58 Middle East Journal of Agriculture Research, 1(1): 58-63, 2012 ISSN 2077-4605

The Use of Tissue Culture Technique for Propagation of ( giganteus Wallich)

Mona A-Amin and Essam El- Atrach

Timber Trees Dep., Hort. Res. Inst., A.R.C. Egypt.

ABSTRACT

The effect of various treatments on the behavior of consecutive micropropagation stages of Bamboo (Dendrocalamus giganteus Willich) was studied. The statistical analysis of data revealed that, for culture establishments stage, the middle microcutting cultured on full strength MS medium induced the highest number of shootlets / explant, shootlets length (cm), length and number of leaves/ shootlet than the other treatments. Concerning to the multiplication stage, using MS medium supplemented with (1.0 mg/L BA and 0.1 mg/L NAA), recorded the highest number of shootlets/explant, shootlets length (cm), leaves number, fresh and dry weights of shoot. The various treatments of BAand NAA caused a significant effect on chlorophyll (a,b ) and carotenids in the shootlets. For rooting stage, the highest root length and the highest root formation were recorded by using MS medium at half strength supplemented with 1.0 mg/L NAA. During acclimatization stage, soil mixture (peat, sand, vermiculate) at a ratio of (1:1:1)v/v/v was more better than the other treatments, by inducing an increase in the length of shoot, length of root, as well as number of leaves.

Key words: Bamboo, Micropropagation, BAP, NAA, Dendrocalamus giganteus.

Introduction

Bamboos are often long-lived, woody, evergreen grass, members of the Family Gramineae, trib Bambuseae. The giant bamboo often reach a height of 20-30 m, while most are shrublike, medium or dwarf species with a few exceptions as a climbers (Lies, 1987). There are about 75 genera and over 1250 species in the world, which occur mostly in natural vegetative in tropical, subtropical, and temperate regions and are found in great abundance in tropical Asia (Saharia and Sen 1990). Bamboos are known to have strong and long fibers which are widely used for pulp and paper . Bamboos are forest grasses, it makes wonderful in door companions and in the landscape, (Susanne, 1999). stated that Bamboos are used in the construction of dome structure which could meet requirements for school, marketplace, shopping centers and even hospitals. (Mishra, et al., 1988), reported that bamboos are used extensively for house construction because of their strength properties, preferential costs and easy availability and rural areas. Large number of species are consumed by wild animals (Saxenas and Bhojwaniss, 1993). The long intervals between seedling in many species make propagation in this way impractical for this reason. Vegetative propagation has traditionally been used to grow of the genus. Methods of vegetative propagation are discussed (Chang, 1991). There are number of problems with this type of propagation. The offsets used are bulky, heavy and difficult material is available. Clump, and material is not always available so that large scale vegetative propagation is impossible. Propagation attempts using branch cutting and clump sections have also attempted. These propagules have been found successfully only if roots relatively grew quickly. Rootpromotion substances have little effect on bamboo rooting and planted meretial has been found to develop very slowly (Hassan 1980). Furthermore vegetative, propagation is also made difficult due to seasonal specificity of material (Saxen and Bhojwaniss, 1993). Because of the difficulties with this vegetative propagation, micropropagation and somatic embryogenesis techniques have been developed in order to propagate some dendrocalamus species. Also these problems can be overcome by using micropropagation techniques which allow the rapid multiplication of disease – free and true to type selected clones (Boccon-Gibod,1989). The aim of the present work is to establish a protocol for clonal micropropagation of bamboo to obtain mass production all over year. Attention is also paid to find a conventional technique to obtain a successful acclimatization process leading for field grown plants.

Material And Methods

These studies have been carried out in Tissue Culture and Germplasm Conservation Research Laboratory, Horticulture Research Institute, Agriculture Research center, during the years of (2010-2011).

Corresponding Author: Mona A-Amin, Timber Trees Dep., Hort. Res. Inst., A.R.C. Egypt. E-mail:[email protected] 59 Middle East j. Agric. Res., 1(1): 58-63, 2012

Plant Materials:

The explants were collected from the tree grown in the Department of Woody Trees, Horticulture Research Institute, Agriculture Research Center.

Surface Sterilization:

The explants were surface sterilized under aseptic condition in laminar air flow cabinet by ethanol (70%) for 2-minutes and followed by Chlorox (sodium hypochlorite NaOCL active gradient 5.25%) at 20% and few drops of tween-20 (polyoxyethylene sorbitan as a liquid detergent) for ten mintutes-agitation. Mercuric chloride (o.1%W.V) was then used for ten minutes. Each disinfection process was followed by three rinses with steril distilled water.

Establishment stage:

Three types of medium MS (Murashige and Skoog, 1962), WPM (Lloyd and McCown,1980) and SH (Schenk and Hildebrandt.1972). Media were supplemented with sucrose (3.0%) and agar (7g/L). Three parts of explants (terminal, middle and basal part),were cultured on different types of media and incubated for four weeks. Four explant portion were cultured in each jar ,ten jars were employed for each treatment as replicates . At the end of this stage, shootlet number/explant shootlet length (cm) and leaves number/shootlet were calculated.

Multiplication stage:

Shootlets obtained from establishment culture were employed as a source for shootlets multiplication. Explants were cultured on MS basal medium supplemented with different concentrations of BAP (benzyl amino purine) (0.0,1.0 and 2.0 mg/L) combined with NAA (Naphthalene acetic acid) at (0.0 and0.1mg/L).In each treatment forty explants in ten replicate were cultured for one month. After four subcultures shootlets number/explant, shootlet length (cm), leaves number/shootlet fresh weight and dry weight (g), Chlorophyll A and B and carotenoids were determined.

Determination of pigments:

Plant pigments determination were adopted during multiplication with dimethyl formamide as described by Nornaj (1982).

Rooting stage:

Shootlets taken from multiplication stage were separated and cultured on MS–half strength medium supplemented with NAA at concentrations (0.0, 0.5 and 1.0 mg/L),combined with BA at concentrations (0.0,0.5 and 1.0 mg/L) and supplemented with 2g/L activated charcoal.. All treatments comprised four explants in each jar, ten jars for each treatment. Several parameters were recorded (rooting percentage, number of roots, and root length (cm).

Culture conditions:

All media were adjusted to pH 5.75±0.1 prior to autoclaving for 20 minutes at 121oC and 1.2 kg/cm2. The cultures were incubated in a growth chamber at 24± ˚C1 under 16 hrs, photoperiod (day light fluorescent tube) (Phillips, 40 MES-1 cm-1).

Acclimatization of plantlets:

Rooted shootlets were removed from agar medium and potted directly into potting mixtures [peatmoss, peatmos + sand (1:1, v/v), peatmoss + vermiculate (1:1, v/v) and peatmoss + sand + vermiculate (1:1:1, v/v/v) ] under controlled conditions for 2-3 weeks. After this period, the plantlets were transferred out to PVC greenhouses, and maintained for 2month to be ready to the field.

60 Middle East j. Agric. Res., 1(1): 58-63, 2012

Experimental design and data analysis:

The lay-out of the experiment was designed in completely randomized design and least significant difference L.S.D at P<0.0.5 was used for comparison among means according to Steel and Torrie (1980).

Results and Discussion

1-Effect of different medium types and explant parts during establishments stage of Bamboo In Vitro:

Data in Table (1) indicated that, MS medium recorded the highest significant values of (shootlets/explant 10.02 ),( length of shootlet 10.34(cm) and number of leaves (12.68 leaves/shootlet) compared with the others media. Concerning the type of explant parts, results, indicated that using the middle part significantly recorded the highest number of shootlets (9.69 shootlets/explant), length of shootlet (9.45 cm) and number of leaves (17.20 leaves/shootlet). Regarding the interaction between medium type and explant parts, data showed that significantly increased the number of shootlet, length of shootlet and number of leaves (11.46 shootlets/explant, 11.55(cm) and 19.30 leaves/explant) were recorded when the middle explant parts cultured on MS medium. Also it could be concluded that, the terminal parts cultured on SH medium recorded the lowest values in all parameters under investigation(5.76shootlets/explant,6.98 (cm) and 5.60 leaves/explant). These results can be ascribed to that MS full strength medium has a higher mineral elements concentration than the other media under investigation. Similar results were obtained by Scarpa et al (1996.)

Table 1: Effect of different medium types and explant parts on number of shootlets/explant, length of shootlet (cm)and number of leaves during establishment stage of Bamboo . Number of shootlets/explant Explant parts(B) Terminal Middle Basal Mean (A)

Medium type (A) MS 18.91 11.46 9.70 10.02 WP 5.91 9.1 6.85 7.29 SH 5.76 8.51 6.25 6.74 Mean (B) 6.76 9.69 7.60 L.S.D. 5% A 0.67 B 0.18 A.B 0.96 Length of shootlet (cm) MS 10.43 11.55 9.05 10.34 Wp 7.58 8.70 7.65 7.98 SH 6.98 8.10 7.05 7.38 Mean (B) 8.33 9.45 7.92 L.S.D.5% A 0.55 B 0.15 A.B 0.78 Number of leaves MS 9.05 19.30 9.68 12.68 WP 6.20 16.45 6.83 9.83 SH 5.60 15.85 6.22 9.22 Mean (B) 6.95 17.20 7.58 L.S.D. 5% A 0.68 B 0.19 A.B 0.97 A= medium type B. explant part A.B= interaction

2-Effect of different types and concentrations of plant growth regulators on shooting behaviour of Bamboo, during multiplication stage:

Data in Table (2) showed that the different types and concentrations of plant growth regulators had significant effect on shootlets number/explant, shootlet length (cm), leaves number/shootlet,fresh and dry weights (g). The explants cultured on MS medium supplemented with 1.0 mg/L BA +0.1 mg/L NAA gave the largest number of shootlets (25.60 shootlets /explant), longest shootlet (8.55cm), largest number of leaves (20.11 leaves/explants) and highest fresh and dry weights (0.86 and 0.35g) respectively. On the other hand, MS medium free hormones (control) gave the lowest value in all parameters (12.0 shootlets/explant, 5.20 (cm), shootlet length and (0.15g) for fresh weight and (0.06g) for dry weight. This enhancing effect could be ascribed to a stimulatory effect of this modification on cell division and enlargement which was accompanied by metabolic activities producing metabolic products. These results are in harmony 61 Middle East j. Agric. Res., 1(1): 58-63, 2012

with Sinha et al (1993), on Albiza Foliataria ,Serres et al (1994) on Vaccinium vitis indea ,Franca et.al (1995) on Poly phythum, Arafa (1992 and Bouza et.al (1994).

Table 2: Effect of different types and concentrations of plant growth regulators on shooting behaviour of Bamboo, during multiplication stage In Vitro Growth characters Number of shoot Length of Number of D.W. of F.W. of shoot(g) lets /explant shootlet (cm) leaves/shootlet shoot(g) Treatment (mg/L) Control 12.0 5.20 15.0 0.15 0.06 NAA(0.1) 12.33 6.40 17.35 0.20 0.08 BA(1.0) 12.90 6.49 18.20 0.66 0.23 BA(1.0)+NAA (0. 1) 25.60 8.55 20.11 0.86 0.35 BA(2.0) 12.20 6.20 15.30 0.40 0.15 BA(2.0) + NAA (0.1) 18.30 5.40 17.0 0.57 0.23 Mean 15.56 6.37 17.16 0.47 0.18 L.S.D .5 % 3.55 1.70 1.12 N.S 0.02

3-Effect of different types and concentrations of plant growth regulators on plant pigments concentration (mg/g F.W.) during multiplication stag:

Data in Table (3), demonstrated that the various treatments of the cytokinin (BA) and the auxin NAA treatments caused significant effects on shootlets content of chlorophyll (a,b) and carotenoids . The maximum chlorophyll a and b values (0.50 and 0.20 mg/g) were observed as affected by the supplement MS medium with( 1.0mg/L BA+0.1mg/L NAA) compared with medium free hormones (control) which gave the lowest values (0.16 and 0.01 mg/g). For carotenoids, medium containing 0.1mg/L NAA gave the highest value of carotenoids (0.12mg/g) compared with medium supplemented with 2.0mg/L BA which gave the lowest value (0.06mg/g). The obtained results revealed that 0.1mg/L NAA treatments promoted the synthesis of chlorophyll a,b while the carotenoids were diminished. Confirming results were reported by Fadl and Sari El-Deen (1979) on olive seedling. They found that BA treatment retained higher concentration of chlorophylls than untreated plants. Similer results were obtained by Hanafy et al (2002) on Myrtus communis.

Table 3: Effect of different types and concentrations of plant growth regulators on plant pigments concentration (mg/g F.W.) during In vitro multiplication stage of Bamboo. Plant pigment( mg/g F.W.) Chlorophyll(A) Chlorophyll(b) Cartenoids

Treatment( mg/L) Control 0.16 0.01 0.09 NAA (0.1) 0.20 0.17 0.12 BA(1.0) 0.19 0.13 0.11 BA(1.0) + NAA (0.1) 0.50 0.20 0.08 BA(2.0) 0.25 0.18 0.06 BA(2.0)+ NAA (0.1) 0.19 0.14 0.07 Mean 0.25 0.14 0.08 L.S.D.5 % 0.05 0.02 0.05

4-Effect of plant growth regulators on In vitro rooting behaviour of Bamboo:

The data in Table (4) revealed that, explants cultured on full MS medium containing 1.0mg/L NAA produced the highest significant rooting percentage (88.20%), largest number of roots (1.25 roots/shootlet) and longest root (32.30 cm). On the other hand, MS free hormones medium, MS medium supplemented with( 0.5mg/L NAA +0.5mg/L BA),( 0.5mg/L NAA +1.0mg/L BA) and (1.0 mg/L NAA +1.0 mg/L BA) resulted in no root formation (0.0). The main role of auxin in tissue culture is stimulating cell division and root development.( Rao and Lee, 1986) reported that, higher auxin and lower cytokinin levels promote root development. Similar results were obtained by Aydieh et al (2000), and Sawsan et.al (2010). They found that increasing the concentration of both IBA and NAA mostly enhanced root initiation and elongation. These results refer to the biological activities of IBA and NAA belonging to auxin synthesis which promote cell division and elongation. Similar results were obtained by Koriesh and AL- Manie 2000).

5-Effect of different soil mixtures on plant acclimatization of Bamboo:

Concerning the effect of soil mixture on acclimatization, data in Table (5) indicated the superiority of the soil mixture. (peat: sand: vermiculate) than the other soil mixtures. In concern with the morphological characters of the acclimatization plants, each of shoot length, root length, number of leaves, data observed that peat moss alone gave the lowest values in all parameters under investigation. However the mixture of peat + sand + 62 Middle East j. Agric. Res., 1(1): 58-63, 2012

vermiculate significantly recorded the longest plantlet length (12.20 cm), longest roots (16.30cm) and largest leaves number (16.0 leaves/explant,). Similar results were obtained by HO et.al (1987) and Sawsan et.al (2010).

Table 4: Effect of plant growth regulators on In vitro rooting behaviour of Bamboo. Characters Rooting (%) Number of roots Length of roots (cm) Treatment( mg/L) Control 0.00 0.00 0.00 NAA(0.5) 30.76 0.49 3.70 NAA(1.0) 88.20 1.25 32.30 BA(0.5) 1.00 0.47 1.30 BA(1.0) 1.45 1.00 2.70 NAA(0.5)+BA(0.5) 0.00 0.00 0.00 NAA(0.5)+BA(1.0) 0.00 0.00 0.00 NAA(1.0)+BA(0.5) 30.00 1.25 30.00 NAA(1.0)+BA(1.0) 0.00 0.00 0.00 Mean 16.82 0.49 6.77 L.S.D. 5% 8.20 2.03 0.35

Table 5: Effect of different soil mixtures on plant acclimatization of Bamboo . Characters Plantlet Root length No. of leaves/ shoot Length (cm) (cm) Treatments Peat moss 5.30 8.20 9.00 Peat +sand 11.60 12.80 11.80 Peat+ vermiculate 9.20 12.30 12.00 Peat + sand + vermiculate 12.20 16.30 16.00 Mean 9.58 12.40 12.20 L.S.D. 5 % 3.90 3.11 2.81

Conclusion:

In this investigation, MS medium and middle explant part were superior to other media and other explant parts, which recorded the highest significant values in all morphological characters. Moreover the combination between BA (1.0mg/L) + NAA (0.1 mg/L), added to MS medium allowed to improve the multiplication of plantlets and enhancing morphological characters. MS half strength medium supplemented with NAA (1.0mg/L) enhanced root formation as well as number and length of roots. Concerning with hardening of plants on soil mixture consisted, of peat + sand + vermiculate at rate of (1:1:1 v/v/v) gave the best results during acclimatization stage. This may be considered as a protocol to mass propagation of Bamboo (Denderocalamus giganteus) In Vitro.

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