The Effect of Gamma Irradiation on the Growth and Multiplication of the In Vitro Shoot of Patchouli (Pogostemon cablin Benth.)

The objective of this research was to evaluate the effect of gamma irradiation on shoot growth and multiplication of Patchouli (Pogostemon cablin Benth.) Two weeks-old in vitro shoots were irradiated gamma-ray, at doses of 0, 15, 30, 45, 60, and 75 Gy. The control shoot was not irradiated. The irradiated shoots were cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg.L-1 NAA and 0.3 mg.L-1 BA and incubated in a growth room for eight weeks at a temperature of 25±2°C. The results showed that the gamma irradiation inhibited the growth and multiplication of shoots. Explants irradiated with high-dose gamma-ray (45-75 Gy) had not formed shoot in four weeks of culture, while 58.3-83.3% of the explants without irradiation or irradiated at low doses 15 and 30 Gy formed shoots. The higher irradiation doses increased percentage of browning explants and reduced the percentage of forming shoots. Within the eight weeks of culture, explant without irradiation was able to form shoots at the percentage of 100% with 24 shoots per explant, while explants irradiated at 15-45 Gy were able to grow form shoots at the percentage of 77.7-95.5%. The high doses-irradiated explants (60 and 75 Gy) were only able to form shoots less than 13-20%, with 2-3 shoots per explant.


INTRODUCTION 
Patchouli (Pogostemon cablin Benth.) is one of the essential oil-producing plants. The oil is mostly found in its leaves [1]. Essential oil forms this plant is universally recognized as the best because it is the material for the food, pharmacy, and cosmetic industry [2]. Indonesian Plantation Service recorded that patchouli plantation in the country covers an area of 18,841 ha, producing 2,115 tons patchouli oil [3]. The annual demand for the oil is 60%, around 700-2,800 tons. Indonesian export is estimated to be 80% of the world's export [4].
Industrial development has made both domestic and international demand for Patchouli oil increase [5]. One of the ways to increase oil production is developing genetic variations for quality seeds through mutation induction; one of the techniques is gamma-ray mutagen treatment [6]. Successful experiments to increase essential oil content and secondary metabolites are those in Boswellia carterii [7], orange peel [8], and ginger (Zingiber officinale Roscoe) [9]. Gamma irradiation of 30-100 Gy on in vitro shoot explants can produce variations in Eustoma grandiflorum and banana (Musa spp.) [10,11]. Gamma-ray also affects shoot growth of Strawberry [12] banana [13] and inhibited root growth in Rubus fraxinifolius [14] under in vitro conditions. The objective of this research was to evaluate the effect of gamma irradiation on the growth and multiplication of in vitro patchouli shoot explants.

MATERIALS AND METHODS Plant material
In vitro shoots of Patchouli cv. Lhokseumawe were derived from laboratory's collection. In vitro shoots were propagated on MS mediumsupplemented 0.1 mg.L -1 NAA and 0.3 mg.L -1 BA. Two weeks old in vitro shoots were used as explant for gamma-ray irradiation.

Gamma irradiation of in vitro shoot cultures.
The gamma irradiation was carried out at National Nuclear Energy Agency, Jakarta, Indonesia. Two weeks old in vitro shoot of approximately 1 cm in length were exposed to gamma radiation at different doses of gamma-rays (0, 15, 30, 45, 60, and 75 Gy). These doses based on previous research on Coleus blumei to obtain new Coleus variances in a relatively short time [15]. The source of gamma rays was 60 Co gamma irradiator-Gamma Cell 220 at a dose rate of 4585.5 Grey/minute. After irradiation, treated shoots were immediately transferred to a fresh MS medium containing 0.1 mg.L -1 NAA and 0.3 mg.L -1 BA. Fives shoots were cultured in each culture bottle, and ten replicated were conducted for each gamma-ray dose (Fig. 1).
The culture was incubated at a temperature of 25±2°C and 16h day/8h dark photoperiod at the light intensity of 600 lux in a growth room for 8

ISSN. 2087-2852 E-ISSN. 2338-1655
Gamma Ray Irradiation on In Vitro Shoot of Patchouli (Banyo, et al.) 145 weeks. Percentage of browning explants and shoot formation; number and length of shoot were observed every four-week. Analysis of variance (ANOVA) followed by the LSD test was used to determine the differences in mean number of all tested parameters between irradiation doses.

RESULTS AND DISCUSSIONS
The Gamma-irradiation on in vitro shoot explants affected the shoot growth and multiplication of patchouli. Irradiation at the doses 15-75 Gy caused browning on the shoot tip apical meristem and made the leaf yellowish. The higher the dose of irradiation, the higher was browning of explant. Gamma-ray irradiation on shoot explants also inhibited the growth of explants and the formation of new shoots. The inhibition of growth and multiplication of irradiated shoot increased as the gamma dose increased (Fig 2).
Gamma irradiation on in vitro shoot can increase the percentage of shoot browning and decrease shoot-forming explants. The effects of doses of gamma rays on shoot growth and formation of the new shoot during eight weeks in culture are shown in Figure 3. Gamma irradiation on shoot explant increased the percentage of shoot browning and decreased shoot-forming explant percentage, number, and length of the shoot. The browning percentage of irradiated shoot increased as the gamma dose increased.
Irradiation of gamma-ray also reduced the number of regenerated shoots per explant. The number of regenerated shoots reduced prominently with the increase in gamma irradiation dose. The number of regenerated shoots per explant was significantly lower at higher doses (60-75 Gy). However, at lower doses (15-45 Gy) was slightly decreased compared to control (Fig. 3C). In eight weeks of culture, 100% of non-irradiated explant produced shoot with 24 shoots per explant, while irradiated explant at lower doses of gamma rays (15-45 Gy) formed shoot was 67-77.7% with 7-20 shoots per explant.  Shoot forming percentage of higher irradiated explant (60-75 Gy) was only 13-20%, with 2-3 shoots per explant ( Fig. 3B and 3C). Beside shoot formed was lower, shoot growth of irradiated explant was also inhibited. The shoot length reduced with an increase in gamma irradiation doses (Fig. 3D). Shoot height growth at 8 weeks in the culture of explant without gamma-ray irradiation was about 1 cm, while shoot height growth of explant irradiated with gamma rays at doses of 15-45 Gy was between 0.3-0.6 cm. Shoot growth of explants that were irradiated by 60-75 Gy was only about 0.2 cm (Fig. 3D).
Analysis of the correlation between gamma-ray irradiation and the percentage of browning explant and the percentage of shoot forming obtained correlation coefficient R values of 0.9143 and 0.8431, respectively. There was a positive correlation between the dose of gamma-ray irradiation and the percentage of browning explants and a negative correlation between the dose of gamma-ray irradiation and the percentage Gamma Ray Irradiation on In Vitro Shoot of Patchouli (Banyo, et al.) 147 of shoot forming. This indicated that the increasing in the percentage of browning explant and the decreasing in the percentage of shoot formation were in line with the increase of gamma doses (Fig.  4). Gamma-ray irradiation on plant tissue caused browning and inhibited the growth and formation of a new shoot. Previous research found that explant browning caused by gamma irradiation was due to the degradation of indole acetic enzyme, which plays a role in the synthesis of IAA. The enzyme degradation produced browning of explants [16]. Browning is caused by phenol oxidation after cell membrane degradation or cell disorganization followed by chlorophyll degradation. This process is an indicator of the formation of quinones as a result of the enzyme activity [17]. Effect of gamma irradiation on explant browning also observed on shoot explant of Colocasia esculenta (L.) Schott [18], callus of Ferula gummosa [19], and plantlet of Gerbera jamesonii [20]. A previous study observed the effect of gamma irradiation on in vitro total phenolic content of Ferula gummosa. The research showed that the gamma irradiation-induced phenolic compound in Ferula gummosa callus and phenolic content increased with the increased gamma irradiation dose [19].
Cell damage from high doses of irradiation is thought to be the cause of the growth and development of Robusta BP 436 coffee to be inhibited. The inhibition of growth and development is indicated by the black callus, which indicates necrosis (cell death). High doses of gamma-ray irradiation produce free radicals in the hydroxyl form. Hydroxyl radicals or hydrogen peroxide will cause physiological damage in the form of inhibition of cell division and differentiation processes and gene damage if these hydroxyl radicals attach to the nucleotide chains causing DNA damage [21].
Callus of Sugarcane (Saccharum officinarum L.) irradiated with gamma-ray doses 10-80 Gy cause browning at a dose of 40 Gy, and the extent of browning also increased with the increase of gamma irradiation dose [22]. Callus of Cucumis irradiated with a high-doses of 100-200 Gy experienced browning due to the absorption of gamma-ray irradiation that could increase free radicals, which damaged many cells in the callus [23]. High irradiation doses can change the ratio of the phytohormones auxin and cytokinin. It leads to the pattern change of cell differentiation, which could cause delays production of new shoots [24].
Irradiation-induced inhibition of shoot growth was caused by damage in meristem cells. The effect of irradiation was the inhibition of cell division, which results in inhibition of explant growth. Inhibition of shoot growth at higher doses of gamma rays is due to reduced mitotic activity in the meristematic tissue and reduced moisture contents of explants [25]. Chauduri also reported that irradiation at higher doses killed meristematic cells and result in the inability of the cells to absorb nutrients [26]. The inhibitory effect of the high dose of gamma rays on shoot growth was due to disruption of hormonal balance and enzymatic activities [27]. Irradiation does influence water intake to cells and the synthesis of endogenous hormones [20]. Inhibition of shoot growth due to the high dose of gamma rays was derived from cell cycle arrest at G/M phase during cell division and/or various damages in the entire genome [28].
Gamma irradiation inhibited in vitro shoot elongation of patchouli. Effect of gamma irradiation on inhibition of shoot length was observed in Triticum aestivum L. [29] Cicer [30], Corn [31], and Chrysanthemum [32]. Gamma irradiation prevented the shoot elongation of Cicer. The effect of gamma irradiation on shoot elongation was depending on the dose of gamma and genotype, respectively. The higher irradiation, shoot length was more affected [30]. Previous research found that gamma irradiation decreased the number of leaves and branches on Chrysanthemum [32]. Gamma-ray irradiation also had a significant impact on the shoot length of Triticum aestivum L. The shoot length decreased by 46% as the gamma dose increased [29]. It was observed that a decrease in shoot length is in proportion with increasing gamma dose in corn [31]. It was assumed that suppressed growth of explant tissue was caused by damage from irradiation treatment.

CONCLUSION
Gamma-ray irradiation on patchouli in vitro shoot inhibited shoot growth and multiplication. The irradiation of gamma rays at doses 15-75 Gy increased the browning percentage of explant and inhibited shoot formation. The percentage of shoot-forming explant, the numbers of shoots per explant, and shoot length decreased as the gamma-ray dose increased.