogenous bacterial contamination.
From these observations and results, it can be inferred that sucrose sensitivity impinges on
cryopreservation success. This would suggest that for each cultivar, the optimum conditioning
treatment has to be determined. Thinh (1997) found that one month preconditioning on 120 g/1
sucrose medium achieved the highest post-LN recovery rates with the varieties investigated.
However, in this research seven weeks preconditioning oif 90 g/1 sucrose medium gave the
best results for cultivar TNS, whereas with cultivars E399 and CPUK, the overnight preculture
on 0.3 M sucrose medium using shoot-tips excised from three-month old plants proved best.
Lastly, the age of shoot-tip donor plants also influences post-LN recovery rates. Thinh (1997)
found that increasing the age of in vitro shoot-tip donor plants from one to three months was
directly proportional to higher post-LN recovery rates. The results presented in section 5.5
indicate that maintaining the in vitro ST donor plants without subculturing for longer times is
likely to enhance the post-LN recovery rates. Three months appears to be the best for ST donor
plants when over night preculture conditions were used for cultivars CPUK and E399, while
seven weeks preconditioning of explants on 90 g/1 sucrose medium obtained the highest
recovery rates for cultivar TNS.
The longer culture times are thought to enhance the physiological status of the shoot-tips.
Yongjie, et al. (1999) and Zhao et al. (1999) reported that increased duration between the last
59
subculture of ST donor plants and the cryopreservation of apices had a positive effect on the
post-LN regeneration rate. It is believed that longer subculture time of mother plants lowers
the water content of apices. In this study, it was also observed that such shoot-tips were larger,
more robust and less succulent than their younger counterparts. However, very long culture
times could stress the plants due to lack of growing space and minerals and increased levels of
carbon dioxide. The results presented in Table 5.6 show that cultivar TNS had the best
recovery rates of all cultivars for all conditions. This could be due to cultivar TNS having been
initiated into tissue culture more recently than the others and therefore had under gone the
least number of in vitro culture cycles. This relationship between age of ST donor plants and
post-LN recovery rates needs to be investigated.
As described in section 5.4.2.2, three cryopreserved shoot-tips had one axillary bud each
appearing from their bases (Figure 6.2). These buds could be adventitious ones induced by the
multiplication system or could be from two apical meristems as produced by some taro
cultivars (Figure 6.2) (Taylor, 2001:pers. comm.).
6.2 Effects of PVS2 Exposure Time on Post-LN Recovery Rates
The investigations carried out to determine the most effective PVS2 dehydration time for the
different culture conditions showed 12 min exposure as optimal (section 5.1). Thinh (1997)
optimized PVS2 exposure time for taro at 10 min. There is great variability in optimum PVS2
dehydration times for different species (Appendix 1). Takagi (2000) and Thinh et al. (2000)
have considered screening for PVS2 sensitivity as an important step in optimization of the
vitrification protocol for any vegetatively propagated tropical monocot. If shoot-tips are not
adequately dehydrated prior to LN freezing, they will die due to freezing injuries resulting
from intracellular water crystallization (Thinh et al., 2000).
60
On the other hand, too long PVS2 exposure would kill the plant cells due to high osmotic
pressure and biochemical phytotoxicity (Merymann and Williams, 1985). Prolonged PVS2
exposure of 30 min at 25°C resulted in up to 40% survival but 0% recovery as none of these
shoot-tips produced plantlets (Table 5.5). All had one green leaf expanding on the recovery
medium that turned brown and died after about 4 weeks. The greening observed was not of the
meristems themselves, but of the outer leaves only, hence they turned brown after a month.
This suggests PVS2 toxicity. 30 min PVS2 time exposure was too long for the meristematic
cells of the apical dome to survive. Consequently, the ST did not rejuvenate into plantlets.
No success was achieved with prolonged PVS2 dehydration at 0°C and proline pretreatment
prior to loading solution exposure. Hence, from the work carried out to date, it appears that
improving the quality of ST donor plants and using preconditioning treatments are more
effective in improving recovery after LN exposure with the taro varieties investigated.
6.3 PVS2 Timing and Correct Technique
The Initial Method described in section 4.4.2 had certain drawbacks. Shoot-tips were more
vulnerable to physical damage from the physical processes of being sucked into and expelled
from the Pasteur pipette, followed by handling with the needle. In addition to this, the
processes of eluting the loading solution from the cryovial before pouring in PVS2 and eluting
PVS2 before pouring in unloading solution would result in longer PVS2 exposure than
desired. These could lead to lower recovery rates arising from poor technique rather than
shortcomings of the vitrification protocol itself. Using the tissue paper for wrapping the shoot-
tips (section 4.4.3.3) enables PVS2 treatment timing to be more accurate and also reduces
physical damage. This technique has the added advantage in that a greater number of shoot-
tips can be cryopreserved daily.
61
The immersion of the shoot-tips into LN, and the subsequent transfer of the shoot-tips into the
water bath at 40°C should be a very fast process (within one second), to avoid devitrification
in the plant cells (Sakai, 1993). Such a fast wanning ensures that the cytosol changes from its
glass phase directly to liquid phase without first forming damaging ice crystals. This process
can be speeded up by first transferring the shoot-tips after treatment with cryo solutions to a
container of LN with a wide neck. The wide neck facilitates the transfer process. Furthermore,
the shoot-tips have to be in constant contact with water at 40°C during thawing (Sakai et al.,
1990). To ensure this, the cryovial is shaken rapidly in the waterbath.
There were no survivors for trials where the thawing method described in section 4.4.2.6 was
used. The post thaw recovery process should involve culturing shoot-tips overnight on MS
medium supplemented with 0.3 M sucrose followed by transfer to MS medium supplemented
with 0.1 M, rather than plating shoot-tips directly on the latter medium. This ensures that the
dehydrated shoot-tips absorb water more gradually. Transferring them directly onto 0.1 M
sucrose medium after they have been subjected to 1.2 M sucrose unloading solution would
cause the cells to absorb too much water and burst. In addition to this, the shoot-tips should be
transferred from the unloading solution onto a layer of sterile filter paper on the surface of the
0.3 M sucrose plate. The filter paper blots the excess unloading solution and any toxins
released by the recovering shoot tips.
6.4 Explaiit Variability
Explant variability is a factor that cannot be controlled by the tissue culturist, and can
obviously influence the success of cryopreservation. This variability was observed between
different plants of the same cultivar growing with different vigour on high sucrose medium
(section 5.6). As discussed in chapter 3, the taro var. esculenta is a more difficult plant to
micropropagate as compared to var. antiquorum. Common experience is that plants difficult to
62
micopropagate are also difficult to cryopreserve (Reed, 2001:pers. comm.; Thinh, 2001:pers.
comm.). Thinh (1997) had lower eryopreservation success rates with var. esculenta than var.
antiquorum. Tropical plants can be more recalcitrant to eryopreservation than their cold and
temperate counterparts. Since the latter types of plants are periodically exposed to low
temperatures, they have developed natural physiological mechanisms that confer them freeze
tolerance. This adaptation is absent in tropical plants rendering them more susceptible to
freeze damage. The variability that always exists between labs could also have implications
for the rates of eryopreservation successes achieved when using a technique established in
another laboratory. For example, an International Plant Genetic Resources Institute (IPGRI)
coordinated slow growth storage experiment for sweet potato using the same eultivars from
the same source, gave different results when earned out in different labs in SPC, PNG, Tonga
and Samoa (Taylor, 2001:pers. comm.).
(
6.5 Proposed Vitrification Protocol
From this study, it can be postulated that the vitrification method of eryopreservation has
potential for the eryopreservation of the tropical taro (Colocasia esculenta var. esculenta). The
proposed protocol for the eultivars E399 and CPUK is using ST donor plants cultured on solid
MS in large jars for three months as sources of shoot-tips which consist of the apical dome
surrounded by two leaf primodia, preculturing these shoot-tips overnight (16 hr) on 0.3 M
sucrose medium, loading with liquid MS supplemented with 2 M glycerol + 0.4 M sucrose for
20 min, dehydration with PVS2 for 12 min followed by rapid immersion in LN. Thawing
should be done by shaking rapidly for one and half minutes in waterbath at 40°C, followed by
rehydration in liquid MS medium supplemented with 1.2 M sucrose for 15 min. The shoot-tips
should then be plated on a layer of filter paper on MS medium supplemented with 0.3 M
sucrose and left overnight in the dark. Next day, they are transferred onto MS medium
supplemented with 0.1 M sucrose and maintained in the dark for three days, then transferred to
63
dim light (10 umolnf s"') for one week before exposure to normal culture conditions as stated
in section 4.2. Plantlets are produced after about two months. It is recommended that cultivar
TNS explants should be preconditioned on solid MS supplemented with 90 g/1 sucrose for
seven weeks prior to dissecting and cryopreserving the shoot-tips without any preculture. The
vitrification procedure should be the one described in section 4.4.3.
6.6 Future research
There is certainly need to optimize the vitrification protocol for taro. As shown in this study,
varietal difference has a significant contribution to cryopreservation success. Charoensub et al.
(1999) also emphasized genotypic differences in the responses of cultured plants to cryogenic
techniques and recommended optimizing each step of procedure for deciding an optimal
protocol for each cultivar. The myriad of characteristics depicted by different cultivars in in
vitro culture illuminate their variability. Consequently, different responses to cryopreservation
conditions such as different sucrose concentrations and PVS2 exposure are almost imminent.
Optimizing the preconditioning sucrose concentrations for individual cultivars would be a
good starting point. Thinh (1997) found that steadily increasing the sucrose levels in overnight
preculture medium (0.3M-0.5M-0.7M) helped shoot-tips to gradually acclimatize to high
sucrose concentrations. The converse was true for recovery medium. This could also be
investigated for taro from PICs.
Secondly, effects of the age of shoot-tip donor plants on post-LN recovery rates ought to be
experimented on. There are two implications here. Firstly, effects of the lengths of time the in
vitro plants are maintained without subculture before they are used as sources of shoot-tips
needs investigating. Secondly, the recovery rates for shoot-tips derived directly from field
conns as compared to those from freshly initiated cultures and those that have been through a
number in vitro culture cycles needs assessment.
64
Cryopreserving shoot-tips from plants that have already survived cryopreservation to assess if
they are more amenable to withstand LN cooling stress would be an intriguing investigation. It
would also be worthwhile to investigate other protocols of cryopreservation with taro.
Recently, it has been found that another cryoprotectant, PVS3 (50% sucrose + 50% glycerol
prepared in liquid MS) enhances the recovery rates of material with which PVS2 was not
effective (Nishizawa et al, 1993; Wu et al., 1997; Wu et al, 2000; Zanetta et al., 1999).
Furthermore, Thinh (1997) obtained high recovery rates of 80% for two taro cultivars using
the method of encapsulation-dehydration. This method could also be tried with taro cultivars
ofPICs.
65
3"1 petiole
7T petiole
apical dome
Shoot tip Transversed cut through the shoot tip
Figure 6.1 Diagram showing the structure of taro shoot-tip (Thinh, 1997)
Figure 6.2 Two apical meristems on an explant dissected from an in vitroplant of cultivarTNS
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APPENDIX 1
Recent successful cryopreservation of shoot apices by vitrification
Botanical name{common name)
Non cold-hardy species(herbaceous plants and tropicalwoody plants)Ailium sativum (garlic)
Ananas comosus (pineapple)
Asparagus officinalis (asparagus)
Colocasia esculenta (taro)
Colocasia esculenta (taro)
Cymbidium spp. (Cymbidium)
Dioscorea alata (graeter yam)
Dioscorea rotundata (white yam)
Greviliea scapigera (tropical treespecies)
Ipomoea batatas (sweet potato)
Pregrowth(meristem-donor
plants)
-
-
-
Sucrose <120g/L~~enriched medium;lino
-
-
-
-
-
Preculture(dissected
meristems)
3% sucrose; 2d
0.3M sucrose;2d
-
0.3M sucrose; 16h
0.3M sucrose; 16h
0.3M sucrose; 16h
0.3M sucrose;24h
0.3M sucrose;72h
0.6M sorbitol; 2d
0.09M sucrose;24hi 0.6M sucrose ;24h
Loading
L2; 25 min
-
Ll;25°C;20min
LI; 25°C; 20min
Ll;25°C;20min
-
-
-
Dehydration
PVS2;25°; 10-15 min
PVS2; 0°C; 7h
PVS2; 25 °C; 45min
PVS2; 25°C; 20min
PVS2; 25°C; 20min
PVS2; 25°C; 15mm
PVS2; 25°C; 20min;PVS2; 0°C; 30min
PVS2; 25°C; 30min
PVS2; 0°C; 30min
S; 0°C; 100 min
Post-thaw shootrecovery
12cvs.: 71-100% (av.82%)
3 cvs.: 25-65% (av.42%)
90%
2 cvs.: 66,77%
6 cvs.: 67-100% (av.87%)
76%
85% (cv. OrientalLisbon); 47% (cv.UM68O);91%(cv.UM680)6 cvs.: 43-68% (av.63%)
65%
47%
Reference
Niwata 1995
Gonzalez- Arnao etal. 1998bKohmura et al.1992
Takagi et al. 1997
Thinh 1997
Thinh 1997
Kyesmu 1998
Kyesmu et al. 1997
Touchell and Dixon1996Plessis andSteponkus 1996
89
Botanical name(common name)
Musa spp. (banana)
Wasabia japonica (wasabi)
Wasabia japonica (wasabi)
Xanthosoma spp. (tannia)
Encapsulation / vitrificationWasabia japonica (wasabi)
Minimally cold-hardy species (herbaceous plants)Allium wakegi (Japanese shallot)Lilium japonicum (Japanese pioklily)Trifolium repens (white clover)
Encapsulation / vitrificationFragaria x ananassa (strawberry)
Pregrowth(meristein-donor
plants)Sucrose (60g/L) -enriched mediumImo
-
-
-
-
-
Cold-hardening;0°C; 7-30 d-
Cold-hardening;4°C; 2 weeks
Preculture(dissected
meristems)
0.1M sucrose; 16h
0.3M sucrose; 16h
0.3M sucrose + 0.5MGly; 16h
0.3 M sucrose; 16h
0.3M sucrose; 16h 4encapsulated in 0.4Msucrose0.8% sucrose;7 d;4°C i 0.4M sucrose;1 d; 4°C0.3M sucrose;! d;25°Cl,2Msorbito!;2d;4°CEncapsulated in 2Mglycerol + 0.4Msucrose
Loading
LI; 25°C; 20min
LI; 25°C; 20min
-
Ll;25°C;20min
Ll;25°C;30min
-
Ll;25°C;20min-
Dehydration
PVS2; 0°C; 20-30min
PVS2; 25°C; lOmin
PVS2; 25°C; lOmin
PVS2; 25°C; 20min
PVS2; OX; lOOmin
PVS2; 25°C; 45min
PVS2;0°C; HOmin
PVS2; 0°C; 15min
PVS2; 0°C; 2h
Post-thaw shootrecovery
10 cvs.: 4 1 - 92% (av.69%
4 cvs.: 79-92% (av.84%)
85%
2 cvs.: 62%, 67%
4 cvs.: 87 - 93% (av.92%)
80%
83%
83%
95%
Reference
Thinhefe/,1999
Matsumoto et al.1994Matsumoto andSakai 1995
Thinh 1997
Matsumoto et al.1995b
Kohmura et al.1994
Matsumoto et al.1995aYzmaA&.etal 1991
Hirai et al. 1998
f Loading solution ~ LI: 0.4M sucrose + 2M glycerol; L2: 0.75M sucrose + IM glycerol.Vitrification solution - PVS2: 30% ( w/v) glycerol + 15% (w/v) ethyiene glycol + 15% (w/v) DMSO; S: 50wt% ethyiene glycol + 15wt% sorbitol + 6wt%bovine serum albumin.
(Takagi, 2000)
90
APPENDIX 2
Bacteriological 523 Medium (Viss)
To the appropriate amount of deionized water in a beaker add
SucroseCasein hydrolysateYeast extractKH2PO4MgSO4.7H2O
lOg/L8g/L4g/L2g/L
0.15 g/L
Adjust the pH to 6.9. transfer to Erlenmeyer flask and add
Gelrite 6 g/L
Bring the mixture to a boil and autoclave. Cool to 45-50°C in waterbath and pour intosterile petri dishes.
(Viss et al., 1991)
91