In vitro somatic embryogenesis and plantlet regeneration from immature male inflorescence of adult dura and tenera palms of Elaeis guineensis (Jacq.)

The regeneration protocol reported here via indirect somatic embryogenesis has three
major developmental stages: induction of callus, induction of embryogenic callus,
somatic embryo maturation, and plantlet regeneration. Auxins are critical for the
callus induction from explants of oil palm which is the first step in the oil palm
tissue culture process. There was tissue swelling and profuse growth within 3–4 months
and callus induction was observed from the cut edges (Figure 1c) in all the four different auxin treatments viz., A1, A2, A3 and A4. The overall
callus induction percentage ranged from 54.67 to 82.44 percent in dura palms (Figure 2a). Callus induction obtained with 2,4-D was not significantly different from that
obtained with picloram and the combination of 2,4-D + picloram, which indicated that
the effect of picloram as auxin was similar to 2,4-D in inducing callus. The auxin
NAA also could induce callus but it was significantly less as compared to other auxins
(Figure 2a). There were genotypic differences in responses to auxins. Data analysis of callus
induction is given in Figure 2b. In D1 palm the callus induction ranged from 45.67 to 80%. In this palm the best
callus induction was obtained with picloram. In D2 palm both 2,4-D and picloram are
able to induce the maximum callus induction of 83.3% and were on par to each other.
In D3 palm the best callus induction was 86.67% obtained in media with 2,4-D and it
was not significantly different from that obtained with picloram (84%). Callus induction
with NAA was significantly lower in all the palms. In case of two tenera palms, the callus induction obtained was ranging from 42 to 72% on Y3 media with
both 2,4-D and picloram (Table 1). The callus induction obtained with T1 tenera palm was better than T2 palm on the
same media and was significantly different. Teixeira et al. (1994]) reported a callus induction percentage ranging from 6 to 50 from immature female
inflorescence of oil palm when the 2,4-D concentration ranged from 400 to 500 µM in
MS (Murashige and Skoog 1962]) media. The callus induction obtained in our experiments using Y3 media was much
higher than reported earlier. Activated charcoal is the main antioxidant that is used
for absorption of phenolic compounds in palm tissue culture and its use has been reported
in all palms (Valverde et al. 1987]; Karun et al. 2004]; Huong et al. 1999]; Steinmacher et al. 2007]; Luis and Pereira 2014]). However the use of activated charcoal should be coupled with the use of high concentration
of auxins since most of these auxins are also absorbed by activated charcoal. Thuzar
et al. (2012]) reported that use of 100–140 mg/l of 2,4-D in the presence of activated charcoal
will effectively supply only 2–2.8 mg/l of the auxin and the remaining will be absorbed
by activated charcoal. In our experiments we found that activated charcoal at 0.3%
was effective in controlling oxidation and thereby improving the callus induction
percentage. These calli had to be subcultured after every 4 months to a fresh media
with reduced concentration of auxins. After every subculture it was found that there
was an increase in oxidation of calli and later the tissues again started responding.
The emergence of embryogenic calli was observed after fourth subculture and continued
until sixth subculture. The observations on the percentages of cultures showing embryogenesis
were taken at the end of sixth sub culture. The embryogenic calli induction was characterised
by the appearance of white to yellowish globular or nodular structures with suspensor
region (Figure 1d). By the end of fourth subculture, these embryogenic calli began to multiply and
proliferate rapidly (Figure 1e). Asynchronous development of somatic embryos was observed when observed under the
microscope. Different stages of somatic embryos including the heart shaped and torpedo
shaped embryos were clearly visible (Figure 1f). The data obtained on embryogenic calli was analyzed and the data revealed that
2,4-D + picloram induced the highest embryogenesis of 4.9% followed by picloram at
3.4%. Both 2,4-D and NAA induced low embryogenesis and were not significantly different
from each other (Figure 2c). The overall embryogenesis percentage ranged from 0.33 to 4.98 in dura palms. Genotypic analysis among dura palms revealed that D2 palm was more potent for embryogenesis (7.3%) compared to
other two palms (Figure 2d). In all the palms it was found that the combination of 2,4,-D and picloram could
induce the maximum embryogenesis. Though the auxins 2,4,-D and picloram individually
could induce maximum callus induction in dura palms, maximum embryogenesis was obtained only with the combination of 2,4-D + picloram
in all the three palms. Hence, this was selected as most potent treatment and used
for tenera palms. In case of tenera palms, the embryogenesis ranged from 6.8 to 9.35% with 2,4-D + picloram in Y3 (Table 1). The embryogenesis percentage obtained with tenera palms was better than dura palms on the same media. Among the tenera palms T1 palm showed a better callus induction and embryogenesis percentage.

Figure 2. Effect of different auxins on callus induction and embryogenesis from immature male
inflorescence of dura palms. A1, A2, A3 and A4 represents 2,4-D, picloram, 2,4-D + picloram and NAA, respectively. D1, D2 and D3 represents the three dura palms selected for the study. Means were compared using least significant difference
test (LSD: p  0.05). Means with the same letter are not significantly different from
each other. a Effect of different auxins on callus induction. b Analysis of callus induction obtained with different auxins in dura palms. c Effect of different auxin treatments on embryogenesis in dura palms. d Analysis of embryogenesis percentage obtained with different auxins in dura palms.

Table 1. Callus induction and embryogenesis percentages in tenera palms

Oil palm tissue culture is still a challenging process since it is found that certain
genotypes do not respond to culture conditions and are not embryogenic (Thuzar et
al. 2012]). Also this tissue culture process is affected by numerous culture conditions, including
culture medium composition, genotype, type and concentration of growth regulators
etc. (Feher et al. 2003]). In our study it was found that our genotypes were showing a high percentage of
primary callus induction which has not been so far reported and also they were embryogenic.
Teixeira et al. (1994]) reported that while primary callus induction from immature inflorescence was possible
in MS media, embryogenic calli induction was possible only in Y3 media. The superior
effect of picloram in combination with activated charcoal for induction of embryogenic
callus induction has been reported from many monocots and palms (Karun et al. 2004]; Huong et al. 1999]; Steinmacher et al. 2007]; Luis and Pereira 2014]; Beyl and Sharma 1983]; Fitch and Moore 1990]; Groll et al. 2001]). Superiority of this auxin over the other auxins has been attributed to the effective
uptake and mobilisation of this growth regulator and rapid mobilisation at target
sites (Karun et al. 2004]).

On transfer to light in regeneration media the well developed somatic embryos regenerated
into plantlets (Figure 1g). After sufficient shoot growth, they were transferred to Y3 basal media with reduced
concentration of activated charcoal (0.5 g/l) and increased concentration of sucrose
(60 g/l) along with IAA (23 µM) and IBA (19.6 µM) for rooting. The rooted plantlets
(Figure 1h) of ?10 cm shoot and 5 cm root were transferred to sterile soil in pots covered
with polythene bag and kept in the laboratory conditions for a month. The established
plants were transferred to pots containing sterile soil and transferred to mist chamber
(Figure 1i).

Male inflorescence was chosen since it was found in our preliminary work there was
lesser oxidation in male inflorescence as compared to female inflorescence and also
better response for callusing. These were observed in several repeated experiments.
Male inflorescence apart from producing pollen is not of much use to the palm since
it is the female inflorescence that forms the fruits and bunches. Hence collection
of male inflorescence will not be a loss to the palm and also it can be collected
with minimal damage to the palms.