Embryogenic callus induction of Indonesian Cassava (Menti and Adira 4) on different picloram concentrations

Cassava (Manihot esculenta Crantz) is an important staple crop in tropic countries. A reproducible somatic embryogenic callus production will be useful in the biotechnology breeding program for plants including cassava. Menti and Adira-4 are two types of high yield cassava genetic resources in Indonesia. Immature leaf lobes from cassava in vitro were used as explants on MS basal medium with five level concentrations of Picloram: 0 mg L−1, 3 mg L−1, 5 mg L−1, 10 mg L−1, and 12 mg L−1. Initial embryogenic callus were seen on the explants in 2 weeks after treatment. Explants were then subcultured to the same medium every three weeks until three cycles of subculture to proliferate pro-embryos. Result showed that MS basal medium containing picloram 3 – 12 mg L−1 could induce embryogenic callus formation in Menti and Adira 4 immature leaf lobe explants. The highest percentage of explants forming embryogenic callus was on D medium (MS + 3 mg L−1 Picloram). These findings would suggest that at all picloram concentrations tested both varieties of cassava showed a high embryogenic callus production. High frequency on embryogenic callus formation is a key step in cassava regeneration system specifically to support the modern breeding program.


Introduction
Cassava (Manihot esculenta Crantz) is a root crop that has potential as a source of food, biofuel and industrial raw materials.Cassava productivity may decrease due to frequent pest and disease attacks and unfavourable environmental factors.Therefore, it is necessary to use healthy seedlings or conduct plant improvement.Tissue culture is used to produce many plants by means of asexual reproduction, in which plants are grown from plant cells or tissues that are placed in conditions suitable for growth and differentiation.This technique may accelerate plant propagation and breeding program.In addition to environmental conditions, such as temperature and light, genetic factors affect the success of tissue culture [1].
The embryogenic callus is the cell with strong division ability and the potential of differentiation into somatic embryos, which can be divided into two types: the compact embryogenic and the loose embryogenic calluses [2].Picloram is a type of synthetic auxin commonly used in plant tissue culture to induce callus formation and embryogenic development.The differentiation of embryogenic callus into intact plants depends on various factors, including the concentration of plant growth regulators used.Regeneration is a process for redevelop callus into plant tissue or a complete plant.Regeneration in plant tissue culture can be done in several ways, including through organogenesis and somatic embryogenesis [3].
The aim of this study was to determine the effect of different concentration of picloram on embryogenic callus induction of two types of Indonesian cassava, Menti and Adira 4. The optimal concentration of picloram for differentiation of embryogenic callus may vary depending on the variety and the experimental conditions used.This research could provide information regarding effective tissue culture media in mass cassava propagation and support the process of plant breeding through biotechnology.

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Material and method

Time and place
The experiment was carried out from January to April 2023 in GMO Plant Tissue Culture Laboratory, Genomic Research Building, KST (Integrated Science Area) Soekarno -Cibinong.

Plant material
This research used two types of cassava, Menti and Adira-4 which were grown at the cassava collection garden of KST (Integrated Science Area) Soekarno -Cibinong.Menti is the local cassava genotype of Indonesia, while Adira 4 is the released national variety of Indonesia.The plantlet of Menti and Adira-4 were maintained and micropropagated on hormone-free MS medium.

Induction of Embryogenic Callus Cultures
Immature leaf lobes (ILL) (Figure 1) were excised from six weeks old cassava plantlet then planted on MS basal medium containing 3% sucrose, 2 µM CuSO4, 0.3% phytagel, and varied concentrations of the Picloram.There were five level concentrations of Picloram used in this research: 12 mg L -1 (A), 10 mg L -1 (B), 5 mg L -1 (C), 3 mg L -1 (D), and 0 mg L -1 (O).Three petri dishes per cultivar, per treatment were used with each petri dish consisted of 3 -5 explants.The observation was conducted every two weeks under a stereomicroscope (Olympus SZX7).The variables observed were when the callus formation begin, the percentage of explant form callus, and the callus type.
(A) (B) Figure 1.The immature leaf lobe from cassava plantlet (A) and the excised leaf lobe explant (B) for embryogenic callus induction.

Subculturing
The embryogenic callus formed was subcultured onto the same medium up to three times with an interval of three weeks for proembryonic proliferation.Explants were incubated at 28°C in the dark.

Statistical Analysis
Experiments were arranged in a completely randomized design with two factors of experiment, variety and concentration level of picloram.Experiments were replicated three times.Data were analyzed using an ANOVA test followed by the Least Significant Difference (LSD) test at a 5% level.Analysis was performed with the Statistical Tool for Agricultural Research (STAR) 2.0.1 software.

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Result and discussion Somatic embryogenesis is a pathway of regeneration that is important for genetic improvement [6].In this experiment, callus formation began at two weeks after planting the explant on the treatment medium.Callus formation of immature leaf began with the development of a swollen mass of tissue at the cut edges of the leaf and extended to the entire leaf surface.The resulting callus characters on callus induction medium showed mostly embryogenic callus forma compact non-embryogenic callus and a fibrous callus.The efficient production of somatic embryogenesis was evaluated based on both the percentage of callus for each treatment, and the percentage of embryogenic callus for each treatment (Table 1).Somatic embryogenesis in cassava begins with the induction of primary embryos from explants by culturing on cassava induction callus medium, followed by the production of secondary embryos.Mature cassava somatic embryos are regenerated into cotyledonary plantlets via organogenesis [8].
The result of the ANOVA test showed that the interaction between the genotype and the treatment media had no significant effect on the percentage of callus and embryogenic callus.The genotype also had no significant effect while the treatment medium had a significant effect on the percentage of callus and embryogenic callus.The MS basal medium with picloram 3 -12 mg L -1 could induce embryogenic callus formation in Menti and Adira 4 immature leaf lobe explants.These results might indicate that both types of cassava tested showed a high embryogenic callus formation frequency at all picloram concentrations.Embryogenic callus subcultured on the same media showed an increase in size and also showed more proembryo formed.
In general, Menti and Adira 4 explants showed an early stage of embryogenic callus formation on treatment medium with picloram.The potential of somatic embryogenesis, as indicated by the percentage of embryogenic callus formation, was evaluated in Menti and Adira 4. The highest percentage of explants forming embryogenic callus was on D medium (MS + 3 mg L -1 Picloram) but not significantly different with A, B, C medium (Table 1).It means that the D medium was the most efficient to induce embryogenic callus from the immature leaf lobe of Menti and Adira 4.
Picloram has been reported to induce embryogenic callus in many studies [9,10,11,12], which is in line with this experiment.Zainuddin et al. [4] recommended an embryogenic callus induction medium with 12 mg L -1 picloram to produce the embryogenic callus of cassava.However, in this study, medium containing 12 mg L -1 and 3 mg L -1 of picloram had no significant difference in the percentage of callus and embryogenic callus.The A, B, C and D medium produced a higher percentage of embryogenic callus (78.9-84.4% range) compared with the O medium (15.5%).All explants that produced callus on C medium had embryogenic callus characteristics, so the percentage of callus was the same as the percentage of embryogenic callus.However, in other medium, the percentage of callus was not the same as the percentage of embryogenic callus.For example, the explant on A medium had 100% of explant produced callus but only 83.3% of it showed embryogenic callus characteristics.
Rossin and Rey [8] state, somatic embryos potential should be evaluated from 4 to 6 weeks after induction, as was carried out in this study.The ability of cassava genotypes to produce somatic embryos is influenced by the explant type.The most commonly used explants type to induce SE is ILL [4,8,9,10,13,15], followed by shoot apical meristem [9,10,15] and axillary bud [8,16].
Figure 2 shows the embryogenic callus formation of cassava three weeks after culture on the treatment medium.Explant changed colour to yellow and became larger in size after six weeks of culture on an embryogenic callus induction medium.The colour of the callus was white in the first weeks which later turned dark yellow and ceased to grow.Yelli et al. [13] and Mahadi et al. [17] reveal this decrease in growth is due to the callus reaching optimum cell division, its physiological degradation and/or lack of nutrients.In this study, both cultivars showed high SE potential that could be exploited for genetic modification for important traits such as cassava mosaic disease resistance and drought tolerance.

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Conclusion Production of embryogenic callus has been tested in the Indonesian cassava, Menti and Adira 4. Picloram at the wide range concentration (3 -12 mg L -1 ) could effectively induced the formation of embryogenic callus.The ability of Menti and Adira 4 to produce embryogenic callus is an important factor for initiating the cassava regeneration system through embryogenesis.This result will be beneficial for seedling production and genetic transformation in these species which will improve the plant quality and productivity.

Figure 2 .
Figure 2. The embryogenic callus formation at 3 weeks after embryogenesis induction on treatment medium D in Menti (A) and Adira 4 (B) varieties.

Table 1 .
Percentage of explants forming embryogenic callus on MS medium containing different picloram concentration.