Influence of Media Composition and Genotype on Potato (Solanum tuberosum L.) Microtuberization

Potato (Solanum tuberosum L.) is one of the most important crops in the world. Providing virus free, early generation seeds is a major potato production problem. Production of early generation potato minitubers under greenhouse conditions still have the risk of insect contamination and virus transmission. In vitro microtuberization provides an alternative method in the production of clean seed. The purpose of this study was to determine the optimum media for microtuber production in four potato genotypes, i.e. AOTX98202-1RU, ATX9202-3RU, ATTX98468-5R/Y and ATTX98518-5P/Y. The four genotypes were cultured in Murashige and Skoog (MS) media with addition of 6% sucrose, with or without 2 g L phytagel, and with or without 10 mg L kinetin. Two nodal cuttings were cultured in each vessel with five replications. Culture were incubated with 16 h/day light and 23 °C for two weeks, and then moved to a cooler growth room at 16 °C for two weeks, followed by incubation in the dark at 16 °C for 6 weeks, and observed for 10 weeks. Result shows that genotypes responded differently to the media. 2 g L phytagel + 10 mg L kinetin treatment (T4) was an effective treatment to enhance microtuber growth in genotype ATTX98468-5R/Y and ATTX985185P/Y. ATTX98468-5R/Y in T4 media produced the highest number of microtubers (4.2 microtubers/vessel), while ATTX98518-5P/Y produced the heaviest average fresh weight (363.6 mg). Results suggest the importance of developing specific protocols for each genotype for optimum production of microtubers.


INTRODUCTION
Potato (Solanum tuberosum L.) is one of the most important crops, ranks in fourth after wheat, rice and maize grown all over the world (Zhang et al., 2017). At present, potato growing areas in Indonesia ranging between 60 000 and 70 000 ha with a total production of about 1.2 -1.3 million tons per year. The Indonesian Agricultural Research Development Agency (IAARD) considered potato as a priority crop to develop, because of its potential as an alternative source of carbohydrates in food diversification and for export markets (Gunadi et al., 2017;Putra et al., 2019).
Providing virus free, early generation seed is the major potato production problem in developing countries. In Indonesia, mostly use of late generation seed decrease potato productivity less than 20 ton ha -1 . In vitro microtuberization provides an alternative method in the production of clean nuclear seed in potato (Hoque, 2010). Microtubers have advantages in terms of diseases free, storage, and transportation. It can be directly sown into the field and can be produced all the year, also have similar morphological and biochemical characteristics. Therefore, mass production of potato microtubers to be the key of potato production in the world (Gudeva et al., 2016). Important factor for in vitro growth and microtuber production were genotype use, media component such as carbohydrate source, growth regulators (Hossain et al., 2017;Ali et al., 2018;Teng et al., 2018).
Uses of kinetin as growth regulator in microtuberization have been reported in a number of studies. Kinetin significantly stimulated tuber formation in both wild type potato and D-12 cultivar (Aksenova et al., 2009). Nasiruddin et al. (2013) reported that kinetin is more effective than BA for microtubers production. Garcia et al. (2019) described that the independent action of kinetin in potato microtuberization increased number and weight, diameter in Alpha genotype.
Phytagel is solidifying agent that produced from a bacterial substrate that is composed of rhamnose, glucose and glucuronic acid. Phytagel produce clear culture media and colorless growth matrix for plants (Sigma-Aldrich, 2019;Jacques et al., 2020). Arregui et al. (2003) reported that phytagel can promote more rapid tuberization and larger microtubers compared to medium containing Difco Bacto-agar when cultured in medium with kinetin and placed in the dark or under an 8-h photoperiod. Phytagel also increased in vitro multiplication rate in majority of Indian potato cultivars (Sharma et al., 2011). The aims of experiment was to investigate the ability to produced microtubers of four potato genotypes by using combination of phytagel and kinetin.

Plant material
The experiment used potential breeding lines from potato breeding program, Horticulture Department, Texas A&M University, i.e. AOTX98202-1RU, ATX9202-3RU, ATTX98468-5R/Y and ATTX98518-5P/Y. Plant materials were four weeks after planting in vitro potato shoot, cultured in Tissue Culture Laboratory, Horticulture Department, Texas A&M University, College Station, Texas, USA. The plant growth regulator used for this experiment was kinetin, and Phytagel TM Sigma Chemical Co. (St. Louis, MO, USA) as the gelling agent.

In vitro production of microtubers
Two nodal cuttings (0.5-1.0 cm) of four potato genotypes were cultured in each vessel with media consisted of Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog 1962) with 6% of sucrose. The pH of the media was adjusted to 5.8. Cultures were maintained with 16 h day -1 light and 23 °C for two weeks, and then moved to a cooler growth room at 16 °C for two weeks, followed by incubation in the dark at 16 °C for 6 weeks.
Observation were recorded until 10 weeks i.e. total microtuber produced/vessel and average fresh weight of microtube. Microtubers were harvested from the stems. Callus formation was also recorded. After harvest microtubers were then placed into falcon tubes for storage. Data were analysed using Analysis of Variance on SPSS Statistical and means of all treatments and cultivars were compared by Duncan's Multiple Range Test (DMRT) at 5% level of significance (P ≤ 0.05).

RESULT AND DISCUSSION
The results showed that all variables (genotypes, media combination and interaction between genotype x media) were significantly different (Table 1). Genotype and treatment of phytagel and kinetin were significantly affected the number of microtuber/vessel produced and average fresh weight of microtuber. Differences in the number of microtuber and average fresh weight between treatments depended on the genotype.

Discussion
Microtubers are usually induced on medium containing growth regulator. Growth regulator can create physiological imbalance causing formation of microtubers and induce acids in the microtuberization medium (Nistor et al., 2013). It was found in this experiment, the highest number of microtuber/vessel produced was on genotype ATTX98468-5R/Y cultured in media T4. The heaviest average weight was ATTX98518-5P/Y also cultured in media T4. Addition of 10 mg L -1 kinetin has positive effect on microtuberization and increased total microtuber/vessel produced. Romanov et al. (2000) and Sota et al. (2020) reported that kinetin induced cell elongation and tuberization. Kinetin is needed to stimulate the process of translocation of sugar into developing tubers. The amount of starch required to form microtubers appears to depend on the amount of sucrose available in the tissue culture medium. Thus, higher sucrose together with kinetin stimulates microtuber formation. Kinetin is thought to have a stimulatory effect by increasing starch synthetase activity and suppressing starch hydrolase activity (Mohamed and El-Sherif, 2014). Momena et al. (2014) reported that the use of kinetin in microtubers production media increased the microtuber formation rate of potato cultivars Diamond. Ali et al (2018) also revealed that addition of kinetin in culture media induced earlier microtuber formation.
High concentration of kinetin induced callus formation on genotype AOTX98202-1RU. Kumar et al. (2014) explained that in theory, equal amounts of auxin and cytokinin promote callus induction. However, in practice, this can vary due to variations in the endogenous levels of phytohormones in individual plants. Genotype AOTX98202-1RU may have high level of endogenous auxin in which together with addition on kinetin in the media resulted in callus induction.
Successful plant tissue culture depends on properties of growth medium, especially the mechanical property. Phytagel belongs to a complex fluid (Zhou et al., 2019). The addition of phytagel in media clearly support number of microtubers and weight of microtubers produced. This may be due to present of glucose in phytagel together with sucrose in the media, induced microtuber production. Phytagel was commonly added at 0.5% -2.0% for optimum plant growth in vitro (Zhou et al., 2019). In this experiment, phytagel provided support for the shoots to grow upward and performed photosynthesis in the first four weeks, followed by microtuber formation for six weeks. Previous study by Veramendi et al. (1997) reported that phytagel allowed faster in vitro tuberization and larger tubers of potato than those cultured in Difco Bacto-agar. Phytagel as compared to agar may change the tissue sensitivity to cytokinins such as kinetin. Nevertheless, the in vitro development is dependent on the explants and the medium interaction.
Sucrose concentration used in this study was 6%, and the optimum sucrose concentration has been reported between 6% and 8% for tuber induction (Aslam and Iqbal, 2010;Hossain et al., 2017). In plant tissue culture, most plant requires an exogenous carbohydrates source because limited photosynthesis occurred in vitro (Lian et al., 2014). Tuberization is known to be regulated by carbohydrates availability such as sucrose, which is the transported form of sugar required for starch synthesis (Abelenda et al., 2019). The amount of sugar were positively correlated with the size of potato. The result of this experiment showed that three genotypes (AOTX98202-1RU, ATX9202-3RU and ATTX98468-5R/Y) produced microtubers even in media without addition of plant growth regulators. Similar results from Aksenova et al. (2009) found that microtubers can be induced even without using growth regulator. Garcia et al. (2019) also reported that the heaviest weight of microtuber in potato cultivar Atlantic was in a growth regulator-free culture medium. Uses of media without plant growth regulators revealed that the genotype has an innate capacity to produce microtubers. Apart from effect of growth regulator, microtuberization also depends on plant genotype and amount of sucrose in culture medium (Romanov et al., 2000).
In this experiment, for microtuber induction incubated in the dark for six week successfully induced microtuber formation. Same results was found on Ali et al. (2018), who reported that dark condition provides better response on microtuberization as compared to light condition. Potatoes are known as short day plant, so that dark photoperiod would affect tuber formation. In such condition, in vitro potato shoots will change its growth direction from upper to lower meristem, including micotuber formation (Dwiati and Anggorowati, 2011).
Results of this study suggested the importance of developing specific protocols for each genotype for optimum microtuber production, as each genotype response differently to media (Table 2). This experiment did not consider the effects of the hormone and phytagel on microtuber dormancy. How the microtubers respond to long term storage, reintroduction in vitro, greenhouse conditions, and could be directly planting microtubers in the field will have required further investigation.
In the future, mass production of microtuber could be a promising option to provide good quality, disease free seed potatoes. This in vitro technique increase rate of multiplication process, resulting in more seed potato can be produced in shorter time, all year round and economically cheaper than other method (Herrea-Isidron et al., 2021). Other advantage include easy storage and transportation (Hossain et al., 2017).