Effects Of Different Sterilization Regimes & Growth Regulators On Micropropagation Of Female Date Palm (Phoenix dactylifera L.)

The success of in vitro culture techniques is always hampered by microbial contamination. The present study was carried out to develop an efficient protocol for date palm explants sterilization for successful somatic embryos induction and plantlets formation of some date palm varieties. The shoot tips were treated with different sterilizing agents at different concentrations and durations of exposure. The use of ethanol (70%), sodium hypochlorite (3.5% & 70%) and mercuric chloride (0.2%) with or without addition of Tween-20 had different effects on decontamination of the date palm explants. The percentage of explants contaminated with bacteria for sterilants 1, 2 and 4 was 18.8%, 6.3% and 6.3% respectively while 25%, 37.5%, 31.25% and 6.25% were contaminated with fungi for sterilants 1, 2, 3 and 4 respectively. Under the conditions used, a combination of antioxidants (Citric and Ascorbic acids at 100mg/l), 0.2% mercuric chloride and 3.5% sodium hypochlorite solution with 3 drops/100ml of Tween-20 helped in the reduction of chlorosis, contamination and die-back in the shoot tip explants. The explants were further cultured in appropriate media for callus initiation and subsequent somatic embryo induction. Optimal embryogenic callus was obtained from the shoot explant of sterilant number 4 which had the minimal contamination and die-back of all the cultures. After 3 subcultures, the somatic embryos formed were multiplied for shoot development. From this study, we established that the use of appropriate surface sterilant at suitable concentration and duration of exposure of date palm explant to it is indispensable for maximum responses of in vitro cultures.


Introduction
Date palm (Phoenix dactylifera L.) grows well in the hot sahelian zone of Northern Nigeria, where it is an important agricultural commodity . The high demand for female date palms on the local market in Nigeria requires expansion of increased production. High-yielding elite genotypes are needed for cultivation, but cannot be met by conventional breeding alone. Micropropagation through tissue culture is one of the most widely accepted tools for the large scale production of genetically improved plant materials (George and Sherrington, 1984). Date palm explants are exposed to microbial infection at all stages of tissue culture. Contamination might come with the explants or occurs during the propagation procedures. Different explanations have been proposed about the causes of microbial contamination of date palm tissue culture (Abass, 2013). One explanation is the method used to sterilize the explants, tool and equipment. Improper methods and insufficient amounts of disinfectants are the most common problems in tissue culture. Another problem is external contamination of the explants which comes from contaminated tools, equipment and workers in the preparation and culturing of media (Abass, 2013). The composition of the tissue culture medium is a good source of nutrients for microbial contaminants, with all the essential requirements to support their growth and development (Abass, 2013).
Culture initiation is the first and most critical stage in micropropagation (Daud et al., 2012;Vujovic et al., 2012). The major adverse effects of microbial contamination on date palm tissue culture are degradation and browning of the infected tissues caused by the release of substances into the medium, such as degrading enzymes (cellula se, phenol oxidase and others); microbes such as viruses, bacteria, yeast and fungi are found on the surface and inside the plant body, as well as toxins (Abass, 2013). Microbial contamination leads to waste of time, effort and material and contributes to severe economic losses (Hameed and Abass, 2006;Omamor et al., 2007). There is competition between the microbes and the plants for nutrients.
The success of plant tissue culture protocol depends largely on sterilization of the explants. Selection of sterilizing agents and time of exposure is also critical because the living material should not lose their biological activity, and only contaminants should be eliminated during sterilization (Tiwari et al., 2012). Utilizing a proper and precise sterilization can be vital to save time and effort. The different sterilants used on explant materials for in vitro multiplication affect tissue responses. Therefore, suitable concentration, exposure duration of the explants to the various sterilants and the sequence of using these sterilants have to be standardized to minimize explants injury and achieve better survival (Rady et al., 2018). The present study was undertaken to unveil the effects of different types of sterilizing agents by varying their concentrations and time of exposure on date palm explants for induction of somatic embryos and plantlets formation.

Sample collection
Explant materials (offshoot of 2 to 3 years) were excised from selected special palms of female dates with good fruit quality at the date palm experimental substation of the Nigerian Institute for Oil Palm Research (NIFOR), Dutse, Jigawa State.

Explants Preparation
The method of Khan and Bibi (2012) was used for explants preparation. This was then treated with different sterilizing agents at different concentrations and timing (Plate 1a and1b).

Plate 1a: Excised offshoots under
Plate 1b: Explant sterilization using laminar flow condition before sterilants at different concentrations commencement of the sterilization and timing before inoculation into the procedure media.
Sterilization Procedure Sterilant 1: The explant was kept in 70% ethanol (EtOH) for 10 min, rinsed 2-3 times in sterilized distilled water. It was then immersed in 10 % sodium hypochlorite (NaOCl) solution mixed with three drops/100ml of Tween-20 for 15 min and rinsed thrice in sterile distilled water.
Sterilant 2: The explant was kept in 70% ethanol (EtOH) for 10 min, rinsed 2-3 times in sterile distilled water, then kept in 10% Sodium hypochlorite (NaOCl) solution mixed with 3 drops/100ml of Tween-20 for 15min and rinsed 3 times in sterile distilled water. It was finally kept in 0.1% mercuric chloride for 5min and then rinsed thrice in sterile distilled water.
Sterilant 3: The explant was sprayed with 70% ethanol (EtOH) and left for 5 min after which it was further reduced and kept in 0.2% Mercuric Chloride (HgCl2) for 10min, rinsed 3 times in sterile distilled water. It was then dipped into 3.5% sodium hypochlorite (NaOCl) solution containing 3drops/100ml of Tween-20 for 20min and thereafter rinsed 3 times in sterile distilled water.
Sterilant 4: The explant was kept in an antioxidant (Citric and Ascorbic acids at 100mg/l) for 5min, rinsed once in sterile distilled water. Then it was immersed in 0.2% Mercuric Chloride (HgCl2) and placed on an orbital shaker for 10min, rinsed 3 times in sterile distilled water. It was finally kept in 3.5% sodium hypochlorite (NaOCl) solution containing 3 drops/100ml of Tween-20 for 20 min and then rinsed 3 times in sterile distilled water.

Culture Medium
Full strength of Murashige and Skoog (MS, 1962) culture medium was used for this experiment. The following supplements were incorporated in the culture medium 0.02% glutamine, 0.0125% myo-inositol, 0.0075% citric acid and ascorbic acid 3.0% sucrose and 0.8% agar. Combination of growth regulators (Table 1) with 1.5 g/l activated charcoal were tested for callus initiation. The pH was adjusted to 5.8 using potassium hydroxide (KOH) after addition of all compounds. Further trimming of the shoot tips was done in order to remove the upper and lower regions exposed to sterilants (Plate 2). It was then sectioned longitudinally and inoculated into the media (Plate 3). The cultures were incubated in the dark at 27±1°C with a proper and regular record for contamination and browning taken for 30 days. Only one factor was studied in this research because of limited explant materials. The surviving explants with the different sterilizing agents were subcultured every 4 weeks into the same media composition to evaluate embryogenic callus and somatic embryos for plantlets formation.  (3) KEY: NAA = 1-naphthalenacetic acid; 2, 4-D = 2, 4-dichlorophenoxyacetic acid and 2-iP = 6dimethylallylamino purine Plate 2: Trimmed Shoot tip explants Plate 3: Inoculation of shoot tips immediately after sterilization to expose the meristem.
after dissection into culture media containing growth regulators at different concentrations

Explants Sterilization
The result of the various effects of sterilizing agents on the contamination and colour change (browning) of the explants is presented in Figure 1. Percentage contamination was considerably higher in the explants sterilized with sterilants 1 and 2 than those that received sterilants 3 and 4 involving the use of Mercuric chloride and antioxidant solutions. The percentages of the explants contaminated with bacteria (Plate 4a and 4b ) for sterilants 1, 2 and 4 were 18.8, 6.3 and 6.3 % respectively, while 25, 37.5, 31.25 and 6.25% were contaminated with fungi (Plate 4c) for sterilants 1, 2, 3 and 4, respectively.
The explants sterilized using sterilant 4 proved better than those of steilants 1, 2 and 3. In contrast to contamination, the percentage chlorotic colour or necrosis developed by the explants was highest in the second sterilized explants with about 25% of the explants turning chlorotic after 4 weeks in the cultures. The fourth sterilized explants showed the least colour change with only 6.25% of them turning chlorotic with the highest percentage of survived cultures (Figure 1). Based on the result of this experiment, the explants were consistently sterilized in subsequent studies using the fourth method of sterilization which involves treatments with antioxidants (Citric and Ascorbic acids at 100 mg/l), 0.2% Mercuric chloride and 3.5% Sodium hypochlorite solution containing 3 drops/100ml of Tween-20.

Callus and Somatic embryos induction
Embryogenic callus and somatic embryos were observed after 4 to 12 weeks in the initiation media from the remaining surviving cultured explants. A maximum response was observed from the cultures of sterilant number 4 due to minimal contamination and die-back. The culture medium supplemented with 2, 4-D (100mg/l) and 2-iP (3mg/l) gave the highest callus and somatic embryogenic response (Plate 5a and 5b). pre-acclimatization process.

Plant Acclimatization
The regenerated plantlets were transferred to a mixture of peat moss and soil at equal volumes (v/v) and then kept in a plastic bag (Plate 13) under green house conditions and wetted with 10% MS once every week. Plantlets were sprayed with 2.5% benlate solution every 2 to 3 days to prevent crown and leaf rot. After 8-12 months, well acclimatized and hardened plants (Plate 14) were transferred to the open field for further monitoring.
Plate 13: Plant acclimatization stage Plate 14: Hardening plant in nursery after under controlled humidity and successful acclimatization process. temperature.

Discussion
The present investigation was carried out to optimize sterilization protocol on date palm shoot tip for induction of somatic embryos and plantlets formation in vitro. Under the conditions used for the sterilization of the explants, a combination of antioxidants (Citric and Ascorbic acids at 100mg/l), 0.2% mercuric chloride and 3.5% sodium hypochlorite solution containing 3 drops/100ml of Tween-20 proved to be the best of all the four sterilizing agents (Figure 1) as it greatly reduced chlorosis, contamination and die-back in the explants.
The use of ethanol, sodium hypochlorite and mercuric chloride had different effects on decontamination of the explants. The ineffectiveness and harmful effects of ethanol on decontamination of plant tissues have been observed by other researchers like Hammond et al. (2014) who recorded 100% contamination when 70% ethanol was used alone in the sterilization of sweet potato explants. Similarly, Sen et al. (2013), studying seed germination of Achyranthes aspera in vitro reported 100% contamination when ethanol alone was used for sterilization and that ethanol was more harmful to the explants. Furthermore, Osterc et al.  Acheampong et al. (2015) in their study reported that the duration of exposure of pineapple explants to sodium hypochlorite improved sterilization; however, exposure for long durations beyond 20 minutes were detrimental to the explants. Sodium hypochlorite has been reported to be very effective against many types of bacteria (Tiwari et al., 2012). Very small concentrations can reduce bacterial populations (Nakagawara et al. 1998 In this study, the auxin-cytokinin interactions enhanced the formation of somatic embryos from the embryogenic calli for plantlets formation. The culture medium supplemented with 2, 4-D (100mg/l) and 2-ip (3mg/l) gave the highest calli and somatic embryos. Most researchers suggest 2, 4-dichlorophenoxy acetic acid (2, 4-D) as the most effective auxin to induce embryogenic callus (Al-Khayri and Naik, 2017). Asemota et al., (2010) reported the use of 100mg/l NAA or 2, 4-D and 3mg/l 2ip to induce embryogenic callus from the meristem and leaf of date palm explants.
The somatic embryos from the embryogenic calli were transferred to growth regulatorenriched media for germination and multiplication (Plates 8 and 9). The cultures with shoots development were separated into multiplication and elongation media supplemented with NAA, BAP and 2-ip.  also obtained several plantlets on a growth regulator-enriched medium. The role of plant growth regulator in increasing the shoot length of date palm was also reported by Khierallah and Bader (2007).

Conclusion
The success of plant tissue culture depends on sterilization of explants and therefore selection of sterilizing agents and time of exposure are very critical. This study identified the combination of sterilizing agents at an appropriate concentration, combination and duration of exposure for reduction of contamination and tissue browning for successful micro propagation of date palm.