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In Vitro techniques for the proliferation of axillary shoots to establish complete plants of Artocarpus chaplasha Roxb.

INTRODUCTION

Artocarpus chaplasha Roxb. is a tall deciduous plant of about 40 m in height and 3.0-4.5m in girth from the family of Moraceae. It is found scattered in the moist deciduous and evergreen forests. In Bangladesh, it is mainly distributed in Chittagong and Chittagong hill tracts. Recently it is planted in different areas of the country. It requires a well drained loamy soil and should not be planted in areas that are water logged. In its natural habitat the maximum shade temperature varies from 35-40[degrees]C and absolute minimum from 3-16[degrees]C, and the normal rainfall is 200-500cm [5].

The wood is yellow brown to golden. It is light 480-512 kg/[cm.sup.3] and seasons well without any serious defect. It is regarded as first class general utility timber and extensively used for various purposes. For rapid height growth it is good for pole production; it can also be used for beams of houses, rafters, purloins, trusses and columns [5]. The lumber of Chaplasha is recommended as substitute of Teak and Shal after suitable treatment and preservation treatment. It is resistance to white ant and insect attack so it is first choice for frame work, door and windows. It works well by hand or machine finishing to a good lustrous surface. It's straight and shallowly interlocked grained and even-textured. It takes satisfactory polish, so it is used for making various furnitures.

Advantages of In vitro propagation:

In recent years, propagation of numerous woody plants by tissue culture has become accepted for commercial practice. One of the most important problems in woody perennials is the establishment of high quality propagules for regeneration. In vitro vegetative propagation has a number of advantages over the sexual one in a large scale reforestation program [1,27,8]. These are-(1) superior gene combinations are propagated unaltered by cloning superior trees, which are lost through genetic recombinations in sexual method; (2) Improvement of gene pool of planting stock in sexual means is slow because of long breeding cycles of trees whereas with cloning it is easy and fast to get a true to mother type of a desirable clone; (3) Genetic uniformity of a clone is maintained; (4) In some species clonal propagules initially grow much faster than seedlings; (5) Often the juvenile phase of development can be by passed if desired and some valuable hybrid and polyploid trees are infertile in general, but can be propagated by cloning through tissue culture; (6) In a relatively short time and space a large number of plants can be produced starting from a single individual.

Most forest species do not readily root from cuttings. The standard method for their propagation is seedling. However there are numerous problems associated with seed propagation: (a) It is extremely difficult to obtain large quantities of seed for many economically important species [13]; (b) While heterosis is one of the hybrid qualities that should be taken advantage during material selection for reforestation, the difficulty in mass cross pollination of forest species makes the cost of hybrid seed production too high to be practical; (c) Tree species have long generation times which prohibit carrying out the type of breeding program used in annual crop plant; (d) Seeds lose their viability during storage.

Problems in micropropagation of woody plants:

Compared to herbaceous plants, the micropropagation of woody species has lagged far behind. It is usually more difficult to establish shoot cultures from mature trees than from juvenile plants (Bonga, 1987; Hackett, 1987). The greatest difficulty is experienced in rooting, especially when explants are taken from mature trees. Difficulty during the primary culture establishment is also frequently encountered. This is partially due to the existence of large quantities of phenolic compounds in the tissue of many woody species and partially due to the difficulty of breaking the physiological quiescent state of the axillary buds. Another difficulty is contamination of cultures especially when explants are taken from mature trees.

Browning of explants during in vitro culture:

Many plants are rich in polyphenolic compounds. After tissue injury during dissection such compounds will be oxidized by polyphenoloxidases and tissue will turn black or brown. The oxidation products are known to inhibit enzyme activity, kill the explants and darken the tissue and culture media. Such phenomena impose a serious block on the establishment of primary cultures, especially in woody plants. Some of the procedures used by various workers to combat this problem are: (1) Adding antioxidants like ascorbic acid, polyvinyl pyrrolidone (PVP) dithiothreitol or bovine serum albumin into culture medium (McComb and Newton, 1981); (2) Presoaking explants in antioxidant before inoculating culture [In Tectona grandis bud culture, explants were suspended in various solutions of different antibrowning agent, performed by Gupta et al, [11]; (3) Incubating the initial period of primary cultures in reduced light or darkness; (4) Frequently transferring the explant into fresh medium whenever browning of the medium is observed.

Disinfection of explants for in vitro culture:

Complete disinfection of many woody species can be extremely difficult, especially when the explants are taken from the mature plant collected from field. Explants must be free from microorganisms when placed on nutrient media and this has usually been achieved by surface sterilization with solutions of sodium or calcium hypochloride, mercuric chloride, or 75-90% ethanol. But such treatment has failed with some adult trees, presumably because of penetration of tissue by microorganism [10,15].

Objectives of the experiment:

Artocarpus chaplasha Roxb. produces seeds but seeds lose their viability only 5-7 days after removal from fruits. There is no report on vegetative propagation of the tree. Application of in vitro propagation techniques could play an important role in propagation and conservation of such an unexploited tree. However the plants propagated from seeds show wide range of variability in its internal physiological state and plant obtained from seeds may not give desired off springs. The objective of this study was to formulate a protocol for micropropagation of Artocarpus chaplasha Roxb. in order to obtain a 'true to type' plant genotype.

Material and Methods

Initiation of aseptic culture:

Explants were collected from rejuvenated tissues of mature Artocarpus chaplasha plants. Rejuvenation was induced after 25-30 days of shoot pruning of mature twigs. The shoot tips and nodal segment were cut and collected in a beaker containing water to avoid desiccation, then brought to the laboratory.

Surface sterilization:

For surface sterilization the shoots were defoliated and apices of two three nodes were taken. They were then washed thoroughly under running tap water for 30-40 minutes, washed with liquid detergent for another 15-20 minutes, and then with a solution of 5% W/V Aseptic Savlon for 10 minutes. After repeated washing with distilled water the explants were finally treated with 0.2% Hg[Cl.sub.2] for 10 minutes in the laminar air flow cabinet and washed for three times with autoclaved double distilled water.

Culture method:

After surface sterilization, shoot apices were excised at the base and divided into pieces of the explants of size 25-30 mm with at least one node in each explant. The basal medium used for all the experiment were Murashige and Skoog [18] mineral formulation, MS containing standard salts and vitamins, 3% sucrose and 0.7% agar (W/V). The media were supplemented with benzyl aminopurine (BAP), kinetin (KIN) either individually or in combination with auxins, naphthalene acetic acid (NAA) and indole acetic acid (IAA). Coconut water was added to the medium at different concentration. Polyvinyl pyrrrolidone (PVP) was also added to avoid the existence of phenolic compounds. The pH of the medium was adjusted to 5.8 before adding agar and the media were then autoclaved (1.1kg/[cm.sup.2] for 20 minutes at 120[degrees]C).

Incubation:

The culture tubes or flasks were incubated in light of 12 hour duration and 12 hour duration of dark at 25 [+ or -] 1[degrees]C and the light intensity of the growth chamber was 2000 Lux. The cultures were regularly subcultured at four-week intervals. Observation was recorded over 5 days of inoculation and subculture. All experiments were repeated twice with at least ten cultures per treatment.

Multiplication of shoots:

The primary shoots regenerated from explants cultured in MS basal medium supplemented with 2.5 mg/L BAP + 0.5 mg/L NAA were isolated and subcultured on same media for four weeks. For the further improvement of the media, 20% (v/v) coconut water + 300 mg/L Casein Hydrolysate were used.

Rooting:

For rooting 25-30 mm long shoots were excised from multiplication cultures and implanted to rooting medium consisting of half strength of MS macro and micro nutrients in the glass tubes. The media were supplemented with IAA and ABA (0.5-1.5mg/l) either alone or in combinations.

Transplantation:

Rooted shoots of four-weeks-old cultures were transferred to pots after in vitro hardening. For in vitro hardening the culture tubes were kept in a room at ambient temperature (30 [+ or -] 2[degrees]C) and normal day light for 7 days. Plantlets were then taken out from the culture tubes, the agar was washed out from the root and plantlets transplanted to small trays (each tray contained ten plantlets) filled with compost, soil and sand (1:1:1). The trays were kept in a shady place, covered with transparent polythene sheet and were watered daily. After 2 or 3 weeks when the plants were fully acclimatized to the outdoor conditions, they were again transplanted individually in clay pots containing compost and soil (1:1) and watered on alternate days.

Results and Discussion

Initiation of shoot culture:

The surface sterilization procedure described in Material and Methods yielded 90% aseptic cultures. MS medium containing 3.0 mg/L KIN + 0.5 mg/L NAA showed 64% shoot induction with 5.8 shoots per culture, whereas MS medium containing 2.5mg/L BAP + 0.5mg/L NAA showed highest 75% shoot induction with 6.4 shoots per culture.

BAP-induced shoot proliferation from the shoot apices has also been reported in Artocarpus heterophyllus [3]. A combination of BAP and NAA resulted in proliferation of shoots up to 5.2 [+ or -] 0.17 cm in Artocarpus heterophyllus [21]. Similar result was described by Roy et al. when they used 2.5mg/l BAP with 0.5 mg/L NAA in micropropagation of Artocarpus heterophyllus. Adiga [2] had also obtained similar results in jackfruit. Roy and Datta [22] used BAP and kinetin for micro-propagation of Albizia procera tree.

Shoot Multiplication:

For further improvement of the medium, casein hydrolysate (CH, 50-300 mg/L) and coconut water (5-20%) were added individually or in combination to the medium. Thus the medium established was MS basal medium with BAP 2.5mg/L + NAA 0.5mg/L + 20 % CW + 300 mg/L CH. When sub-cultured to this medium, the shoots continued to proliferate (10 shoots per transfer). In a number of plant species regeneration improvement was achieved by augmenting culture medium with coconut water [7,14,16].

Nasib et al. [19] found number of shoots, shoot length and number of nodes to be increased when 20% coconut water was added to the medium for Actinidia deliciosa plant.

According to Quraishi et al. [20], addition of adenine sulfate appears to supplement cytokinin requirement of the nodes that is not fulfilled by BAP or kinetin. The nodes placed on MS supplemented with 0.44 microM BAP and 25.0 mg/l CH showed shoot number 1.2 [+ or -] 0.0, shoot length (mm) 12.6 [+ or -] 0.81 and number of nodes per micro shoot to be 2.0 [+ or -] 0.16.

Saxena and Dhawan [25] used 100mg/L casein hydrolysate in the medium as additives for the propagation of Anogeissus pendula. Deb [9] used 200 mg/l of casein hydrolysate for the induction of embryogenic callus from 3-4 days imbibed seeds of Melia azedarachta.

Rooting:

Well developed shoots, excised from the culture flask were implanted individually on root induction medium. 1.0 mg/L IBA+0.5mg/L NAA was found to be the best combination of auxins for proper rooting in which 80% shoots rooted within 4 weeks of culture.

A hundred percent rooting efficiency has been recorded in jackfruit with IBA and NAA (1.0 mg/L each) [3].

Transplantation:

About 75% of the transplanted plants survived when the plants in rooting culture tubes were kept at normal room temperature for 7 days before transplanting in trays where plants were reared for three weeks. At the time of rearing shoots elongated and the plants were very healthy. After transplanting in the clay pots, plants grew more vigorously.

References

[1.] Abbot, A.J., 1978. Practice and promise of micropropagation of woody species. Acta Horticulturae, 79: 113-127.

[2.] Adiga, D.J., 1996. Clonal Propagation of Jackfruit (Artocarpus heterophyllus Lam) cv. Singapore Jack through tissue culture. Current Science, 78: 1231-1234.

[3.] Amin, M.N., 1992. In vitro enhanced proliferation of shoots and regeneration of plants from explants of jack. Plant Tissue Culture, 2: 27-30.

[4.] Amin, M.N., and V.S. Jaiswal, 1993. In vitro response of apical bud explants from mature tree of Jackfruit (Artocarpus heterophyllus). Plant Cell Tissue and Organ Culture, 33: 59-65.

[5.] Anonymous, 1985. The wealth of India. In: Raw Materials, vol-1 : A Dictionary of Indian Raw materials and Industrial products. Publication and Information Directors, CSIR, New Delhi. pp: 444-453.

[6.] Bonga, J.M., and D.J. Durzan (Eds), 1987. Cell Tissue culture in Forestry, Vols 1, 2 and 3. Martinus Nijhoff Publishers, Dordrecht/Boston/Lancaster.

[7.] Bose, M.R., S. Wright, and P.L. McLeay, 1993. Coconut milk enhancement of auxillary shoot growth in vitro of Kiwifruit. New Zealand Journal of Crop and Horticultural Sciences, 21: 171-176.

[8.] Boulary, M., 1980. La micropropagation des arbres forestiers. Comptes Rendus des Seances de I Academic de Agriculture de France, 66(8): 697-708.

[9.] Deb, C. R., 2001. Somatic embryogenesis Plantlets regeneration of Melia azedarach L. (Ghora neem) from cotyledonary segments. Journal of Plant Biochemistry and Biotechnology, 10: 63-65.

[10.] Franclet, A., 1979. Rejeunissement des arbres adultes en vue de leur propagation vegetative. In: Annales de Recherches Sylvicoles, AFOCEL. Etudes et Recherches No. 12, 6/79. Micropropagation d. Arbres Forestiers, 3-18.

[11.] Gupta, P.K., A.F. Mascarenhas, and V. Jagannathan, 1981. Tissue culture of forest trees: Clonal multiplication of mature trees of Eucalytus citriodora Hook. by tissue culture. Plant Science Letters, 20: 195-201.

[12.] Hackett, W.P., 1987. Juvenility and maturity In: J.M Bonga and D.J Durzon (Eds) Cell and tissue culture in forestry, Lancaster pp 216-231.

[13.] Hu, C.Y., 1979. Propagation of Sassafras canadiense (Hay). Rehd. Taiwan Forestry Journal, 5: 30-31.

[14.] Jayasih, Y., and D.I.G. Wattimeana, 1994. The effect of coconut water and Zeolite on micropropagation of potato shoot. Acta Horticulturae, 369: 451-454.

[15.] Jones, O.P., M.E. Hopgood, and D. O'Farrell, 1977. Propagation in vitro of M. 26 apple rootstocks. Journal of Horticultural Science, 52: 235-8.

[16.] Mandal, A.B., M. Aarna, and R. Elanchezhian, 2002. In vitro micropropagation of Ananas comosus L. (Merr.)Var. Qeen. Journal of Applied Horticulture, 4:107-112.

[17.] Mc Comb, T.A., and S. Newton, 1981. Propagation of Kangaroo paws using tissue culture. Journal of Horticultural Sciences, 56: 181-183.

[18.] Murashige, T., and F. Skoog, 1962. A revised medium for rapid growth and bioassay with tobacco tissue culture. Physiologia Plantarum, 15: 473-497.

[19.] Nosib, K., K. Ali, and S. Khan, 2008. An optimized and improved method for the in vitro propagation of Kiwifruit (Actinidia deliciosa) using coconut water. Pakistan Journal of Botany, 40: 2355-2360.

[20.] Quraishi, A., and S.K. Mishra, 1998. Micropropagation of nodal explants from adult trees of Cleistanthus collinus. Plant Cell Reports, 17: 430-433.

[21.] Roy, P.K., M.A. Rahman, and S.K. Roy, 1996. Clonal propagation of Syzygium cumuni. Bangladesh Journal of Botany, 25: 159-164.

[22.] Roy, S.K., and S.K. Datta, 1985. Clonal propagation of a legume tree Albizia procera through tissue culture. Bangladesh Journal of Botany, 14: 127-131.

[23.] Roy, S.K., M.S. Islam, J. Sen, and S. Haiduzzaman, 1993. Propagation of flood tolerant jackfruit Artocarpus heterophyllus by in vitro culture. Acta Horticulturae, 336: 273-276.

[24.] Roy, S.K., and S. Hadiuzzaman, 1991. Micropropagation of two species of Artocarpus through in vitro culture. Bangladesh Journal of Botany, 20: 27-32.

[25.] Saxena, S., and V. Dhawan, 2001. Large-scale production of Anogeissus pendula and A. latifolia by micropropagation. In Vitro Cellular and Developmental Biology - Plant, 37: 586-591.

[26.] Sen, J., M.S. Islam, S.K. Roy, and S. Hadiuzzaman, 1992. Micropropagation of juvenile and adult Gmelina arborea. Plant Tissue Culture, 2: 89-95.

[27.] Vasil, L.K., M.R. Ahuja, and V. Vasil, 1979. Plant tissue cultures in genetics and plant breeding. In: Advances in Genetics, 20: 127-215.

Md. Atiqur Rahman, Farhana Rahman, A.K.M. Mahmudul Haque, Md. Meherab Hossain, Mohammed Rahmatullah

Faculty of Life Sciences, University of Development Alternative, Dhanmondi, Dhaka-1209, Bangladesh

Received: 25 June 2014; Received: 8 July 2014; Accepted: 25 July 2014; Available online: 20 August 2014

Corresponding Author: Dr. Mohammed Rahmatullah, Pro-Vice Chancellor University of Development Alternative House No. 78, Road No. 11A (new) Dhanmondi R/A, Dhaka-1209 Bangladesh

Phone: 88-01715032621; Fax: 88-02-8157339; E mail: rahamatm@hotmail.com

Table 1: Effect of growth regulators in MS medium on
shoot induction, number of shoots per explants and
length of longest shoot of Artocarpus chaplasha.

Plant        Days to      % of            No of       Length of
growth       Shoot        shoot           shoot per   longest
regulators   initiation   proliferation   culture     shoot (cm)

Without growth regulators

0.0          --           05              0.4 (.11)   0.05 (.09)

BAP (Mg/L) 0.5

0.5          30-35        50              2.0 (.50)   3.04 (.12)
1.0          30-32        52              2.2 (.24)   3.08 (.12)
2.0          26-28        55              3.4 (.37)   3.20 (.13)
2.5          25-28        60              3.8 (.50)   3.40 (.14)
3.0          20-25        64              4.2 (.41)   3.60 (.11)

KIN (Mg/L)

0.5          32-36        50              1.8 (.37)   3.16 (.13)
1.0          32-34        52              2.0 (.31)   4.02 (.13)
2.0          28-30        55              2.6 (.31)   3.30 (.09)
2.5          25-28        58              3.8 (.37)   3.40 (.14)
3.0          22-24        60              4.0 (.24)   3.50 (.14)

BAP + NAA (Mg/L)

1.0+0.1      30-35        20              2.0 (.40)   3.02 (.12)
2.0+0.1      30-32        40              3.0 (.37)   3.40 (.24)
2.5+0.1      26-28        60              5.0 (.24)   3.00 (.14)
3.0+0.1      30-32        50              4.0 (.40)   3.60 (.31)
1.0+0.5      30-32        35              3.0 (.31)   3.80 (.24)
2.0+0.5      20-22        55              2.5 (.21)   4.00 (.12)
2.5+0.5      18-20        75              6.4 (.20)   5.00 (.12)
3.0+0.5      26-28        70              3.0 (.31)   4.12 (.12)
2.0+1.0      30-32        60              2.8 (.24)   3.20 (.12)
2.5+1.0      25-28        55              3.2 (.40)   3.60 (.15)
3.0+1.0      30-35        25              2.0 (.21)   3.40 (.11)

KIN+NAA (Mg/L)

1.0+0.1      30-35        20              2.0 (.20)   2.22 (.12)
2.0+0.1      30-32        30              3.0 (.31)   2.60 (.24)
2.5+0.1      26-28        45              4.8 (.37)   3.00 (.12)
3.0+0.1      30-32        40              3.4 (.40)   3.60 (.15)
1.0+0.5      30-32        30              2.2 (.21)   4.00 (.11)
2.0+0.5      30-32        50              2.9 (.37)   4.10 (.12)
2.5+0.5      28-30        55              4.0 (.31)   4.20 (.11)
3.0+0.5      22-24        64              5.8 (.21)   4.80 (.15)
2.0+1.0      30-32        54              2.2 (.31)   3.00 (.11)
2.5+1.0      32-34        50              2.0 (.21)   3.20 (.14)
3.0+1.0      25-28        25              4.0 (.37)   4.10 (.12)

Standard error is shown in parentheses

Table 2: Statistical analysis of different treatments
on various parameters.

Treatment                  Percent       No of         Length
                           of shoot      shoot         of
                           regenerated   regenerated   longest
                           per           per           shoot
                           explants      explants      (cm)

1           3.0 Mg/LBAP    64 (a)        4.2 (a)       3.60 (a)

2           3.0 Mg/L KIN   60 (a)        4.0 (a)       3.50 (a)

3           2.5 Mg/L BAP   75            64            5.00 (b)
            + 0.5 Mg/
            LNAA

4           3.0 Mg/L KIN   64 (a)        5.8 (c)       4.80 (b)
            + 0.5 Mg/
            L NAA

Values within the same column with same letters are not
significantly different at 5% level of significance by DMRT.
Data Analysis was done by SPSS 15.0 Computer Software.

Table 3: Effect of growth regulators in 1/2 strength
MS medium on root induction, number of roots per
shoot and days to root initiation from shoots of
Artocarpus chaplasha.

Plant growth   Days         % of            Number
regulators     to root      root            of roots
               initiation   proliferation   formed
                                            per
                                            shoot

Without growth regulators

0.0            --           No root         No root

IBA (mg/l)

0.5            32-34        65              3.2 (.15)
1.0            30-32        60              3.0 (.12)
1.5            35-40        50              2.8 (.09)

IAA ( mg/l)

0.5            30-32        58              3.0 (.21)
1.0            30-32        45              2.6 (.24)
1.5            48-50        35              2.5 (.11)

IBA + NAA ( mg/l)

0.5+0.5        30-35        60              3.5 (.19)
1.0+0.5        26-28        80              4.6 (.21)
1.5+0.5        28-30        55              3.4 (.21)
0.5+1.0        30-32        25              3.0 (.37)
1.0+1.0        34-36        30              3.5 (.12)
1.5+1.0        40-42        10              2.5 (.24)

IAA+NAA (mg/l)

0.5+0.5        32-34        40              3.5 (.21)
1.0+0.5        30-32        50              3.4 (.11)
1.5+0.5        36-38        25              3.2 (.31)
0.5+1.0        32-34        25              2.0 (.24)
1.0+1.0        30-32        15              2.1 (.19)
1.5+1.0        45-48        10              1.2 (.12)

Standard error is shown in parentheses
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Title Annotation:Research Article
Author:Rahman, Atiqur; Rahman, Farhana; Haque, A.K.M. Mahmudul; Hossain, Meherab; Rahmatullah, Mohammed
Publication:American-Eurasian Journal of Sustainable Agriculture
Article Type:Report
Geographic Code:9BANG
Date:Jun 1, 2014
Words:3643
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