Plant tissue culture is the aseptic growth of cells, tissues, organs and whole
plants on artificial or definable media. The advantages of plant tissue culture
are enormous and strategic in plant biotechnology (Okonko
et al., 2006).
Tissue culture techniques have become an attractive field of biotechnological research and their roles are particularly appreciated in the areas of large scale clonal propagation and crop improvement. The success of tissue culture among other things relies on shoot regeneration and its efficiency requires a better understanding of the influence of culture conditions on shoot regeneration and development. Plant cells and tissues in vitro culture do not have autotrophic
ability and so require exogenous carbon source for their energy needs. Sucrose
has been the main source of carbon for such in vitro culture of plants
(Ahmad et al., 2007; Hilae
and Te-Chato, 2005). The optimal concentration of sucrose in a medium should
be enough to satisfy the energy needs for cell division and differentiation
without having any negative osmotic effect on shoot formation (Javed
and Ikram, 2008).
The in vitro culture of plants is a technique that has caught up in
Ghana with crops like cassava, coconut, plantains/bananas, yam, potatoes, pineapple
and many more now being multiplied in vitro as a way of producing large
quantities of healthy planting materials. Although in vitro multiplication
of plants started some 40 years ago (Bud, 1993) and is
promising, extensive use of this technology is limited by the high cost of media
components. Media chemicals account for less than 15% while the carbon sources
such as laboratory grade sucrose contributes about 34% of the production cost
(Demo et al., 2008).
For most of the developing countries like Ghana to benefit from the direct
use of tissue cultured material, the cost of commercial micro propagation has
to be drastically reduced without compromising the quality of micro propagules
(Demo et al., 2008). This can be done through
identifying cheaper alternatives to some of the expensive media components.
Sugarcane has been identified as a plant that has high amount of sucrose and
other sugars (Walford, 1996) which could possibly be
used as an alternative source of carbon. Prakash et al.
(2002) and Alkhateeb (2006, 2008)
have used date palm syrup as a carbon source for the in vitro culture
of date palm.
Sugarcane grows abundantly in Ghana in seven out of the ten regions of the country. The canes are consumed raw for its juice and the bulk is used in the preparation of local gin. The price of the raw sugarcane is very cheap compared to ordinary sugar, therefore if sugarcane juice can be used as an alternative carbon source for the in vitro culture of plants, it will reduce the cost of commercial tissue culture. This study therefore seeks to investigate the possibility of using sugarcane juice as an alternative carbon source for the in vitro culture of plantains and bananas.
MATERIALS AND METHODS
Plant materials and explant preparation: The study was conducted at the tissue culture laboratory of the Plant Genetic Resource and Research Institute Bunso, Ghana from November 2009 to June 2010. Two plantain cultivars Oniaba, Apantu (Musa paradisiaca) and one Dwarf Cavendish banana cultivar (Musa accuminata) were used as the plant materials.
Plant materials were taken from sword suckers of the three cultivars of Musa sp. that had been grown in the open field at Bunso in the Eastern Region of Ghana, under good watering regime. The materials were taken early in the morning by using earth chisel to separate the sucker from the parent at the point of attachment. Before washing with running tap water, the roots and the top of the shoots were trimmed off.
The sheaths that form the pseudostem were carefully removed to reduce the size
of the material to about 4 leaf sheaths. They were then sterilized with 70%
ethanol for 3 min and washed three times in sterilized distilled water (Buah
et al., 2000).
More leaf sheaths were then removed aseptically in a clean bench until about two leaves covered the shoot meristem. This process was followed by sterilization with 1% Sodium hypochlorite solution containing a drop of polyoxyethylenesorbitanmonolaurate (Tween 20) for five minutes with occasional shaking and there after washed three times with sterilized distilled water. Prior to their inoculation on the medium, each shoot tip (about 1cm) was longitudinally divided into two halves and again sterilized with 1% Sodium hypochlorite (NaClo) as above for 1 min.
Preparation of sugarcane juice: Sugarcanes were obtained from a farmers
field early in the morning. They were cut into pieces and washed thoroughly
under running tap for several minutes. The canes were then peeled and further
cut into smaller pieces and washed again. They were then placed in a previously
cleaned and dried sugarcane crusher to extract the juice. The phenol-sulphuric
acid assay spectrophotometer was used to check the sucrose content of the juice
(Masuko et al., 2005).
Media composition: MS medium (Murashige and Skoog,
1962) supplemented with 4.5 mg L-1 6-Benzylaminopurine was used.
Three different media were prepared according to their carbon source and each
treatment was replicated 10 times for each of the three Musa sp. used.
The culture bottles were arranged in a completely randomized fashion. As 10%
sugarcane juice, 5% sugarcane juice, 30 g L-1 sucrose and No carbon
source added (control).
Thirty grams per liter sucrose was used because it had been the optimal sucrose
concentration from previous work with Musa species (Buah
et al., 2000).
pH of the media used was adjusted at 5.8 and 0.9 g L-1 gerlite was then added to the media before autoclaving for 15 min at 121°C.
The explants were inoculated into the media and kept under a temperature of 26°C, 16 h photoperiod with an intensity of 3000 Lux and a relative humidity of 60%. The initial sub culturing was done 4 weeks after placing the explants in the media and subsequently at two weeks interval. During subculturing, materials with multiple shoots were separately removed and placed into different vessels. In all, seven subcultures were done during which data were taken.
The isopiestic psychrometer (Tang et al., 2002)
was used in measuring the water potential of the various media before inoculating
them with the explants. Prior to the measurements, the jointed parts of the
psychrometer were made airtight by applying vaseline to prevent the escape of
water vapor from the samples. The thermocouples were then cleaned with 90% ethanol
and rinsed in sterilized distilled water before drying with an air stream. About
0.4 g of each of the media samples were placed one at a time in the chamber
and covered with the cylinder containing the thermocouple. A drop of sucrose
at a designated concentration was placed at the tip of the thermocouple before
inserting it into the cylinder that covers the chamber.
Data were taken from the first subculture on Number of shoots, Fresh weight (g), Shoot height (cm), Shoot dry weight (g) and Media water potential (Mpa).
Data was analyzed with Genstat version 7.1 for analysis of variance and Excel
2007 for the plotting of graphs (Hilbe, 2007).
The various sources of carbon used in this experiment affected some physical properties (water potential) of the medium as well as the growth of the explants. Sugarcane juice was found to be a good alternative to the laboratory sucrose and was evidenced in almost all the parameters measured.
The explants were taken through seven cycles of sub culturing from the 4th
to the 16th week after inoculation. Generally, explants of the three Musa
sp. cultured on medium supplemented with 5% sugarcane juice had higher fresh
weight values than all the other treatments (Fig. 1a-c).
On the average, all the three types cultivars of Musa sp. obtained a
fresh weight of 16 g per plant at the end of sixteen weeks, on medium supplemented
with 5% sugarcane juice. This was followed by plants cultured on medium supplemented
with 30 g L-1 sucrose which had fresh weight of 15 g per plant.
||Fresh weight g/plant of Musa sp. (a) Apantu, (b) Oniaba
and (c) Dwarf cavendish cultured for 16 weeks on media supplemented with
different carbon sources. SCJ: Sugarcane juice NCS: No carbon source
These were better than those cultured on the 10% sugarcane juice which had
average fresh weight of 8 g/plant. Plants that were cultured on the medium without
a carbon source had the least fresh weight values of between 6.1 and 6.4 g per
plant for all the Musa sp.
Oniaba had a slightly higher fresh weight 15.6 and 16.6 g on the 30 g L-1
sucrose and the 5% sugarcane juice respectively (Fig. 1b)
compared with Apantu and Dwarf cavendish (Fig. 1a, c).
Even though the shoot height of plants cultured on media supplemented with
the different carbon sources increased throughout the experimental period, there
were clear differences among the various types of carbon treatments (Fig.
Similar to the pattern found with the fresh weight, plants cultured on 5% sugarcane
juice and 30 g L-1 sucrose grew taller than those cultured on 10%
sugarcane juice and the control medium. Plants cultured on 5% sugarcane juice
had higher values of 17.9, 16.9 and 14.6 cm for Apantu, Oniaba and Dwarf Cavendish,
respectively (Fig. 2a-c) compared with 17,
16.0 and 14.2 cm for
||Shoot height (cm) of Musa sp. (a) Apantu, (b) Oniaba
and (c) Dwarf cavendish cultured for 16 weeks on media with different carbon
sources. SCJ: Sugarcane juice NCS: No carbon source
Apantu, Oniaba and Dwarf Cavendish that were cultured on 30 g L-1 sucrose. Among the three Musa sp., Apantu and Oniaba grew slightly taller than the dwarf Cavendish on media supplemented with 5% sugarcane juice and 30 g L-1 sucrose.
Media supplemented with 10% sugarcane juice and the control gave the least shoot height among the various sugar concentrations. However, plants on 10% sugarcane juice had higher values of about 11 cm compared to plants on the control medium which had values of about 9.5 cm. It was observed that plants cultured on 10% sugarcane juice grew taller initially than the other treatments and this however declined after the second sub culture.
All the three Musa sp. produced more shoots on medium supplemented with 5% sugarcane juice than those on the other media. The highest number of shoots per explant was recorded with Oniaba which produced 20.72 shoot per explant, followed by Dwarf Cavendish and Apantu with 20.10 and 15.53 shoot per explant respectively as shown in Table 1. The number of shoots produced by Oniaba (20.72) and Dwarf Cavendish (20.10) on medium supplemented with 5% sugarcane juice were statistically different from those produced by Apantu (Table 1). Also, the number of shoots produced by the three Musa sp. on 5% sugarcane juice were different statistically, from the other media treatments.
|| Effect of carbon source on mean number of shoots for three
cultivars of Musa sp. cultured for 16 weeks
|| Effect of carbon source on mean dry weight for three cultivars
of Musa sp. cultured for 16 weeks
|| Water potential of media supplemented with different carbon
The dry weight of plants cultured on 5% sugarcane juice was higher than those obtained from the three other media. Oniaba had the highest dry weight of 7.25 g per plant compared with 6.72 per plant and 6.07 g per plant for Cavendish and Apantu, respectively (Table 2). The dry weight values for plant cultured on 5% sugarcane juice were statistically different from the other treatments (Table 2). Five percent sugarcane juice was followed in terms of shoot dry weight by the 30 g L-1 sucrose which had dry weight values of 4.10, 4.00 and 5.82 g per plant for Apantu, Dwarf Cavendis and Oniaba, respectively and these were also statistically higher than the values obtained for 10% sugarcane juice and the control treatments.
The water potential of the culture medium is very important for the growth
of plants in vitro. The various carbon sources and their concentrations
in this study, affected the water potential of the media (Fig.
3). Medium supplemented with 10% sugarcane juice had the lowest water potential
of -0.8 MPa. The highest water potential of -0.1 MPa was obtained from the control
medium whilst the 5% sugarcane juice and 30 g L-1 sucrose all had
water potential values of -0.3 MPa.
The type of sugar and their concentrations in a culture medium affect the growth
of plants in vitro. The plant materials that were cultured on the different
carbon sources and their concentrations exhibited differences in their growth.
This observation has also been made by Sul and Korban (1998)
who reported that the type of carbon source affects the in vitro growth
of plants and this depends on the plant and the part of the plant that is used
as the explant.
Generally, medium supplemented with 5% sugarcane juice and 30 g L-1
sucrose produced plants with higher fresh weight compared to the 10% sugarcane
juice and the medium without a carbon source. The superiority of sugarcane juice
as reported in our work is also corroborated by Sul and
Korban (1998) who found sugarcane juice superior to laboratory grade sucrose.
Sugarcane juice has other reducing sugars apart from sucrose, known to speed
up cell division thus leading to an increase in the volume and weight of tissues.
Moreover, the sugars in sugarcane juice are better translocated and assimilated
than pure sucrose. The juice also contains other elements like iron, phosphorus,
potassium and sodium compared to the scant traces in laboratory sucrose. Such
additional nutrients may further boost growth of in vitro plantlets (Demo
et al., 2008).
Kodym and Zapata-Anas (2001) also confirmed this by
comparing table sugar, sugar beet and sugarcane juice as carbon substitutes
for the in vitro culture of Grand Naine banana and found sugarcane juice
and table sugar to be superior.
On the contrary however, other researchers have found laboratory grade sucrose
as the best carbon source for the in vitro culture of plants (Bahmani
et al., 2009; Esan et al., 2009).
Similarly, Rahman et al. (2004) have also found
30 g L-1 laboratory grade sucrose as the best carbon source for the
in vitro shoot growth and development. The difference between our work
and theirs however, are the plant materials. Whilst Musa sp. were used
in this study, Arariae lata was used in the case of Rahman
et al. (2004). Their cultures were also placed under different culture
conditions and this could explain the differences in our findings with sucrose
and theirs. Gawad et al. (2010) and Shatnawi
et al. (2006) have also reported the superiority of fructose over
laboratory grade sucrose for the in vitro growth of banana plants.
It is believed that the addition of sugar to the culture medium promotes plant
growth in vitro and compensate for the low or negative net photosynthetic
rate as a result of poor photosynthetic ability, thus increasing the survival
rates of tissue sections cultured in vitro (Demo
et al., 2008; Kubota, 2002).
The differences in the suitability of the various carbon sources as reported by the various researchers is indicative of the fact that one carbon source may not be suitable for the in vitro culture of all plants but rather the type of plant and the culture conditions may play a role.
The pattern of fresh and dry weights of plants cultured on the various sources of carbon were similar since they all decreased with increasing concentration of sugarcane juice from 5% to 10%.
Similar observation have been made by Asemota et al.
(2007) and Bahmani et al. (2009) who reported
a decrease in fresh weight of date palm on high concentrations of sucrose and
date palm syrup. The detrimental effects of high levels of sugars in culture
medium on the development of in vitro plants have also been confirmed
by Hilae and Te-Chato (2005) and Yildzil
et al. (2007). It is possible that the accumulation of phenolic compounds
in the medium had inhibitory osmotic effect which adversely affected shoot growth
and development as a result of stress (Karim et al.
On the other hand however, Baksha et al. (2003)
and Shatnawi et al. (2006) have obtained better
shoot formation and higher fresh weights in Lilium species that were
cultured on higher concentration of sucrose.
Plants which were cultured on our control medium also had low fresh and dry
weight values which could be attributed to the unavailability of sufficient
energy to carry out the metabolic processes in the no sugar culture environment.
In other cases, shoot have failed to form on medium that has no added sugar
as was observed by Yildzil et al. (2007).
The water potential of a medium is very important to the growth and development
of plants in vitro since it determines the movement of water and mineral
elements into the plant material and also maintains a better turgor for the
plant cells. One of the factors that affects the water potential of media is
the type and concentration of sugar used (Buah et al.,
2000). Water potential of the medium without sugar was the highest -0.1
MPa whilst the medium with the 10% sugarcane juice had the least water potential
of, -0.8 MPa. This difference could only be attributed to the concentrations
and types of carbon source since they were the only variables. In previous work
by Buah et al. (2000), medium supplemented with
30 g L-1 sucrose had a water potential of -.03 MPa and the value
obtained in this work confirms this. It is possible that the differences in
the growth of the Musa sp. on the various media could be due to the effect
these sugar sources and concentrations had on the media water potentials.
Daozhi et al. (2005) and Tang
et al. (2002) have reported a decrease in leaf conductance in various
plants due to decreased leaf water potential which consequently led to a decrease
in the growth of the plant in vitro. Similarly, Tang
et al. (2002) have reported a decrease in the fresh and dry weight
of rose when leaf water potential decreased from -0.5 to -0.8 MPa. This was
attributed to decreased photosynthetic activity which may be the result of decreased
leaf conductance. This phenomenon may explain the better growth of plants cultured
on 5% sugarcane juice and 30 g L-1 sucrose all of which had a medium
water potential of -0.3 MPa.
Even though cost analysis was not done, it is believed that the replacement
of laboratory grade sucrose with sugarcane juice could have a positive effect
in reducing the cost of in vitro rapid multiplication of plantains and
bananas. Preeti et al. (2009) successfully reduced
the cost of banana tissue culture by 90% by replacing the tissue culture grade
sucrose with table sugar.
It has been demonstrated that sugarcane juice as a carbon source for the in vitro culture of plantains and bananas, is comparable or better than laboratory grade sucrose in terms of the growth of the plantlets in vitro. Plants which were cultured on 5% sugarcane juice gave higher fresh and dry weights as well as plant heights compared to those cultured on 30 g L-1 laboratory grade sucrose. Therefore the use of sugarcane juice in place of laboratory grade sucrose could reduce the cost of in vitro multiplication of Musa sp. without compromising on the quality of growth. Comparing the two concentrations of sugarcane juice used (5 and 10%), the 5% was optimal for the growth of Musa sp. in vitro.
Many thanks to the Biochemistry laboratory of the University of Cape Coast for making their laboratory instruments available for the analysis of sugarcane juice used in this work. Also the use of the laboratory of the Plant Genetic Resources and Research Institute of Ghana is much appreciated. Finally, we thank Dr. (Mrs) Van der Puije who took pains to edit this study.