Enhancing the regeneration of TIS-derived somatic embryos of oil palm (Elaeis guineensis Jacq.) with CaCl2 and GA3

Micropropagation of oil palm through somatic embryogenesis (SE) technique provides clonal and uniform seedlings as high-quality planting materials for plantation. However, a problem associated with oil palm SE is the low regeneration rate of the somatic embryos. It was revealed that nutrient balance played a vital role in the success of SE regeneration. Therefore, a study was conducted to determine the balance between a macronutrient, calcium, and the germination-inducing hormone, Gibberellin (GA3) in enhancing the regeneration of the oil palm somatic embryos derived from the Temporary Immersion System (TIS). Treatments consist of various concentrations of CaCl2 (440, 880, or 1320 mg L-1) and enrichment of GA3 (2 or 4 mg L-1) applied into SE regeneration media, followed by the observation of regeneration rate and shoot tip necrosis after five weeks of culture. The study revealed that CaCl2 in the concentration of 880 mg L-1 was the best in stimulating oil palm TIS-derived SE regeneration. A higher concentration of GA3 (4 mg L-1) was needed in the first transition of SE from TIS into SE regeneration media in solid culture, while further SE-derived shoots development needed a lower concentration of GA3.


Introduction
Somatic embryogenesis (SE) has been commonly applied for the propagation of oil palms [1,2].The main benefit of propagation through SE is producing clonal planting materials.Conventional clonal propagation of oil palms through vegetative propagation is impossible due to the lack of axillary shoots.The use of clonal planting materials, especially derived from superior mother trees, could increase plantation yield by at least 20% [3].Therefore, SE can accelerate the multiplication of trees with particular traits such as high yielding or resistance against pests or diseases.Moreover, SE is an important tool for molecular breeding [4].
Many researchers have worked on the optimization of oil palm SE protocols [1,5,6].However, there are still some bottlenecks in the oil palm SE, such as low efficiency of SE induction and regeneration [6], and the risk of somaclonal variation resulting in abnormal planting materials [7].Although many improvements in the initial steps of SE have successfully enhanced callus formation and somatic embryos expression [1,2], the somatic embryos' germination into shoots, and further development into plantlets is often problematic, such as low germination rate and occurrence of shoot tip necrosis [8].Monteiro et al. [9] reported that the maximum regeneration rate of oil palm somatic embryos was around 50% for the best treatments.Nutrient imbalance including macronutrients and hormones is cited as one causing problems in somatic embryo regeneration [10,11].
Taken together, all the bottlenecks hamper large-scale oil palm propagation without implementing a proliferation phase, whether callus proliferation or somatic embryo proliferation.However, proliferation does increase the risk of somaclonal variation due to intensive exposures of explants to the medium [12].Therefore, a system called a temporary immersion system (TIS) is a way to scale up plant propagation while preventing somaclonal variations.TIS implemented a semi-automated system to control the duration and period of the medium-immersing explants temporarily.The system was revealed to be beneficial for plant growth and multiplication because it composes a balance between nutrients and gas for plant cells in the system [13].Moreover, the limited exposure between plant and medium can minimize the risk of somaclonal variation [14].
Protocols for the propagation of oil palm through SE utilizing TIS have been reported [5,14].Typically, the callus initiation step was conducted through a solid culture system, while TIS was implemented for callus proliferation until somatic embryo expression or further somatic embryo regeneration.Therefore, in this study, TIS was utilized to proliferate the oil palm callus, then induce the callus to emerge somatic embryos.Afterward, somatic embryo regeneration was conducted in the solid culture system with treatments of balancing a macronutrient (calcium) with hormone (GA 3 ), to cope with some problems in somatic embryo regeneration, especially low germination rate, shoot slow growth, and shoot tip necrosis.

Callus initiation
Callus was initiated from immature leaves of Tenera oil palm obtained from the Indonesian Oil Palm Research Institute -Bogor Unit plantation.The callus initiation phase was performed in jar bottles containing 30 mL solid callus initiation medium (CIM).CIM consisted of Murashige & Skoog (MS) basal minerals supplemented with 2,4-Dichlorophenoxy acetic acid (2,4-D), Kinetin, 30 g L -1 sucrose, 2 g L -1 activated charcoal, and 3 g L -1 gelling agent.Callus initiation culture was incubated in the dark room at 25 °C until callus emerge from explants.The initial callus was subcultured 2 -3 times in the same composition medium to achieve embryogenic callus as materials for the next phases in TIS.

TIS culture: callus proliferation, somatic embryos expression and maturation
SE phases conducted in TIS were callus proliferation, somatic embryo expression, and maturation.About 1 gram of embryogenic callus was transferred aseptically into the TIS flask in the Laminar Air Flow (LAF) cabinet.The upper containment of the TIS flask contained explants (callus), while the lower containment contained liquid callus proliferation medium (CPM).CPM is composed of a similar composition with CIM omitting gelling agent and with a lower concentration of 2,4-D.After 2 -3 cycles of proliferation, most of the embryogenic calli turned into embryoid or globular somatic embryos.The globular somatic embryos were then maturated by replacing the medium with the embryo maturation medium (EMM) containing CPM enriched with abscisic acid (ABA).All TIS cultures were incubated in the culture room with 12 hours of photoperiod under LED lamps (20 µmol m -2 s -1 of light intensity), at 25 °C of temperature.The immersion period of TIS was programmed following the method of Saptari et al. [15]

Optimization of somatic embryos 11regeneration
Phases of regeneration are somatic embryos germination into shoots and shoots maturation into plantlets.Matured embryos (scutellar -coleoptelar embryos) derived from TIS were transferred into jar bottles containing regeneration medium (RM).The composition of RM was MS basal minerals with various concentrations of CaCl 2 in the form of CaCl 2 .2H 2 O (440; 880; 1320 mg L -1 ), supplemented with Kinetin, 30 g L -1 sucrose, 2 g L -1 active charcoal, and 3 g L -1 gelling agent, and Gibberellin (GA 3 ) (2; 4 mg L -1 ).Regeneration was conducted in three cycles, 5 weeks for each cycle.The first cycle was applied to TIS-derived mature embryos.The second cycle was conducted by subculturing explants from the first cycle to the same composition medium.Afterward, growing shoots from the second cycle were transferred to RM to perform the third cycle.All regeneration cultures were incubated in the culture room with 12 hours of photoperiod under LED lamps (20 µmol m -2 s -1 of light intensity), at 25 °C of temperature.

Data collection and analysis
The experiment for the optimization of somatic embryo regeneration was designed following a completely randomized design (CRD).Treatments applied in the experiment (various doses of CaCl 2 .2H 2 O and GA 3 ) have each ten technical (number of jar bottles) with 2 and 4 biological (clump of somatic embryos, and 4 shoots clump respectively) replicates.Data were collected in the transition of each cycle, including the number of growing germinants/ shoots, shoots height, and occurrence of shoot tip necrosis (STN).The data were then adjusted to the analysis of variance followed by Tukey's posthoc tests to evaluate the effectiveness of treatments.

Results and Discussion
The oil palm callus emerged from the explants starting from 6 weeks of incubation.Initial callus was typically translucent with a soft watery texture (Figure 1.a).The initial callus must be subcultured 2 -3 times to achieve embryogenic potential.Callus with embryogenic potential was typically hard nodular or friable, with smaller nodular sizes to the initial calli (Figure 1.b), and fast-growing.
Furthermore, the proliferation of embryogenic callus in TIS (Figure 1 c, d) resulted in 8 -13 multiplies of biomass.The embryogenic calli then expressed somatic embryos after 2 -3 cycles of TIS.The early phase of somatic embryos was whitish globular in structure (Figure 1.e: GE), while the next phase was elongated and scutellar embryos (Figure 1.e: EE, SE).The scutellar embryos were further developed into colleoptelar embryos characterized by the greenish elongated shoot apical meristem (Figure 1.f), expected to develop towards coleoptile/ shoots.
The well-matured embryos from TIS were induced to germinate in the RM.Table 1 showed that in the first cycle, germination was better in the medium containing 880 mg L -1 CaCl 2 and 4 mg L -1 GA 3, resulting in the highest number of growing shoots, highest shoots heights average, and relatively low incidence of shoot tip necrosis (Table 1).Each clump of somatic embryos produced ± 3.2 shoots with an average height of 1.20 cm in the optimum treatment.Meanwhile, in the next cycle, lower concentration GA 3 (2 mg L -1 ) resulted in better germination and shoot growth (Table 2).After two cycles of regeneration, the oil palm TIS-derived somatic embryos have developed into 1 -2 cm in vitro shoots (Figure 2).
The 1 st and 2 nd cycles of regeneration were focused on obtaining more shoots, while the further regeneration cycle was focused on the shoots' development, mainly reflected by the increase in the shoots' height.According to Table 3, the use of 1320 mg L -1 CaCl 2 combined with 2 mg L -1 GA 3 produced the highest number of new growing shoots.However, as for the shoots height increament was better with the media containing 880 mg L -1 of CaCl 2 and 2 mg L -1 GA 3 , the same composition as the optimum medium in the 2 nd cycle.It achieved ± 0.66 cm of shoots height increament in the 3 rd regeneration cycle (Table 3).
Generally, the optimum concentration of CaCl 2 in the regeneration medium was 880 mg L -1 .A balanced amount of calcium (in the form of CaCl 2 .2H 2 O in the tissue culture medium) was commonly ascribed to play a role in the in vitro shoot development, particularly in relieving shoot tip necrosis.Machado et al. [16] reported halved incidence of shoot tip necrosis in the in vitro shoots regeneration of true lavender (Lavandula angustifolia Mill.), by increasing the threefold of CaCl 2 concentration of the MS medium (from 440 to 1320 mg L -1 ).Mubina et al. [17] eliminated shoot tip necrosis in the shoot regeneration of chickpea (Cicer arietinum L.) by doubling the MS standard concentration of CaCl 2 and KNO 3 .Similarly, results in Table 1 -3 also showed that shoot tip necrosis incidence was relatively lower with the use of a higher concentration of CaCl 2 from the standard MS medium (440 mg L -1 ).In the first transition between TIS culture to a solid culture of regeneration phases, the shoot tip necrosis incidence with the MS standard concentration of CaCl 2 reached 30% (Table 1).This might be due to      In plant cells, Ca 2+ serves as a universal secondary messenger molecule.Fluctuation of Ca 2+ level upon external stimuli, such as biotic and abiotic stresses, functions as a signal for initiating downstream responses [18].Therefore, in this research, more levels of Ca 2+ released by CaCl 2 .2H 2 O in the medium might help the better response of somatic embryos in tolerating stress due to culture transition, resulting in better regeneration performance.Nevertheless, Ca 2+ was also correlated with some hormone signaling [19].Okada et al [19] reported that gibberellin induced an increase in cytosolic Ca 2+ .Previously, Moll & Jones [20] investigated the relationship between calcium ions and GA 3 in inducing the hypocotyl growth of lettuce, revealing that GA 3 balanced the CaCl 2 effect on the hypocotyl growth.Therefore, determining the optimum combination between GA 3 and CaCl 2 is necessary for promoting the shoot development process.Taken together, a combination between 880 mg L -1 CaCl 2 and 4 mg L -1 GA 3 in the RM was optimum to be used in the first cycle of oil palm somatic embryo regeneration, while for further shoot development, GA 3 level could be reduced.However, has all phases of SE cultivated in the same culture system, it might need further optimization.
The occurrence of shoot tip necrosis in the shoot maturation phase is relatively high (Table 3).The highest occurrence reached 44.2%.The optimum treatment (CaCl 2 combined with 2 mg L -1 GA 3 ) was able to halve the incidence.However, the occurrence of ± 20% shoot tip necrosis might still significantly affect the efficiency of oil palm seedlings production through SE, as also reported by Sha et al. [21].Shoot tip necrosis, also called apex necrosis or shoot tip damage, was a sign of browning and death during the development process of the shoot tip (Figure 1.f: STN).The symptom appears from the shoot tip and then spreads to the shoot's lower parts.Although it is not always fatal to the plant, the occurrence of shoot tip necrosis still inhibited or delayed shoot growth.In the woody plant species, shoot tip necrosis mostly encouraged axillary branching [21].However, for the oil palm species with no axillary branch architecture, this shoot tip necrosis was a severe problem that should be maximally suppressed.

Conclusion
Low germination of the oil palm somatic embryogenesis could be enhanced by optimizing the nutrient balance in the medium, such as the macronutrients and hormones.CaCl 2 in concentration the of 880 mg L -1 was the best in stimulating oil palm TIS-derived SE regeneration.A higher concentration of GA 3 (4 mg L -1 ) was needed in the first transition of SE from TIS into SE regeneration media in solid culture, while further SE-derived shoots development needed a lower concentration of GA 3 .Further study is still necessary to better conquer the problem of shoot tip necrosis in the oil palm somatic embryo regeneration.

Acknowledgment
The research was a part of the project of Research, Development, and Utilization of Oil Palm Clonal Seedlings, funded by Holding Perkebunan Nusantara RI.

Figure 2 .
Figure 2. The oil palm shoots after 5 weeks cultured in the optimum regeneration media.

9 *
Different letters in the same column indicate means are significantly different according to Tukey's post hoc test wit a significance level of 0.05.

Table 1 .
The 1 st cycle of oil palm somatic embryo regeneration.Explant: Matured embryos from TIS Different letters in the same column indicate means are significantly different according to Tukey's post hoc test wit a significance level of 0.05.

Table 2 .
The 2 nd cycle of oil palm somatic embryo regeneration.Explant: Germinants from the 1 st cycle

Table 3 .
The 3 rd cycle of oil palm somatic embryo regeneration.Explant: Shoots from the 2 nd cycle