Abstract: The study was undertaken to examine banana (Musa sp. cv. Kanthali) floral bud apex as an alternative source material for in vitro propagation because huge number of explants die due to microbial contamination in case of shoot tip explants. Contamination free cultures were established by treating the floral bud explants with 0.1% HgCl2 for 6 min. For reducing phenolic compounds secretion, explants were treated with 0.125% potassium citrate: citrate solution. Compact, white/greenish white callus was formed in different amount at all concentrations of BA+Kn+IAA+15% CW and BA+Kn+IBA+15% CW from terminal floral bud explants after 3 weeks of culture. All were again subcultured at same medium and after another 30-35 days at 2.0 mg L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1 IAA+15% CW some callus showed embryogenic structure.
Introduction
Banana (Musa sp.) is the 4th largest food crop in the world and affects lives of 400 million people (Vuylsteke, 1989). It is a nutritious fruit rich in carbohydrates and a good source of vitamins. Musa sp. cv. Kanthali (genome AAB) is a traditional table banana cultivar of Bangladesh (Chattopadhyay and Hasan, 2000). The plant is only found in the southern part of the country and its population is continuously decreasing due to lack of commercial cultivation. Its production rate is relatively lower but the plant is more salt tolerant and disease resistant than other commercial cultivars.
The development of micropropagation techniques has been a major focus of Musa research during the past two decades and such techniques have now been well established (Banerjee and De Langhe, 1985; Vuylsteke, 1989; Israili et al., 1995). Micropropagation of banana has been achieved using shoot tip (Cronauer and Krikorian, 1984) and from male floral apices (Doreswamy and Shijiram, 1989). Gupta (1986) did meristem culture for clonal propagation and virus eradication. There are also reports of somatic embryogenesis and regeneration in liquid medium (Novak et al., 1989). Utilization of whole flowers, buds, ovary sections and inflorescence sections as primary explant source material has been reported (Slabbert et al., 1995; Wildi et al., 1998; Amomarco and Ibanez, 1998; Richwine et al., 1995; Holme and Petersen, 1996). Bunn and Dixon (1997) demonstrated that adventitious shoot formation arises directly from the perianth or external ovary tissues in Blandfordia grandiflora, with this technique proving advantageous for rapid shoot regeneration with minimal explant material. This protocol is also useful for conservation of endangered species as it is a nondestructive method utilizing seasonal structures of the plant therefore allowing for the preservation of the mother plant (Amomarco and Ibanez, 1998). This method of in vitro propagation demonstrates that many hundreds of clones can be obtained successfully from a single inflorescence of date palms (Loutfi and Chlyah, 1998) and regeneration of recalcitrant species in vitro can be overcome (Verdeil et al., 1994).
For in vitro propagation of banana, bacterial contamination is a great problem. Although initially surface sterilization works, later on microbial contamination at the base of the explant is observed within 7 to 15 days after inoculation. Bacterial growth is also observed around the explants in the culture media. Huge number of explants is destroyed in the culture due to endogenous bacteria (Hadiuzzaman et al., 2001). In this aspect, utilization of immature inflorescence tissue as explant material can favors minimal contamination rates compared to other tissues. Another problem of in vitro cultured explants, accompanied by darkening of culture medium has been attributed to phenolic compounds exuded from tissues and accumulating in the culture medium. This process is initiated by browning of the surface of plant tissues due to the oxidation of phenolic compounds resulting in the formation of quinones which are highly reactive and toxic to plant tissue (Zaid, 1987; Salisbury and Ross, 1992; Taji and Williams, 1996). Understanding the processes contributing to the oxidation of phenols and how these can be minimized when initiating e xplants is critical for successful in vitro culture.
However, high productivity in agriculture output has been mainly achieved through breeding programs and genetic manipulation. Therefore, present investigation was carried out to establish in vitro rapid clonal propagation of Musa sp. cv. Kanthali from its floral bud apices.
The aims of the experimental design addresses the following aspects of in vitro development:
| • | That sterilization procedure can be developed which will optimize tissue survival in vitro. |
| • | That antioxidant treatment minimizes phenolic exudation. |
| • | That cytokinin and auxin ratios will facilitate tissue regeneration. |
| • | That certain tissue types will respond to successful regeneration. |
Materials and Methods
Collection and Preparation of Explants
Banana cv. Kanthali (Genome AAB), a Cavendish type leading traditional cultivar
of southern part of Bangladesh was the investigating subject. The source material
used for culture establishment was banana floral bud explants. The plant materials
were collected from a village named Amtola in Batiaghata Thana, Khulna and was
very near from Khulna University campus.
Terminal floral apices of banana were collected from mature plants after they produced all possible hands. The terminal bud was cut from the peduncle and the bracts with their associated hands of male flowers were removed in a stepwise manner until they became too small to remove by hand. Working with a dissecting microscope, scalpel and forceps, the remaining bracts and minute hand of flowers were removed until the rounded growing point was exposed. The floral apex and approximately 1 cm long subtending peduncle tissue were excised.
Disinfection Procedure
The surface sterilization procedure began with dissection of explant material
into manageable units. Stem sections containing axillary buds and immature inflorescences
were treated by initially removing the small leaflets and cleaning away surface
detritus under running tap water for 1 to 2 min. A plastic vessel (130x320x120
mm) was used for treatments with sterilant solution. Sterilization was undertaken
for 6 min using 0.1% (w/v) HgCl2. Explants were transferred to a
separate vessel for the washing phase in three changes of sterile distilled
water.
Techniques Applied for Reducing Phenolic Compound Secretion of Explants
One of the most common problems associated with the in vitro establishment
of many monocotyledonous and woody species is the deleterious effects of oxidized
phenols (Vasil and Thorpe, 1994; Teixeira
et al., 1994; Khatri et al., 1997;
Zweldu and Ludders, 1998). The results of the detection
of phenolic compounds experiment (Table 2-4) clearly indicate
that Musa sp. cv. Kanthali has phenolic compounds present in inflorescence
tissue. This experiment provides a simple technique for detecting the presence
of phenolic compounds in banana inflorescence tissue. This procedure assists
in early detection of phenols (Panaia, 1998) and assists
in preparation of explant source material to reduce injury associated with phenolic
oxidation. George (1996) describes an antioxidant as an
electron donor (reducing agent) which inhibits the oxidation of labile substrates.
The antioxidant compounds utilized in the experimental work in this chapter
were selected because they have been used successfully in the past to delay
browning in other arborescent monocotyledonous species (Drew,
1986; Zaid, 1987; Khatri et
al., 1997). George (1996) details the use of citric
acid and ascorbic acid combinations to delay browning. The successful prevention
of browning in explants of Musa textilis by using a mixture of ascorbic
acid, citric acid and cysteine are reported by Mante and
Tepper (1983). The behavior of the citrate in citric acid works as a chelating
agent bonding to ions responsible for activating polyphenol oxidative enzymes
(George, 1996). Ascorbate behaves as a reducing agent
and is converted to dehydro-ascorbic acid (Panaia, 1998).
Ascorbate is able to scavenge oxygen radicals produced when tissue is damaged
and radicals are attributed to exacerbating oxidative injury. Antioxidants containing
citrate and ascorbate reduce browning of tissue by detoxifying these free radicals.
The results provide evidence that K-C: C combination is a useful antioxidant
for explant preparation for Musa sp. cv. Kanthali.
Musa sp. cv. Kanthali stems are susceptible to tissue browning and elimination or minimization of this process is an essential prerequisite to successful culture establishment. Therefore identification of a suitable treatment to minimize tissue browning in the explant source material of Musa sp. cv. Kanthali will be the focus of this chapter. The specific aim of this component of the study was to:
| • | Research methods for reducing phenolic induced injury in Musa sp. cv. Kanthali during explant preparation with particular emphasis on the use of appropriate antioxidant treatments. |
Antioxidant Treatment
The extracted buds were placed in petri dishes containing an antioxidant
wash of 0.125% potassium citrate: citrate (K-C: C in a ratio of 4:1 w/w) solution.
A concentrated stock of the antioxidant wash was filter sterilized (with 0.22
μM disposable filter) and frozen in 10 mL units until required. The concentrate
was later thawed and further diluted with SDW to give the final 0.125% concentration.
Petri dishes (90x14 mm) were filled with sufficient antioxidant solution to
fully cover the explants. Peduncle sections were cut into discs under the antioxidant
solution to minimize browning during initial preparation. Each explant was placed
in a test tube containing 20 mL of media after five min in the antioxidant treatment.
Growth Responses
After antioxidant treatment the floral bud explants of banana were cultured
on MS solid medium supplemented with cytokinins, auxins and coconut water for
initiating vegetative growth. After 3 weeks of culture compact, white/greenish
white callus was formed more or less at all treatments. All were again subcultured
at same medium and after another 30-35 days at a specific concentration some
callus showed embryogenic structure but others remained unchanged. These were
observed at the conclusion of this research and were not able to be further
analyzed.
Results and Discussion
The experiment was conducted at Plant Biotechnology Laboratory of Khulna University, Bangladesh during January to November 2005. One indigenous banana plant (Musa sp. cv. Kanthali) of Bangladesh was studied in order to establish suitable protocols for in vitro plant regeneration. In the present investigation in vitro growth responses of floral bud apices from mature plants was studied for large scale plant propagation.
For culture initiation all the experimental explants were cultured on MS medium supplemented with different concentrations of cytokinins and auxins for promoting the morphogenic responses. The results of the experiment are described as follows:
To overcome contamination problem surface sterilization of explants was done with 0.1% (w/v) HgCl2 for different durations to assess the contamination percentages and viability of the explants used for in vitro culture.
Contamination free cultures with elegant survivability (100%) were achieved by treating the explants with 0.1% HgCl2 for 6 min (Table 1).
Detection of Phenolic Compounds Using Sodium Hydroxide (Naoh)
Detection of phenolic leakage was tested in the study plant Musa
sp. cv. Kanthali. Flowering stems of Musa sp. cv. Kanthali were collected
from a village named Amtola in Batiaghata Thana, Khulna for assessment. Concentrations
of sodium hydroxide (NaOH) were made at rates 1 , 0.1 , 0.01 and 0.001 M to
treat the various plant tissues. NaOH oxidizes phenols which causes darkening
of affected or damaged tissue (Panaia, 1998). Peduncle
sections and pedicel slices of Musa sp. cv. Kanthali were cut in 2 mm
thick discs and submerged in petri dishes containing the concentrations of NaOH
for 5 min. Similarly, bract tissue was cut into squares approximately 15x15
mm and also placed in the NaOH solutions. Observations were made of any browning
on the surface or cut edges of the various plant tissue to assess if phenolic
leakage had occurred.
Antioxidant Experiment
Pedicel and peduncle disc sections from Musa sp. cv. Kanthali were
collected from a mature flower stem and treated with various antioxidant solutions.
Disc sections were selected as they have a large surface area and have been
shown to be prone to oxidation.
A stock solution of potassium citrate and citrate (K-C: C) was made up using 1 g K-C and 0.25 g citrate and dissolved in 10 mL of SDW. The concentrate was then diluted and used at a final concentration of 0.125%. For treatments 5 and 6 0.02 g L-1 L-cysteine HCl was added to the 0.125% solution of K-C: C and 0.25 g L-1 ascorbic acid was added to treatment 6. One hundred milliliters of the various solutions were used to fully cover the disc sections with the control treatment cut on filter paper and exposed to air. All other material was cut under the various treatments to avoid exposure to the air. The prepared disc sections were placed onto water agar petri dishes and results were recorded at time at intervals of 0, 7, 30, 60 and 120 min. Observations of the extent of browning were recorded (Table 2).
| Table 1: | Standardization of HgCl2 treatment period for surface sterilization of the explants |
| Indicates no contamination, * Indicates explant death due to tissue killing, * = 5-25%; ** = 26-50%; *** = 51-75% and **** = 76-100% | |
| Table 2: | Antioxidant treatments |
| Table 3: | Relative browning of disc sections of Musa sp. cv. Kanthali treated with antioxidants over a 2 h period |
| O: No oxidation, + Low oxidation, ++ Medium oxidation, +++ High oxidation | |
| Table 4: | Degree of tissue discoloration after incubation in NaOH solutions for Musa sp. cv. Kanthali |
| O: No discoloration, + Low discoloration, ++ Medium discoloration, +++ High (darkly stained) | |
| Fig. 1: | Comparison between untreated (A) and treated (B) floral bud apex explants for excessive phenolic compound secretion |
All cut surfaces in the control appeared to oxidize rapidly once exposed to air as evidenced by tissue browning. Subsequently all other tissues were prepared under each of the antioxidant treatments. Pedicel sections showed similar rates of browning to excised flower bud material when exposed to air without antioxidant treatment. T4, T5 and T6 initially reduced browning of the disc sections and after 2 h, T4 (K-C: C) was visually better than the other 2 antioxidant treatments (Table 3 and Fig. 1).
Detection of Phenolic Compounds Using Sodium Hydroxide (Naoh)
Pedicel and peduncle sections produced a large degree of discoloration after
having been sliced into discs and placed in the NaOH. The bract tissue of Musa
sp. cv. Kanthali developed a distinct green/brown line, approximately 2
mm wide around the cut edges (Table 4).
Potassium Citrate-citrate Combinations as Antioxidant Treatments for Excised
Tissue
All tissues initiated into culture were treated with the K-C: C treatment
as it proved to be the best treatment type from experimental results. The antioxidant
treatment reduced browning in all tissue types after 24 h in culture. The cut
surfaces and any damaged areas of untreated tissue (particularly peduncle sections)
turned brown within 15 min after the excision. These explants continued to oxidize
under culture conditions and were completely brown after 1 h and were subsequently
discarded.
After 3 weeks of culture the phenolic leakage in treated tissues was minimized and in most cases controlled. Many of the explants had remained pale while others had started showing signs of greening. Some minor staining of the media was evident in some explant tissues predominantly in peduncle sections. The results from this study indicate the browning phenomenon in Musa sp. cv. Kanthali tissue can be greatly reduced by pre-soaking of explants in antioxidant solution of 0.125% (w/v) potassium citrate and citrate prior to culture. Also, incubation in the dark for the first 1 week may arrest the rate of tissue browning by slowing the enzymatic activity responsible for tissue oxidation. Frequent subculturing to fresh medium may also assist so that toxic phenolic compounds do not hinder the activity of plant growth regulators on tissues. The combined effects of the treatments outlined above proved beneficial to explant survival in vitro of Musa sp. cv. Kanthali inflorescence tissue.
Organ Formation from Floral Bud Explants
Floral bud apices of banana (Musa sp. cv. Kanthali) were isolated
aseptically. Then after successful sterilization and antioxidant treatment they
were cultured on MS medium supplemented with cytokinin, auxin and coconut water
for initiating vegetative growth.
Effect of Different Concentrations and Combinations of BA, Kn, IAA and 15%
CW for Callus Induction
Floral bud explants of banana were cultured on MS solid medium supplemented
with different concentrations and combinations of BA, Kn, IAA and 15% coconut
water for initiating and observation of vegetative growth. After 3 weeks, cultures
showed enlargement of the floral primordial and compact, whitish/greenish white
callus was formed more or less at all treatments. But at MS+2.0 mg L-1
BA +1.0 mg L-1 Kn+1.0 mg L-1 IAA+15% CW and MS+2.0 mg
L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1 IAA+15% CW
excellent degree of callus was formed. And very poor degree of callus was formed
at MS+0.5 mg L-1 BA+0.5 mg L-1 Kn+0.5 mg L-1
IAA+15% CW and MS+1.0 mg L-1 BA+0.5 mg L-1 Kn+0.5 mg L-1
IAA+15% CW.
The floral bud explants of banana were again cultured on MS solid medium supplemented with different concentrations and combinations of BA, Kn, IBA and 15% coconut water for observation of their vegetative growth. After 3 weeks, cultures showed enlargement of the floral primordial and compact, whitish/greenish white callus was formed more or less at all treatments just like previous treatment. But at MS+2.0 mg L-1 BA+0.5 mg L-1 Kn+0.5 mg L-1 IBA+15% CW and MS+2.0 mg L-1 BA+1.0 mg L-1 Kn+1.0 mg L-1 IBA+15% CW excellent degree of callus was formed (Table 5 and Fig. 2).
Subculture for Somatic Embryo like Structure Development
All the callus were again subcultured at same medium and after another 30-35
days at 2.0 mg L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1
IAA+15% CW some callus showed embryogenic structure. The embryogenic callus
showed several developed embryos on the surface. However, conversion of these
embryos into plants did not occur, probably due to a lack of development of
the shoot apical meristem (Fig. 3).
| Table 5: | Effect of different concentrations of BA in combination with Kn, IAA and 15% CW for callus induction from banana floral bud explant. There were 10 explants for each treatment and data were taken after 3 weeks of culture |
| +++ Indicates excellent, ++ Indicates good, + Indicates poor degree of callus | |
| Fig. 2: | Degree of callus formation from banana floral bud explants on MS medium supplement with 2.0 mg L-1 BA+1.0 mg L-1 Kn+1.0 mg L-1 IAA+15% CW (A) and 2.0 mg L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1 IAA+15% CW (B) |
| Fig. 3: | Embryogenic callus formation from floral bud explant of banana on MS+2.0 mg L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1 IAA+15% CW |
Conclusion
This study examined the application of micropropagation protocols to assist germplasm conservation of a traditional cultivar table banana [Musa sp. cv. Kanthali (Genome, AAB)] of Bangladesh. This has implications for commercial explant production in large scale as it generally produce 5-6 suckers from a mature mother plant per year. For in vitro propagation of banana, bacterial contamination is a great problem. So, the aim was to investigate the floral bud apices as an alternative explant source material because it favors minimal contamination rates compared to other tissues. Key factors investigated in this study includes sterilization technique to establish contamination free culture, control of phenolic exudates in explant preparation and the selection of appropriate growth regulator levels to achieve successful in vitro regeneration.
One of the most commonly encountered problems in in vitro culture establishment is the contamination by microbial contaminants. One hundred percent contamination free culture was established by soaking the floral bud explants in 0.1% HgCl2 for 6 min followed by several washes in sterile water obviated the need to develop extensive and complicated surface sterilization protocols.
This study found that inflorescence tissues of experimental plant was high in phenolic compounds. The oxidation of tissues was severe and proved deleterious to all tissues in the initial stages of explant preparation. So, for reduction of phenolic compounds explants were pre-soaked in antioxidant solution of 0.125% (w/v) potassium citrate and citrate. Then they were placed in dark for 1 week so that rate of tissue browning was arrested. The effect of the treatments outlined above proved beneficial to explant survival in vitro of Musa sp. cv. Kanthali inflorescence tissue.
For successful explant establishment, a wide range of cytokinin and auxin combinations were investigated. The floral bud apices were cultured for 3 weeks on MS basal medium supplemented with various concentrations and combinations of cytokinins, auxin and additives. Hard, compact, white/greenish white callus was formed in different amounts at all concentrations of BA+Kn+IAA+15% CW. All were again subcultured at same medium and after another 30-35 days at 2.0 mg L-1 BA+2.0 mg L-1 Kn+2.0 mg L-1 IAA+15% CW some callus showed embryogenic structure. These were observed at the conclusion of this research work and were not able to be further analyzed. So, long time continuous investigation is prescribed to establish full protocol.
Acknowledgments
First of all we would like to express our deepest gratitude for the personnel of Plant Biotechnology Laboratory of Khulna University, Khulna-9208, Bangladesh for their invaluable support and instructions they all gave us during this experiment. We would also like to thanks Khulna University for financing of this research.