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Jour Pl Sci Res 33 (2) 181-199 2017
Low Cost Tissue Culture : An Overview
S. K. Datta, Debasis Chakraborty1 and T. Janakiram2

DBT-NER Visiting Research Professor, Department of Life Science and Bioinformatics, Assam University,
Silchar; Retd. Sci.’G’, CSIR-NBRI, Lucknow; Ex CSIR Emeritus Scientist, Bose Institute, Kolkata.
1
Genetics and Molecular Biology Div., CSIR-National Botanical Research Institute, Lucknow-226001,
India,
2
Assistant Director General (Hort. Sci), Indian Council of Agricultural Research, Horticultural Science
Division, Room No. 423, Krishi Anusandhan Bhawan - II, Pusa, New Delhi- 110012
*Corresponding author e-mail: subodhskdatta@rediffmail.com, debasis1972@rediffmail.com

Plant tissue culture is a very important technology which is used for production of large scale quality
planting materials of selected novel high yielding varieties to boost production. The technique has
certain demerits like high production cost which have limited the use and exploitation at industrial
level. The technology is capital, labor and energy intensive. Low cost tissue culture technology may
be considered as a high priority in agriculture, horticulture, forestry and floriculture of any country.
Various strategies have been adopted by different researchers intelligently and seriously to simplify
various operations involved in in vitro propagation to reduce the costs without compromising the
quality of the micropropagules and plants. This article reviews the recent advancement in the
development and utilization of different cost effective methods/options by improving process efficiency
and better utilization of resources.

Key words: Tissue culture, Cost management, Quality product, Industrial use.

INTRODUCTION basically to ornamental plants only (Ammirato et al.,

1989a), but now it has been extended to about 1000
The conventional propagation practices for clonal
plant species including various vegetable and fruit
propagation of plants are time consuming and labor
crops such as potato, strawberry, oil palm, banana,
intensive. Micropropagation, popularly known for large-
etc., medicinal and aromatic plants and trees (Bajaj,
scale clonal propagation, is the first major and widely
1986; Bajaj et al., 1988). Plant tissue culture is now
accepted practical application of plant biotechnology.
a very important technology which can be used for
It is an alternative method of vegetative propagation.
production of large scale quality planting materials of
It offers many unique advantages over conventional
selected novel high yielding varieties to boost
propagation methods such as rapid multiplication of
production. It is a very successful technology which
valuable genotypes, expeditious release of improved
helps quick transfer of technology from laboratory to
varieties, production of disease-free quality planting
field and then to market. There are many advantages
materials, non-seasonal production (round the year),
of this in vitro technique of plant propagation as
higher yield, earlier and more vigorous sucker
compared to the conventional procedure (Murashige,
production, uniform and mass production in relatively
1974, 1978; Murashige and Skoog, 1962; George and
short periods of time, germplasm conservation and
Sherrington, 1984; Pennell, 1989). This technology
facilitating their easy international exchange (Deberg
can also be used to solve many practical problems in
and Zimmerman, 1991; Kozai et al., 1997; Levin and
economic plants. In spite of these merits, the
Vasil, 1989). Initially, the technique of micropropagation
micropropagation technique has certain demerits also
for large-scale production of plants was employed
182 S. K. Datta, Debasis Chakraborty and T. Janakiram
which have limited the use and exploitation of this
technique at industrial level.
Need for low cost technology
Commercial application of tissue culture technology
is restricted due to high production cost (Kozai et al.,
1997; Babbar and Jain, 2006). Hence, the most
challenging aspect at present is to reduce the
production cost, thereby improving the production
efficiency (Ahloowalia and Prakash, 2004; Ahloowalia
et al., 2004; Anderson and Meagher, 1977; Sluis and
Walker, 1985; Donnan, 1986; Levin and Vasil, 1989;
Aitken-Christie, 1991; Savangikar, 2004; Savangikar
and Savangikar, 2004). During the past couple of
decades, there has been an increased interest in
problems related to the large-scale plant production
as well as in its cost reduction for commercial
micropropagation (Debergh and Mane, 1981; Donnan,
1986; Jain and Babbar, 2005; Seko and Nishimura,
1997; Anonymous, 2002; Dessai et al., 2002; Namdeo
et al., 2006; Binita et al., 2005; Deb and Pongener,
2010; Chu and Kurtz, 1989; Andrea-Kodym and
Zapata-Arias, 2001; Raju, 1994; Raju et al., 2002;
Muralidharan, 1995, 1997; Muatafa and Kumar, 2012;
Xiao et al., 2000; Sharafi et al., 2010; Zobayed et
al., 2000; Zapata, 2001). Although conventional plant
tissue culture has been applied for decades, the high
production cost is a drawback for laboratories with
limited resources, especially in the developing
countries. A number of cost reduction strategies have
now been developed to overcome this limitation.
Appreciable literature/knowledge generated by
different researchers on low cost tissue culture have
been published which are spread in the form of
proceedings of symposia, review articles, chapters in
books, books, bulletins, catalogues, scientific journals,
popular magazines etc. It is difficult for all researchers,
teachers, students, amateurs, florists, gardeners,
commercial growers, business houses, nurserymen
and farmers to get an overview of earlier and recent
developments regarding low cost tissue culture.
Attempt has been made to put together important
information to develop a complete documentation of
the results of the research and demonstrations
conducted by different scientists for low cost tissue
culture. The document has been prepared only from
The Journal of Plant Science Research
published information as a review document. Attempt
has been made to cite maximum important publications
suggesting cost reduction in tissue culture. The main
objective of the review is to create awareness among
the plant tissue culture utilizing community to make
the technologies simple.
Although micropropagation protocols have been
developed for a large number of species, only a limited
number of plants are being produced on a large scale
through micropropagation. This may be due to the fact
that conventional methods of propagation are cheaper
than tissue culture technology or there is a selective
market demand. Ornamentals account for
approximately 80 percent of the world trade in the
tissue culture industry (Ahloowalia and Prakash, 2004).
Therefore, to overcome this limitation, a number of
cost reduction strategies have now been developed.
Low cost tissue culture is very useful not only for
farmers (Thro et al., 1999) but also for routine large
scale commercial multiplication. Micropropagation
generally involves following distinct stages: initiation
of cultures, shoot multiplication, rooting of in vitro
grown shoots, and acclimatization. Different plant
tissue culturing components are, namely, nutrients/
media chemicals (plant growth hormones, vitamins and
minerals nutrients), plant materials, equipments
(culture containers, autoclave, laminar flow,
instruments used for micropropagation, pH meter etc)
and the infrastructures (media preparation, inoculation,
growth and hardening rooms) and all these factors
are subjected to play important roles in cost reduction
as noted by Ganapathi et al. (1995).
Research institutions and universities have played very
important role in developing and refining the tissue
culture technology. Contribution of Department of
Biotechnology (DBT), Government of India is highly
appreciable. DBT has developed complete technology
package for more than 65 plant species of economic
importance through a network programme among
nearly 80 research institutes/universities/laboratories.
This package of information would provide valuable
data and also be a reference material for future
research activities in this area (http://
dbtmicropropagation.nic.in/).

Low cost option should lower the cost of production
without compromising the quality. The adoption of
wrong low-cost options may make the production
process prone to disasters. Low cost techniques will
succeed only if the basic conditions for tissue culture
are scrupulously adhered to maintain propagule quality.
It is not the sophistication but the procedures that
ensure the quality of tissue cultured plants. It can be
told that low cost technology means an advanced
generation technology, in which cost reduction is
achieved by improving process efficiency, and better
utilization of resources. Low cost technology is not
only required for developing countries but also
developed countries require this technology to
progressively reduce the cost of propagule
productions. Low cost tissue culture technology may
be considered as a high priority in agriculture,
horticulture, forestry and floriculture of any country.
Various strategies may be adopted intelligently and
seriously to simplify various operations involved in in
vitro propagation to reduce the costs in a tissue culture
facility.
Washing and Sterilizing operations
One should be always careful to increase the efficiency
of production and how to bring down the cost of
production. Various operations can be simplified and
cost may be reduced by adopting low-cost alternatives
(Pennell, 1989; Ahloowalia, 2000; Ahloowalia and
Prakash, 2004). Considering the labour cost, culture
containers can be washed manually without using
costly machines and can be dried in sun. Considering
the quantum of work large size pressure cookers can
be used without using costly autoclaves. Costly
aluminium foil is used for wrapping the instruments
before sterilization. Autoclavable stainless steel
containers may be used as a substitute.
Medium Substrate / Media Composition
Proper refinement of protocol will help in cost
reduction in tissue culture. A high degree of purity is
justified in case of basic studies in tissue culture. But
several ingredients of tissue culture can be replaced
by low cost options commercial grade chemicals of
lower purity than the analytical grades (Dhamankar,
1992).
Low Cost Tissue Culture : An Overview 183
Since agar was introduced as gelling agent more than
100 years ago, it has been extensively used as for
microbial and plant tissue culture media (Babbar and
Jain, 2006). Agar is useful for the purposes due to its
stability, high clarity, nontoxic nature and resistance
to its metabolism (McLachlan, 1985; Henderson and
Kinnersley, 1988). In the recent past several attempts
have been made to look for suitable substrata that
could possibly replace agar in culture medium because
of doubts about its inertness and nontoxic nature, fear
of over-exploration of its sources and above all, the
high cost of tissue culture grade agar (Babbar and
Jain, 1998, 2006; Jain and Babbar, 2002;
Temjensangba and Deb, 2005; Deb and
Temjensangba, 2006; Deb and Sungkumlong, 2008;
Sungkumlong and Deb, 2009). In the recent past
agarose (Johansson, 1988), alginates (Scheurich et al.,
1980), gelrite (Pasqualetto et al., 1988), isubgol
(Babbar and Jain, 1998; Jain et al., 1997), xanthan
gum (Babbar and Jain, 2006), guar gum (Babbar et
al., 2005; Jain et al., 2005), starch (Zimmerman et
al., 1995; Nene et al., 1996) etc. have been used with
reasonable success as substitutes for agar. Betel-nut
coir, coconut coir, chopped forest leaf litters are natural
and eco-friendly and have potential application in plant
tissue culture. Reports are available on screening some
possible low cost, eco-friendly raw materials for use
as alternative substrates against agar in plant tissue
culture. Mohan et al. (2005) attempted to reduce the
cost and improve the quality of micro-cuttings of
strawberry cv. Dover by using a natural support
based on sugarcane bagasse as a substitute for the
traditionally used agar gelled medium. A comparison
with agar-grown micro-cuttings showed that the
sugarcane bagasse yielded better result. Low cost
media composition have been reported for
multiplication of orchids (Hossain, 1995; Pervin, 1997;
Komol, 1998; Hoque et al., 1994, 1999; Zahed, 2000).
Ranaweena et al (2002) did experiments with coir
dust, sand, refuse tea, sago, cassava flour, rice polish,
compost, kithul flour etc. to detect the low cost
alternative medium substrate. They have
recommended the method as cost effective, time
saving and practical feasible micropropagation method
for production of field plantable tea plants. Deb and
Pongener (2013) successfully attempted to use ‘betel-
The Journal of Plant Science Research
184 S. K. Datta, Debasis Chakraborty and T. Janakiram
nut coir, coconut coir and polyurethane foam disk’ as
alternative to agar for non-symbiotic embryo culture
of Cymbidium iridioides. The outcome of the study
showed that the production cost of the tissue cultures
raised plants could be reduced by ~24 percent by
incorporating ‘polyurethane foam’, betel-nut coir,
coconut coir as alternative to agar (Aggarwal et al.,
2006; Deb and Temjensangba, 2005; Temjensangba
and Deb, 2005; Deb and Imchen, 2010).
Prakash et al. (2004) suggested low cost options to
replace expensive gelling agents, sugars and water.
Agar is available commercially in various brands and
grades. Their price, performance and composition vary
widely. There is no need to use high purity agar for
large-scale micropropagation (Boxus, 1978). Agar is
most common gelling agent for preparation of solid
and semi solid media (Nene and Shiela, 1997; Debergh,
1983). A number of low cost agar alternatives have
been reported for routine use in commercial
micropropagation (Pierik, 1989; Kodymand and
Zapata, 2001; NRDC, 2002; Nagamori and Kobayashi,
2001). Alternatives are starch-Gelrite, white flour,
laundry starch, semolina, potato starch, rice powder,
sago etc. The use of laundry starch, potato starch
and semolina (2:1:1) reduced the cost of gelling agent
by 70-82 percent (Prakash, 1993). Corn starch has
been used with low concentration of Gelrite for
propagation of fruit trees, sugarcane, ginger and
turmeric (Zimmerman, 1995; Stanlex, 1995).
Disadvantages of starch gelling agents have been
reported, which need further standardization (Powell
and Uhrig, 1987). Tapioca starch has been used as a
good substitute for ‘Bact-agar’ for potato shoot culture
(Nene and Shiela, 1997; Getrudis and Wattimera,
1994). Barley starch has also been used for culturing
potato-tuber discs (Sorvari, 1986; Sorrari and Schieder,
1987). Sago was used as a substitute for agar in MS
medium for multiplication of chrysanthemum
(Bhattacharya et al., 1994). ‘Isobgol’ has been tested
as a good gelling agent for the propagation of
chrysanthemum (Babbar and Jain, 1998, Bhattacharya
et al., 1994).
The potential of cassava starch as an alternative and
cheap gelling agent for potato in vitro culture micro-
propagation media was investigated (Ogero et al.,
The Journal of Plant Science Research
2012; Kasanadze, 2000; Maliro and Lameck, 2004;
Mbanaso et al., 200). The use of 10 percent cassava
starch reduced cost by 42.5 percent in comparison
with use of agar (Kuria et al., 2008). Kwame et al.
(2012) developed cost-efficient protocol for cassava
micropropagation using locally available materials.
They used Easygro vegetative fertilizer as an
alternative source of both macronutrients and
micronutrients which reduced the cost by 92.2 percent.
Gum katira (derived from the bark of Cochlospermum
religiosum) has been successfully used as a gelling
agent in tissue culture media for in vitro shoot
formation and rooting in Syzygium cuminii and somatic
embryogenesis in Albizzia lebbeck (Jain and Babbar,
2002). Production cost could be substantially reduced
(about a fourth) by using foam as agar alternative.
Deb and Pongener (2013) and Deb and Arenmongla
(2014) made successful attempt to use ‘betel-nut coir,
coconut coir and polyurethane foam disk’ as
alternative to agar for non-symbiotic embryo culture
of Cymbidium iridioides. The processed substrata
were incorporated in the culture vials against agar in
the medium. Results showed that the production cost
of the tissue cultures raised plants could be reduced
by ~24 percent by incorporating ‘polyurethane foam’,
betel-nut coir, coconut coir as alternative to agar.
For culturing callus, cell clusters, buds and somatic
embryos normally suspension cultures without gelling
agents are used. Sterilized, non-chlorine bleached,
rolled, pure cotton fibre has been successfully used
for culturing callus and proliferating shoots of Texus,
Agrotis and Artemisia (Moraes-Cerderia et al., 1995).
Use of cotton fibre in liquid media has been reported
in commercial propagation of orchids, banana,
chrysanthemum and potato. Use of some more
alternative culture supports like foam-plastic, filter
paper bridges, nylon cloth, glass beads, ‘viscose’
sponge, glass wool and rock wool in liquid media have
been reported (MacLeod and Nowak, 1990). Strips
of glass wool, nylon, filter paper and polystyrene foam
slocks have been used as supporting bridges for the
propagation of chrysanthemum on MS medium
(Bhattacharya et al., 1994). Glass bead based liquid
media showed higher multiplication rate and better
growth for ginger, turmeric, vanilla, Ficus, Saintpaulia,
Syngonium, Philodendron and Spathiphyllum (Prakash,

1993). Advantage of glass bead based liquid media
have been reported (MacLeod and Nowak, 1990;
McCulloch et al., 1994). Luffa sponge and coir have
been used for enhancing multiplication rate and rooting
in orchids (Gangopadhyay et al., 2002, 2004).
Carbon sources
The most common carbon source in the
micropropagation is sucrose. Household sugar and
other sugar sources have been used for culturing
ginger, turmeric, potato, banana, orchids,
chrysanthemum, lentil, peanut, chickpea, medicinal
plants, fruit trees etc. to reduce the cost of the medium
(c.f. Prakash et al., 2004, Prakash, 1993). Banana
plant production via low cost technology in which cost
reduction is achieved by improving process efficiency
and better utilization of resources has been reported
by Savangikar (2002). Low cost options should lower
the cost of production without compromising the quality
of the micropropagules and plants (Prakash et al.,
2002). A low cost protocol for multiplication of healthy
banana seedlings has also been reported by Gitonga
et al. (2010). The carbon source such as grade
sucrose that is often used in the micropropagation of
plants at laboratory contributes about 34 percent of
the production cost (Demo et al. 2008). Sucrose has
been reported as a source of both carbon and energy
(Bridgen, 1994). There are reported success in
reducing 90 percent cost of tissue culture banana
plants by replacing sucrose. Adoption of this protocol
can empower farmers to set up low cost tissue culture
laboratories in their localities to increase banana plant
production. In the plant propagation medium Kaur et
al. (2005) substituted sucrose with table sugar which
reduced the cost of medium considerably by 96.8
percent. The observation is in agreement with that of
Prakash et al. (2002) who reported the reduction in
the cost medium by 78 to 87 percent using common
sugar. Kodyam and Zapata-Arias (2001) could
successfully reduce about 90 percent resource cost
in tissue culture of banana (Musa ‘Grande Naine’)
by replacing tissue culture grade sucrose and Gelrite
in the medium with locally available commercial sugar
and a starch/Gelrite mixture and by using sun light
instead of artificial light. Thirteen commercial sugars
from different countries were tested. Best results
Low Cost Tissue Culture : An Overview 185
were achieved using white and light brown sugars
with low electrical conductivity. Sugars of cane or
sugar beet origin were suitable. Starches of corn or
potato could partially substitute for Gelrite and agar.
In all experiments, micropropagation rates under
natural light conditions were equal to or higher than
under the controlled conditions of a growth room with
PPFD of 65 mol m–2 s–1 and a 16-h photoperiod.
Plants were exposed to average PPFD levels of 58–
96 mol m–2 s–1 and photoperiods ranged from 8–16
hours. Goel et al. (2007) reported use of sugar in glass
beads supported liquid medium caused up to 94
percent reduction in the cost of the medium used for
culturing of Rauwolflora seperpentina. The purpose
of using sugar in the study was to reduce the overall
cost of micropropagating and testing the response of
the local banana. The use of market sugar instead of
sucrose has been reported to reduce the cost of in
vitro conservation of banana, Karpura chakkarakeli
cultivar, with no significant effect on regeneration
compared to sucrose (Agrawal et al., 2010).
The use of table sugar as an alternative source for
carbon reduced the cost by 97.1 percent. This led to
savings of 96.3 percent in the cost of nutrients and
carbon source used in preparing a litre of the medium.
This study has shown that it is possible to use locally
available salts as low cost sources of tissue culture
nutrients. Santana et al. (2009) used different
concentrations of a locally available fertilizer to
micropropagate cassava for low cost. Escobar et al.
(2006) also tried different kinds of fertilizers at
different concentrations and realized a cost reduction
of 24.4 percent for the medium prepared. This
research has shown that it is possible to reduce the
cost of plantlet production during tissue culture. The
low cost medium evaluated here can be adopted easily
in the production of cassava planting material. This
will greatly enhance availability of cassava planting
materials at an affordable cost which will boost its
production. Ogero et al. (2012) evaluated the
possibility of using locally available fertilizers as
alternative nutrient sources for cassava
micropropagation. A Low Cost Medium (LCM)
whereby the conventional sources of four Murashige
and Skoog (MS) macronutrients had been replaced
with locally available fertilizers was developed. Stanes
The Journal of Plant Science Research
186 S. K. Datta, Debasis Chakraborty and T. Janakiram
Iodized Microfood was used as the alternative source
of micronutrients. A reduction of 95.50 percent in
nutrient cost was achieved. Ogero et al. (2012)
developed a low cost protocol for cassava tissue
culture using Easygro vegetative fertilizer as an
alternative source for conventional MS salts.
Dhanalakshmi and Stephan (2014) developed a low
cost media options for propagation of two banana
varieties. They replaced conventional sources of MS
media by mixed nutrients containing both macro and
micronutrients. The mixed nutrients were
supplemented with 30 g/L of table sugar and 8 g/L
agar. The successful regeneration of the two Banana
cultivars indicates that locally available salts like mixed
nutrients can be used as an alternative source which
can greatly reduce the cost of media and hence, the
cost of shoot production which in turn will lower the
production of shoots. There was 61.4 percent
reduction in the cost of the nutrients used in the media
preparation. Gabriel et al. (2013) reported better
growth, quality and quantity of plantlets in Musa on
formulating culture media using low-cost materials as
substitute to MS. The establishment of in-vitro
techniques and nursery management for the
production of cv. Lacatan banana seedlings provided
some benefits both to the researchers and the farmer-
clientele. The objective of this study is to reduce the
cost of banana tissue culture nutrients by using
alternative nutrients sources. The conventional
sources of MS media were replaced by mixed
nutrients containing both macro and micronutrients.
The mixed nutrients were supplemented with 30 g/L
of table sugar and 8 g/L agar. There was 61.4 percent
reduction in the cost of the nutrients used in the media
preparation. A 90 percent resource cost reduction in
tissue culture of banana was achieved by replacing
tissue culture grade sucrose and Gelrite in the medium
with locally available commercial sugar and a starch/
Gelrite mixture and by using sun light instead of
artificial light. Thirteen commercial sugars from
different countries were tested. Best results were
achieved using white and light brown sugars with low
electrical conductivity. Sugars of cane or sugar beet
origin were suitable. Starches of corn or potato could
partially substitute for Gelrite and agar. In all
experiments, micropropagation rates under natural
The Journal of Plant Science Research
light conditions were equal to or higher than under the
controlled conditions of a growth room (Kodym and
Zapata-Arias 2001). Vora and Jasraj (2012) studied
the impact of various fruit juices on in vitro shoot-
multiplication of banana var. Grand naine for the
purpose of cost-reduction. Sweet-lime juice was found
useful as the substitute of such costlier synthetic
cytokinin.
Gebre and Sathyanarayana (2001) and Demo et al.
(2008) evaluated alternative cheap sources of carbon
and energy in potato culture media in order to reduce
the overall cost of micro-propagation. They used
laboratory grade sucrose with two types of local
commercial table sugar (white and brown sugar).
Brown sugar enhanced significantly higher mean
number of roots per plantlet after four subculture
generations for all cultivars. Results also showed that
table sugar not only enhanced micro-propagation but
also significantly lowered the production input costs
by 34 to 51 percent when compared with the analytical
grade sucrose.
Sharma et al. (2013) reported that there was 85
percent reduction in cost of media for plant
caulogenesis by using inexpensive carbon source,
water source and gelling agent. Laboratory reagent
grade sucrose was replaced by locally available
commercial sugar (market sugar and sugar cubes) as
carbon source, bacteriological grade agar by isabgol
(Plantago ovata), sodium alginate, starch as gelling
agent and distilled water, tap water and packaged
drinking water as a water source. Based upon cost
analysis the use of isabgoal as a gelling agent, use of
sugar cubes and packaged drinking water for
preparation of media can be used to boost the
caulogenesis in Stevia rebaudiana. Up to 73 percent
decrease in cost of media for plant regeneration and
in vitro conservation was achieved in Curcuma longa
cv Prathibha by using inexpensive carbon source and
gelling agent. Laboratory reagent-grade sucrose was
replaced by locally available commercial sugar (market
sugar or sugar cubes) as carbon source and
bacteriological grade agar by isabgol (also named
isubgol) as gelling agent. No adverse effects on shoot
regeneration and conservation on isabgol-gelled low-
cost media were observed as compared to that on

agar-gelled control medium (CM) (Tyagi et al., 2007).
Prakash (1993) investigated and standardized less
expensive methods for production of ginger and
turmeric. Bonaobra et al. (1994) studied the effects
of table grade sugars on growth and development of
coconut (Cocos mucifera L.) embryos in vitro.
Water
Tap water (free from heavy metals and contaminants)
can be substituted for distilled water to lower the cost
of the medium (Ganapathi et al., 1995; Sharma and
Singh, 1995). Tap water may be used after autoclaving
in small facilities rather than distilled water. Use of
RO water for stock solutions and hormone
preparations and distilled water for media preparation
will reduce the cost most effectively. RO water also
is used for washing plants prior to sterilization and
also for the purpose of added sterilants for cleaning.
Glasswares
Glass test tubes are normally used in plant tissue
culture. Conical flasks, glass and plastic petri dishes
are also used. These are expensive. A wide variety
of containers have been tested at different stages of
micropropagation. Pre-sterilized disposable-plastic
petri dishes are cheaper. Glass bottles and baby food
jars with polypropylene caps have been tested and
found most economic and low cost option.
Autoclavable transparent plastic containers and
containers made of polypropylene, polycarbonate and
polystyrene are used in many countries. Gamma ray
sterilized non-autoclavable food containers,
polystyrene sandwich boxes, plastic bags, PVC pots
and jars are being used for large scale
micropropagation (c.f. Prakash et al., 2004). Culture
vessel like ‘StarPac’ disposable bag, ‘Watson Modules
(plastic type container) are being used at different
stages (hardening, multiplication, soil growing) of plant
growth (Prakash et al., 2000, Ahloowalia, 1999a,
1999b; Tanaka et al., 1988). Juice, Jam and jelly bottles
and even old whisky bottles are used in Bangladesh,
India, Thailand, Singapore, Indonesia, Malaysia. Vessel
closures and lids play important role for growth of in
vitro plants. In normal practice non-absorbent cotton
plugs, polyurethane foam plugs, plastic plugs,
aluminium foil, stainless steel caps, polypropylene
Low Cost Tissue Culture : An Overview 187
caps, PVC film, polythene film, silicon rubber etc. are
used. For large scale production such caps have been
replaced by autoclavable screw caps made of stainless
steel or polypropylene (Kozai and Iwanami, 1988;
Crisp and Walkey, 1974; Mahlberg et al., 1980;
Bateson et al., 1987; Osmotec, 2002).
Light management
The production cost of tissue culture plants can be
reduced by 50-90 percent by using such low cost
alternatives (c.f. Prakash et al., 2004). The
conventional method of downward illumination of
culture racks can be replaced by sidewise lighting
system. This reduces the number of lights and provide
uniform illumination to the cultures. The electrical
lighting systems can be replaced by sunlight in tropical
and Mediterranean regions. Proper planning is
necessary for commercial laboratories so that one can
use the facilities maximum throughout the year.
Artificial lighting of cultures in the growth rooms is
one of the most expensive methods in tissue culture
technology. Use of natural light is a positive low cost
option in tissue culture. Natural light can be used in
different ways. In vitro cultures can be grown in
different natural light under plastic or glass in
temperate climates. Existing laboratories in tropical
and sub-tropical regions can be redesigned and
artificial lights can be replaced by ‘Solatube’
(Anonymous, 2001; Kodym and Zapata-Arias, 1999,
2001; Kodyam et al., 2001). Glass windows of growth
rooms may be facing Southwest which allow indirect
diffused natural day light. Muslin or Venetian curtains
(made of bamboo or plastic) helps to diffuse the light
to the desired intensity (Baezas-Lopez, 1995). In vitro
cultures in plastic bags (polypropylene) can be grown
favourably under natural light. In this process plants
undergo primary hardening when they are still
developing in the culture. Such plants have well-
developed stems, leaves and good growth vigour, and
survive better on transfer to soil. Such modified system
reduces cost of air filtration and air conditioning
normally used in the sophisticated facilities. Similarly
artificial lights in media preparation, transfer and
hardening rooms can be reduced (c.f. Ahloowalia and
Savangikar, 2004). Electrical cost can be reduced
significantly by hardening plants in open net-shades
The Journal of Plant Science Research
188 S. K. Datta, Debasis Chakraborty and T. Janakiram
after transfer to soil or by placing containers in shade
under thatched or plastic covered open huts. It has
been tested that plants hardened under natural light
are hardy and withstand better in the field after
transplantation (c.f. Ahloowalia and Savangikar, 2004).
Solar illuminated tissue culture rooms are currently in
use in Cuba (Anonymous, 1998).
Low cost micropropagation procedure for vetiver
(Vetiveria zizanioides) has been developed (Be et
al., 2006, 2007, 2008). A liquid MS medium,
supplemented with 2-4 mgBAl-1 of medium yielded
the best multiplication, with average eight new shoots
during a culture interval of six weeks. Proliferation
and rooting stage in vitro can be carried out under
the natural ambience of a nethouse instead of growth
chamber conditions. No statistical differences were
observed between the two environmental conditions
for propagation and rooting, and survival cluster after
ten weeks of acclimatization approximated 100
percent. Nethouse plants were assessed to be about
22 percent cheaper to produce than growth chamber
plants.
As mentioned above, energy cost is one of the major
concerns for commercial applications when using
artificial lights. Various efficient light sources were
investigated worldwide. Various types of light source
can be used in horticulture including incandescent bulb,
tubular fluorescent, compact gas-discharge, high-
pressure mercury, metal halide and high-pressure
sodium lamps. A microwave-powered lamp, developed
by an American company (MacLennan et al., 1995),
has many advantages over conventional lamps for use
in artificial lighting of plants (Kozai et al., 1995; Kozai
and Kubota, 1997). Recent developments have resulted
in greatly increased light output for red and blue light-
emitting diodes (LEDs). LEDs with the characteristics
of high energy-conversion efficiency and low thermal
energy production, thus, making it a promising light
source for plant growth in confined environment (Bula
et al., 1991; Hoenecke et al., 1992). A good amount
of experiments have been conducted and the subject
of promising new light source - LEDs. has been
reviewed. Use of light-emitting diodes (LEDs) is
potentially one of the biggest advancements in
horticultural lighting in decades. LEDs can play a
The Journal of Plant Science Research
variety of roles in horticultural lighting, including use
in controlled environment research, lighting for tissue
culture, and supplemental and photoperiod lighting for
greenhouses. LED lighting systems have several
unique advantages over existing horticultural lighting,
including the ability to control spectral composition,
the ability to produce very high light levels with low
radiant heat output when cooled properly, and the
ability to maintain useful light output for years without
replacement. LEDs have the potential for significant
cost savings over current horticultural lamp types.
LEDs provide many advantages as plant lighting, but
there are difficulties that are slowing their
implementation for horticultural applications. Green
Power TLED (25-100 mol/m2/s) offers an extremely
effective and efficient alternative to traditional
fluorescent lamps, delivering energy savings of up to
70 percent (Sager et al., 1982; Morrow and Tibbitts,
1988; Morrow et al., 1989; Nijssen et al., 1990;
Morroe et al., 1995; Batra et al., 1991; Bula et al.,
1991; Bula et al., 1991; Batra et al., 1992; Hoenecke
et al., 1992; Philips lighting, 1992; Tennessen et al.,
1994; Brown et al., 1995; Kozai et al., 1995;
Tennessen et al., 1995; MacLennan et al., 1995; Goins
et al., 1997; Kozai and Kubota, 1997; Schuerger and
Brown, 1997; Miyashita et al., 1995; Wei Fang and
Jao, 2000; Dougher and Bugbee, 2001; Goins et al.,
2001; Yorio et al., 2001; Heo et al., 2002; Steigerwald
et al., 2002; Emmerich et al., 2004; Kim et al., 2004;
Narisada and Schreuder, 2004; Massa et al., 2005;
Fujiwara and Sawada, 2006; Massa et al., 2005;
Caldwell and Britz, 2006; Massa et al., 2006; Ono
and Watanabe, 2006; Philips Lumileds, 2006, 2007,
2008; Schuerger and Richards, 2006; Kim et al.,
2007; Robert C. Marrow, 2008; Rajesh Pati, 2014).
Labour management
Tissue culture is a labour intensive system and it is a
major cost factor in micropropagation in developed
countries. The typical profile of a tissue culture plant
production system shows that 40 percent costs are
for labour, 10 percent for materials, 20 percent for
overheads, and 30 percent for sale, general and
administrative activities (Walker, 1986). It is very
important to maintain high labour efficiency to reduce
the cost of tissue-cultured plants. The production cost

may be reduced if the duration of hardening facility
can be reduced but increasing the survival rates. The
plants need relatively shorter duration for hardening
if natural light is used during multiplications and
rooting. Loss of plants at the hardening stage
contributes to increased costs of production. A cost-
effective system based on natural sunlight for in vitro
acclimatization gives high survival (Ziv, 1986,
Savangikar et al., 2002). Low cost alternatives
reduces the capital cost of hardening by saving
laboratory space, labour and time. This procedure has
been used on a commercial scale for a number of
plants such as Gerbera, Spathiphyllum, Syngonium,
African Violets, Chrysanthemum, Ficus, Philodendron
and other ornamentals in Netherlands and Israel
(Prakash and Peirik, 1991; Prakash, 1996; Torres,
1989; Lim et al., 1998; Tanaka et al., 1991; Roy et
al., 2006; Zahed, 2000).
A double phase culture system was standardized and
compared with conventional micro propagation system
by taking Rauwolfia serpentine L. Bent. as a model.
The culture period was reduced by 44 percent in DPS
compared to the CMS. In DPS nutrient cost/plantlet,
energy cost/plantlet and labour cost also reduced by
35.36 percent, 40.66 percent and 33.33 percent
respectively in comparison to CMS. Thus DPS has
many advantages and can be used for large scale
quality production of commercial plants in reduced
time, cost and labour, which ultimately reduced the
production cost of the farmer (Senapati, 2015).
One should always try that the price of tissue culture
raised plants must be competitive with those obtained
from conventional propagation. Elite materials may
be grown through tissue culture and these can be used
as nuclear material. These nuclear materials can be
further multiplied by conventional vegetative
propagation to reduce the cost of plants. In Thailand,
tissue cultured chrysanthemum plants are sold to
farmers who multiply them further for conventional
cuttings for the production of cut flowers. This
procedure helps quick introduction of new varieties
to meet the market demand. On-farm conventional
multiplication of the tissue culture propagated material
is the cheapest and the most sustainable method for
cost reduction (Ahloowalia, 2004).
Low Cost Tissue Culture : An Overview 189
Hormones substitute
Jaisy and Ghai (2011) reported the use of charcoal
other than rooting hormones will reduce the cost of
the media. For multiplication, BAP, ascorbic acid and
Gelrite agar media yielded higher multiplication rate
when compared to other media composition. In the
rooting media, addition of charcoal at 2 percent
concentration instead of hormones (IAA, IBA)
showed 95 percent success ratio.
Autoclaving require a three-phase connection. But in
small facilities it is sensible to operate the units on a
single-phase electrical connection.
Substitution of macronutrients and
micronutrients
Gitonga et al. (2010) realized to evaluate the potential
for developing a low cost micropropagating protocol
for local bananas in Kenya. The substitution of
macronutrients and micronutrients with the alternatives
reduced the cost by 94.2 and 97.8 percent,
respectively. Substitution of gelling agents (agar and
gelrite) with support matrices (glass beads, cotton wool
and vermiculite), conventional equipments (autoclave,
culture bottles, micropipette and measuring cylinder)
with easily accessible alternatives (pressure cooker,
jam jars, insulin and vet syringes) reduced costs by
94.2 and 85.9 percent, respectively. Support matrix
(glass beads) and plastic equipment (jam jars, insulin
and vet syringes) were used repeatedly after
maintaining and washing them thoroughly before re-
autoclaving, thus reducing the cost significantly.
Support matrices have also been used successfully
as a low cost alternative to gelling agents (Andrea-
Kodym and Zapata-Arias, 2001; Bhattacharya et al.,
1994; Goel et al., 2007; Vora and Jasrai, 2011, 2012).
Gitonga et al. (2010) evaluated a micropropagating
protocol for local banana (Musa spp.) in Kenya as an
alternative to reduce the unit cost of tissue culture
micropropagation. Use of support matrices, locally
available macronutrients, micronutrients, sugar,
equipment and facility reduced the cost of consumable
material for banana tissue culturing by about 94
percent. Putting into account energy, labour and capital
investments, the cost dropped from approximately US
$ 1.5 to 1.0 per plantlet. Use of plastic syringes instead
The Journal of Plant Science Research
190 S. K. Datta, Debasis Chakraborty and T. Janakiram
of glass cylinders and micropipettes, to measure
volumes reduced the cost of the equipment by 96
percent.
Das et al. (2010) developed an efficient
micropropagation protocol using shoot apical
meristem as explants of Aloe vera L. The protocol
involves induction, multiplication and in vitro rooting
of the regenerated shoots and their acclimation
under ex vitro conditions. Combination of 8.87 M
BAP and 2.46 M IBA produced highest number
of shoot buds (22.0 ± 0.14) and enhanced bud
proliferation within one two weeks after first
subculture. For induction of in vitro rooting, Aloe
gel as an alternative to conventional rooting medium
used for the first time resulted in 100 percent rooting
and highest number of roots per culture (10.90 ±
0.17). The plantlets were successfully hardened and
cent per cent plants survived in the field condition.
Cost analysis showed that the cost of regenerated
plants in one jam bottle does not exceed Rs. 58.47.
Keeping in mind that jam bottles can be recycled
many times, price of one tissue culture plant
produced through present protocol cannot be over
Rs. 2.65, whereas the current market price of one
single Aloe plant is Rs. 5. Use of Aloe gel for root
induction may further reduce the cost price. This
efficient, reproducible and cost effective protocol
can be adopted for commercialization of Aloe
cultivation. An efficient micropropagation protocol
has been developed using shoot apical meristem as
explants in a high barbaloin content ¹bitter¹ cultivar
of Aloe vera L. The protocol is highly cost effective
and economically viable (Das et al., 2010).
Kieffer et al. (2001) standardized an improved and
cost effective protocol for in vitro mass propagation
of cauliflower from fractionated and graded curd. The
propagule unit cost was drastically reduced.
Micronutrient requirements for maximization of
sugarcane yield has been standardized (Savangikar
et al., 1999).
The commercial production of ornamental plants is
growing worldwide. Its monetary value has
significantly increased over the last two decades
and there is a great potential for continued further
The Journal of Plant Science Research
growth in both domestic and international markets
(Jain, 2002). Major pot plants such as Begonia,
Ficus, Anthurium, Chrysanthemum, Rosa,
Saintpaulia, and Spathiphyllum are being produced
in the developed countries (Anonymous, 2003).
Ornamental industry has applied immensely in vitro
propagation approach for large-scale plant
multiplication of elite superior varieties (c.f. Rout
et al., 2006). Deb and Imchen (2010) reported an
alternative approach to acclimatize the microshoots
of orchids by using an alternate substratum. Tissue
cultured raised orchid seedlings are acclimatized
and hardened in vitro by using 10 percent MS basal
medium with no carbon source or any plant growth
regulators. In the culture vials different types of
matrix as an alternative substratum have been used
for epiphytic and terrestrial orchids. A combination
of charcoal pieces (5–7 mm in size), small brick
chips and mosses at (1:1) ratio was found suitable
for epiphytic orchids and (1:1) ratio of moss and
decayed wood/forest litter along with charcoal
pieces, brick chips was preferred for terrestrial
orchids. Similarly, Agnihotri et al. (2004) also
reported 80 percent transplant success of Carica
papaya plantlets when transferred from culture
tubes along with soilrite plugs to the potted mix of
garden soil and soil rite in the ratio of 1:1.
Deb and Arenmongla (2014) screened some low cost
substrata as alternative to agar for in vitro
regeneration of orchid (Malaxis acuminate D. Don).
Among the three alternative substrata incorporated
in the regeneration medium, regenerative response on
‘foam disk’ as substratum was competitive with agar
gelled medium.
Experiment indicated that 100 percent sucrose
supplementation during proliferation reduces the
chemical cost by 15.12 percent without causing any
significant difference among the number of multiple
shoots. During rooting no significant difference was
observed up to 66 percent sucrose supplementation,
and liquid media can be used with Luffa as supporting
matrix, which reduced the chemical cost by 61.92
percent. Results support almost 42.5 percent of total
chemical cost reduction for efficient

micropropagation of pineapple (Firoozabady and
Gutterson 2003, Dutta et al. 2013, Be and Deberg,
2006). A low cost novel plant tissue culture medium
“KFA and KFA plus” (patented) has been developed
for Mentha arvensis and M. spicata using ‘Flyash”
(FA) as the main source of inorganic constituent in
the medium. Fly ash in the KFA and KFA plus culture
medium resolves the aim of low cost plant production
and also the disposal problem of the thermal power
plant waste upto a large extent (Biswas et al., 2014).
Muralidharan (1988) simplified micropropagation of
Kaempferia galangal for routine plant tissue culture
procedures. A simple sterile hood was used instead
of a laminar flow bench and a pressure cooker
instead of the autoclave. Laboratory grade chemicals
and tap water were used for preparation of tissue
culture media. Plantlets could be raised in low-cost
polypropylene bags modified in shape and sue using
a heat sealer. Liquid medium was found to be
adequate for culturing multiple shoots, Incubation of
cultures was also carried out at room temperature
using sunlight as the only light source. Plantlets were
rooted in the multiplication media itself and
transferred to soil after a few days of hardening.
Ogero et al. (2012) experimented to reduce the cost
of sweet potato tissue culture nutrients by using
affordable alternative nutrient sources. The
conventional sources of MS salts were substituted
with Easygro vegetative fertilizer containing both
macro and micronutrients. Two grams of the fertilizer
were supplemented with 30 g/L of table sugar and 9
g/L of agar. Conventional MS medium supplemented
with 30 g/L of sucrose and 3 g/L of gelrite was used
as the control. Two farmer preferred sweet potato
varieties, Kemb-36 and Tainurey were initiated on
the two media. There was 96.9 percent reduction in
the cost of the nutrients used in media preparation.
The developed low cost medium can be used to boost
the production of affordable disease-free sweet
potato seedlings.
Explant
Leafy or chlorophyllous explants currently
micropropagated have been found to have high
photosynthetic ability. Their growth and development
have been promoted on sugar-free medium rather than
Low Cost Tissue Culture : An Overview 191
on sugar-containing medium, provided that the
environmental factors, such as CO2 concentration,
light intensity and relative humidity, are controlled for
promoting photosynthesis and transpiration of explants/
shoots/plantlets in vitro. Several types of sugar-free
(photoautotrophic) culture systems for large-scale
micropropagation of plants have been developed
which could reduce the cost and plant quality could
be improved significantly with photoautotrophic
micropropagation (Kozai et al., 1997). The concept
of photoautotrophic micropropagation is derived from
research that revealed the relatively high
photosynthetic abilities of chlorophyllous cultures such
as leafy explants and plantlets in vitro. To meet the
ever-increasing demand for quality transplants, the
scaling-up of photoautotrophic micropropagation
systems, for commercialization, has become
necessary. This article reviews the recent
advancement in the development and utilization of large
culture vessels for photoautotrophic micropropagation
with special emphasis on the feasibility of the system
for the commercial-scale propagation (Zobayed et al.,
2004).
A cost effective micropropagation protocol has been
developed successfully using both conventional and
unconventional low cost alternatives for the
conservation of important medicinal plants resources.
The addition of low cost alternatives can reduce cost
by 50 percent of the medium without compromising
the production rate, quality and survival percentage
of the micropropagules (Sharma et al., 2008).
The costs of these methods are higher than for
traditional propagation and preservation, but they may
be necessary for species under higher threat. The
multiplication rate of a culture, as well as the rates of
rooting and acclimatization, has a major effect on the
number of transfers needed for producing plants or
tissue for banking, and improvements that will increase
the efficiency of these steps can help lower costs.
Further research into factors affecting the growth of
tissues in vitro, as well as coordination of efforts
among institutions with infrastructure for in vitro work,
should facilitate the application of in vitro methods to
the endangered species that cannot be propagated or
preserved using seeds (Pence, 2011).
The Journal of Plant Science Research
192 S. K. Datta, Debasis Chakraborty and T. Janakiram
Management of scientific temperament
This is most important in low cost tissue culture.
Tissue culture means scientific temperament towards
sophistication. But now all above experiments / results
clearly indicate that low cost protocol of tissue culture
can be developed for all economic crops for
commercial exploitation. Therefore, motivation of
scientific temperament is most essential for developing
low cost tissue culture protocol.
Large-scale culture of plants in bioreactors
To reduce the intensive labor requirement along with
the production cost during plant propagation by tissue
culture technique, there is an immense need of
developing scale-up systems and automation
(Ammirato, 1983b; Aitkin-Christie, 1991; Paek et al.,
2001; Zobayed et al., 2004; Paek et al., 2005).
Progress in tissue culture automation will depend upon
the use of liquid cultures in bioreactors (Sakamoto et
al., 1995). The use of shake cultures utilizing liquid
culture medium alone (Weathers and Giles, 1988) or
in combination with solid culture medium (Debergh
and Maene, 1981; Aitken-Christie and Jones, 1987)
have been developed and used by various workers
(Earle and Langhans, 1975; Takayama and Misawa,
1981; Takayama, 1991; Paque et al., 1992; Chu et
al., 1993). Use of liquid culture medium for the purpose
of micropropagation has been proposed as one of the
important methods for cost reduction. Liquid media
reduce the cost of multiplication. Increasing the
multiplication rate through enhanced axillary bud
proliferation helps in cutting cost of production. In
liquid medium, the close contact of the tissue with the
medium may stimulate and facilitate the uptake of
nutrients and phytohromones, leading to better shoot
and root growth (Ziv, 1989; Smith and Spomer, 1994;
Sandal et al., 2001; Mehrotra et al., 2007). To reduce
the intensive labor requirement along with the
production cost during plant propagation by tissue
culture technique, there is an immense need of
developing scale-up systems and automation (Aitkin-
Christie, 1991; Paek et al., 2005). Progress in tissue
culture automation will depend upon the use of liquid
cultures in bioreactors (Sakamoto et al., 1995). The
major advantage of using bioreactor culture system
for micropropagation of economically important plants
The Journal of Plant Science Research
is the potential for scaling-up in lesser time limit,
reduction in the production cost as well as an
automated control of physical and chemical
environment during growth phase of the plant cultures.
CONCLUSION
Sophisticated and costly greenhouses can be
substituted by cost-effective techniques for hardening
in vitro derived plantlets. For this, it is necessary to
understand the changes that tissue-cultured plantlets
go through during the transfer from in vitro to in vivo
conditions.
The aim of this review is to report scientific
investigations that have been made during the recent
past on standardization of low cost tissue culture. The
progresses obtained on the knowledge of this in vitro
technology are very impressive and mainly achieved
due to concentrated efforts of researchers. The
knowledge extends from culture initiation to field
transfer of in vitro raised plants. These scientific and
technological achievements are scattered among
science media. We gathered the information scattered
world wide in a sort of summary of what has been
achieved on low cost tissue culture. A compilation is a
best way to faithfully transmit the view of experts. It is
clear from the review that cost of tissue culture plants
can be reduced appreciably by wisely and intelligently
substituting costly ingredients with low cost ingredients.
Protocols standardized and reported above are not
expected to be accepted for any crop for various
reasons. Agarose is also cost prohibitive for many
operators. Starch has poor gelling ability as well as a
metabolizable nature that can result in softening of
media. While, though isubgol has the potentiality for
good gelling agent due to its polysaccharide nature, gel
clarity and resistance to enzymatic activity, but due to
its high melting point (~70 oC) it needs pH adjustment
and rapid dispensing after autoclaving (Babbar and Jain,
2006). One has to be determined from beginning to
standardize the cost effective protocol for the desired
material. It requires only confidence and trial. It is
possible. This compilation will be a good source of
inspiration not only to the scientists and students working
in this area, but also to the industries and other end
users involved in the production and commercialization
of tissue culture technology.

ACKNOWLEDGEMENTS
Working facilities and financial support provided by
CSIR, ICAR and DBT are sincerely acknowledged.
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Received: 09-06-2017

Accepted: 12-06-2017

The Journal of Plant Science Research

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