Mass propagation of Bambusa bambos (L.) Voss through in vitro culture

Protocol for mass propagation of Bambusa bamboos (L.) Voss was developed through in vitro culture. Nodal segments containing pre-existing axillary bud, after surface sterilization, were inoculated on liquid Murashige and Skoog’s (MS) basal medium containing different concentrations and combinations of cytokinins (BAP, TDZ and Kn). The highest direct shoot induction (90%) was obtained in the MS liquid medium supplemented with 2.0 mg/l BAP and 1.0 mg/l TDZ with maximum average number of shoots (3.14 ± 0.06) per explant. Highest shoot multiplication (16.58 ± 0.24 shoots per culture) with highest average shoot length (9.21 ± 0.13 cm) was obtained when in vitro raised shoots were cultured in gelrite gelled MS medium in conjunction with 2.0 mg/l BAP and 1.0 mg/l TDZ. Incorporation of 10% coconut water with 4% sucrose in the above mentioned medium resulted satisfactory shoot growth and development with an average 26.7 ± 0.60 shoots per culture. For root induction, in vitro raised shoots were divided into clumps of 4-5 shoots in each clump and transferred onto both liquid and gelled half-strength MS medium containing different concentrations and combinations of auxins (IBA and NAA). Maximum rooting (86.67%) was achieved in half-strength of MS medium fortified with 2.5 mg/l IBA and 2.5 mg/l NAA with an average 8.72 ± 0.42 root per shoot. The rooted plantlets were then transferred to polybags containing garden soil, sand and compost mixture with 1:1:1 ratio. After a month the hardened plantlets were then transferred to the larger pots containing garden soil and compost with 1:1 ratio for sufficient growth and finally transplanted to the field. In this process, the highest 100% survivability was recorded from well-established rooted plantlets. The regenerated plants showed well developed root and shoot systems in field condition.


INTRODUCTION
Bamboo is a giant grass which is one of the most fascinating and versatile group of plants known to mankind. These are long-lived, woody, evergreen, members of subfamily Bambusoideae of family Poaceae (Singh et al., 2012a). Most of the bamboos are shrub like, medium or dwarf with a few exceptions as a climber (Liese, 1987). It is the fastest growing plant on the planet with a growth rate of up to 1.2 meters a day. Its roots can reduce soil erosion by up to 75 percent. As bamboos are fast growing plants, recently they are considered as a prime renewable resource for biomass production (Sharma and Sarma, 2011).
There are about 75 genera and over 1250 species of bamboo in the world. Bamboo species occurring in Bangladesh were recorded as 27 in 10 genera in which Bambusa spp. are mostly common (Alam, 1992). Approximately 157 species from the genus Bambusa were documented worldwide, in which the most widely used species is B. bambos (L.) Voss also known as 'Giant Thorny Bamboo' or 'Indian Thorny Bamboo' which is native to Southeast Asian country (Sharma et al., 2014). It prefers humid tropical climate, moist soil and grows along perennial rivers and valleys as well as in other moist sites.
Bambusa bambos is cultivated for building and scaffolding material. It is used as a raw material for pulp and paper industry. The spiny branches are used for fencing. It has been widely used in Indian folk medicine. In Ayurveda, the entire plant is used as astringent, laxative, for inflammatory conditions and as diuretic (Joshi, 2000). It may be used for reforestation and stabilization of eroding banks. It is often planted as wind-breaks and is recommended in agroforestry.
Current methods of bamboo propagation rely on culm cutting, rhizome division, offset cuttings, branch cuttings, air layering or seed (Prutpongse and Gavinlertvatana, 1992). Rhizome cutting and culm cuttings are the most widely used methods of vegetative propagation. There are number of problems with this type of propagation. The offsets used are bulky and heavy. Clumps are not always available due to seasonal specificity, so that large scale vegetative propagation is impossible (Saxena and Bhojwani, 1993). Because of the difficulties with these traditional propagation methods, in vitro methods of propagation (micropropagation) provides an alternative to meet the demand of the market, which allow the rapid multiplication of disease-free and true to type selected clones. In this study, an efficient and reproducible micropropagation protocol has been established mass production and generating uniform clones for the bamboo species, Bambusa bambos (L.) Voss.

MATERIALS AND METHODS
Nodal segments containing unsprouted axillary buds of Bambusa bambos (L.) Voss were collected from the lateral branches of young culm thicket grown in the campus of Jahangirnagar University, Savar, Dhaka. The nodes containing pre-existing axillary buds were used to initiate and establish the in vitro culture. Leaf sheath of nodal segments were removed with sharp blades and nodes containing lateral spines were removed by scraping carefully with scalpel. These nodal explants were thoroughly washed under running tap water to remove the dust particles. They were then washed with few drops of liquid detergent for 15 minutes followed by 3-4 times washing with distilled water. Further surface sterilization of the explants was done under laminar airflow by giving fungicide treatment (Bavistin) for 5-7 minutes followed by 3 times washing with autoclaved distilled water. Finally these explants were surface sterilized with (0.05%, 0.1% and 0.2%) HgCl 2 solution for 3-10 minutes to ensure contamination free culture and then thoroughly washed with autoclaved distilled water.
Then both ends of the sterilized nodal segments were trimmed and cultured singly in liquid MS medium (Murashige and Skoog, 1962) supplemented with various concentrations and combinations of Cytokinins (BAP, TDZ and Kn). The pH of the medium was adjusted to 5.8 with the help of pH meter by adding 0.1N NaOH or 0.1N HCl accordingly prior to autoclaving at 15 lbs pressure and 121°C for 15 mins. Axillary buds of nodal segments were sprouted within 18-25 days. The sprouted buds were then sub-cultured in another bottle containing same gelled media. Within 4-weeks induced shoot elongated and developed into a number of multiple shoots. The elongated cluster (4/5 shoots) of shoots were used as explants for further shoot multiplication and root induction. At the end of each stage, shoot number per explant and shoot length (cm) were calculated.
The effect of different concentrations of source (1-5%) and coconut water (5-20%) on shoot multiplication were also studied. An aseptic condition was maintained throughout the whole operation. Cultures were incubated at 24±2°C temperature under illumination with florescent light for 16-hours light and 8-hours dark per day.
In vitro induced shoots (4/5 shoots) in multiplication medium were then used for root induction. Half-strength of both solid and liquid MS medium with various concentrations and combinations of IBA and NAA were used. After 30-45 days of culture, the number of roots per shoot cluster and rooting percentage was recorded. The plantlets with welldeveloped root system were acclimatized.
For acclimatization the plantlets with sufficient root inside the test tubes were kept in the room temperature for 5-7 days to bring them in contact of normal temperature. The roots of the plantlets were gently washed under running tap water to remove any chemical attached to the root area. Immediately after that they were transferred to small polybags containing the soil mixture garden soil, sand and compost with 1:1:1 ratio. The plants along with pots were covered with transparent polythene bags to prevent sudden desiccation. The inner side of the polythene bags was sprayed with water at every 8 hours to maintain high humidity around the plantlets. The polythene bags were gradually perforated to expose the plantlets to the outer normal environment and subsequently removed after 14 days.

RESULTS AND DISCUSSION
In the present investigation, nodal segments containing pre-existing axillary bud from mature clumps of Bambusa bambos were mainly preferred as explants due to their availability all the year round in sufficient numbers to initiate the in vitro cultures. Similarly Arya and Sharma, (1998);Nayak et al., (2010); Anand et al., (2013) and Brar, (2014) also used nodal segments as explant to initiate the in vitro cultures of B. bambos. Most suitable result on surface sterilization was obtained when nodal segments of B. bambos were surface sterilized with 0.2% HgCl 2 for 7 min which resulted in 92% contamination free explants (Table 1). The present results corroborates with the findings of Kheng and Lee, (2011) in B. ventricosa;Waikhom and Louis, (2014) in B. tulda and Melocanna baccifera; Sharma and Sarma, (2011) in B. balcooa;Jha et al. (2013) in Dendrocalamus hamiltonii; Arya and Arya, (2015) in D. asper, D. hamiltonii and Drepenostachyum falcatum. In the present investigation, for in vitro shoot induction MS medium was used. The use of MS basal medium for micropropagation of B. bambos has been reported in earlier reports (Arya and Sharma, 1998;Nayak et al., 2010;Anand et al., 2013 andBrar, 2014).  (Table 2). Whereas Kabade, (2009) reported that MS liquid medium containing 0.1 mg/l NAA and 0.25 mg/l TDZ was the best for direct shoot induction (100%) with an average 6.53 shoots per explants within 3 weeks in case of B. bambos. TDZ was found to be suitable in some other previous studies on D. strictus (Singh et al., 2000); B. edulis (Lin et al., 2004) and B. oldhamii (Lin et al., 2007). Arya and Sharma, (1998); Anand et al., (2013) and Nayak et al., (2010) investigated that the cytokinins (BAP and Kn) were effective in direct shoot induction of the axillary buds of B. bambos as well as Devi and Sharma, (2009) in Arundinaria callosa, and Chaudhary et al., (2004) in D. strictus. BAP has also been found to be essential for direct shoot induction in bamboo (Nadgir et al., 1984;Banik, 1987 andSaxena, 1990).  In the present study, directly induced shoots were excised from nodal segments after 4 weeks of growth on initiation medium and cultured on MS gelled medium supplemented with different concentrations of BAP alone or in combination with TDZ, Kn and NAA. Among the different cytokinins BAP has been found the best cytokinin for shoot multiplication of B. bambos. The combined effect of BAP with another cytokinin such as TDZ and Kn, has been found to be more effective for axillary shoot multiplication of B. bambos than using BAP singly. In this regard, the effective combined concentration was 2.0 mg/l BAP and 1.0 mg/l TDZ, which produced maximum (16.58±0.24) average shoots per explant as well as maximum average shoot length (9.21±0.13 cm) in B. bambos ( Table 3).

Table 3. Effects of different concentrations and combinations of Cytokinins (BAP, TDZ and Kn) and Auxin (NAA) on shoot multiplication of Bambusa bambos
The shoot multiplication was best on MS medium supplemented with 3.0 mg/l BAP in a previous study on B. bambos conducted by Arya and Sharma, (1998). These results were also supported by the earlier reports on micropropagation of bamboos, where BAP had been used extensively for shoot multiplication (Ramanayake et al., 2006;Yasodha et al., 2008;Ramanayake et al., 2008;Arya et al., 2008;Mudoi and Borthakur, 2009). TDZ has been proved an effective cytokinin for shoot proliferation in B. oldhamii (Lin et al., 2007); B. edulis (Lin and Chang, 1998); D. giganteous and B. vulgaris (Ramanayake et al., 2001 and. Combined effect of BAP, TDZ and NAA in shoot multiplication was also tested in the present study, in which a moderate multiplication rate of axillary shoots of B. bambos was found. There are some reports that addition of auxins (IAA or NAA) has either no effect on multiplication or decreased the multiplication of bamboo (Saxena, 1990;Das and Rout, 1991;Prutpongse and Gavinlertvatana, 1992).
A significant increase in the shoot multiplication rate with more healthy cultures was observed, when 10% coconut water and 4% sucrose was adjuvated with 2.0 mg/l BAP and 1.0 mg/l TDZ, gave a maximum rate of multiplication with an average 26.7±0.60 shoots per culture which were 11.2±0.32 cm long. The effect of coconut water on bamboo shoot multiplication has been reported by different authors. Nadgauda et al. (1990) and Rajapakse, (1992) induced shoot proliferation in the medium supplemented with coconut water in seedling derived culture of B. bambos and D. brandisii and in D. giganteus respectively, for which in vitro flower were also induced. Saxena and Bhojwani, (1993) reported to use 10% coconut milk (CM) as additive for better shoot proliferation in D. giganteus. Ramanayake et al. (2001) reported that a high level of sucrose (4%) adversely affected the shoot multiplication in D. giganteus. 3% sucrose are most commonly used by many workers as originally used by Murashige and Skoog (1962). Nadgir et al. (1984) and Brar, (2014) used 2% sucrose for shoot multiplication in B. bambos as well as Yasodha et al. (2008) in B. nutans;Agnihotri et al. (2009) in D. hamiltonii andNadha et al. (2013) in D. asper.
In vitro rooting was attempted by transferring clusters (4-5) of shoots in a half-strength of both liquid and gelled MS medium fortified with different concentrations and combinations of auxins (IBA and NAA). A significant rooting was obtained within 15-22 days in B. bambos when 2.5 mg/l IBA was added in conjunction with 2.5 mg/l NAA with 86.67% rooting efficiency (Table 4) whereas, Arya and Arya, (2009) achieved 80-85% rooting within 3-4 weeks of subculture on MS + 3.0 mg/l NAA and on MS + 10 mg/l IBA in case of B. bambos. However, 100% rooting was observed by Singh et al. (2012b) in Dendrocalamus asper by using combination of 5 μM of each of the IBA and NAA. The combined use of IBA and NAA for rooting was also reported by Islam and Rahman, (2005); Arya et al. (2006) and Rathore et al. (2009) in many important bamboo species.
In vitro raised plants need to be acclimatized before transferring them from in vitro condition to the field conditions, because they were grown under aseptic conditions with high humidity, diffused light and constant temperature. So, it is necessary to transfer the plants to field through various hardening stages to increase the survival percentage.
Finally the acclimatized plants, after a month, were transferred to the larger pots containing garden soil and compost with 1:1 ratio for sufficient growth and transplanted to the field. This micropropagation technique can be established as a practical method for high frequency plant regeneration of B. bambos at a faster rate than any conventional method of propagation.
The present study provides an efficient and cost effective protocol for in vitro propagation of Bambusa bambos (L.) Voss from the nodal segments of the field grown mature culm, with high multiplication efficiency, proper rooting and easy establishment in the field condition with normal growth performance of the in vitro propagated plants. This protocol will be helpful for large scale production of edible bamboo as well as for gene pool conservation.