Plant regeneration of Amomum tsaoko Crevost & Lemarié in vitro

Cardamom (Amomum tsaoko Crevost & Lemarié) is a valuable medicinal herb in both traditional and western medicine. The pharmacological properties of the A. tsaoko seeds make it a useful cure for such ailments as abdominal pain, bloating, hiccups, vomiting, diarrhea, malaria, bad breath, and tooth decay. It has even further value for its use in food processing. Therefore, it is necessary to develop protocols that reduce the time required for the multiplication of cardamom using tissue culture techniques. The research was conducted to initially build up the process of in vitro propagation of cardamom from the rhizome buds. An 8 minutes treatment of HgCl2, with a concentration of 0.1%, is the most efficient for the disinfection of the rhizome buds, resulting in the rate of the disease-free survival explants reaching 18.29%. MS medium supplemented with 1.0 mg/l BAP is the most suitable medium in the fast multiplication phase, with a multiplier of 4.54 shoots/explant, average shoot height of 5.45 cm, and good shoot quality after 6 weeks of culture. A suitable rooting medium for cardamom invitro-shoots was MS medium supplemented with 0.5 mg/l IBA with a rooting rate of 100%, the average number of roots was 5.6 roots/shoot and the average root length was achieved 6.2 cm, after 8 weeks of culture.


Introduction
Amomum Roxb. (Family: Zingiberaceae Lindl.) as currently circumscribed is a plant genus of some 150 to 188 [1] species. In Vietnam, 21 species have been recorded of this genus [2]. The generic name was first used by Linnaeus (1753) but, as explained by Burtt & Smith (1968), none of the species Linnaeus included is now in Amomum. The name now used is Amomum Roxb. which is a conserved name. Roxburgh (1810) defined Amomum by its labellum, anther, and fruits. Amomum tsaoko is one of Amomum's 188 species. It was established by Crevost & Lemarié (1917) and distributed in China, Laos, and Vietnam [1].
In Vietnam, Amomum tsaoko Crevost & Lemarié commonly known as "Cardamom" is a perennial herb and monocotyledon. It is one of the valuable non-timber forest products and is an important medicinal plant with excellent export potential in herbal drug trade. In traditional medicine, A. tsaoko seeds are used as a cure for abdominal pain, bloating, hiccups, vomiting, diarrhea, malaria, bad breath, and tooth decay [3][4][5]. In addition, they are widely used in many dishes as a spice [6].
For these reasons above, there is an urgent need to scale up this biotechnology for the agricultural sector. Currently, the large and long-standing cardamom growing areas of Vietnam, such as Ha Giang, Lao Cai, and Lai Chau, are mainly propagated by seeds and rhizome segments [10]. However, these methods have not brought about high efficiency and have not met the input needs of production. The propagation method using plant tissue culture technology promises to produce a large number of plants in a short time, with seedlings free of disease and uniform quality compared to traditional methods.
In the world, in vitro propagation has been carried out on some species of Amomum genus such as A. longiligulare [11], A. krekrevanh [12] and A. subulatum [13][14][15]. In Vietnam, plant tissue culture method has also been used to propagate some species of this genus such as A. longiligulare [16], Amomum sp. [17]. The research was conducted to initially build up the process of in vitro propagation of cardamom (A. tsaoko Crevost & Lemarié) from the rhizome buds.

Sterilization and culture initiation
In the first step, rhizome segments of A. tsaoko Crevost & Lemarié were cut into 3-5 cm single nodal segments. In the second step, they were washed under running tap water for 15-20 min; outer scales of them were excised by a sharp blade. In the third step, they were soaked in a thin soap solution [1% (v/v)] for 10 minutes, washed directly under running tap water. In the fourth step, they were surface sterilized in 70% ethanol for 30 sec, followed by immersion in a 0.1% (w/v) aqueous mercuric chloride [0.05% or 0.1% (w/v)] or sodium hypochlorite [5% or 10%, (w/v)] for 4, 8 or 12 minutes. Lastly, after sterilizing with chemicals, they were removed from the damaged tissue at both ends after being rinsed 4-5 times with sterile distilled water and were placed on solid MS medium (Murashige and Skoog, 1962).

Culture media and culture conditions
All solid media consisted of 0.8% (w/v) agar and 3% (w/v) sucrose, pH was adjusted to 5.6-5.8 by 1N NaOH before being autoclaved at 121ºC and at 1

Experimental design and statistical analysis
The experiments were arranged completely randomly and repeated three times with 28-45 explants per treatment. Analysis of variance (ANOVA) was performed using Sirichai Statistics 7.0 and means were compared using LSD at a 0.05 level of probability.

Explants sterilization
Explants sterilization has an important role in an in vitro propagation process. Rhizome segments are in the ground. Therefore, they often contain many bacteria and fungi, making it difficult to sterilize explants. In this study, HgCl2 or Ca (ClO)2 at different concentrations were used to sterilize explants. Research results show that, when using Ca(ClO)2, increasing the concentration and sterilization time increases the sterilization efficiency of explants with the rate of survival and disease-free changes from 1.8% to 6.73%. The concentration of 10% Ca(ClO)2 for better sterilization results, survival rate, and disease-free explant were 5.22% after 8 minutes and 6.73% after 12 minutes, respectively. However, this ratio is still lower than when sterilized with HgCl2. Using HgCl2, depending on the different concentrations and sterilization time, the survival and disease-free explant rate varied from 5.57% to 18.29% (еable 1). Thereby, it can be concluded, the sterilization of the rhizome buds with 0.

Effect of cytokinin on the shoot regeneration
Among growth regulators of the cytokinin group, BAP and kinetin are commonly used in in vitro shoot multiplication in general as well as species of the genus Amomum [13][14][15][16]. This study evaluated the effects of kinetin and BAP separately on the ability to rapidly multiply in vitro cardamom shoots.
Research results show that by adding kinetin at a low concentration (0.5-1.0 mg/l) to the culture medium, the number of shoots/explant increased compared to when cultured on a medium without kinetin supplementation. At a concentration of 1.0 mg/l kinetin, the number of shoots/explant was the highest, with 3.45 shoots/explant, and the quality of shoots was good with the shoots being fat and strong with thick leaves of a dark green color (figure 1). When the concentration of kinetin was increased to 1.5-2.0 mg/l, the number of shoots was reduced to 2.56-2.75 shoots/explant (еable 2). The shoots obtained had a small stem diameter with thin and light green leaves. The use of kinetin in vitro shoot  [14], on A. subulatum Crevost & Lemarié, the highest shoots/explant coefficient was 9.34 in the culture medium supplemented with 3.0 mg/l kinetin. The medium suitable for rapid in vitro multiplication of A. longiligulare shoots is the medium with 1.0 mg/l kinetin with a multiplier of 5.67 [16].
On the other hand, when adding BAP, the shoot multiplier factor increased markedly, the quality of the shoot was good, the shoots were fat and the leaves were thick and of dark green color (figure 1). At the concentration of 1.0 mg/l BAP, the shoot multiplier was the highest with 4.54 shoots/explant. When the concentration of BAP was increased from 1.5-2.0 mg/l, the shoot multiplier decreased to 3.48-3.76 shoots/explant (table 2). This shoot multiplier was also higher which was the highest achieved using the kinetin in the above experiment. Furthermore, shoot height in the experiment using BAP also improved significantly compared to the experiment using kinetin. This result has been completely consistent with previous studies on A. subulatum Roxb. [13][14][15] and A. longiligulare T.L.Wu. [16].

Effect of auxin on rooting of in vitro-propagated shoots
The in vitro rooting stage is an important stage to prepare the tree to absorb water and photosynthesis in the natural environment. IBA and α-NAA that are two phytohormones in the auxin group were used in the in vitro rooting stage of Amomum genus [14,16,17]. In this experiment, IBA and α-NAA were also used to stimulate in vitro rooting from A. tsaoko shoots.
The addition of IBA to the culture medium stimulates the in vitro rooting process, increasing the rooting rate, the number of roots/shoot, as well as the length of the root, compared to the medium without IBA supplementation (figure 2). When IBA was added, rooting shoots were 100% and explants did not form calluses. The highest number of roots obtained 5.6 roots/shoot on medium supplemented with 0.50 mg/l IBA. In this environment, the roots are of good quality, fat, and many hairs. However, when the IBA concentration increased to 0.75-1.00 mg/l, the rooting rate remained at 100%, but the roots formed were thinner and less hairy, and the number of roots reduced to only 4.2-4.4 roots/shoot (table 3). This result has been completely consistent with the the previous study of Dang Ngoc Phuc et al. (2011) [16] on A. longiligulare T.L.Wu. According to Poudel et al. (2018) [15], the appropriate IBA concentration for rooting for Large cardamom shoots (A. subulatum Roxb.) was 1.0 mg/l; the highest number of roots at 4.8 roots/shoot.   Similar to the IBA, the addition of α-NAA also stimulated rooting with a 100% rate of root shoots. The number of roots/shoot also increased, ranging from 3.2 to 3.8 roots/shoot compared to 2.1 roots/shoot on the medium without α-NAA supplementation. The number of roots as well as root length was highest in the medium supplemented with 0.50 mg/l α-NAA with 3.8 roots/shoot and a mean root length of 4.1 cm (table 3). On this medium, the roots are of good quality, fat, and many hairs (figure 2). When the concentration of α-NAA increases to 0.75 -1.00 mg/l, the roots become thinner, the number of roots decreases to 3.2 -3.5 roots/shoot with less absorbent hair of a light yellow color. This result has been completely consistent with the previous study of Poudel et al. (2018) [15] on Large cardamom (A. subulatum Roxb.). In contrast, according to studies of Truong Thi Bich Phuong et al. (2017) [17] on Amomum sp. showed that MS medium supplemented with α-NAA was more effective in the induction of number of roots and their length than MS medium supplemented with IBA.

Conclusion
We have reported the initial results in the development of in vitro propagation of cardamom from the rhizome buds. An 8 minutes treatment of HgCl2, with a concentration of 0.1%, is the most efficient for the disinfection of the rhizome buds, resulting in the rate of the disease-free survival explants reaching 18.29%. MS medium supplemented with 1.0 mg/l BAP is the most suitable medium in the fast multiplication phase, with a multiplier of 4.54 shoots/explant, average shoot height of 5.45 cm, and good shoot quality after 6 weeks of culture. A suitable rooting medium for cardamom invitro-shoots was MS medium supplemented with 0.5 mg/l IBA with a rooting rate of 100%, the average number of roots was 5.6 roots/shoot and the average root length was achieved 6.2 cm, after 8 weeks of culture.