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Clonal propagation of bamboo (Dendrocalamus strictus)

Bamboo grows naturally in many types of forests. About 50% of the annual production of bamboo in our country is used by various industries like pulp, paper, rayon, mat boards, besides agricultural implements. It is also used for making baskets, bridges, coffins, beds, toys and weapons. A grove of bamboo at ground zero in the area that destroyed Hiroshima in 1945 sprouted new shoots within months 1. The distribution of bamboo in India is largely governed by rainfall, temperature, and altitude and soil types. In recent years bamboos are in great demand; but nonavailability of sufficient quantity of saplings and seeds are the major problem. Dendrocalamus strictus is most commonly called as solid or lathi bamboo. The species is widely distributed in dry deciduous forests and grows rapidly in all climatic conditions. It grows better in the drier parts and on sandstone, granite and coarse grained soils with low moisture-retaining capacity and soils with pH 5.57.6. It grows more than 8 feet in 6months. The pulp is used for making quality paper and the clumps, being strong and elastic, are used for lathies, shafts, axe handles, walking sticks, agricultural, and industrial implements and therefore it is rightly called as poor mans timber. The major problem in the propagation of D. strictus is the erratic flowering and non-availability of seeds on regular basis, besides low viability of seeds. Moreover the seeds have to be stored in 3 to 5C after reducing the moisture (8%) or stored in a desiccator with anhydrous calcium chloride. Under these circumstances, other techniques of propagation become important. Development of novel methods of propagation by tissue culture techniques, micropropagation and macropropagation including clonal propagation is desirable for large-scale application. In recent years vegetative propagation techniques are standardized and adopted to improve self-incompatible bamboos with poor seed set2. Attempts to develop lowcost planting stocks of bamboo using culm-cuttings 3 and in vitro techniques 4,5 are of paramount importance in bamboo improvement. We describe here an efficient technique of clonal propagation of bamboo.
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Mature stem cuttings of 210 cm girth from 5 to 6 year-old plants were clonally propagated. The stem cuttings of 40 100 cm height with 37 nodes were directly planted in polybags. About 170 explants of different sizes were used in the present study. Mature explants (56 years old) were classified into four categories according to size, i.e. girth 23 cm, 36 cm, 69 cm and 912 cm. These explants were planted in different sizes of polybags with red soil + cocopit + farm manure (1 : 1 : 1). The polybags were placed in a mist chamber. Seeds collected were dehusked and washed thoroughly under running tap water for 20 min. Seeds were sterilized with Bavistin (0.1%) for 15 min and further treated with HgCl 2 (0.1%) for five min, finally washed with sterile distilled water 56 times and were inoculated initially on MS media supplemented with 2 mg/l 2,4-D.

The explants propagated through stem cuttings showed response in about two weeks. Nodal branching was observed and the pattern varied considerably depending on the basal or apical node (Table 1). Each stem cutting had four to six nodes (Figure 1). In general, younger axillary nodes gave better response when compared to the basal nodes of the same stem. The rooting pattern differed considerably depending on the age of the stem cuttings of explant. The older explants exhibited bushy type of rooting compared to top younger cuttings, where the tap root system with rhizomes were observed (Figure 2). The mature explants also exhibited tillering after 34 months presumably by rhizome. Since rhizomes can be selected and propagated as superior clones, they were separated from seedlings (Figure 3) grown in polybags and new seedlings were obtained. Culms are

Figure 1.

Clonal propagation of Dendrocalamus strictus through stem cuttings.

Figure 2.

Pattern of rooting of stem cuttings.

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Table 1. Girth (cm) 23 36 89 912 Height (cm) 5072 4087 5582 5283 Initiation of root/nodal branches of different stem cuttings Response (%) 71.1 75.5 67.7 71.1 Nodal branches/node 23 35 34 36 Nodal branches (average height) 17.8 13.1 19.1 83 Nodal branches/ node (average) 3 5 3 5

No. of explants 78 49 31 21

Figure 4.

Induction of multiple shoots from seeds in tissue culture.

Figure 3.

Propagation through rhizomes. Figure 5. Tissue culture rooted plantlets in polybags.

generally produced from young rhizomes, but under exceptional conditions, two-yearold rhizomes also produced culm-beds. More than one culm was found growing in one year from the rhizome which required turgescent conditions of high relative humidity. Dense shade and heavy weeds were detrimental to its growth. In this particular experiment 2000 rhizomes were transferred. The seeds inoculated on MS media germinated after 10 days. The embryos

were collected in sterile condition and inoculated initially on half strength MS media supplemented with 2 mg/l BAP. From the explant, multiple shoots (130 180) were derived in 4050 days and these shoots were subcultured for every 15 days for better multiplication rate (Figure 4). The shoots were then transferred onto the half strength MS medium with 2 mg/l IBA for rooting. After 15 days the rooted plants were transferred to polybags (Figure 5). This cost-effective technique in bam-

boo micropropagation has added advantage by rapid multiplication of superior germplasm efficiently. Clonal propagation through matured stem cuttings will provide definite advantage in immediate age transfer and will save time in propagation compared to seedlings. The added advantage is that the superior clones can be directly propagated. This technique can be adopted for large-scale plantation of wastelands and saline soils to meet the growing needs of D. strictus .
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1. Hutchinson, J., Readers Digest, April 2002, pp. 8388. 2. Koshy, K. C. and Gopalkumar, B., Curr. Sci., 2005, 89 , 14741476. 3. Kumar, R. and Pal, M., J. Bamboo Rattan , 2004, 3 , 401409. 4. Nadgauda, R. S., John, C. K., Joshi, M. S., Parasharami, V. A. and Mascarenhas, A. F. Plant, Cell, Tissue Organ. Cult., 1997, 48 , 181188. 5. Saxena, S. and Dhawan, V., Plant Cell Rep., 1999, 18 , 483443. 6. Mehta, Usha, Rao, I. V. R. B. and Mohan Ram, H. Y., In Proceeding of Vth International Congress of Plant Tissue and Cell Culture. Plant Tissue Culture, Tokyo, Japan, 1982. ACKNOWLEDGEMENT. I thank NATP, CRIDA, Hyderabad for financial support. Received 20 January 2006; revised accepted 25 August 2006

G. M. REDDY Prof. G.M. Reddy Research Foundation, 4-123/E, Swaroop Nagar, Uppal, Hyderabad 500 039, India e-mail: gmrrf@sify.com

Scale structure of a cobitid fish, Cobitis linea (Heckel, 1849) using different modes of SEM
Detailed structure of the fish scale can be helpful in the identification of fishes up to major groups 13, species levels 46, phylogeny7,8, sexual dimorphism9, age determination1013 and pathology of fish scale due to water pollution of the water body1416 and for growth studies 1726. Studies on the scale structure of commercial fishes 1721 have been undertaken and these studies have been successfully used for growth studies2226 and calculation of minimum harvestable size27 so that legal fishable size can be prescribed for the determination of old age in the commercial fishes11 and also the pollution status of the waterbody1416,28. Due to some subjective reasons, in the past most of the lepidological studies are made on the commercial fishes 11,1921,23,24,26,27. Recently it has been reported that due to loss of fish habitat as a result of water management practices, release of effluents into the natural waterbodies and several anthropogenic activities, most of the fish species are under different types of threats as is evidenced from the squeezing of geographical limits and the reduction in the stocks of most of the fish species 29. In some cases, the fish community structure is completely disrupted. Due to the reduction in the fish stocks, fish biologists are unable to get a large number of specimens for studies relating to fish bionomics. Under these circumstances, lepidological study is the best alternative as fish scale is considered to be the best tool in fish biology30,31. During a literature survey, it has been found that age and growth studies on minnows are rarely attempted and cobitids are completely ignored11. These
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two groups of fishes form one of the important links in the fish community structure in hillstreams3234, hence standardization of the scale structure of the fishes belonging to these two categories help in understanding their bionomics. Further, cobitids live at the bottom and are considered to be the natural scavengers. Their presence indicates that the water is regularly cleaned. Hence, their presence is encouraged in the fish aquaria for the natural cleaning of the glass walls. An attempt has been made here to study the ultrastructure of the scale of an cobitid, Cobitis linea (Heckel, 1849) employing different modes of SEM collected from the streams of south-west Iran. In Iran, this group of fishes is generally known as sag mahi, meaning dogfish in Persian and equivalent to loach in English. The fishes were collected by one of the authors (HE) using cast-net, scoop-net and ordinary insect net having mesh size not more than 5 mm during March 2005. The scales were gently removed with fine forceps from the left side of the body between the dorsal fin and lateral line in such a way that while removing the scales no damage is done to the scale. Immediately after their removal, the scales were cleaned mechanically using a fine brush and rinsed with triple distilled water. The cleaned scales were dehydrated in 30, 50, 70% ethanol and dried on filter paper. The scales were not put in absolute alcohol as 100% ethanol causes the scale margins to curl. To avoid curling, after 70% ethanol, the scales were kept between the microslides for 23 days. The cleaned and dried scales were mounted on metallic stubbs by double adhesive

tape with dorsal surface upward and ventral surface sticking to the tape and coated with a 100 thick layer of gold in a gold coating unit. The gold coating overcomes the problem of charging and beam damage. An additional advantage of gold coating is to improve the strength of secondary electron signals from the specimens surface. The scales were viewed under vacuum in a JEOL JSM6100 scanning electron microscope at an accelerating voltage of 20 kV at low probe current. Various images of the scales were recorded on the photographic film. When goldcoated scales were not being viewed, the stubbs were stored in a desiccator to avoid moisture. The different modes of SEM such as secondary electron image (SEI, low energy electrons), back scattered electron image (BEI, high energy electrons), mixed signals of both SEI and BEI and reverse polarity (negative of mixed signals) have been employed for the study of scale morphology35 (Figure 1 ad). For the details of the circuli, X and Y modulations have been use 35 (Figure 1 ef ). The length and breadth of the scale of C. linea (Heckel, 1849) from Iranian freshwaters is almost the same having nearly circular shape (Figure 1 ad). The scales maintain the same morphological proportions located on the different parts of the body. The scales present below the dorsal fin, above the lateral line are largest and those on the other parts of body are smaller in size. As the scale from this region depicts all the features, this scale has been designated as key scale. The distinct focus which lies in the posterior part of the scale divides the scale into an-

CURRENT SCIENCE, VOL. 91, NO. 11, 10 DECEMBER 2006