Biotechnology of Bamboos

7 Biotechnology of Bamboos

Pooja Thapa, Devinder Kaur, Priyanka Sood, Rupali Mehta, Jasmine Brar, Harleen Naddha, R.K. Ogra, Om Prakash, Amita Bhattacharya and Anil Sood

1. Introduction Bamboos are perennial, evergreen woody monocots that belong to the family Poaceae, subfamily Bambusoideae and tribe Bambuseae. The plant is distributed at latitudes from 46°N to 47°S and at altitudes up to 4,000 m amsl in the subtropical and temperate zones of all continents except Europe (Banik, 1995; McNeely, 1995; Kharlyngdoh and Barik, 2008). There are over 75 genera and 1250-1500 species distributed across the globe (Sharma, 1980; FAO, 2001; Qing et al., 2008). Asian countries such as Nepal, Vietnam, Laos, Thailand, China, India, Bhutan, Bangladesh and Myanmar account for about 1000 species, covering an area of over 180,000 km2. Among these, India and China together contribute more than half of the total bamboo resources of the world. Bamboo is aptly called as the 'Green Gold' of the 21st century in several countries of Asia. The plant is an important structural component of many types of vegetation and plays a major role in ecosystem dynamics (Keeley and Bond, 1999). It has gained importance in social forestry programmes due to its short rotation cycle, fast growth and ease of progressive harvesting on a sustainable basis (Godbole et al., 2002). Bamboo housing is especially popular in earthquake prone areas. Being a good source of fibre, proteins, and minerals with anti-oxidant, anti-ageing, anti-bacterial and anti- viral functions, bamboo shoots constitute an important food crop in the international as well as national markets. The shoots can also be processed into beverages, medicines, pesticides, and other household items like toothpaste, soap, etc. Bamboo is the main food source of the giant pandas in China. Bamboo foliage is reportedly nutritious forage for grazing cattle. It is a low-calorie source of potassium and is used for healing and treating infections. The silicious concretion called Banslochan or Tabashir or Tawashir obtained from the culms of the bamboo stem is used as a tonic for respiratory diseases. Bamboo leaf (Herba Lophatheri) has been widely used in a variety of foods and drugs. This has led to approximately 1,500 commercial 148 Pooja Thapa et al. applications of bamboos including construction and reinforcing fibres, paper, textiles, board, food, combustion, carbon sequestering activity, environment management and other bioenergy applications. The plant is indeed a major player in the economies of many Asian countries (Qiu, 1992; SCBD, 2001; Ghosh et al., 2011). An estimated 2.5 billion people of the world depend on or use bamboos. In India, the present bamboo- dependent economy is worth Rs. 2,043 crore and is expected to reach Rs. 26,000 crore by 2015 (Mahapatra and Shrivastava, 2013). In view of the future uses of bamboos and their increasing role in different economies, it is anticipated that there will be a huge shortage of bamboo planting material in the coming years (Subramanian, 1995). However, there has been rampant dwindling of natural stands due to extensive anthropogenic pressures and over- exploitation of this immensely versatile, natural resource. Thus, there is an increasing need to enhance the productivity and quality of bamboos worldwide.

2. Micro-Propagation In nature, bamboos propagate both vegetatively through rhizomes and sexually through seeds. New sprouts emerge from rhizomes during the rainy season and rapidly grow into tall culms within a year. Plants are also propagated by culm cuttings, branch cuttings, offsets, rhizomes and seedlings. However, these methods suffer from various limitations such as high cost, labour intensiveness, low quality planting material, difficulty in transportation for establishing large scale plantation and low seed viability (Banik, 1987). Vegetative propagation has proved useful for only small scale production of clonal planting material and is of limited value. In contrast, micro-propagation is one of the most effective supplementation to conventional methods of vegetative propagation. This method is one of the fastest ways of getting healthy, disease-free and genetically uniform planting material en masse. It is the only reliable method for mass scale propagation of plants where >500,000 plants yr-1 are involved (Gielis et al., 2002). However, tissue culture of bamboos had received rather scant attention until the last two decades (Huang and Murashige, 1983). Initial reports on regeneration of bamboo plantlets through embryo culture appeared only in the late 1960's (Alexander and Rao, 1968). But major strides in bamboo tissue culture were made in the 1980s. The complete protocol on micro-propagation of Bambusa arundinacea syn. B. bambos from seeds (Mehta et al., 1982) laid the foundation of bamboo micropropagation for replenishment of natural stands and conservation. Thereafter, micropropagation of bamboos gained considerable impetus (Yeh and Chang, 1986a; Paranjothy et al., 1990; Rao and Rao, 1990; Saxena, 1990; Sood et al., 1994a, b; Saxena and Dhawan, 1999; Bag et al., 2000; Arya et al., 2002, Godbole et al., 2002; Ramanayake and Wanniarachchi, 2003; Yeh and Lin et al., 2004; Agnihotri et al., 2009). Several efficient protocols have been successfully developed for many genera/species where various explants were employed (Table 2.1.). However, factors Biotechnology of Bamboos 149

Table 2.1. Summary of micropropagation work done on different bamboos

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such as season and stage of explant collection, cultivars/genotypes/ecotypes, process of surface sterilization, culture media and process of hardening and acclimatization had to be extensively standardized for each of these bamboos. At CSIR-IHBT, highly efficient micro-propagation protocols were developed from nodal segments of important bamboos such as B. balcooa, B. bambos, B. tulda, B. nutans, D. asper, D. hamiltonii, D. membranaceus, D. giganteus, Guadua angustifolia and Phyllostachys pubescens (Table 2.2.). Besides, shoot cultures, somatic embryogenesis was also attempted by various workers; and successful protocols were established (Table 2.1.). At CSIR-IHBT, somatic embryogenesis was induced in nodal segments of D. hamiltonii and basal MS medium containing 2 per cent sucrose, 4.52 μM 2, 4-D and 4.44 μM BAP was used. When 2, 4-D was removed from this medium, high frequency of both recurrent somatic embryogenesis and germination were recorded (Godblole et al., 2002). In B. nutans also, callus induced from nodal segments led to somatic embryogenesis on MS medium supplemented with 3 per cent sucrose, 4.52 μM 2, 4-D and 4.44 μM BAP. While the use of 20 mg l-1 ascorbic acid was important for the proliferation of the somatic embryos, they germinated when 0.499 μM TDZ and 10.74 μM NAA were used (Mehta et al., 2010). Indirect regeneration from nodal segments was also successfully established in important bamboos such as B. bambos, B. nutans, B. tulda, D. asper and D. membranaceus. MS medium supplemented with 2,4-D invariably induced callusing in the nodal Biotechnology of Bamboos 155 . . . e g a p

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segments of these bamboos. However, their concentrations differed from genus to genus and species to species. In D. asper, 14.61 μM 2,4-D induced callusing followed by rhizogenesis and caulogenesis, whereas, 22.29 μM 2,4-D along with 10 per cent coconut milk induced callusing followed by plant regeneration in B. tulda (Nadha, 2012). In case of D. membranaceus, however, callusing occurred when 4.53 µM 2,4-D was combined with 5.37 µM NAA. Indirect shoot bud developed on these calli upon transfer to light but elongated only when 4.4 µM BAP and 1.15 µM Kn were used. In B. bambos, callus was initiated in dark when 4.53 µM 2, 4-D and 5.37 µM NAA were used along with 3 per cent sucrose. Only rhizogenesis but no plant regeneration occurred in these calli when they were shifted to 2.26 μM 2,4-D and 1.14 μM NAA (Brar, 2014) (Fig. 2.1.-2.2.). At CSIR-IHBT, the major bottlenecks of rooting, acclimatization and survival under field conditions were also overcome in important bamboos (Table 2.2., Fig. 2.3.). Rooting was more effectively induced when clusters of three to four shoots were used as compared to individual shoots. However, the strength of the MS medium and the concentration of different auxins governed high percentage of rooting. Among the different genera/species that were studied, maximum rooting (92.5%) was recorded in B. balcooa, whereas, a minimum of 80 per cent rooting was recorded in D. asper. In all the species that were studied, rooted plantlets were first acclimatized in plastic pots containing river bed sand and covered with plastic sheet

Fig. 2.1. Somatic embryogenesis in different species of Bambusa. (a-b) Induction and maturation in B. tulda, (c-d) induction and rhizogenesis (arrows) in B. bambos and (e-f) induction and germination in B. nutans. 158 Pooja Thapa et al.

Fig. 2.2. Somatic embryogenesis in different species of Dendrocalamus. (a-b) Induction along with rhizogenesis in D. asper, (c-f) induction and germination in D. membranaceus (c- d) and D. hamiltonii (e-f).

Fig. 2.3. Shoot multiplication, rooting, hardening and survival under field conditions during micropropagation of different bamboo species. (a-e) B. bambos, (f-i) P. pubescens and (j-m) D. asper. Biotechnology of Bamboos 159 for maintenance at 25-30°C and 80-85 per cent RH under green house condition. After an average of 21 days, well rooted plantlets were successfully transferred to sand : soil : farmyard manure (1:1:1) for further growth and acclimatization. After 55-60 days, the plants were finally shifted to outdoor conditions in pits (2 ft × 2 ft × 2 ft) at a plant to plant and row to row distance of 6 m. In general, the plants showed up to 95 per cent survival under field conditions. The plants had to be protected from the rodents/ monkeys by either putting thorny branches or using waste pieces of agro net around the young plants.

3. In vitro Conservation by Slow Growth and Cryopreservation In commercial tissue culture units, efficient stockpiling is often required to avoid the wastage of costly propagules during periods of low production schedule (Cervelli and Seneranta, 1995). Slow growth is often induced in in vitro cultures for medium term storage of propagules. This ensures an additional back-up collection while allowing steady availability of planting propagules during active production schedules (Withers, 1991; Sutton and Polonenko, 1999). The method is also used for germplasm conservation (Chaturvedi et al., 2004; Hassan et al., 2007). In this regard, rapidly multiplying somatic embryos of D. hamiltonii were successfully stored for >365 days under slow growth conditions imposed by liquid paraffin overlay (Kaur et al., 2012). Importantly, the stored somatic embryos showed high frequency of germination upon retrieval (Fig. 3.1.).

Fig. 3.1. Slow growth and storage of D. hamiltonii (a) somatic embryos under LPO, (b) germination of somatic embryos retrieved after 365 days under LPO, (c) cryopreserved somatic embryos encapsulated in sodium alginate film and (d) cracking of alginate film and growth of somatic embryos after retrieval from cryo-preservation. 160 Pooja Thapa et al.

3.1. Cryopreservation Besides production of juvenile planting materials for replenishment of dwindling stands, attempts have been made to cryopreserve different bamboos. Workers such as Krishnapillay et al. (1993) reported that cryopreservation of seedlings of B. bambos, D. membranaceus, D. brandisii and Thyrsostachys siamensis was better when they were dried for five hours at 25+2°C with 55 per cent relative humidity in a laminar flow cabinet. At CSIR-IHBT, however, the somatic embryos of D. hamiltonii were cryopreserved by the vitrification as well as encapsulation-vitrification methods. The ones subjected to the vitrification method failed to survive, irrespective of vitrification temperature or the duration of cryopreservation. However, the somatic embryos subjected to the encapsulation-vitrification method cracked after 15 days on MS basal media supplemented with 4.44 μM BAP and 8 per cent sucrose. These proliferated 30 days onwards (Fig. 3.1.). The somatic embryos were influenced by vitrification- temperature and showed 50 and 29 per cent survival at 0 and 25°C, respectively after 1 hr of cryopreservation. However, their survival percentage decreased with further increase in the duration of cryopreservation (Kaur et al., 2014).

4. In vitro Flowering and Its Implication in Germplasm Conservation Flowering has been a major deterrent to the conservation and propagation of bamboos. Most genera/species have a long intermast period of juvenile stage that varies from 40-120 years. At the end of this period, bamboos flower gregariously, irrespective of geographical locations and the entire culms of all clonal individuals (from a single mother) flower simultaneously and die en masse (Jackson, 1987). Gregarious flowering results in the production of a large number of viable seeds that cannot be stored for long. Moreover, the seeds are rich in nutrients and attract rodents that increase in population and cause mass devastation to the plants (Jackson, 1987). This results in huge loss of valuable germplasm (Janzen, 1976; John and Nadgauda, 2002; Ramanayake, 2006; Austin and Marchesini, 2012; Shukla et al., 2012), and is a continuous but unpredictable threat to all standing populations of bamboos (John and Nadgauda, 2002). In addition to gregarious flowering, some culms of bamboos also flower sporadically and set a few seeds annually, and die (Jackson, 1987). However, the seeds are mostly non-viable. Although the process of flowering in bamboos demands attention, it is poorly understood and is considered to be an enigmatic process (Ramanayake, 2006). The age of the plants growing in wild is not known and their flowering age cannot be predicted in advance. Thus, no measures can be taken to check the loss of valuable germplasm. Moreover, the plants are very tall and slender, often reaching to a height of 15-18 m. This renders routine and extensive studies extremely difficult. Therefore, a reproducible system of in vitro flowering was established in D. hamiltonii. MS medium supplemented with 2.22 µM BAP, 1.23 µM IBA and 2 per Biotechnology of Bamboos 161 cent sucrose invariably supported 80 per cent flowering. The system proved effective for tagging, sampling and tracking the process of flowering under controlled environmental conditions (Kaur et al., 2014). Using this system, six distinct stages of development were identified, and the flowers were comparable with in planta sporadic flowers (Fig. 4.1.). Since the pollen viability of the in vitro flowers was also higher than those of in planta ones, it had the potential to serve as propagules for medium- or long-term storage and manipulation of in vitro fertilization (Kaur et al., 2014). In vitro flowering has been also reported by Rao and Rao (1990), Nadgauda et al. (1990, 1997), Chambers et al. (1991), Rout and Das (1994), Gielis (1995), Ansari et al. (1996), Gielis et al. (1996), Joshi and Nadgauda (1997), Lin and Chang (1998), John and Nadgauda (1999), Ramanayake et al. (2001) and Lin et al. (2003; 2004a, b; 2007a, b) for different bamboos such as B. arundinacea, B. bambos, B. edulis, B. ventricosa, B. vulgaris, D. brandisii, D. giganteus and D. strictus, etc. A low frequency of seed set has also been reported in B. arundinacea, B. vulgaris, D. brandisii, D. giganteus and D. strictus (Nadgauda et al., 1990; Rout and Das, 1994).

Fig. 4.1. In vitro flowering in D. hamiltonii. (a) Shoots, (b) bunch of mature flowers and (c-d) distinct stages of flower development (c) in vitro and (d) in plant.

5. Genetic Modification through Transgenics Despite the multiple utilities of bamboos, various factors hamper their extensive and complete utilization in emerging industries. Its wood is available only in two colours and is resistant to colouring. The presence of high amounts of lignin incurs expensive processing by paper industry. Moreover, the plants are prone to various kinds of insect attacks (termite and post powder beetle) and fungal pathogens such as white rot and 162 Pooja Thapa et al. brown rot. Besides these serious problems, the plant is sensitive to frost and cannot be successfully grown at high altitudes. As a result, improvement of bamboos is required for harnessing its full potential. In this regard, transgenic technology has proved to be an effective and useful approach for genetic improvement/modification of different plants (Dale et al., 1993; Christou, 1994). However, till now, there are only three reports on genetic transformation of bamboos. For the first time, Douglas et al. (1985) showed attachment of A. tumefaciens to bamboo cell walls. However, it was only after 14 years that Wu and Feng (1999) reported the use of electroporation method of transformation for transient expression of gus gene in intact root-callus cells of Bambusa beecheyana Munro var. beecheyana. It took about 12 years thereafter for Ogita et al. (2011) to obtain transformed Phyllostachys nigra suspension cells by the particle bombardment-mediated transformation. However, they failed to develop transgenic plants from these cells. It was only at CSIR-IHBT that extensive optimization was carried out using somatic embryos of D. hamiltonii. Parameters that prevented Agrobacterium mediated transformation of these woody monocots were finally identified. Necrosis due to polyphenol oxidation, lack of differentiation due to cell wall thickening at wound sites and most importantly, waxy surfaces of the somatic embryos were found to prevent Agrobacterium attachment and infection. Therefore, a simple and effective method was devised for transforming the somatic embryos with fresh overnight grown bacterial cultures containing 500 mg/l polyvinyl pyrrolidone (PVP). Use of 0.01 per cent Tween-20 as surfactant during infection was extremely important along with co-cultivation of the infected somatic embryos on MS medium in the presence of both 100 μM acetosyringone and 4.44 μM BAP for 2 days. Histo- chemical GUS assay, PCR, slot blot and Southern hybridization confirmed successful genetic transformation of the somatic embryos, thereby, confirming the production of first transgenic D. hamiltonii plants in the world (Sood, 2013). The method paved the way for genetic transformation of other important bamboos.

6. Nutritional Profiling for Use as Fodder and Food

6.1.Fodder Bamboos are traditionally used as fodder for livestock during lean winter months. Thus bamboos growing in Palampur, Himachal Pradesh such as B. bambos, B. nutans, B. tulda, B. ventricosa, B. vulgaris, D. asper, D. hamiltonii, D. hookerii, D. strictus, M. baccifera, P. aurea and Sasa auricoma were evaluated for their leaf nutritive composition, in-vitro digestibility, in vitro gas production, short chain fatty acid (SCFA) profile, efficiency of microbial protein synthesis and partitioning factor (PF) (Sahoo et al., 2010). The results revealed M. baccifera as the most superior fodder Biotechnology of Bamboos 163

followed by D. hamiltonii > D. hookerii > D. asper > B. bambos > B. nutans > P. aurea > B. vulgaris >B. ventricosa > D. strictus > B. tulda. However, on the basis of in vitro digestibility, gas production, DM degradation, in vitro ruminal fermentative attributes and nutritive profiles, M. baccifera, D. hookerii, D. hamiltonii and D. asper were considered as promising fodder bioresource.

6.2.Food Young shoots of bamboos are rich in nutrients and are either eaten raw as salad, cooked or fermented. Since they take the flavour of the ingredients in which they are cooked, they are used in soups, snacks, curries, stir fries, pickles, canned vegetable and also as extenders. The shoots have high contents of water (about 90%), proteins (1.49-4.04 g), carbohydrates, minerals such as potassium, calcium, manganese, zinc, chromium, copper, iron, phosphorus and selenium. They also contain tyrosine (57-67%), edible fibres (6-8%), vitamins like thiamine, niacin, vitamin A, vitamin B6, and vitamin E (Xia, 1989; Shi and Yang, 1992) and phytosterol (Qiu, 1992; Ferreira et al., 1995; Kozukue et al., 1999). Besides being very low in fats (0.26-0.94%), sugars (2.5%) and calories, they are naturally organic (Nirmala et al., 2007; 2011). Thus, bamboo shoots with their cholesterol lowering activity surely qualify as nutraceuticals (Brufau et al., 2008). Thus, bamboos rank among the five most popular health care foods in the world. Eleven Indian species (B. balcooa, B. nutans, B. tulda, D. giganteus, D. hamiltonii, D. hookerii, D. longispathus, D. sikkimensis, Melocanna baccifera, Phyllostachys bambusoides and Teinostachyum wightii were found to have the potential for commercialization as food products (Bhat et al., 2005). Out of these, D. giganteus and D. hamiltonii are nutritionally the best ones (Nirmala et al., 2007). At CSIR-IHBT, nutritional profiling of P. pubesense, D. asper, D. hamiltonii and B. bambos shoots revealed significant variations in fat (0.29-0.39%), fibre (1.20 to 1.50%) and protein contents (3.43 to 3.70%). Furthermore, the shoots were processed into edible products such as preserve, candy, chutney, nuggets, cracker (papad) and chukh. These were also analyzed for nutritional and organoleptic qualities. The results revealed nutritional richness and sensory acceptability of the products.

7. Conclusion The future goal of bamboo biotechnology is to provide high quality plants in very short time while overcoming the problems of hybridization and developing bamboos with added-values and improved nutritional and health characteristics. In this regard, the cryopreservation and slow growth methods developed at CSIR-IHBT can be used for preservation of endangered and other bamboo germplasm. Similarly, the efficient micropropagation protocols are being utilized to replenish denuded sites and to 164 Pooja Thapa et al. reclaim degraded lands. While the in vitro system of flowering has opened the way for understanding the enigmatic process of flowering and reproductive biology in bamboos, the efficient genetic transformation methods is a step towards time effective improvement of bamboos. The achievements in nutritional profiling and development of food products open the possibilities of providing livelihood opportunities to the local population while ensuring food security in traditional areas.

Tissue Culture Protocol for Dendrocalamus membranaceus I.D. Arya, Sushma Sharma, Sudhir Sharma, Sudhir Chouhan and Sarita Arya

1. Introduction per cent mercuric chloride solution for 10-15 Dendrocalamus membranaceus is a minutes and rinsed 3-4 times with sterile moderate sized, strong bamboo forming distilled water. The surface sterilized axillary loose clumps. Culms developed are straight buds were cultured on semisolid and liquid and around 20-24 meter in height, 6-10 cm in Murashige and Skoog's (MS) medium diameter and are covered with white supplemented with a cytokinin like 6- powdery substance, deciduous when benzyladenine (BA) and kinetin (Kn). The pH young, green in colour and the nodes are of medium was adjusted to 5.8 prior to strongly ringed. Internodal length is around autoclaving the medium at 121°C for 15 20-40 cm. Culm sheath is glabrous outside minutes. Cultures were maintained at and with dark brown hairs. Leaves are hairy 25±2°C with 14 hours illumination with a on the mid rib base, ligule is very short and photon flux density of 2500 lux, from hairy. It is a native of Myanmar, and in India it fluorescent tubes. is cultivated in Calcutta, Dehradun and also been introduced in Kerala. It is reported to 3. Multiplication of Shoot Cultures have flowered gregariously in Dehradun in Axillary buds cultured on liquid and 1973 (Lohani, 1973; Naithani and Biswas, semisolid MS medium supplemented with 1992). D. membranaceus is mainly used for cytokinin, proliferated number of axillary building purpose (construction and poles). It shoots. These axillary shoots were excised is said to be one of the most promising and subcultured on fresh liquid as well as species for pulp. Kennard and Freyre, in semisolid medium for further in vitro shoot 1957 considered this bamboo to be an multiplication. In 3-4 weeks these shoots excellent from the processing point of view were again subcultured on liquid and because young shoots are almost smooth semisolid medium supplemented with BA and easy to handle. In China, it is used for (2.0-5.0 mg l-1). Different sets of experiment making bamboo chopsticks, shreds and conducted to obtain the maximum in vitro paper (Bennet and Gaur, 1990). shoot multiplication rate. For each experiment a minimum of 30 replicates were 2. Explant Source taken. Observation was recorded after an Young and juvenile shoots were collected interval of 4 weeks. Once the optimum of from nursery raised 2 years and 3 years old shoots were multiplication medium was plants (Fig. 2.1a-e). The nodal segments established, the in vitro shoots produced with single axillary buds were used as were excised in propagules of three shoots explant for micropropagation. The axillary and were subcultured every 3-4 weeks. This buds were first washed with 5 per cent shoot multiplication was regularly conducted cetrimide (ICI Ltd. India) solution for 5 and maintained under 20-30 µ EM-2 S-1 minutes and then surface sterilized with 0.1 photon flux density for 16 hours photoperiod Biotechnology of Bamboos 165

at 25 ± 2°C. The number of propagules solution (without organics) twice a week for cultured to the number of the propagules three weeks. After three weeks, these derived at the end of subculture was bottles were shifted to mist chamber having regarded as the rate of multiplication or fold relative humidity of 80-90 per cent with a multiplication. temperature at 30±2°C, the mouth caps of the bottle were opened and plantlets were 4. In vitro Rooting allowed to remain in the bottles for 3-4 days before they were transferred into polybags The in vitro regenerated shoots (measuring containing a mixture of sand, soil and 2.5-3.5cm) were cultured on full, half, and farmyard manure (FYM) in a ratio of 1:1:1. In quarter strength of MS medium containing mist chamber the plants were kept for three v a r i o u s c o n c e n t r a t i o n o f a u x i n s weeks and were irrigated with tap water. naphthylacetic acid (NAA), indole acetic acid Later these polybags were shifted to shade (IAA), indole butyric acid (IBA) (1.0-10.0 mg house for two months. After one month l-1) for root induction. The shoots cultures (2- plants were shifted to bigger polybags (14”) 4 shoots) were placed on both liquid and containing same putting media composition. semisolid supplemented with different Hardened and acclimatized plants were concentration of auxins. Three propagules directly planted in the field preferably during (shoot clusters of different size) were monsoon or after monsoon season (July- cultured per conical flask (100-150 ml). After December). Simple silviculture practices four weeks, plantlets were taken out and were followed during plantation with 5m x 5m rooting percentage, numbers of roots per meter spacing. The field plants were propagule were counted. irrigated every 15 days for first three months.

5. Hardening and Acclimatization 6. Results Procedures Rooted shoots from four weeks old cultures 6.1. Initiation of Shoot Culture were transferred to soil under shade house Axillary buds cultured on cytokinin (BA and either directly or after in vitro hardening the Kn) supplemented MS medium showed plantlets were taken out from the flask, axillary bud break in two weeks. The number agar/medium was washed and were of shoots proliferated and the shoots transferred to 250 ml screw cap bottles elongated were more in BA supplemented containing ¼ volume of soilrite. These medium as compared to Kn supplemented plantlets were supplied with half strength MS medium (Table 6.1.1.) BA concentration

Table 6.1.1. Effect of cytokinin (BA and Kn) concentration on shoot bud proliferation from axillary bud in D. membranaceus . Data recorded after three weeks from 20 replicate each Cytokinin Concentration No. of shoots Average shoot Average (mg l -1) proliferated length shoot (Min-Max) (cm) number 6-Benzyladanine (BA) 1.0 3-5 3.5 3.8 2.5 4-5 3.0 4.2 5.0 6-8 2.5 7.1 7.5 6-8 2.0 7.0 10.0 6-10 1.0 7.4 Kinetin (Kn) 1.0 3-4 1.5 3.2 2.5 3-4 1.8 3.4 5.0 4-6 1.5 4.9 7.5 4-6 1.5 5.3 10.0 4-8 1.5 5.2 166 Pooja Thapa et al.

(1-10 mg l-1) in MS medium had a marked subculture the shoots were carefully effect towards axillary shoot proliferation. At multiplied and were subculture on MS +2.0 high concentration of BA (5-10.0 mg l-1), mg l-1 BA. In three weeks, these in vitro maximum number of axillary shoot shoots were further multiplied and were proliferation was achieved (6-10 used for different experiment carried out for shoots/axillary bud) in three weeks (Fig. 2.1a) obtaining maximum shoot multiplication with the increased concentration of BA in the rate. It was observed that cytokinin BA had a medium, length of proliferated axillary significant effect on shoot multiplication rate. shoots decreased. However, the thickness With the increased concentration of BA (1.0- of axillary shoots remained independent of 4.0 mg l-1) the shoot multiplication rate BA and was closely related to the thickness increased from 6.91 to 15.55 fold (Table of culture nodal segment. 6.2.1.). A further increase in BA concentration (5.0-10.0 mg l-1) reduced the 6.2. In vitro Shoot Multiplication shoot multiplication rate in term of In vitro shoots after their initial proliferation propagules derived. Generally, the shoots from axillary buds on medium containing produced in these experiments were different concentrations of BA and Kn were observed to be thin with small leaves. The excised and subcultured on MS medium effect of Kn on in vitro shoot multiplication supplemented with 3.0 mg l-1 BA. BA showed similar results for in vitro shoot concentration of 2.0 mg l-1 gave a greater multiplication but at higher concentration in gain in the number of shoots than either 1.0 MS medium (Table 6.2.2.). Thus, the optimal or 5.0 mg l-1 supplemented MS medium shoot multiplication in terms of number of (Table 6.2.1.). After three weeks of the first shoots obtained was MS+2 mg l-1 BA

Table 6.2.1. Effect of BA concentration in MS medium and size of propagule on shoot multiplication rate. Data recorded after 4 weeks. BA conc. Propagule size Average no. of shoots Average multiplication rate (mg l-1) (no. of shoot) produced (no. of times) 1.0 3 20.10 6.91 4 24.50 6.12 2.0 3 45.00 15.01 4 30.86 7.72 3.0 3 36.75 12.25 4 32.41 8.10 4.0 3 30.30 10.10 4 29.32 7.33 5.0 3 27.00 9.00 4 34.84 8.71

Table 6.2.2. Effect of Kn concentration on shoot multiplication in MS medium (Propagules of three shoots were cultured. Data recorded after four weeks) Kn (mg l-1) Average no. of shoots produced Average multiplication rate (no. of times) 1.0 15.35 5.10 2.0 40.01 13.31 3.0 42.30 14.01 4.0 45.09 15.02 5.0 30.45 10.14 Biotechnology of Bamboos 167

supplemented medium. On this medium However, due to a number of problems the sizable and distinct shoots were proliferated culture were maintained and multiplied on in four weeks (Table 6.2.1.). Thus, all shoot semisolid MS medium. multiplication was carried out on MS The auxin, NAA when used in -1 medium+2.0 mg l BA. On this medium the combination with cytokinin BA (2.0 mg l-1) shoot multiplication rate reached to a showed a reduced multiplication rate. The maximum of 15.55 fold in 3-4 weeks (Fig. 2.1b). multiplication rate constantly decreased with Effect of subculture period on shoot the increase in NAA concentration in MS multiplication rate was studied. It was found medium from 1-2 mg l-1 in the MS medium that three to four week incubation time was supplemented with BA. necessary for complete in vitro shoots Effect of pH of the medium on in vitro multiplication and shoot development. A shoots multiplication was studied in liquid subculture of one to two weeks only resulted MS (2.0 mg l-1 BA) medium. It was observed in incomplete multiplication and shoot that good shoot multiplication occurred even development. in acidic medium. However, the shoot The in vitro shoots cultures when kept for multiplication rate declined on basic medium longer duration (five weeks and above) on and died on 7.5 pH of the medium (Table 6.2.3.). medium started dying. The shoot propagules Effects of different strength of MS salt of such cultures showed reduced potential were also studied on in vitro shoot for in vitro shoots multiplication. No shoot multiplication and it was observed that multiplication was obtained on cytokinin free maximum in vitro shoots were produced on MS medium only. A slight increase in shoot full strength of MS salts. As the strength of multiplication rate (17.50 fold) was observed MS salts reduced (75-25%) in the medium a in the liquid MS medium (Table 6.2.1.) as sharp decline in the shoot multiplication rate compared to the semisolid medium (15.55). was observed (Table 6.2.4.). Table 6.2.3. Effect of pH of the medium (liquid) MS+ 2.0 mg l-1 BA on shoot multiplication (Data recorded after four weeks) pH of medium Size of No. of shoots Multiplication rate propagules obtained (no. of times ) (shoot) 3.5 3 43.5 14.5 4.0 3 46.5 15.5 4.5 3 47.0 15.66 5.0 3 45.0 15.0 5.5 3 50.0 16.66 5.8 3 552. 17.50 6.0 3 40.72 13.57 6.5 3 30.10 10.17 7.0 3 24.35 8.10 7.5 3 4 1.33 Table 6.2.4. Effect of strength of MS medium on in vitro shoot multiplication (Propagules cultured on 2 mg l-1 BA. Multiplication recorded after four weeks) MS medium Size of propagules Average no. of Multiplication strength (shoot) shoots obtained (no. of ti mes) Full 3 46.20 15.40 Three fourth (¾) 3 30.1 10.03 Half (½) 3 21.34 7.11 One Fourth (¼) 3 16.20 168 Pooja Thapa et al.

7. In vitro Rooting 70 per cent, in MS medium supplemented -1 The in vitro raised shoots failed to root on with 70 mg l IBA. hormone free basal MS medium. Various Shoots cultured on MS medium auxin in the medium were attempted for supplemental with different concentration of rooting trials. Both liquid and semisolid MS IAA did not produce roots, in all the medium were used. In vitro shoots when experiments carried out (Table 7.1.). transferred on NAA supplemented medium produced 70-98 per cent roots from the 8. Hardening and Acclimatization cultured shoots. MS medium supplemented -1 Four-week-old cultures on rooting medium with 2 mg l NAA produced 98 per cent roots develop healthy root and shoot system, from the shoots in three weeks in liquid hardening of these plantlets was done in two medium and it takes 4-5 weeks in semisolid different methods. In the first methods, the medium. To complete in vitro rooting in NAA rooted plantlets were kept in the culture concentration (3-5 mg l-1) in the MS medium vessels until the nutrients of the medium reduced the rooting percentage (Table 7.1.). were completely exhausted. This was done Usually propagules of 3-4 shoots were used to strengthen the plants under physiological for rooting experiments. stress conditions. These in vitro rooted Generally, 8-14 roots per propagule plantlets were washed with tap water to developed (Fig. 2.1c) when the length of remove adhered agar-agar and then directly shoots cultured in a propagules, ranged from transferred into polybags containing sand: 1.5- 3.5 cm. Root initiation starts from 8-12 soil: FYM in 1:1:1 ratio and kept in shade days of shoot cultured on rooting medium house. These plants were supplied with half and completes in 5 weeks. In vitro rooting strength MS solution (twice a week) for three -1 was also obtained on IBA (5-10 mg l ) weeks. Later, these plants were irrigated supplemented MS medium. However, with tap water and kept in shade house for rooting percentage varied from 30-70 per another one month before they were filed cent with a maximum rooting percentage of transferred. In the second method, the in

Table 7.1. Effect of different concentration of NAA on in vitro rooting of D. membranaceus shoots (propagules of three shoots cultured for rooting) Treatment Average no. Rooting percentage Plantlet survival of roots ( after 5 weeks) ( after 5 weeks) NAA (mg l-1) 1.0 8.72 73.33 70.36 2.0 14.02 98.26 96.42 3.0 13.41 97.08 95.35 4.0 13.45 91.66 96.31 5.0 12.72 73.33 96.03 IBA (mg l-1) 1.0 - - - 3.0 - - - 5.0 4.32 30.02 50.35 7.0 6.41 56.26 86.32 10.0 6.36 70.14 85.01 IAA (mg l-1) 1.0 - - - 3.0 - - - 5.0 - - - '-' indicates no rooting response. Biotechnology of Bamboos 169

vitro rooted plantlets were washed with tap with water. In the next two months these water so as to remove adhered agar- plants developed rhizome in polybags agar/medium and then transferred to conditions in the shade house. At this stage autoclaved soilrite in the culture bottles. the plants were field planted. The These plants were supplied strength MS micropropagated field plants showed a high medium (without organics) twice a week. field survivability. The field plants were The culture bottles were kept in the culture irrigated every 15 days for three months. rooms for three weeks and later transferred Those plants which were not irrigated to mist chamber with relative humidity of 80- showed casualties due to overgrowth of 90 per cent and 30ºC temperature. The caps weeds and grasses. Up to now nearly two of the culture bottles were open and the thousand plants of D. membranaceus have bottles were kept (open mouth) for 3-4 days been micropropagated and field planted later the plants were transferred into successfully. The field plants develop polybags containing sand : soil : FYM in rhizome and later sprouted new culms. equal volume (Fig. 2.1d and 2.1e). These plants were kept in mist chamber 9. Summary and Conclusion for three weeks. After mist chamber stage, The present investigation was undertaken to the hardened plants were shifted to open develop the methods of propagation by shade house conditions for acclimatization developing an efficient, reproducible, to outer environmental conditions. In shade reliable and routinely available technology house the plants were further transferred for mass propagation of D. membranaceus. into bigger polybags (14") and were irrigated The study was considered necessary since

2.1a. Axillary shoot proliferation 2.1b. In vitro shoot multiplication 2.1c. In vitro rooting from a propagule

2.1d. Tissue culture plantlets 2.1e. Hardening of tissue culture plants in soilrite Fig. 2.1(a-e). Micropropagation of Dendrocalamus membranaceus. 170 Pooja Thapa et al.

bamboo is very useful to the Indian people Cytokinin (BA). A high rate of shoot and has been a source of economically multiplication was obtained due to BA in the important valuable to the people. The normal medium. This stimulated the growth of vegetative propagation through cutting in multiple shoots during shoot multiplication. rooting is not possible for commercial Similar results reported in D. strictus (Nadgir venture. The present study deals with the et al., 1984), B. ventricosa (Dekkers, 1989), application of plant tissue culture technology D. giganteus and D. strictus (Das and Rout, for mass propagation from selected mature 1991), Madhuca longifolia (Das and Rout, clumps and 2-4 years old fast growing 1993), D. asper (Arya and Arya, 1996a, b), seedlings of D. membranaceus. An efficient Syzygium cumini (Yadav et al., 1990) and reproducible protocol has been Eucalyptus (Gupta et al., 1983) and developed for micropropagation of this Anogeissus pendulla (Joshi et al., 1991). bamboo. Nodal segments containing axillary The results showed that in case of D. bud was found to be the best explant for membranaceus, shoot multiplication also micropropagation. occurred in acidic medium but multiplication The suitability of the nodal segments has rate sharply declined in basic medium. already has been established in Similar results were obtained in case of B. micropropagation of D. strictus and betula (Saxena, 1990). In the present study, D. giganteus (Das and Rout, 1991), D. 3 per cent sucrose was found to be essential strictus (Nadgir et al., 1984), Bambusa for shoot multiplication. On sucrose free vulgaris (Tikiya, 1984), D. asper (Arya and medium or at 1-2 per cent sucrose in MS Arya, 1996a, b). The uses of dormant axillary medium, instances of albinism with death bud or nodes ensure allocation of reserved and decay of shoot and leaves are observed. food materials. The technique offers the However, (Saxena, 1990) found 2 per cent potential to raise thousands of plantlets from sucrose was ideal for shoot multiplication of the nodal region in existing clumps. In the B. tulda. past also, nodal segment containing axillary In the present investigation, high shoot buds were largely used to micropropagate multiplication rate was obtained when the numerous tree species, like Wrightia size of propagules carrying 3-4 shoots were tomentosa (Purohit et al., 1994), Betula used in shoot multiplication cycle. Das and pendula (Arya and Arya, 1995), Syzygium Rout (1991) also reported maximum shoot cumini (Yadav et al., 1990) Eucalyptus multiplication rate in propagules of D. (Gupta et al., 1983), Azadirachta indica strictus containing at least 6-10 shoots. Our (Arya et al., 1995) Anogeissus acuminata observation that single isolated shoot did not and Capparis decidua (Deora, 1993). multiply is in complete agreement with the The frequency of bud break on control earlier reports. (MS medium without growth regulators) was In the present study, multiplication rate of -1 very low. Incorporation of BAP (1-10 mg l ) 15 fold in D. membranaceus in 3-4 weeks into the medium improved the incidence of has been achieved. There are only few other bud break and promoted the multiple shoot reports mentioning the rate of multiplication formation. The frequency of bud break, in Bamboo. In seedling material, number of shoot developed on Explant and multiplication rates of 8.4 fold in 30 days in shoot multiplication rate were higher on MS bamboo was achieved. Das and Rout (1991) medium than WPM medium. Similar effect of also claimed that multiplication rate of 45 fold BA on axillary shoot proliferation was was achieved in three weeks by axillary observed in D. strictus (Nadgir et al., 1984), branching in B. tulda. B. ventricosa ( Dekkers, 1989), D. giganteus In the present study, the in vitro shoots and D. stritcus (Das and Rout, 1991), obtained from axillary bud and multiple Tecomella undulata (Rathore et al., 1991), shoots were successfully rooted. The ability D. asper (Arya and Arya, 1996a, b). of plant tissue culture to form adventitious Multiple shoot produced from single roots depends on interaction of many axillary buds were harvested and rapidly endogenous and exogenous factors. The subculture on MS medium containing role of auxin in root development is well Biotechnology of Bamboos 171

established which has been reviewed by References Scot (1972) and Torrey (1976). Nemeth Arya, I.D.; Arya, S. 1995. Rapid in vitro 1986 suggested that optimal endogenous multiplication of silver birch (Betula and exogenous auxins/cytokinin balance pendula Roth) through axillary bud is required for root formation. During the culture. Annals of Forestry, 3(1): 65-71. present study, it was recorded that shoots A r y a , I . D . ; A r y a , S . 1 9 9 6 a . formed root in auxins medium. These Micropropagation protocol for exotic shoots also showed simultaneously shoot edible bamboo (Dendrocalamus asper). elongation, which is due to 'cytokinin carry ICFRE FORTIP Technical Bulletin. over effect' in the shoots. In the present Arya, I.D.; Arya, S. 1996b. Introduction, study, NAA, IBA in MS medium was effective mass multiplication and establishment for in vitro rooting. Similar results were of exotic bamboo Dendrocalamus asper reported in other bamboo (Das and Rout, in India. Indian Journal of Plant Genetic 1991). Resources, 9(1): 115-121. In earlier publications concerning Arya, S.; Rathore, T.S.; Arya, I.D. 1995. rooting of different plant species, MS In vitro propagation of neem from macro and micro elements at full strength seedling and mature tree. Indian were the component of the media usually Journal of Plant Genetic Resources, used (Boxus, 1971; Mehra and Mehra, 8(2): 247-252. 1974). Later, the full salt concentration of Bennet, S.S.R. and Gaur, R.C. 1990. macro elements of this formulation was Nomenclature of Burmese bamboo: lowered by half, one third and quarter as Melocanna humilis Kurz. Indian basal rooting medium. (Quoirin et al., Forester, 116(8): 648-649. 1977; Lane, 1979a, b; Skirvin et al., 1980). In the present case, full strength Boxus, P. 1971. La culture meristemes de MS medium with auxins yielded optimal prunus. Note preliminaire relative a l' rooting response. However, during espece P. Pandora. Bulletin des hardening phase half strength ms medium Recherches Agronomiques de Gembloux, was used to nourish the developing roots 6: 3-5. and plants. Although the rooting was Das, P.; Rout, G.R. 1991. Mass observed with the auxins (IBA/NAA) multiplication and flowering of bamboo tested at various concentration (1-15 mg l- in vitro. Orissa. Journal of Horticulture, 1) but the best rooting was obtained in full 19(1-2): 118-121. strength in MS medium supplemented Dekkers, A.J. 1989. In vitro propagation with 2.0 mg l-1 NAA in D. membranaceus. and germplasm conversation of certain These roots grew up to 8-15 cm. bamboo ginger and costus species. A very high success rate of (90-100%) Ph.D. thesis. National University of transplantation and plant survival was Singapore, Singapore. obtained. The success limits to the Deora, N.S. 1993. Regulation of gradual procedure of hardening and differentiation and regeneration as acclimatization of the plantlets. Earlier related to clonal propagation. Ph.D. low transplantation success rate of 70-80 thesis. J.N.V. University, Jodhpur, India. per cent have been reported only in B. Gupta, P.K.; Mehta, U.J.; Mascarenhas, tulda (Saxena, 1990) and in D. strictus A.F. 1983. A tissue culture method of (Nadgir et al., 1984). Large number of rapid propagation of mature trees of plant transfer in the field is, however, not Eucalyptus torelliana and Eucalyptus mentioned in these publications. The camaldulensis. Plant Cell Report, 2(6): present protocol is efficient and rapid 296-299. method (160 day cycle) for large scale Joshi, R.; Shekhawat, N.S.; Rathore, T.S. c o n t i n u o u s m u l p l i c a t i o n o f D . 1991. Micropropagation of Anogeissus membrabaceus which otherwise takes a pendula Edgew – An arid forest tree. long intermast period of several years for Indian Journal of Experimental Biology, seed production. 29: 615-618. 172 Pooja Thapa et al.

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