An Efficient In vitro Hardening Technique of Tissue Culture Raised Plants
Micropropagation has been extensively used for the rapid multiplication of many plants species. However, its wider use is restricted often by the high percentage of plant loss or damaged when transferred to ex vitro condition. To acclimatize the micropropagated plants, different workers have employed different approach toward successful establishment of in vitro raised plants under ex vitro condition. In the present study, a successful attempt has been made to acclimatize the tissue culture raised plants which is cost effective compare to other existing hardening technique. Tissue culture raised orchid seedlings were acclimatized and hardened in vitro by using 1/10th liquid MS basal medium subsequently replaced by tap water with chips of charcoal, bricks and decayed wood as an alternate substratum. The newly formed roots under this condition firmly attached to the charcoal chips with the passage of time, similar to velamenous root attach to the trunk of a tree. Charcoal and moss substratum was more suitable than bricks for epiphytic species and decayed woods for terrestrial. The researchers report the novel and efficient one step hardening technique for tissue culture raised orchid seedlings which will reduce the production cost.
Micropropagation has been extensively used for the rapid multiplication of
many plants species. However, its wider use is restricted often by the high
percentage of plant loss or damaged when transferred to ex vitro condition.
This is due to regenerates has to adjust to many abnormalities in ex vitro
environment like high level of irradiance, low humidity and water is limiting
due to low hydraulic conductivity of roots and root-stem connections (Fila
et al., 1998). Acclimatization of regenerates will overcome this
threat with gradual lowering in air humidity (Bolar et
al., 1998; Lavanya et al., 2009). These
days many acclimatization units have been developed with temperature, humidity,
irradiance, CO2 concentration and air flow controlled by computer
(Hayashi et al., 1988; Pospíšilová
et al., 1999), but this comes at a price.
The ultimate success of in vitro propagation lies in the successful
establishment of plants in the soil (Saxena and Dhawan, 1999).
To acclimatize the micropropagated plants, different worker have employed different
approach toward successful establishment of in vitro raised plants under
ex vitro condition. The concentration of sucrose and agar in the medium
is also said to have an effect on subsequent acclimatization to ex vitro
conditions (Genoud-Gourichon and Sallanon, 1996; Hazarika
2003; Synkova, 1997; Lavanya
et al., 2009). A major part of the production cost; labour cost,
rooting and acclimatization of plantlets accounts approximately 60% (Hazarika
2003), therefore an efficient acclimation technique is necessary for in
vitro raised plants. So, in quest of a new efficient and cost effective
technique of in vitro hardening (one step process), the authors demonstrate
the effectiveness and viability of the new technique on three different orchid
species (Arachnis labrosa (Lindl. ex Paxt.) Reichb.f., Cleisostoma
racemiferum Lindl. and Malaxis khasiana Soland ex. Swartz) and its
advantages over the conventional technique. This novel technique of one step
hardening of orchid was successfully employed and established with high degree
MATERIALS AND METHODS
The present study was carried out in the Department of Botany, Nagaland University, Nagaland, India during April 2002 to December 2008. The present investigation was carried out with three different orchids (two epiphytic monopodial orchids viz. Arachnis labrosa (Lindl. ex Paxt.) and Cleisostoma racemiferum (Lindl.) Garay and one terrestrial orchid viz., Malaxis khasiana Soland ex. Swartz.
The plantlets (size: ~3-4 cm long with 3-4 roots) were raised from different
explants sources of all the three species following the protocols given by Deb
and Temjensangba (2005, 2006a, b)
and Temjensangba and Deb (2005a-c,
2006). The fully differentiated plantlets with 3-4 roots
from the axenic culture condition were selected for the present investigation.
The plantlets were maintained in a highly reduced level of MS (Murashige
and Skoog, 1962) salt solution (1/10th strength) devoid of any plant growth
regulators and sucrose or any other organic carbon sources. In the culture vials
different types of matrix/substratum were used as an alternative substratum.
Alternatively plantlets were also maintained without MS salt solution i.e.,
with only water. The different matrix/substrate like charcoal pieces (~5-7 mm
size), small brick chips (~5-7 mm size) and mosses were used for the epiphytic
orchids whereas, decayed wood/chopped forest litter was used along with charcoal
pieces, brick chips and moss for terrestrial orchid. The matrix was either used
singly or in combination (in 1:1 ratio). The substratums were chopped and air
dried before use. In the culture vials different substratum were added and mixed
with MS salt solution/water [~8-10 mL in each test tube (25x150 mm size) and
~20-25 mL in each conical flask (250 mL size)] and cotton-plugged before autoclaving
at 121°C of 1.05 kg cm-2 for 30 min. In another set of the experiment
the substratum were incorporated without autoclaving. The rooted plantlets from
the axenic sources were taken out washed off the traces of agar using thin brush
followed by the rinsing in luke-warm water. The plantlets were then inoculated
in the culture vials and in each culture vials two plantlets were cultured.
The cultures were maintained at 24±1°C and under normal laboratory
light condition (40 μmol/m2/sec) provided with fluorescent tube
and 12/12 h (light/dark) photoperiod. Initially the cultures are maintained
for about one month and culture vials are plugged and thereafter the cultures
vials were watered wherever culture vials were dried up and culture vials caps
were kept open. The cultures were maintained for about three months to acclimatize
and hardened the plants. A part from the above some cultures were also maintained
on reduced MS medium containing sucrose (2%) (w/v) accompanying no plant growth
regulators and maintained in the normal laboratory condition as practiced in
The hardened plants from all the three species were transplanted to Community Potting Mix (CMP) (charcoal pieces: brick chips: moss at 1:1:1 ratio for epiphytes and charcoal pieces: brick chips: moss: forest litter at 1:1:1:1 ratio for terrestrial orchids) along with the content of the culture vials. The potted plants were maintained in the poly house as well as directly in the natural condition. Initially the transplanted plants were watered at regular interval. The plantlets were transplanted at different seasons of the year both in the natural condition as well as in the poly house. A part of the hardened plants were maintained in the culture vials for last three year and are watered at regular interval. The experimental design was completely randomized all the experiments were repeated at least thrice.
RESULTS AND DISCUSSION
The hardening of in vitro raised plantlets is essential for better survival
and successful establishment. Direct transfer of tissue culture raised plants
to field/wild is not possible due to high rate of mortality, as the regenerates
in the culture condition has been cosseted environment with a very high humidity,
varied light and temperature condition and being protected from the attack of
microbial and other agents. Direct transfer to sunlight also causes charring
of leaves and wilting of the plants (Hiren et al.,
2004; Lavanya et al., 2009). In other words
the survival percentage is determined by the hardening of the plantlets. It
is therefore, necessary to accustom the plants to a drier or natural atmosphere
by a process called acclimatization or hardening.
In the present investigation, the plantlets were raised from different explants
like immature embryos, foliar explants and aerial roots following the protocol
given by Deb and Temjensangba (2005, 2006a,
b), Temjensangba and Deb (2005a-c,
2006). The fully differentiated well rooted plantlets
from the regeneration media were used for the present study (Fig.
1a-c). Among the different types and combination of substratum
used for hardening, a combination of charcoal pieces and mosses (at 1:1 ratio)
was found superior for epiphytic orchids while for terrestrial a mixture of
moss and decayed wood/forest litter (1:1 ratio) was preferred (Fig.
1d-f). Similarly, different matrix or substrates with
manipulation in salt solution were employed for hardening of in vitro
raised plants by various workers viz.: soilrite for Catica papaya (Agnihotri
et al., 2004), soaked cotton for Saccharum offinarum (Gill
et al., 2004), etc. However, no report on the use of other substrate
was recorded as studied in present investigation.
In present study, initially MS salt solution was used as nutrient source for one month thereafter MS salt solution was replaced by normal tap water.
Two significant observations were made during the hardening processes which are as follows:
the culture vials were caped/cotton plugged but at later stages the culture
vials were left unplugged. During this period the cultures were very healthy
and in some cases multiple shoot buds and roots developed (Fig.
1g). The roots were adhered to the substratum. It was observed that
roots gets adhered to the substratum (Fig. 1h, i)
Regenerated plantlets from the regeneration medium used for in vitro
hardening. a: Arachnis labrosa, b: Cleisostoma racemiferum
and cL Malaxis khasiana, (d-f) The plantlets established on
in vitro hardening medium. d: A. labrosa, e: C. racemiferum
and f: M. khasiana, g: M. khasiana plantlets under in
vitro hardening showing formation of secondary shoot buds and roots
(Cultures do undergo dehiscence under in vitro condition and resume
normal growth after the dehiscence period), (h- i) The roots of the fully
established plantlets in the in vitro hardening condition are adhering
to the substratum in the culture vessels (Line drawings), h: The roots
of epiphytic orchids are adhering to the charcoal chips and i: The secondary
roots of the terrestrial orchid established in the chopped forest litters
well dried substratum could also be used with equal efficacy compare to
During the process of hardening the regenerates gets acclimatized for its subsequent
transfer to soil or CPM. The cultures were maintained under normal laboratory
temperature and photoperiod for about three months. The hardened plants were
transferred to the potting mix and maintained in the poly house as well as directly
in the wildand watered regularly at initial phase.
survival frequency of transplanted regenerates of A. labrosa, C.
racemiferum and M. khasiana following two different hardening
techniques with control
The hardened plantlets of both epiphytic and terrestrial orchids from new technique
showed about 95% survival under poly house condition. However, the plantlets
hardened through conventional technique exhibited high rate of mortality on
their transfer to CPM. The survival frequency was as low as 40, 45 and 40% under
conventional technique while, in control it was 13, 15 and 16%, respectively.
And transplants from new technique registered about 80 and 85% (epiphytic and
terrestrial, respectively) survival in natural condition after three months
of transfer (Table 1). A similar response was recorded in
Phoenix dactylifera, where transplants exhibited more than 50% mortality
rate after the 1st week transfer but survival rate increased considerably up
to 92%, when plantlets were passed through pre-acclimation phase (Bhargava
et al., 2003). On the other hand, the survival frequency recorded
a remarkable increase from the plantlets hardened through the new protocol developed
in our study. Agnihotri et al., (2004) also reported
80% transplant success of plants hardened in soilrite.
The newly potted plants were watered near the root systems i.e., avoiding the
leaf with 1/10th MS salt solution at one wk interval, which proves to be beneficial
for better growth. In Rubia cordifolia, the potted plants were reported
to have irrigated with 1/2 strength MS medium without vitamins and sucrose thrice
a week for 15 days for 30 days (Shrotri and Mukundan, 2004).
Feeding the plantlets with nutrient salt solution has been reported to be beneficial
for the promotion of orchid survival and growth (Mukherjee,
1983; Kumaria and Tandon, 1994).
The new one step hardening technique proves to be highly cost effective and efficient alternative to conventional hardening technique for acclimatization of in vitro raised plants. Significantly, the survival frequency of the new technique is considerably higher after transplantation in comparison to the conventional technique. This technique of in vitro hardening will reduce the production cost of tissue culture technique. The technique may also be used for other non-orchid groups with partial modification and works are in progress in our laboratory.
Agnihotri, S., S.K. Singh, M. Jain, M. Sharma, A.K. Sharma and H.C. Chaturvedi, 2004. In vitro cloning of female and male Carica papaya through tips of shoots and inflorescences. Indian J. Biotechnol., 3: 235-240.
Direct Link |
Bhargava, S.C., S.N. Saxena and R. Sharma, 2003. In vitro multiplication of Phoenix dactylifera (L.). J. Plant Biochem. Biotechnol., 12: 43-47.
Direct Link |
Bolar, J.P., J.L. Norelli, H.S. Aldwinckle and V. Hanke, 1998. An efficient method for rooting and acclimation of micropropagated apple cultivars. HortScience, 37: 1251-1252.
Direct Link |
Deb, C.R. and Temjensangba, 2005. In vitro regenerative competence of Cleisostoma racimeferum (Orchidaceae) aerial roots. J. Plant Biochem. Biotechnol., 14: 35-38.
Direct Link |
Deb, C.R. and Temjensangba, 2006. On the regeneration potential of Arachnis labrosa (Lindl. ex Paxt.) root segments: A study in vitro. Phytomorphology, 56: 79-83.
Deb, C.R. and Temjensangba, 2006. In vitro propagation and mass multiplication of Malaxis khasiana Soland ex. Swartz through seed germination. Indian J. Exp. Biol., 44: 762-766.
Direct Link |
Fila, G., J. Ghashghaie and G. Cornic, 1998. Photosynthesis, leaf conductance and water relations of in vitro cultured grapevine rootstock in relation to acclimatization. Physiol. Plant, 102: 411-418.
Genoud-Gourichon, C. and H. Sallanon, 1996. A Effect of sucrose, agar, irradiance and CO2 concentration during rooting phase on the acclimation of Rosa hybrid plantlets to ex vitro conditions. Photosynthetica, 32: 263-270.
Gill, N.K., R. Gill and S.S. Goshal, 2004. Factors enhancing somatic embryogenesis and plant regeneration in sugarcane (Saccharum offinarum L.). Indian J. Biotechnol., 3: 119-123.
Hayashi, M., M. Nakayama and T. Kozai, 1988. An application of the acclimatization unit for growth of carnation explants and for rooting and acclimatization of the plantlets. Acta. Hortic., 230: 189-194.
Direct Link |
Hazarika, B.N., 2003. Acclimatization of tissue cultured plants. Curr. Sci., 85: 1704-1712.
Direct Link |
Hiren, A.P., R.M. Saurabh and R.B. Subramanian, 2004. In vitro regeneration in Curculigo orchioides Gaertn. An endangered medicinal herb. Phytomorphology, 54: 85-95.
Kumaria, S. and P. Tandon, 1994. Clonal Propagation and Establishment of Dendrobium fimbriatum var. In: Advances in Plant Cell Tissue Culture in India, Oculatum, H.K.F. and P. Tandon (Eds.). Pragati Prakashan, India, pp: 21-231.
Lavanya, M., B. Venkateshwarlu and B.P. Devi, 2009. Acclimatization of neem microshoots adaptable to semi-sterile conditions. Indian J. Biotechnol., 8: 218-222.
Direct Link |
Mukherjee, S.K., 1983. Orchids. Indian Council of Agricultural Research, New Delhi, pp: 102.
Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
CrossRef | Direct Link |
Pospisilova, J., I. Ticha, P. Kadleaeek, D. Haisel and S. Plzakova, 1999. Acclimatization of micropropagated plants to ex vitro conditions. Biol. Plant, 42: 481-497.
Saxena, S. and B. Dhawan, 1999. Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis. Plant Cell Rep., 18: 438-443.
Shrotri, M. and U. Mukundan, 2004. Thidiazuran induced rapid multiplication of Rubia cordifolia (L.): An important medicinal plant. Phytomorphology, 54: 201-207.
Synkova, H., 1997. Sucrose affects the photosynthetic apparatus and the acclimation of transgenic tobacco to ex vitro culture. Photosynthetica, 33: 403-412.
Direct Link |
Temjensangba and C.R. Deb, 2005. Factors regulating non-symbiotic seed germination of some rare orchids of Nagaland. Nagaland Univ. Res. J., 3: 48-54.
Temjensangba and C.R. Deb, 2005. Regeneration and mass multiplication of Arachnis labrosa (Lindl. ex Paxt.) Reichb: a rare and threatened orchid. Curr. Sci., 88: 1966-1969.
Direct Link |
Temjensangba and C.R. Deb, 2005. Regeneration of plantlets from In vitro raised leaf explants of Cleisostoma racemiferum (Lindl.) Garay. Indian J. Exp. Biol., 43: 377-381.
Temjensangba and C.R. Deb, 2006. Effect of different factors on non-symbiotic seed germination, formation of protocorm-like bodies and plantlet morphology of Cleisostoma racemiferum (Lindl.) Garay. Indian J. Biotech., 5: 223-228.
Direct Link |