In vitro germination of zygotic embryos of hybrid BRS Manicoré ( E . guineensis X E . oleifera )

The interspecific oil palm hybrid BRS Manicoré ( E . guineensis x E . oleifera ) has superior agronomic characteristics. However, the germination rate is low (30%) and the process is slow when the seeds are sown in a conventional form. The purpose of this study was to optimize the in vitro germination of zygotic embryos of this hybrid comparing seed lots. The viability of zygotic embryos was evaluated by the tetrazolium test (0.075%) for 4 h. The embryos were cultured on MS and Y3 culture media, with and without the addition of NaH 2 PO 4 , as well as on MS, MS½ and N6 medium. In MS medium containing NaH 2 PO 4 , the germination rate was increased from 40 to 70% in comparison with the medium without sodium phosphate. The comparison between the culture media MS, MS ½, N6 and Y3 showed that 75% of zygotic embryos cultured in the Y3 medium formed whole plants (with roots and shoots defined), a higher percentage than embryos cultured on MS, MS ½ and N6 media (46, 35 and 17% respectively). In the same Y3 culture medium, the embryos were larger (36% ≥ 2 cm and 30% ≥ 5 cm) than in the other media. Results obtained by the tetrazolium test were similar to those of germination, showing the effect of the genotype of each seed lot. For the germination and development of plantlets it is essential to add NaH 2 PO 4 to a culture medium containing no phosphate or with a low phosphate concentration.


INTRODUCTION
The hybrid palm BRS Manicoré is the result of a cross between two species of palm trees, the African one (Elaeis guineensis) and caiaué (Elaeis oleifera) that occurs in the Tropical Moist Amazon (Collares 2011). This interspecific hybrid stands out from the other cultivars produced, because it has high production capacity, around 30 ton/bunch/ ha, is immune to fatal yellowing disease (Cunha et al. 2009) and is smaller than the African oil palm, which facilitates manual labor. Moreover, it produces more unsaturated oil, that is clearer, more uniform and contains less saturated fatty acids than the oil of the African palm (Ramos et al. 2006).
However, one of the difficulties of this culture is the availability of seedlings on a large scale, as palms have a single meristem and propagation by conventional methods is impossible. It is therefore produced exclusively from seeds (Kiem 1958), which have slow, irregular and often low germination (Meerow 1991). In some cases, embryos abort within the seed, preventing their development (Alves et al. 2011). Thus, only 30% of the seeds germinate, which complicates the conventional propagation of palms of economic interest (Angelo et al. 2007).
The in vitro culture of zygotic embryos can be a useful tool to reduce the germination time and provide higher rates of embryos developed into plants (Lima 2013), as the in vitro culture allows the removal of physical barriers such as hard endocarp and facilitates the supply of nutrients to the developing embryo (Pádua et al. 2014). In vitro germination can overcome this problem by embryo rescue. In this study we optimized the in vitro germination of zygotic embryos of BRS Manicoré hybrid palm comparing various culture media and various seed lots.

PLANT mATERIAL
The research was conducted in the Laboratory of Micropropagation of the Department of Botany -UFPR, Curitiba, Brazil. The seeds were obtained from mother plants of the hybrid BRS Manicoré (E. guineensis x E. oleifera) after controlled crossing and were supplied by EMBRAPA -Western Amazonia. The seeds were collected 150 days after pollination. The hard endocarps were removed and kernels washed with mild soap and running water. They were then immersed in 70% ethanol for 5 min followed by 20 min in commercial bleach (10% active chlorine v/v) containing 1% Tween-20 and rinsed four times with sterile distilled water. The embryos were isolated from the seeds and then disinfected with commercial bleach (2% active chlorine) for 5 min and finally rinsed three times in sterile distilled water.

tETRAzOLIUM tEST OF SEED vIABILITY
The zygotic embryos were isolated and placed on moistened Germitest paper, packed in plastic bags and placed in an oven at 30°C for 16 h. They were then kept in individual plastic containers (4 replicates of 25 embryos for each seed lot), embedded in a tetrazolium solution 0.075% (w/v), pH 6.5 ± 0.1, and placed in a B.O.D at 40°C for 4 h in the dark. After this period, the embryos were washed with running tap water on a sieve in order to remove excess salt (Maquiné et al. 2014).
The embryos were evaluated under a stereoscopic microscope by observing the degree of coloration of the main parts: tigelo and haustorium and the embryos were classified as either vigorous (capable of germinating) or non-vigorous. This classification followed staining patterns of oil palm seeds based on classes established by Maquiné et al. (2014) and modified by Lima (2013). Class 1: Viables with high vigor, tigelo and haustorium with homogeneous red or pink color ( Figure 1c 1 of myo-inositol. In the second experiment MS medium plus NaH 2 PO 4 (0.17 g/L -1 ) and the same media without NaH 2 PO 4 were compared. The same comparison was made in the case of Y3 culture medium. All media were supplemented with 2 g L -1 activated charcoal (AC), 30 g L -1 sucrose and solidified with 6 g L -1 agar (Vetec). The pH of the culture media was adjusted to 5.8 with NaOH 0.1 N or HCl 0.1 N and the media were autoclaved at 120°C for 20 min. Activated charcoal was added along with agar, after pH adjustment. After inoculation, the cultures were maintained in the dark for 30 d at 25 ± 2°C during the day and 21 ± 2°C overnight, and transferred under fluorescent light (white light) with irradiance of 40 µmol. mˉ².sˉ¹ and photoperiod of 16 h during four weeks. After 30 d the percentages of viable embryos, embryos without response and abnormal embryos were evaluated. Crooked and swollen embryos showing a ligule and primary root were considered germinated. Atrophied, rootless and shootless embryos were considered as abnormal. After 45 days the shoot length was measured. The plantlets with shoots and roots were transferred to 56 cm³ polyethylene tubes containing vermiculite as substrate. After planting, they remained in a greenhouse with artificial lighting (light intensity of 13 μmol.mˉ².s -1 , 12 hours photoperiod and temperature of 24 ± 5°C during the day and 20 ± 5°C overnight) automatically irrigated for five minutes every six hours .
The experimental design was entirely randomized with one embryo per tube and 50 repetitions (tubes) per treatment. The experiment in which four culture media were compared (MS, MS ½, N6, Y3 and MS with and without sodium phosphate) was carried out using two lots of seeds whereas three lots were used for the comparison between Y3 media with and without phosphate. Data were subjected to Bartlett´s test in order to verify the homogeneity of variances and then the means were compared by Tukey´s test at 5% probability.

RESULTS
zygotic embryos of oil palm hybrid Manicoré were classified according to the four classes of vigor and viability, based on the intensity, uniformity and location of color patterns of the two parts of the embryo (tigelo and haustorium). Classes 1 and 2 (Figures 1c, d and e) correspond to viable and vigorous embryos that can turn into plantlets with root and developed leaf sheaths. Class 3 (Figures 1f and g) encompasses viable but weak embryos, that are alive but unlikely to produce a plant. Class 4 (Figures 1h and i) corresponds to unviable embryos which can be dead or unable to give plantlets with roots, because of a lack of metabolic activity in the tigelo portion, where the embryonic axis is located.
As shown in Table I, the viability of the embryos varies with the lot of seeds, which means that it can be influenced by the genotype of each lot. Among six lots, only three (CS 428, CS 1477 and CS 1681) had most of the embryos in class 1 according to the tetrazolium test, in other words, embryos that have the ability to produce welldeveloped plantlets.
Lot CS 736 shows most of the embryos in Class 2 (Table I). They are viable with medium vigor, but still have the capacity to produce welldeveloped plantlets. The viability of the embryos is related to the germination and development of normal plantlets (with roots and shoots) ( Figure  2i). These viable embryos cultured in suitable culture media successfully germinated.
Different culture media were compared for lots CS 428 and 1139 which presented 96 and 73% of viable embryos (Table I). Germination rates of 59 and 90% were observed, respectively, for these lots in Y3 culture medium (Table II) whilst the results obtained in other culture media were lower. The overall average for germination rate in all culture media (MS, MS 1/2, N6, Y3 and MS with and without phosphate) was 37.5% for CS 428 and 49.5% for CS 1139 (Table II ).
Relating the average viability of the embryos of three lots of seeds (735, 1477 and 1681) with germination rates in two Y3 culture media (with and without phosphate), embryos viability of 46, 76.25 and 92.50%, respectively, and mean germination rates of 33.6, 68.6 and 91.6% were observed (Table  II). In both Y3 culture media, lot CS 1681 had the highest percentages of viability and germination, with 92.5 and 91.6% respectively (Table II).
In the first two days of culture, zygotic embryos did not show changes in their morphology in the tested media (MS, MS½, N6 and Y3). In the first week of culture only enlargement and swelling were observed (Figure 2a). Embryos cultured for 10 d were curved (Figure 2b) and after 15 d some Values followed by the same letter do not differ significantly by Tukey´s test at 5% probability. embryos remained curved and presented a plumular hook with ligule and others only root primordia (Figure 2c). On the twentieth day the foliar and root primordia appeared in all of them and a decrease of the haustorium size was observed. At this stage, nutritional supplementation was provided by the culture medium, and the haustorium was no longer needed to supply nutrients to the embryo (Figure 2d). After 30 days, the plantlets already had shoots and roots (Figures 2 and f) and after 50 days they were acclimatized. In all culture media some explants did not respond and some others developed abnormally (Table III). The zygotic embryos germinated in all the culture media, but in the Y3 culture medium the ger-mination process was better (75% of embryos with the first leaf and primary root) and the embryos developed into complete plants, in other words, with defined root and shoots, unlike the embryos cultured in the other media (Figure 2i and Table III).
When comparing the development of the embryos on MS medium supplemented with NaH 2 PO 4 and those on MS medium without NaH 2 PO 4 we observed 67.5% of germination in the first medium after 15 days whereas those on the second medium showed a high percentage of explants with no response (Table IV and Figure 2h). On the thirtieth day, the development continued to be better (49.2% of plantlets with leaf primordia and root) in the MS medium supplemented with NaH 2 PO 4 (Table IV).  In Y3 culture medium a higher percentage of embryos developed into complete plantlets (roots and shoots) than in MS medium (Table III), so, this medium was used again, with or without supplementation of NaH 2 PO 4 . However, there were no statistical differences between the rate of developing embryos in both Y3 media (Table IV). In both treatments embryos developed normally after 15 and 30 days of culture.
After 45 days of culture, the size of the plants ranged among MS, MS½, N6 and Y3 media ( Figure  2i): 36% of plantlets cultured in the Y3 media reached a height of 1 to 2 cm and 30% reached 2 to 6 cm. The MS, MS ½ and N6 media showed 20, 27.4 and 17.7% of plantlets with 1 to 2 cm respectively (Table V and Figure 2i).

DISCUSSION
The tetrazolium test can be used to evaluate the viability of oil palm hybrid embryos. The TTC test is based on the activity of dehydrogenase enzymes which act on respiratory processes of tissues, where the release of hydrogen ions occurs and 2, 3, 5 -triphenyl tetrazolium chloride reacts with H + to form a red, insoluble substance called formazan (Delouche et al. 1976). In the present study, lots submitted to the TTC test showed different rates of viability and, among the six lots, only three (CS 428, 1477 and 1681) had embryos with a uniform color. This indicates that they are able to give rise to well-developed plantlets. This result is similar to that obtained by Lima (2013): 93% of seeds were viable as the staining of the oil palm embryos (Elaeis guineensis) by the TTC test was uniform.
The germination rate of the seeds is not always related to the viability of each lot since zygotic embryos of CS 1681, when cultured in a Y3 culture medium with and without phosphate showed 92% viability and a germination rate of 88.8%. This shows that, while 92% are viable, only 88.8% were able to germinate. zygotic embryos (zE) from seeds of CS 428 and 1139 had a viability of 96 and 73% according to TTC test, while 59 and 90% germinated in Y3 culture medium. This may be because the seeds of these lots were viable, but zE of CS 1139 had more vigour to develop than those of CS 428. Other authors also consider that the TTC test can be used for rapid assessment of the viability of oil palm seeds. For example Mok (1972) reported a mean value of embryo viability of 98.6% and a mean rate of ex vitro germination of 95%. Murugesan et al. (2002) also found a correlation between viability and germination rate: oil palm embryos cultured in MS medium supplemented with IAA and kinetin reached a germination rate of 93% while the percentage of viability of these embryos varied between 88 and 92%, at TTC concentrations of 0.5% and 0.75%, respectively. zygotic embryos began their development after two days in the culture media. This delay can be explained by the fact that when the explants are put into a nutrient media, there may be an initial leakage of ions from damaged cells, especially Na + , Ca 2+ , K + , Mg 2+ (Soares 2011), so that the concentration in the plant tissues actually decreases. After this period, cells begin active absorption and their internal concentration rises slowly.
The addition of sodium phosphate in MS culture medium is critical for in vitro germination. It was observed that when the culture medium contained NaH 2 PO 4 , 49% of the embryos germinated after 30 d of culture, whilst in NaH 2 PO 4 -free MS medium there was a high percentage of non-responding explants. In another study (Cardoso et al. 2010), the addition of sodium phosphate to MS and MS½ media was also critical for converting oil palm zygotic embryos into plantlets and enabled plantlets with a larger stem and root to be obtained. When adding 0.17 g/L -1 NaH 2 PO 4 to MS media, these authors obtained an 85.18% germination rate for seeds of hybrid CN 470. Phosphate is the way phosphorus is absorbed by plant cells from the culture media and this process is more rapid than for other ions (George et al. 2008). According to these authors, the phosphate concentration in MS medium is insufficient for some species and this concentration is reduced to zero in a few weeks. Despite the MS medium contains 170 mg L -¹ KH 2 PO 4 the presence of a higher amount of phosphate is essential in most in vitro cultures due to the role of phosphorus in energy metabolism and regulation of enzymatic processes, being differentiation of shoot one of the consequences of phosphorus addition to the culture medium (Santiago et al. 2001). It also influences height, stem thickness and root size (Barcelos et al. 2001). Furthermore, phosphorus is part of the nucleotides, forming units of nucleic acids such as DNA and RNA, directly involved in the process of protein synthesis (George et al. 2008).
In the present study, Y3 culture medium was the most suitable for the full development of the plantlet into shoot and primary root. With regard to the optimization of in vitro germination, in Y3 culture medium the germination rate was higher (75%) than in other media. This could be due to the composition of this medium which contains nutrients similar to what the plant naturally needs, including sodium phosphate (320 mg/L) that is not present in MS media, having only potassium phosphate (170 mg/L). The concentration of phosphate ions is therefore of great importance for the development of palm plants. The mixture of nutrients such as urea, triple superphosphate, potassium chloride and magnesium sulfate used for natural seed germination enables plants to develop well (Barcelos et al. 2001).
On the other hand, some salts and organic substances of Y3 medium are different of those present in MS medium and some elements have different concentrations when compared to MS medium. It is therefore difficult to assign the better results obtained on Y3 medium only to phosphorus concentration. In other surveys of oil palms or their hybrids, MS, MS ½ and Y3 media were adequate for in vitro germination of zygotic embryos without statistical differences between the media, with 90% of germination in MS ½ and Y3 media, and 85% in MS medium (Chourykaew and Kanchanapoom 1996). The results of germination of the lots CS 428 and CS 1139 obtained in the present study showed that in MS culture medium the germination rates were low (42 and 50%, respectively) when compared to the rates obtained in Y3 medium (59 and 90%, respectively). However, Pádua et al. (2014) found that embryos of Manicoré seeds collected 100 days after anthesis presented a germination rate of 88% in MS or Y3 medium.
This work showed that the culture medium suitable for germination can vary according to the genotype of each lot of seed of oil palm hybrid BRS Manicoré. Similarly, Alves et al. (2011) showed that oil palm embryo development varies with the genotype since 37.93% of CJ 2141 embryos, grown on MS medium supplemented with phosphate, developed roots and shoot, meanwhile there was no embryonic development in the case of CJ 502.

CONCLUSIONS
The tetrazolium test can be used to evaluate the embryos viability. The germination rate of the seed lots is related with their viability which varies for each lot of seeds.
The addition of NaH 2 PO 4 to the culture media is essential for the germination of the oil palm hybrid, especially in the media which do not contain sodium phosphate or have a low phosphate concentration.
The Y3 culture medium is better for the full plantlet development into shoot and primary root. This is due to the composition of this medium that contains all the salts and vitamins that oil palm plantlets need to develop.

ACKNOWLEDGMENTS
The authors thank Wanderlei A.A de Lima and Embrapa Amazônia Ocidental for providing seeds, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for funding the research, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing a grant to Keila A.P. Bonetti and Eileen Bagyary for editing the manuscript.