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STUDYING THE ABILITY OF MUTATION AND SELECTION OF PROMISING LINES OF VERRUCOSA LINDL. BY GAMMA IRRADIATION IN COMBINATION WITH IN VITRO TECHNIQUE

Le Thi Thuy Linh, Le Van Thuc, Dang Thi Dien, Han Huynh Dien and Le Thi Bich Thy. Dalat Nuclear Research Institute, 01 Nguyen Tu Luc, Dalat

Project information:

- Code: CS/14/01-02 - Managerial Level: Institute - Allocated Fund: 70,000,000 VND - Implementation time: 12 months (Jan 2014- Dec 2014) - Contact email: [email protected] - Paper published in related to the project: Le Thi Thuy Linh, Le Van Thuc, Dang Thi Dien, Han Huynh Dien, Le Thi Bich Thy. Morphological changes of Spider Orchid ( Bateman ex. Lindl) growing in vitro culture by Gamma Radiation treatment. Conference: Science and Nuclear Technology - The young staff of atomic energy industry for the third time. 10 - 2014 in Hanoi (in Vietnamese).

ABSTRACT: Mutation research by gamma irradiation is an effective technique in producing new breeding. In this study, we optimized some elements on the propagation process of Warty Brassia (Brassia verrucosa Lindl.) in vitro; determination of lethal dose (LD50) of Warty Brassia bud clusters in vitro; initial evaluating of gamma irradiation effects on the morphology of Warty Brassia in vitro. The results showed that, the appropriate medium for meristem-tip culture was on VW medium (Vacin and Went, 1949) containing 1 g/l activated charcoal (AC), 10% coconut water (CW), 50 g/l potato and 20 g/l sucrose; medium for in vitro shoot multiplication was on VW medium supplemented with 0,5 mg/l NAA, 0.3 mg/l BA, 1 g/l AC, 10% CW, 50 g/l potato and 20 g/l sucrose; medium for in vitro root induction was on VW medium supplemented with 0.5 mg/l NAA, 1 g/l AC, 10% CW, 50 g/l potato and 20 g/l sucrose. The plantlets were acclimatized and transplanted to 50% sphagnum moss and 50% coconut fiber, the survival rate was 94.7%; the LD50 values were in the range of 40 - 50 Gy; the stimulation dose was 10 Gy; the highest variation rate was on the dose of 40 Gy; the absolute lethal dose was 80 Gy. 6 variations of Warty Brassia in vitro were obtained including the variant of morphology, structure and diameter of pseudo-bulb were very difference comparing to control. The following study are evaluating of the growth and the phenotypic changing of potential variant types of Warty Brassia in green house. Keyword: Brassia verrucosa, LD50, in vitro propagation, gamma irradiation.

1. INTRODUCTION Brassia verrucosa Lindl. is a perennial plant with big medium size, grows strongly with narrow egg-shaped, light green and small yellowish blue flowers that is grew on shelves in the in-house gardens, ecological tourist gardens. Brassia verrucosa Lindl. is less diverse in shape and colour flower. Overcoming this shortcoming does not only prove the role of the ionized radiation in the creation of varieties but also improve the value of Brassia verrucosa Lindl.

2. EXPERIMENTAL 2.1. Materials Brassia verrucosa Lindl. with long shoots (5-10 cm) from the healthy original plant. 2.2. Reagents Macro minerals, micronutrients, vitamins, hormones are used in all the experiments of Merck; agar (HaLong Canfoco); sucrose (Bien Hoa Sugar Joint Stock Company).

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2.3. Procedures For formulas without repetition, data is recorded and the actual average value is calculated in Microsoft Excel 2010 software. For the repeated formula, the formula is completely random arrangement between the treatments. Data recorded were processed by software MSTATC, LSD test. - The propagation process of Brassia verrucosa Lindl.

Disinfection and separate samples: samples disinfected with 0.2% HgCl2, time 5-7 minutes Create bud (meristem-tip culture): the effect of different types of environments the ability to create and apical buds from apical meristems Warty Brassia Shoot proliferation (shoot multiplication): survey effect the combination of auxin (NAA) and cytokinin (BA) on shoot proliferation Complete plantlet (root induction): survey concentrations of NAA on rooting the Warty Brassia In vitro plant nursery: the effect of substrate type on survival of Warty Brassia putting the tree nursery - External dose study sample Define criteria for tissue bud clusters irradiation mutagenesis Valuation of LD50 (lethal dose 50%) for tissue bud clusters - Review the growth of bud Spider orchids form after irradiation

3. RESULTS AND DISCUSSION 3.1. The propagation process of Brassia verrucosa Lindl - Disinfection and separate samples

Buds from Spider orchid sterilized with 0.2% HgCl2, time 5-7 minutes The result: 84% living and non-infected samples after 15 days in samples - Create bud (meristem-tip culture) The appropriate medium for meristem-tip culture was on VW medium (Vacin and Went, 1949) containing 1 g/l activated charcoal (AC), 10% coconut water (CW), 50 g/l potato and 20 g/l sucrose - Shoot proliferation (shoot multiplication) Medium for in vitro shoot multiplication was on VW medium supplemented with 0.5 mg/l NAA, 0.3 mg/l BA, 1 g/l AC, 10% CW, 50 g/l potato and 20 g/l sucrose, the number of shoots /cluster is highest, fat buds, not deformed. - Complete plantlets (root induction) Medium for in vitro root induction was on VW medium supplemented with 0.5 mg/l NAA, 1 g/l AC, 10% CW, 50 g/l potato and 20 g/l sucrose for the nursery tree quality, uniformity on balanced trees, growing well. - In vitro plant nursery The plantlets were acclimatized and transplanted to 50% sphagnum moss and 50% coconut fiber, the survival rate was 94.7% after 60 days follow-up.

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Sampling Disinfection and Create bud separate samples Shoot proliferation

In vitro plant nursery Complete plantlets

Picture 1: The propagation process of Brassia verrucosa Lindl. 3.2. External dose study sample - Define criteria for tissue bud clusters irradiation mutagenesis Increases in height cluster bud mite spreads depends mainly on incubation time; standard bud cluster tissue samples for irradiation mutagenesis bud clusters with a height of 0.4 cm in the initial period of 10 - 20 days of culture. - Valuation of LD50 (lethal dose 50%) for tissue bud clusters

Table 1: Valuation of LD50 (lethal dose 50%) for tissue bud clusters.

Gamma dose Survival rates (%) (Gy) 20 days 40 days 60 days 0 (Control) 100 ± 0.0 100 ± 0.0 a 100 ± 0.0 a 10 100 ± 0.0 100 ± 0.0 a 98 ± 0.5 a 20 100 ± 0.0 96 ± 0.6 a 82 ± 1.3 b 30 100 ± 0.0 82 ± 0.8 b 64 ± 0.9 c 40 96 ± 0.6 80 ± 0.7 b 56 ± 0.9 c 50 94 ± 0.9 76 ± 0.6 bc 42 ± 0.8 d 60 94 ± 0.6 70 ± 0.7 bc 26 ± 1.1 e 70 94 ± 0.6 72 ± 0.8 bc 8 ± 0.8 f 80 94 ± 0.9 66 ± 1.1 c 0 f 100 92 ± 0.5 48 ± 0.8 d 0 f ns ** ** F-value 1.94 27.22 120.13 CV (%) 5.39 8.95 16.54

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*Different letters within a column indicate significant differences; **: statistically significant at a probability level of 0.01; ns: not significant by MSTATC, LSD test The different doses of irradiation clearly influence on survival of spider orchid bud clusters in vitro. Survival rates of plantlets were lower when irradiated doses higher. The LD50 values were in the range of 40 - 50 Gy; the stimulation dose was 10 Gy; the highest variation rate was on the dose of 40 Gy; the absolute lethal dose was 80 Gy (Table 1) 3.3. Review the growth of bud Spider orchids form after irradiation - Statistical results and noted the variant Table 2: Statistical results and noted the variant.

In vitro Gamma Total Rate bud Type Type Type Type Type Type generation dose buds mutation 1 2 3 4 5 6 (Gy) obtained (%) (%) (%) (%) (%) (%) (%) (Bud) MV1 0 405 0 0.0 0.0 0.0 0.0 0.0 0.0 10 437 1.4 0.9 0.3 0.2 0.0 0.0 0.0 20 321 5.0 2.5 0.3 2.2 0.0 0.0 0.0 30 239 10.5 0.4 4.6 5.5 0.0 0.0 0.0 40 204 17.6 0.0 4.9 4.4 0.0 0.0 8.3 50 105 37.2 0.0 6.7 7.6 0.0 0.0 22.9 MV2 0 461 0 0.0 0.0 0.0 0.0 0.0 0.0 10 480 2.5 1.5 0.4 0.6 0.0 0.0 0.0 20 276 9.8 4.0 3.3 2.5 0.0 0.0 0.0 30 225 18.2 0.9 6.7 10.2 0.4 0.0 0.0 40 178 27.5 0.0 6.7 7.9 3.9 2.8 6.2 50 95 42.1 0.0 5.3 8.4 3.2 6.3 18.9 MV3 0 498 0 0.0 0.0 0.0 0.0 0.0 0.0 10 425 3.3 1.9 1.2 0.2 0.0 0.0 0.0 20 215 11.2 4.7 3.7 2.8 0.0 0.0 0.0 30 190 29.0 1.6 17.3 16.6 0.0 0.5 0.0 40 160 30.0 0.0 10.6 11.3 5.6 2.5 0.0 50 86 37.2 0.0 15.1 12.8 3.5 5.8 0.0 The variant was recorded in statistics and the selected reference dose, different doses were incurred the phenotypic variation at different levels, with different morphology than the original (Table 2), the typical variant: Type 1: vigorous shoot, large pseudo bulb, dark green leave (2a); Type 2: albino shoot, white tree trunk (2b); Type 3: light green bud, streak mosaic (2h); Type 4: shoot out with fake belt round, with many edge, rip (2c, 2e, 2f);

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Type 5: buds have round pseudo-bulb, dwarf trees, leaves short, strong growth (2g). Type 6: buds deformity, poor growth (remove) (2d);

Picture 2: Variations of Brassia verrucosa. - The growth of the bud variations

Table 3: The growth of the bud variations.

The The number Plant Diameter of Root number of of roots/ Variations height pseudo-bulb length leaves/ plant (cm) (mm) (cm) plant Control 7.6 ± 0.7b 0.5 ± 0.04b 5.4 ± 0.4 8.5 ± 0.5abc 3.2 ± 0.7ab Type 1 9.7 ± 1.0a 0.8 ± 0.03a 5.3 ± 0.5 9.5 ± 1.3ab 2.1 ± 0.6c Type 2 6.8 ± 0.5b 0.4 ± 0.03c 4.8 ± 0.4 7.7 ± 0.9c 4.0 ± 0.3a Type 3 7.6 ± 0.3b 0.4 ± 0.09c 5.3 ± 0.5 7.5 ± 0.7c 2.8 ± 0.4bc Type 4 5.6 ± 0.5 c 0.6 ± 0.07b 5.1 ± 0.3 8.1 ± 0.6bc 2.5 ± 0.5bc Type 5 4.6 ± 0.5c 0.6 ± 0.04b 5.4 ± 0.4 9.8 ± 0.9a 2.1 ± 0.3c F 42.47** 38.82** 1.82ns 5.85** 11.24** CV (%) 8.63 9.95 7.60 9.97 17.48

*Different letters within a column indicate significant differences at 0.01 by MSTATC, LSD test Gamma (Co60) rays irradiation effectively effected to the growth, 6 variations of Brassia verrucosa in vitro were obtained including the variant of morphology, structure and diameter of pseudo-bulb were very difference comparing to control (Table 3)

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4. CONCLUSIONS The topic completed registration content: construction and completion of in vitro propagation process; creation of a diverse group of morphological variation of Brassia verrucosa; contribution to the training of young cadres and building scientific credibility of biotechnology center. In the future, this study is going to evaluate the growth and the phenotypic changing of potential variant types of Brassia verrucosa in green house.

REFERENCES [1] Georgina Flores, Escobar, “Propagacion in vitro de la orquidea (Brassia verrucosa Baterman ex Lindl.)”, Revista Chapingo, Serie Horticultura, Universidad Autonoma Champingo Mexico. Vol. 17(1), pp. 5 – 8, 2011. [2] Internatinal Atomic Energy Agency, Nuclear Technology review 2010. Vienna. pp. 35 – 37, 2013. [3] Murashine T., Skoog F. “A revised medium for rapid growth and bioassays with tobacco tissue cultures”. Physiol. Plant 15: 473 - 497, 1962. [4] Talukder S. K., Nasiruddin K. M., Yesmin S., Hassan L. and Begum R., “Shoot proliferation of Dendrobium orchid with BAP and NAA”, Journal of Biolgical Science 3(11), 2003. [5] Vacin E., Went F., Some pH changes in nutrient solution. Botanic Gardent Conservation News 110, pp. 605-613, 1949.

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