Application of Tissue Culture Techniques in Woody Species Conservation, Improvement and Development in Vietnam: Agarwood (Aquilaria crassna Pierre ex LeComte) via tip Culture Tran Van Minh Institute of Tropical Biology 1 Mac Dinh Chi Str., HoChiMinh City VietNam Keywords: Agarwood, satalum, resin, micropropagation, tok Abstract Aquilaria crassa (agarwood), a Vietnamese forest tree, was micropropagated using shoot explants from 20year old trees known to produce the valuable exudates tok. Either shoot tips or internodes could be used for the initial explants, but in subcultures best results were obtained from internodes. Woody Plant Medium was a better basal medium than Murashige and Skoog, and for initial shoot induction BA at 1mg/l and coconut water at 10% was used. For subcultures, BA at mg/l, NAA mg/l and coconut water at 10% gave highest shoot multiplication. A low level of rooting was obtained using either IBA or NAA at 0.3 mg/l. Plants transferred to the field grew to 2m after 18 months and had normal morphology. INTRODUCTION Agarwood (Aquilaria crassna Pierre ex. LeComte) is one of the special and valuable wood species of the tropical rainforest. It grows naturally in Vietnam s forests, and is classified as an endangered, rare and valuable forest species (Chan, 1996). It is conserved as a gene resource (Nghia, 1996) and prohibited from exploitation (Chanh, 1998). Resin concentrated in wounded sites on the plant form a special fragrant material called agarwood or tok, which has high export value. The species is not related to Santalum. It may be propagated from seed but a limited number of genotypes display tok production. It can also be cloned using cuttings. However, the species has been overexploited and is nearly extinct. The application of tissue culture techniques was necessary to assist the conservation, improvement and development of this valuable endangered plant. MATERIALS AND METHODS Young branches from 20yearold A. crassna trees known to produce tok in the forest of Phu Quoc Island (South Vietnam) were collected, placed on ice in an insulated container and transported by air to the laboratory within a day. were washed with soapwater and water, and cut into young shoot tip and young internodal explants. Explants were sterilized by % HgCl2 for 20 minutes, followed by rinsing three times in sterile distilled water. The explants were cultured on media as detailed under Results. All media contained 20g/l sucrose and 8g/l agar, and were autoclaved for 25 minutes at 121 o C. Media were dispensed in 60ml aliquots into 300ml flash closured with rubber cover, unless otherwise specified. The cultures were incubated at 28 o C, under an 8 hour photoperiod with a light intensity of 34.2µmol/m²/s. Each treatment consisted of 1015 explants and was repeated four times. Variables that were examined to optimize the micropropagation protocol included: (1) a comparison of the basal media WPM (Lloyd and McCown, 1980) and MS (Murashige and Skoog, 1962), (2) the addition of BA (6benzylaminopurine), NAA (naphthalene acetic acid) and CW (coconut water) to shoot multiplication media, (3) the effect of gas exchange levels and their interaction with light intensity, (4) the effect of genotype on shoot multiplication, (5) the effect of auxin on rooting, (6) acclimatization protocols. Details of each experiment are given below. Proc. IInd IS on Biotech. of Trop & Subtrop. Species Eds: W.C. Chang and R. Drew Acta Hort 692, ISHS 2005 37
RESULTS AND DISCUSSION Growth and Multiplication from Tip Explants on WPM and MS tips cultured on WPM and MS with BA (1mg/l) and CW (010%) showed better growth on WPM (Table 1). Explants on WPM supplemented with BA (1mg/l) + CW (10%) regenerated healthy normal shoots and showed initiation of multipleshoots whereas on MS medium with the same supplements, callus formed at the base and morphology was abnormal. Effect of Plant Growth Regulators on Explants Single shoot explants were cultured on WPM basal medium supplemented with BA (2mg/l) and NAA (mg/l) separately or in combination. Results indicated that explants on BA (mg/l) and NAA (mg/l) produced the highest number of shoots (56 shoots) and these had dark green leaves and stems providing ideal material for subculture (Table 2). Effects of Plant Growth Regulators on Internodal Explants were cultured on WPM supplemented with BA (2mg/l) and NAA (0.5mg/l). The combination of BA (mg/l) + NAA (mg/l) again gave highest multiplication of healthy strong shoots (89 shoots) and the shoot number had doubled after 3 weeks (Table 3). Effects of Coconut Water on Micropropagation Explants cultured on WPM with BA (2mg/l), NAA (0.5mg/l) and CW (0 10%) indicated that the combination of BA (0.lmg/l) + CW (10%) gave good results having 8.5 shoots (data not shown). On this medium shoots were healthy, reached double numbers within 2 weeks and appeared more juvenile than shoots on other media. Effects of Gas Exchange and Light Intensity on Micropropagation were cultured in 300ml bottles containing WPM basal medium supplemented with BA (mg/l) and CW (10%). Two gaseous environments were studied: a closed system using rubber covers (R), and a semiopen system by using sterile paper covers (P), and three light intensities. Similar results were obtained using the semiopen system with a light intensity of 34.20µmol/m²/s and the closed system with light at 54.72µmol/m²/s, but leaves were dark green in the semiopen system and pale green in the closed system (Table 4). Greatest shoot growth and multiplication was observed at highest light intensity. Effects of Genotype on Micropropagation Four clones (over 18 years old) of genotypes known to have latex deposits growing in Phu Quoc showed significant differences in multiplication, and in leaf and shoot morphology in vitro (Table 5). Comparison of In Vitro Growth of Tips and Internodal Explants on Optimal Medium Single shoot tips and internodes from in vitro plantlets were assessed 15, 30, 45 and 60 days after culture on WPM supplemented with mg/l BA and 10% CW in containers covered with paper, under a light intensity of fluorescent lamp. tips as explants produced only one shoot and a final plant of 56.2mm; whereas internodes resulted in 9.5 shoots per clump after 45 days rising to 10.5 after 60 days with a final plant of 54.7mm. Using internodes and subculturing at 45 day intervals, should give a multiple rate of 37 (data no shown). 38
Rooting In WPM basal medium supplemented with IAA, IBA, or NAA, roots developed on media with IBA or IAA at 0.3mg/l (Table 6). Roots grew well and initiated root hairs. Acclimatization and Growth in the Field Plantlets were potted into coconut fibre and acclimatized in a nursery bed covered by nylon fabric providing high humidity and low light intensity. The survival rate was over 90% after 3 weeks. Plants were then potted into a substract of soil:manure (1:1). They grew well and reached 4050cm after 56 months in the field. We now have thousands of agarwood plantlets produced by tissue culture techniques in the field. Plant growth and development is faster than for seedlings. Plant reached 2m after years and trees have normal morphology. CONCLUSION In vitro micropropagation of explants from mature trees provides a low cost, fast means of multiplying valuable genotypes of agarwood to conserve, improve and develop the species in Vietnam. Literature Cited Lloyd, G. and McCown, B. 1980.Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoottip culture. Comb. Proc. Intl. Plant. Prop. Soc. 30:421426. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473479. Chan, L.M. 1996. Research on planting some precious and rare forest tree species in the arboretum of the forestry college serving the teaching and contributing to gene conservation of tropical tree species in Vietnam. Results of forest scientific and technological research. Agricultural Publishing House 1991 1995. Chanh, D.T. 1998. Report on rare and valuable forest tree species conservation. Department of Agriculture and Rural Development of Kien Giang Province. Nghia, N.H. 1996. Genetic conservation of forest tree species period 19881995. Results of forest science and technology research. Agricultural Publishing House 19911995. Tables Table 1. initiation from shoot tip cultures on MS and WPM for 60 days. Media MS MS WPM WPM BA CW (%) 10 10 Callus (+/) + + 3.2 6.2 6.2 5.6 29.9 1.4 Clone: Phu Quoc RDCI (clone from north of Phu Quoc Island) 4.2 8.0 6.6 25.4 1.4 22.0 2 18.5 3 24.6 30.3 3.4 8.0 1 1 22.6 3 42.0 3 4 40.0 15.1 3.5 39
Table 2. Effects of BA and NAA on shoot growth and multiplication on WPM for 45 days. BA CV (%) LSD (0.05) NAA 1.0 2.3 71.1 0.8 1.7 8.0 3.7 6.0 4.5 49.3 1.1 11.0 40.0 20.0 26.0 3 31.7 26.7 37.3 5.1 10.7 11.0 7.4 48.3 8.5 50.5 3 14.0 44.2 21.0 28.5 56.3 5.2 Table 3. Effects of BA and NAA on shoot production from internodal explants after 60 days on WPM. BA NAA 5.5 3.5 3.2 4.2 66.4 2.0 1.7 9.5 4.0 3.0 5.3 5 26.0 33.2 21.2 32.5 31.2 25.5 36.3 5.5 47.7 2.6 48.5 21.0 31.0 44.2 23.5 29.5 48.5 5.9 Table 4. Effect of light intensities and vessel closure type on shoot growth after 60 days. Light Intensities (µmol/m²/s) 20.52 20.52 34.20 34.20 54.72 54.72 Cover type R P R P R P 10.0 10.0 12.0 29.9 2.4 R: Rubber cover and P: Paper cover 34.5 35.5 34.0 3 59.5 7 45.3 12.5 3.5 4.5 7.7 8.7 1 3 2.1 23.2 25.5 28.0 3 30.0 44.5 31.3 47.1 6.7 1 13.7 9.5 3 2.6 color pale green dark green pale green dark green pale green dark green 40
Table 5. Effect of clonal genotype micropropagation. Explants cultured for 60 days on WMP supplemented with BA (mg/l), NAA (mg/l) and coconut water (10%). Clones BDC1 BDC2 NDC1 NDC2 10.5 8.8 22.0 2.1 Intenodes 7.7 11.0 1 14.0 7.6 39.4 38.7 26.2 31.2 2 30.3 20.3 4.5 Clones: Phu Quoc BDC1, BDC2 (clones in south of Phu Quoc Island) and Phu Quoc NDC1, NDC2 (clones in south of Phu Quoc Island) 11.7 31.4 2.5 50.0 58.7 31.2 53.7 48.4 24.0 7.8 Table 6. Effect of auxins on root initiation on WPM for 60 days. Auxin Auxin concentration IAA IBA NAA IAA 0.3 IBA 0.3 NAA 0.3 41.2 4 43.7 56.2 51.7 44.0 46.6 14.4 6.3 4.0 6.0 4.2 6.2 6.0 34.7 Leaves 5.2 29.7 Roots 1.0 1.0 Root 3 24.2 Figures (see next page) Figs. 111. Agarwood Micropropagation (1) Mature tree (25 years old) in Phu Quoc Island (Vietnam). (2) The base of a mature tree having " Tok formation. (3) A tree with latex called "Tok". (4) Young shoot samples from mature tree for tissue culture. (5) tip tissue culture after 15 days. (6) Multipleshoot production after 30 days. (7) Multipleshoots with healthy leaves. (8) Healthy plantlet with root of WPM supplemented with IBA (0.3mg/l). (9) Plantlet after 1 month in a pot. (10) Plantlets in a nursery on Phu Quoc Island. (11) Micropropagated Agarwood tree after 2 years. 41
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