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Tissue Culture of Artocarpus heterophyllus L., an Underutilized Fruit of Bangladesh

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Tissue Culture of Artocarpus heterophyllus L., an Underutilized Fruit of Bangladesh
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   1 Tissue Culture of Artocarpus heterophyllus   L., an Underutilized Fruit of Bangladesh F.M. Safiul Azam and Mohammed Rahmatullah Department of Biotechnology & Genetic Engineering University of Development Alternative Dhanmondi, Dhaka, Bangladesh.   Ather-uz-Zaman   Centre for Plant Tissue Culture, PROSHIKA, Dhaka, Bangladesh Key words : Jackfruit, clonal propagation, synergistic effect, malnutrition improvement Abstract Jackfruit ( Artocarpus heterophyllus  ) is available in Bangladesh between June to August; however, a variety exists that fruits throughout the year. The edible portion is considered as a good source of carbohydrate, proteins, vitamins and minerals. In Bangladesh, malnutrition persists among 35-45% of the population and child malnutrition rate is more than 50%. Under this circumstance, wide cultivation of a Jackfruit variety that bears fruit throughout the year can play a significant role towards reducing malnutrition in Bangladesh. Since this particular variety is not widely available, we have developed a method for its clonal propagation. Healthy and juvenile shoot tips and nodal segments were collected from field-grown fruit bearing trees and cultured in Murashige-Skoog (MS) media fortified with different concentrations and combination of 6-benzyleaminopurine and Kinetin following the sterilization with 0.1% HgCl 2 . Sprouting and regeneration of micro shoots geared up when MS was enriched with 3.5 mg/l BAP. Proliferation frequency increased considerably and multiple shoots were regenerated as a clump of 2-3 shoots in pH 5.8 media within four weeks of inoculation through synergistic effect of 6-benzyleaminopurine (3.5 mg/l) and Kinetin (1.5 mg/l). With the increase of subculture (up to 10 th  maximum), frequency of shoot proliferation was enhanced. Addition of 0.1 mg/l Indole-3-acetic acid and 20% coconut water considerably increased shoot elongation and stimulated growth of the shoots. About 80% rooting frequency were observed in ½ MS medium with 1.2 mg/l Indole-3-butyric acid. Rooting percentage and their growth were much better in liquid media. After proper acclimatization, rooted plantlets were transferred to polythene bags containing garden soil, sand and cowdung (1:1:1). After eight weeks of transplantation in open-field more than 80% plants were survived. No morphological variants were observed during the passage of clonal propagation. This clonal propagation technique could play a significant role in improving productivity leading to reduced malnutrition.   INTRODUCTION  Jackfruit (  Artocarpus heterophyllus L.) belongs to the family Moraceae and enjoys the crown as national fruit of Bangladesh. The annual production of jackfruit is around 279.5 thousand metric tons per year in Bangladesh. The edible portions are the pulp and seeds, which are considered as a good source of carbohydrate, proteins, Vitamins B 1  and B 2  and minerals (Morton, 1966; Burkill, 1997). Jackfruit is one of the cheapest fruit and referred to as ‘the poor man’s food’ in Bangladesh (Rahman et al., 1995). A family having five to six   2 members can easily consume one jackfruit at a time and satisfy their hunger. It has been reported that one variety exists in Bangladesh which yield fruits throughout the year. Propagation of jackfruit plant from seeds is not widely accepted because of high heterozygosis (Singh, 1986; Samaddar, 1990). Clonal propagation through grafting of selected genotype is highly desirable but the number of plants produced by these conventional methods is relatively low (Rowe-Dutton, 1976; Samaddar, 1990). The application of tissue culture methods for improvement and large-scale propagation of fruit trees has been well demonstrated (Litz et al., 1985; James, 1988). Successful in vitro   propagation of jackfruit seedlings has been demonstrated (Rahman & Blake, 1988). Regeneration of plantlets from bud and nodal explants of mature jackfruit trees has been reported (Jaiswal and Amin, 1990; Roy et al., 1990). Bangladesh has very high child malnutrition rates. The most recent survey results revealed that 50.5% children aged <5 years are stunted for age and 42.7 % children aged <5 years are underweight for age. Thus jackfruit, especially the variety which is available throughout the year can serve as an efficient means to reduce malnutrition in Bangladesh. The present research study was investigated to develop an efficient protocol using tissue culture technique for the large scale propagation of this year round fruiting jackfruit variety through examining the effects of growth regulators and Murasighe and Skoog (MS) medium (Murashige and Skoog, 1962). MATERIALS AND METHODS This experiment was carried out at the Plant Tissue Culture Laboratory of PROSHIKA, Bangladesh during 2006 to 2007. Explant Collection and Sterilization.  50-70 explants were collected for each experiment from 10-15 day-old newly sprouted healthy shoots from 10-12 years-old fruit  bearing trees growing at the Bangladesh Agricultural Research Institute (BARI). The actual number of explants used for any particular experiment is detailed in the accompanying tables. After removal of expanded leaves, the shoot apices and nodes were washed thoroughly under running tap water for 30 minutes and then treated with Tween 80 plus Savlon for 10 minutes and again rinsed with distilled water. The explants were surface disinfected with 0.1% HgCl 2  (for 5 to 15 minutes) followed by seven times rinsing with sterile distilled water in a laminar air flow cabinet. The explants were finally trimmed, prepared and cultured on to the medium. Shoot Proliferation Stage. Explants were implanted vertically on MS medium supplemented with different (0.2, .05, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 mg/l) concentrations of 6-benzylaminopurine (BAP). All media were supplemented with 3% sucrose and 0.8% Difco Bacto-agar. The pH was adjusted to 5.8 prior to autoclaving the media at 1 kg pressure per cm 2  and 121 o C for 20 minutes. All culture vessels were incubated in a growth room at 26 ± 2 o  C with 16 hrs light provided by Philip white fluorescent tube lights with a photon flux density of 300 lux. Multiple Shooting. In a second experiment,  jackfruit explants were cultured on MS medium fortified with different concentrations and combinations of BAP (3.0, 3.5, 4.0 mg/l) and Kinetin (Kn) from 0.2 to 3.0 mg/l to observe the synergistic effect on multiple shoot formation.   3 Root Induction.  For Root induction, micro-shoots were cultured on ½ MS medium enriched with different concentrations (0.2 to 2.0 mg/l) of Indole-3-butyric acid (IBA). Acclimatization.    In vitro  derived plantlets were gradually hardened through gradual exposure to optimum relative humidity and sunlight for seven days and then transferred to net house. Plantlets were taken out from the culture vessels/tubes, washed carefully under running tap water to remove any traces of agar. Each plantlet was transplanted to small poly  bag containing different composition and combination of soil media to select the best formulation. The soil compositions are shown in Table 3. The plantlets were immediately covered with perforated polythene bag to prevent desiccation. Open Field Observation. Acclimatized plants were observed for proper establishment in soil for fifteen days and field observation was done from January`07 to February`07 and data were recorded in 1-week intervals. Data Assemblage. Weekly growth observations were made up to four weeks after establishment and experimental data were recorded. The parameters were as: i.   Shoot length in cm and number of leaves/shoot ii.   Average number of multiple shoot/explant iii.   Average length of multiple shoots in cm iv.   Average number of root/shoot v.   Average root length in cm. Recorded data were analyzed as mean ± SE according to Mian and Miyan (1984). RESULTS AND DISCUSSION   Surface Sterilization.  A survival rate of 82.22% was observed when the excised explants were surface sterilized with 0.1% HgCl 2 solution for 10 minutes. In previous studies, 0.2% HgCl 2 for 5 minutes was used to treated shoot apices and axillary buds of  A. chaplasha Roxb and  A. heterophyllus Lamk (Roy and Hadiuzzaman, 1991). In a preliminary experiment it was observed that lower concentration of 0.1% HgCl 2  was suitable for sterilization, but  prolonged treatment (more than 10 minutes) manifested in vitro  tissue killing. In another study, 0.1% HgCl 2 for 5 minutes was used for surface sterilization process (Amin, 1992a). In the present study we therefore followed the method of Amin (1992a) except that surface sterilization with 0.1%   HgCl 2 was increased from 5 to 10 minutes. It was observed that this treatment gave better surface sterilization with good survival rates.  Shoot Proliferation. Highest frequency (92.30%) of sprouting and adventitious shoot  proliferation was observed when implanted on MS medium supplemented with BAP at 3.5 mg/l (Figures 1 and 2) and the highest length of shoot was 3.81 cm. (Table 1). The higher concentrations of BAP produced multiple shoots but excessive amount (BAP 4.5 mg/l) had deteriorating effect on shoot proliferation resulting in curled leaves. BAP induced shoot  proliferation from the shoot apices has also been reported in  Artocarpus heterophyllus (Amin and Jaiswal, 1993). However the shoot proliferation rate as observed in the present study was much higher than that reported earlier for jackfruit (about 80% bud explants produced deep-green, mossy inflorescence at the axils of basal nodes) (Amin, 1992a). A combination of BAP and NAA resulted in proliferation of shoots up to 5.2 ± 0.17 cm in  A. heterophyllus  (Roy and Hadiuzzaman, 1991). Superiority of BAP over other cytokinins has been recorded in other tree species (Zaman et al., 1993; Pattnaik and Chand, 1997) .     4 Multiple shoot formation.  After the shoot proliferation phage; for multiple shoot  proliferation, BAP at 3.5 mg/l was used along with Kn at different concentrations (0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l) to observe any synergistic effect (Table 1). Highest percentage (58.06%) of multiple shoot proliferation was observed at Kn (2.0 mg/l) + BAP (3.5 mg/l) having 2 shoots/explant with 1.54 cm shoot length. However, with a decrease of Kn (1.5 mg/l) the percentage of multiple shoot proliferation was decreased (47.50%), but on the other hand number of multiple shoot (3 shoots/explant) was increased (Figure 3). Addition of IAA 0.1 mg/l and 20 % coconut water showed green and expanded leaves. In Tectona grandis  (Gupta et al., 1980) both BAP and Kn were observed to be essential. The superiority of BAP over other cytokines for multiple shoot formation in hardwoods has also been reported (Vieitez and Vieitez, 1980). Root induction.  All concentrations of IBA induced roots, but maximum response (80%) and healthy roots (6.17 roots/shoot and length 3.2 cm) were noticed when shootlets were cultured on ½ MS enriched with IBA 1.2 mg/l (Table 2 and Figure 4). Any increase of concentration of IBA (>1.5 mg/l) induced callus formation at the base of the shoots and showed leaf necrosis. Omission of agar from rooting medium was showed same response. IBA 2.0 mg/l showed about 68% rooting and 83% rooting efficiency has been recorded in  jackfruit with IBA and  -naphthalene acetic acid (NAA), (2 mg/l each) (Amin, 1992b). Healthy roots were induced in  A. heterophyllus  with 1.5 mg/l IBA (Roy and Hadiuzzaman, 1991). In jackfruit, 90% rooting was achieved on a media consisting of ½ MS with IBA and IAA (1.0 mg/l each) (Islam et al., 1993). It was observed that 1-2 mg/l each of IBA and NAA induced maximum rooting (Jaiswal and Amin, 1990) where as the present investigation showed maximum response only using IBA. Hardening & Acclimatization.  After hardening, the in vitro raised plantlets were transplanted in to the potted media and were kept in a net house. After 2-3 weeks of transplantation, highest survival rate (80%) was achieved where the planting media was garden soil, sand and cowdung (1:1:1) (Table 3). On the other hand, Islam et al., (1993) observed about 85% survival of jackfruit plantlets in the field. However, Amin et al., (1993) reported only 50% survival rate after transplantation. Field observation.  After 6-8 weeks, the in vitro  propagated plantlets resembled the general morphological characteristics of the mother plant but showed no detectable variation in growth on the basis of soil types (Table 3). Highest length (5.50 cm) and highest number of leaves (4.60) were observed from soil formula 3 (Figure 5). CONCLUSION With this experiment, a protocol for the micropropagation of a unique variety of  A. heterophyllus , which fruits throughout the year have been established from shoot tip and nodal explants. It may be noted that all previous studies were conducted with varieties of this species, which fruits for only two to three months during January till December of any given year. In our knowledge, this is the first successful description of micropropagation of this unique variety. Successful micropropagation of this variety can lead to widespread cultivation and for obtaining this fruit throughout the year, which in turn can lead to a cheap source of a nutritious food available to the general population and specifically to the   5 nutritionally impoverished population of Bangladesh and other countries where this variety can be cultivated. Literature Cited Amin, M.N. 1992a. In vitro enhanced proliferation of shoots and regeneration of plants from explants of Jackfruit trees. Plant Tissue Cult. 2: 27-30. Amin M.N. 1992b. In vitro rooting of Jackfruit (  Artocarpus heterophyllus) microcuttings derived from mature trees. Plant Tissue Cult. 2: 129-133. Amin, M.N. and Jaiswal, V.S. 1993. In vitro   response of apical bud explants from mature tree of Jackfruit (  Artocarpus heterophyllus ). Plant Cell Tiss. & Org. Cult. 33: 59  –  65. Burkill, H.M., 1997. The Useful Plants of West Tropical Africa. Vol. 4, 2nd Edn. Royal Botanic Gardens, Kew, pp: 160-161. Gupta, P.K., Nadgir, A.L., Mascarenhas A.F. and Jagannathan, V. 1980. Tissue culture of forest tree: clonal propagation of mature trees of Tectona grandis L. (Teak) by tissue culture. Plant Sc. Let. 17: 259-268. Islam M.S., Sen, J., Alam, N. and Roy, S.K. 1993. Propagation of Jackfruit (  Artocarpus heterophyllus ) Through Zygotic Embryo Culture In Vitro . Short Communications, Plant Tissue Cult. 3: 51-55. Jaiswal, V.S. and M.N. Amin. 1990. Micropropagation of Jackfruit. In: Abst. VII. Intl. Cong. Plant Tissue and Cell Culture, Amstertdam, Netherlands, p.107. James, D.J. 1988. Cell and Tissue Culture Technology for the genetic manipulation of temperate fruit trees. In: Biotechnology of Higher Plants. G. E. Russell (Ed.). Intercept. Dorset, England. pp. 33-80. Litz, R.E., Moore G.A and Srinivasan, C. 1985. In vitro system for propagation and improvement of tropical fruits and palms. Hort. Soc. 7: 157-198. Morton, J.F. 1966. The Jackfruit (  Artocarpus heterophyllus  Lam.): Its culture, varieties and utilization. Proc. Fla. State Hort. Soc. 78: 336-344. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497. Pattnaik, S.K. and Chand, P.K. 1997. Rapid clonal propagation of three mulberries,  Morus cathayana  Hemsl,  M. Ihou Koiz and  M. serrata Roxb. through in vitro culture of apical shoot buds and nodal explants from mature trees. Plant Cell Rep. 16: 503  –  508. Rahman, A.K.M.M., Huq, E., Mian, A.J., and Chesson, A., 1995. Microscopic and chemical changes occurring during the ripening of two forms jackfruit (  Artocarpus   heterophyllus L). Food Chem. 52: 405-410. Rahman, M.A. and Blake, J. 1988. Factors affecting in vitro proliferation and rooting of shoots of jackfruit (  Artocarpus heterophyllus Lam.). Plant Cell Tissue and Organ Culture 13: 179-187. Rowe-Dutton P. 1976.  Artocasrpus heterophyllus - Jackfruit. In: Garner RJ & Chaudhuri SA (Eds). The propagation of Tropical Fruit Trees. pp: 269-290. FAO, CAB, London. Roy, S.K., Rahman, S.L. and Majumder, R. 1990. In vitro propagation of Jackfruit (  Artocarpus heterophyllus Lam.). J. Hortic. Sci. 65:355-358. Roy, S.K., and Hadiuzzaman, S. 1991. Micropropagation of two species of  Artocarpu s through in vitro culture. Bangladesh J. Bot. 20:27-32.
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