somatic embryogenesis induction and plant regeneration in

Mazri M. A. et aI. (2021). AFRIMED AJ –Al Awamia (131). p. 1-18

1

Somatic embryogenesis induction and plant regeneration

in date palm (Phoenix dactylifera L.) cv. Bouskri

Mazri Mouaad Amine (1), Meziani Reda (2), Elmaataoui Saida (1),

Anjarne Mohamed (1), Alfeddy Mohamed Najib (3) and Bouchiha Fatima (1)

[email protected]

1 : Institut National de la Recherche Agronomique, CRRA-Marrakech, UR Agro-

Biotechnologie, BP 533, Marrakech, Morocco.

2 : Institut National de la Recherche Agronomique, CRRA-Errachidia, UR Systèmes

Oasiens, Laboratoire National de Culture des Tissus du Palmier Dattier, Avenue

Moulay Ali Cherif, BP 2, Errachidia, Morocco.

3 : Institut National de la Recherche Agronomique, CRRA-Marrakech, UR Protection

des Plantes, BP 533, Marrakech, Morocco.


2

Abstract

The effects of different medium components on somatic embryogenesis induction, and

somatic embryo maturation and conversion were evaluated in date palm cv. Bouskri.

Adventitious bud segments were used to induce somatic embryogenesis. The explants

were cultured on Murashige and Skoog medium at full strength (MS) or half strength

(MS/2), supplemented with two concentrations (10 or 20 mg/l) of either 1-naphthalene

acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or indole-3-acetic acid (IAA).

Somatic embryos were then cultured on either MS or MS/2 medium containing three

concentrations (10, 30 or 50 g/l) of polyethylene glycol (PEG). Afterwards, the effects

of gibberellic acid (GA3), polyvinylpyrrolidone (PVP) and activated charcoal on somatic

embryo germination were evaluated. The findings revealed that the use of full-strength

MS medium supplemented with 20 mg/l 2,4-D results in the highest callogenesis

(100%) and somatic embryogenesis (70%) rates. For somatic embryo maturation, MS

medium containing 30 g/l PEG gave the highest mean number (58.6) of mature

somatic embryos per 100 mg fresh weight callus. On the other hand, it was found that

the use of GA3 significantly increases the germination rate of mature embryos, while

there was no significant difference between the effects of PVP and activated charcoal

on somatic embryo germination. After transferring the plantlets to the glasshouse, the

survival rate observed was 82.35%. The results of this study highlight the optimal

medium components for each step of the somatic embryogenesis process of date palm

cv. Bouskri, and could be used for mass propagation and genetic improvement

purposes.

Keywords: Date palm, plant growth regulators, callogenesis, somatic embryogenesis,

plantlet regeneration.


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Induction de l’embryogenèse somatique et régénération des

plantes chez le palmier dattier (Phoenix dactylifera L.) cv. Bouskri

Résumé

Les effets de différents composants du milieu de culture sur l'induction de

l'embryogenèse somatique, la maturation et la germination des embryons ont été

évalués chez le palmier dattier cv. Bouskri. Des fragments de bourgeons adventifs ont

été utilisés pour induire l'embryogenèse somatique. Les explants ont été cultivés sur

le milieu du Murashige et Skoog (MS) ou MS dilué de moitié (MS/2), additionné de

deux concentrations (10 et 20 mg/l) de trois auxines : l’acide 1-naphtalène acétique

(ANA), l’acide 2,4-dichlorophénoxyacétique (2,4-D) ou l’acide indole-3-acétique (AIA).

La maturation des embryons somatiques a été évaluée sur des milieux MS et MS/2

contenant trois concentrations (10, 30 ou 50 g/l) de polyéthylène glycol (PEG) tandis

que pour la germination des embryons somatiques, les effets de l’acide gibbérellique

(GA3), la polyvinylpyrrolidone (PVP) et le charbon actif ont été évalués. Les résultats

ont montré que la culture des explants sur le milieu MS additionné de 20 mg/l de

2,4-D favorise la callogenèse, avec un taux de 100%, dont 70% présentant des

embryons somatiques. La maturation des embryons somatiques a été observée sur

tous les milieux de culture. Toutefois, l'utilisation du milieu MS additionné de 30 g/l de

PEG a donné le nombre moyen le plus élevé (58,6) d'embryons somatiques matures

par 100 mg de cal. De même, l'utilisation du GA3 a augmenté significativement le taux

de germination des embryons somatiques. Par ailleurs, il n'y avait pas de différence

significative entre la PVP et le charbon actif sur la germination des embryons. Au cours

de la phase d’acclimatation, le taux de survie a atteint 82,35%. Cette étude a mis en

évidence la composition optimale du milieu de culture pour l’embryogenèse somatique

du palmier dattier cv. Bouskri. Le protocole développé pourrait être utilisé pour la

propagation en masse et l'amélioration génétique de cette variété.

Mots-clés : Palmier dattier, régulateurs de croissance, callogenèse, embryogenèse

somatique, régénération.


4

صنف (.Phoenix dactylifera L)التمر نبات نخيل نموالجسدي و يكوين الجنيندراسة الت

بوسكري

فاطمةوبوشيحة محمد نجيب الفضي ،محمد أنجارن ،سعيدة المعطاوي ،رضا مزياني ،معاد أمين مزري

ملخص

ة نمو الأجنة الجسدي، التكوين الجنيني الجسدي ىالنمو علوسط تأثير مكونات في هذا البحث بدراسةقمنا

المنتجة البراعم من مأخوذة قطعصنف بوسكري. تم استخدام نخيل التمر عند إلى نباتات كاملة وتطورهم

Skoog و Murashige على وسط تهاتمت زراع و الجسدي يتكوين الجنينداخل الأنابيب لبدأ عملية ال

أو 10تركيزات مختلفة ) و المحتوية على، (MS/2)أو بنية مخففة إلى النصف (MS) القوة ببنية كاملة

ثنائي كلورو -4،2، حمض (NAA)أستيك النفثالين -1 حمض من الأكسين: أصناف ثلاثةلمجم/لتر( 20

تقييم نضج الأجنة الجسدية . بعد ذلك، تم (IAA)أسيتيك 3-و حمض الإندول (D-2,4) فينوكسي أسيتيك

جم/لتر( من البولي إيثيلين 50أو 30 ،10على تركيزات مختلفة ) المحتوية MS/2 و MS على وسائط

حمض ىعل أوساط نمو محتويةإنبات الأجنة الجسدية على تمت دراسةبينما (PEG)جليكول

الفحم المنشط. أظهرت نتائجبأو (PVP)إما بالبولي فينيل بيروليدون و مستكملة، GA)3 (الجبريليك

أدى إلى أعلى D-2,4 من ملجم/لتر 20على والمحتويكامل القوة MS الدراسة أن استخدام وسط ههذ

تمت ملاحظة الموالية،في المرحلة (. ٪70( والتكون الجنيني الجسدي )٪100)الأنسجة تكوين معدلات

جم/لتر 30على المحتوي MS استخدام وسطإلا أن ،جميع الوسائط المقيمةالأجنة الجسدية على نضج

. فيما الأنسجةمجم من 100لكل الأجنة الجسدية الناضجة من ( 58.6)عدد أدى إلى أعلى PEG من

يزيد بشكل كبير من معدل إنبات الأجنة الناضجة، بينما لم يكن 3GA أن استخدامفقد ثبت يتعلق بالإنبات،

بيت إلى ال النباتات. بعد نقل الأجنة الجسديةوالفحم المنشط على إنبات PVPتأثير بينواضح هناك فرق

لوسط الضوء على المكونات المثلى هذا البحث نتائج سلطت . ٪82.35هو النمو الزجاجي، كان معدل

، ويمكن استخدامها صنف بوسكري الجنيني الجسدي لنخيل التمرالتكوين عملية مراحل خلال كل النمو

.الوراثي أو التحسينالسريع التكاثر من أجل

.التكوين الجنيني الجسدي ،نخيل التمر أنسجة ،هرمونات النمو التمر،نخيل الكلمات المفتاحية:


5

Introduction

Date palm (Phoenix dactylifera L.) belongs to the family Arecaceae and the genus Phoenix (Johnson, 2011). It is a fruit species cultivated since ancient times for its delicious and nutritious fruits (Johnson, 2011; Al-Khayri et al., 2018). Today, date palm is also cultivated for socio-economic and ecological purposes since it generates employment, contributes to desertification control, creates appropriate climatic conditions for agriculture and preserves biodiversity (Jain, 2012; Sedra, 2015). Date palm is widely distributed in the Middle East and North Africa (MENA) region, with Egypt, Iran, Saudi Arabia, Algeria and Iraq being the top producing countries (Al-Khayri et al., 2018). In fact, date palm fruits (i.e. dates) have a religious importance for Muslims since they have been frequently mentioned in the holy Quran and are considered as a major food during the holy month of Ramadan (Iqbal et al., 2014; Fallahi et al., 2015). The recent advances made in the field of plant biotechnology, particularly in plant tissue culture, have played prominent roles in the large-scale propagation, preservation and genetic improvement of economically important species. In Morocco, date palm is the most important fruit species in the oasis areas (Sedra, 2015). During several decades, date palm has suffered from Bayoud, a wilt disease caused by fungus Fusarium oxysporum f. sp. albedinis and that killed millions of date palms in Morocco and Algeria (Saker, 2011). Unfortunately, there is no effective chemical treatment to control Bayoud. Thus, the use of micropropagation techniques seems to be the only practical way to rehabilitate the Moroccan groves, and to preserve sensitive cultivars (Ferry, 2011). Date palm micropropagation can be achieved through either organogenesis or somatic embryogenesis (Mazri and Meziani, 2015). In Morocco, date palm is propagated through organogenesis (Ferry, 2011; Meziani et al., 2015). Organogenesis is the in vitro regeneration system by which adventitious shoots and roots are formed from an explant cultured under appropriate conditions. It comprises the following steps: adventitious bud induction, shoot bud multiplication, shoot elongation and rooting and plantlet acclimatization (Mazri and Meziani, 2013). This regeneration system has been used during the last decade in Morocco to produce 3 million date palm plants (Meziani et al., 2015). On the other hand, developing efficient regeneration systems through somatic embryogenesis will also contribute to the large-scale propagation of the best date palm cultivars. Somatic embryogenesis is the in vitro regeneration process by which bipolar structures, known as somatic embryos, are formed from somatic cells without fertilization (Mazri et al., 2020). This technique has been used for the propagation of many plant species such as banana, avocado and coconut (Nandhakumar et al. 2018; O’Brien et al., 2018; Oropeza et al., 2018). Somatic embryogenesis comprises the following steps: callus induction, somatic embryogenesis expression, somatic embryo maturation and conversion and finally plantlet acclimatization. Somatic embryogenesis can be also used for synthetic seed production, genetic improvement and germplasm preservation (Guan et al., 2016). In recent years, an efficient system that combines both organogenesis and somatic embryogenesis was developed for some important date palm cultivars such as Mejhoul and Najda (Mazri et al., 2017, 2018). This new in vitro regeneration pathway allows to avoid the excessive utilization of offshoots and spathes, and the use of disease-free plant material that is available throughout the year (Mazri et al., 2017). Developing an


6

efficient in vitro regeneration system for date palm is influenced by many factors, the most important among them are the genotype and the culture medium components. The purpose of the present study was to develop an efficient regeneration system through somatic embryogenesis for date palm cv. Bouskri. Accordingly, the effects of medium strength, plant growth regulators (PGRs), PEG concentrations and antioxidants were evaluated.

Materials and methods

Plant material and callus induction

Adventitious buds of date palm (Phoenix dactylifera L.) cv. Bouskri were used as explants to induce callogenesis. The adventitious buds were obtained according to the protocol reported by Beauchesne et al. (1986). Briefly, shoot tips of date palm cv. Bouskri were extracted from 3-year-old offshoots. The shoot tips were surface-sterilized by immersion in a solution of 0.03 % potassium permanganate in commercial bleach for 20 min, followed by three rinses with sterile distilled water. To induce organogenesis, the shoot tip explants were cultured for 9 months on half-strength Murashige and Skoog (1962) medium (MS/2) supplemented with 2 g/l polyvinylpyrrolidone (PVP), 0.2 g/l L-glutamine, 30 g/l sucrose, 8 g/l agar, 0.1 g/l myo-inositol, 1 mg/l indole-3-acetic acid (IAA), 1 mg/l 1-naphthaleneacetic acid (NAA), 3 mg/l 2-naphthoxyacetic acid (NOA) and 0.1 mg/l 6-(dimethylallylamino) purine (2iP). The buds were then cut into segments of 5 mm length and cultured on different culture media to induce callogenesis: MS or MS/2 medium, supplemented with either NAA, IAA or 2,4-dichlorophenoxyacetic acid (2,4-D) at 10 or 20 mg/l (Table 1). All culture media were supplemented with 1 mg/l 2iP, 30 g/l sucrose, 1 g/l activated charcoal and 8 g/l agar. The cultures were kept under dark conditions at 25 + 1°C for 6 months, with monthly subcultures to fresh medium.

Somatic embryogenesis expression

The induced calli were transferred to PGR-free media, either MS or MS/2, depending on the basal formulation used for induction (Table 1). All culture media were supplemented with 30 g/l sucrose, 1 g/l activated charcoal and 8 g/l agar. Calli were maintained in the expression medium for 2 months under dark conditions at 25 + 1°C with a subculture to fresh medium after the first month of culture.


7

Table 1: Culture media used for callus induction and somatic embryogenesis

expression

Culture medium code

Callus induction medium Embryogenesis expression medium

MSD 10 MS medium + 10 mg/l 2,4-D + 1 mg/l 2iP PGR-free MS medium

MSD 20 MS medium + 20 mg/l 2,4-D + 1 mg/l 2iP PGR-free MS medium

MSN 10 MS medium + 10 mg/l NAA + 1 mg/l 2iP PGR-free MS medium

MSN 20 MS medium + 20 mg/l NAA + 1 mg/l 2iP PGR-free MS medium

MSI 10 MS medium + 10 mg/l IAA + 1 mg/l 2iP PGR-free MS medium

MSI 20 MS medium + 20 mg/l IAA + 1 mg/l 2iP PGR-free MS medium

MS2 D10 MS/2 medium + 10 mg/l 2,4-D + 1 mg/l 2iP PGR-free MS/2 medium

MS2 D20 MS/2 medium + 20 mg/l 2,4-D + 1 mg/l 2iP PGR-free MS/2 medium

MS2 N10 MS/2 medium + 10 mg/l NAA + 1 mg/l 2iP PGR-free MS/2 medium

MS2 N20 MS/2 medium + 20 mg/l NAA + 1 mg/l 2iP PGR-free MS/2 medium

MS2 I10 MS/2 medium + 10 mg/l IAA + 1 mg/l 2iP PGR-free MS/2 medium

MS2 I20 MS/2 medium + 20 mg/l IAA + 1 mg/l 2iP PGR-free MS/2 medium

Somatic embryo maturation

Embryogenic cultures (i.e. calli with globular somatic embryos) were disintegrated with a scalpel under the laminar flow then 100 mg fresh weight (FW) cultures were transferred either to liquid MS or MS/2 medium, supplemented with 30 g/l sucrose, 1 g/l activated charcoal and 10 mg/l abscisic acid (ABA) for 3 weeks. Afterwards, the cultures were transferred to ABA-free media, supplemented with 30 g/l sucrose, 1 g/l activated charcoal and polyethylene glycol (PEG) at three different concentrations: 10, 30 and 50 g/l (Table 2). The cultures were kept in these media for 9 weeks, with transfers to fresh medium at 3-week intervals. The embryogenic cultures were agitated (60 rpm) and maintained under dark conditions at 25 + 1°C.


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Table 2: Culture media used for somatic embryo maturation

Culture medium code

First step of embryo maturation (3 weeks)

Second step of embryo maturation (9 weeks)

MS Liquid MS medium + 10 mg/l ABA Liquid MS medium

MS + 10 PEG Liquid MS medium + 10 mg/l ABA Liquid MS medium + 10 g/l

PEG


PEG


PEG

MS/2 Liquid MS/2 medium + 10 mg/l ABA

Liquid MS/2 medium

MS/2 + 10 PEG Liquid MS/2 medium + 10 mg/l ABA

Liquid MS/2 medium + 10 g/l PEG





Somatic embryo conversion and plantlet acclimatization

Mature somatic embryos obtained from the optimal treatment were transferred to MS medium supplemented with 30 g/l sucrose and 8 g/l agar. During this phase, the effects of gibberellic acid (GA3; 0 or 1 mg/l), activated charcoal (0 or 1 g/l) and PVP (0 or 1 g/l) were evaluated. The embryos were maintained in the germination medium for 3 months, then transferred to PGR-free medium for 2 months. The germination-conversion phase was conducted under a 16h photoperiod (40 μmol m-2 s-1) at 25 + 1°C. The developed plantlets were transferred to the glasshouse for acclimatization according to the protocol described by Mazri et al. (2016). Briefly, the plantlets were removed from the culture medium, their roots were washed with tap water then soaked in an antifungal solution for 15 min. The plantlets were then potted in plastic bags containing a mixture of peat and gravel (1/1; w/w) and placed under a polyethylene tunnel for 15 days. Afterwards, the polyethylene bag was removed progressively to allow acclimatization to the glasshouse conditions (70 % relative humidity; 27 + 1°C; natural light conditions).

Culture conditions

In all culture phases, the medium pH was adjusted to 5.7 with NaOH and/or HCl before autoclaving at 121°C for 25 min.

Data collection and statistical analysis

For callus induction, 10 explants were placed per jar, each jar was containing 25 ml of culture medium and was considered as a replicate. For each treatment, 10 replicates were used. For somatic embryo maturation, 100 mg embryogenic culture was used per jar containing 25 ml of culture medium, which was considered as a replicate, and


9

each treatment was replicated 10 times. For embryo germination and conversion, 25 mature somatic embryos were placed per jar, and for each treatment, 5 jars were used. At the end of the induction and expression phases, the percentages of callogenesis and embryogenesis were calculated, respectively. At the end of the maturation phase, the number of mature somatic embryos per 100 mg embryogenic culture was estimated. At the end of the germination phase, the percentages of successful germination, callogenesis and necrosis were calculated. After 3 months in the glasshouse, the survival rate of the plantlets was calculated. All experiments were conducted in a completely randomized design (CRD). Data were subjected to analysis of variance (ANOVA). Means were compared by using the Student-Newman-Keuls (SNK) test at 5% level of significance. Before analysis, all percentage data were arcsine transformed. SPSS (v. 26) was used for analysis.


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Results and discussion

Callus induction and somatic embryogenesis expression

In MS medium supplemented with NAA or 2,4-D, callus formation started during the fourth week of culture. Calli appeared in the wounded areas and were white and friable. In MS medium containing IAA and in MS/2 media supplemented with IAA, NAA or 2,4-D, we noticed explant swelling during the first month of culture while callogenesis started after the first subculture (during the second month of culture). This indicates that the beginning of callus formation depends on medium strength and the auxin used. After 6 months of culture, the callogenesis percentages varied significantly depending on the medium strength and auxin type and concentration (Fig. 1). The highest callogenesis rate (100%) was observed on MS medium supplemented with either 10 or 20 mg/l 2,4-D (Fig. 1; Fig. 2a), with no significant difference with MS media containing NAA (92-96%). This was followed by MS/2 medium supplemented with 20 mg/l 2,4-D (88%). Generally, MS/2 medium gave lower callus formation rates than MS medium when the same auxin type and concentration were compared (Fig. 1). On the other hand, the culture media containing IAA showed the lowest callogenesis rates (33-51%).

Figure 1: Effect of medium strength and auxins on callus induction and somatic embryogenesis expression in date palm cv. Bouskri.

100d 100d92cd

96cd

42a51a

74b

88c

68b75b

33a38a

64cd 70d 52bcd 61bcd 0a 0a 42b 49bc 43bc 45bc 0a 0a0

20

40

60

80

100

120

Callogenesis (%) Somatic embryogenesis (%)


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Figure 2: Somatic embryogenesis in date palm cv. Bouskri. (a) Callus induction on MS medium supplemented with 20 mg/l 2,4-D. (b) Globular embryo formation on

PGR-free MS medium (arrows show globular embryos). (c) Somatic embryo maturation on liquid MS medium containing 30 g/l PEG. (d) Somatic embryo

germination on MS medium containing 1 mg/l GA3 and 1 g/l PVP

The significant effect of auxins on callus induction was observed in other date palm cultivars. For example, in cv. Najda, the highest callogenesis rate (90%) was observed in MS medium supplemented with 45 μM 2,4-D (Mazri et al., 2017). Generally, 2,4-D is the most used auxin to induce callogenesis in date palm (e.g. Eke et al. (2005), Kumar et al. (2020), Metwali et al. (2020)). Besides, high 2,4-D concentrations were suggested. For example, in cvs. Nabout Saif, Khanizi and Mordarsing, Al-Khayri (2011) and Eshraghi et al. (2005) used 100 mg/l to induce callogenesis. However, El Hadrami et al. (1995) and Othmani et al. (2009) showed that lower 2,4-D concentrations can be employed to induce callogenesis and somatic embryogenesis in date palm, and recommended the concentrations of 5 and 10 mg/l for cvs. Jihel, Iklane and Boufeggous. In previous studies by our group, it was found that callogenesis and somatic embryogenesis can also be achieved by using 45 μM of picloram (10.86 mg/l) (Mazri et al., 2017, 2018). In this investigation, 10-20 mg/l 2,4-D gave high callogenesis rates. Regarding the auxin NAA, very few studies were reported on its effect on


12

somatic embryogenesis in date palm. This auxin has been successfully used to induce somatic embryogenesis in date palm cv. Najda (Mazri et al., 2017). After transferring the induced calli to the expression media (PGR-free MS or PGR-free MS/2), the development of globular somatic embryos was observed (Fig. 2b). The use of PGR-free media to induce embryo formation was reported by several authors. Indeed, in many plant species such as Carum copticum (L.), Gentiana utriculosa (L.) and Gentiana macrophylla (Pall), calli were induced in media containing PGRs then transferred to PGR-free medium to promote embryo formation (Wu et al., 2011; Vinterhalter et al., 2016; Niazian et al., 2017). After 2 months of culture in the expression medium, the rate of somatic embryogenesis expression varied significantly, from 0 to 70%, depending on the induction conditions (Fig. 1). In fact, no globular embryo was observed when the induction medium was supplemented with IAA, whereas MS/2 medium exhibited lower embryogenesis rates (42-49%) than MS medium (52-70%). The highest somatic embryogenesis rate (70%) was observed when the explants were cultured on MS medium supplemented with 20 mg/l 2,4-D for induction, with no significant difference with those cultured on MS medium supplemented with 10 mg/l 2,4-D (64%) or 10-20 mg/l NAA (52-61%). These results highlight the significant impact of medium strength and auxin type and concentration on the induction and expression of somatic embryogenesis in date palm cv. Bouskri. The different effects of PGRs on somatic embryogenesis could be explained by their interaction with endogenous hormones. In fact, the added PGRs affect the concentration of the endogenous hormones, which results in cell division and differentiation, and in the regulation of plant growth and development in vitro (Gaspar et al., 1996; Feher et al., 2003; Gaspar et al., 2003; Gaj, 2004). The impact of the interaction between exogenous PGRs and endogenous hormones varies depending on the PGR used and its concentration (Teale et al., 2006). In fact, PGRs have different degrees of activity (Davies, 2004).

Somatic embryo maturation

The embryogenic cultures obtained on MS and MS/2 media containing 20 mg/l 2,4-D were used for maturation experiments. It was found that medium strength and PEG concentration significantly affect the maturation of somatic embryos of date palm cv. Bouskri (Fig. 3). The highest average number of mature somatic embryos per 100 mg FW callus (58.6) was observed on MS medium supplemented with 30 g/l PEG (Fig. 2c), and significant differences were revealed with respect to the other treatments, which showed a range of 25.7-50.8 mature embryos per 100 mg FW callus (Fig. 3). In cv. Najda, the effects of different medium strengths and textures on somatic embryo maturation were evaluated, and the promotive effect of full strength and liquid MS medium was highlighted (Mazri et al., 2019a). The use of liquid medium for date palm somatic embryo maturation was also recommended by Fki et al. (2003). Likewise, the promotive effect of PEG on somatic embryo maturation was observed in other Moroccan date palm cultivars. For example, in cvs. Najda, Al-Fayda and Mejhoul (Mazri et al., 2018, 2019a, b). According to Al-Khayri and Al-Bahrany (2012), the use of PEG at a high concentration resulted in the production of large size somatic embryos. According to Zhang et al. (2007) and Yaseen et al. (2013), the beneficial effect of PEG on somatic embryo maturation is due to the creation of a desiccated environment that improves storage material accumulation in somatic embryos. Along


13

this line, PEG was used for the maturation of somatic embryos of many plant species such as walnut, jabuticaba and rapeseed (Yadollahi et al., 2011; Jalali et al., 2017; Silveira et al., 2020).

Figure 3: Effect of medium strength and PEG concentrations on somatic embryo maturation of date palm cv. Bouskri.

Somatic embryo conversion and plantlet acclimatization

Based on the results of the previous experiment, mature somatic embryos obtained on MS medium containing 30 g/l PEG were used in germination experiments. After 3 months of culture, the highest germination rate (34.4%) was observed on the medium supplemented with 1mg/l GA3 and 1 g/l PVP (Fig. 2d), with no significant difference with that containing 1 mg/l GA3 and 1 g/l activated charcoal (33.6%; Table 3). Besides, statistical analysis revealed a significant effect of GA3 on somatic embryo germination (Table 3). The somatic embryos that failed to germinate showed either callogenesis (44.8-65.6%) or became necrotic and died (14.4-24.0%). In cv. Najda, it was found that the use of activated charcoal significantly improves the germination rate of somatic embryos (Mazri et al., 2017). The findings of this study showed that PVP has a similar impact to that of activated charcoal on the germination of cv. Bouskri somatic embryos. Regarding GA3, this PGR was used in many plant species to promote somatic embryo germination. For example, in grapevine, olive and magnolia-vine (López-Pérez et al., 2005; Yang et al., 2011; Mazri et al., 2020). According to Gana (2011), GA3 acts by breaking the dormancy of somatic embryos. The findings of this experiment showed that the use of GA3 at the concentration of 1 mg/l promotes the germination and conversion of somatic embryos of date palm cv. Bouskri.

33.1 b 41.9 c 58.6 e 50.8 d 25.7 a 29.3 ab 34.6 bc 41.0 c0

20

40

60

80

No

of

mat

ure

so

mat

ic e

mb

ryo

s

Maturation medium


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Table 3: Effect of GA3, PVP and activated charcoal on somatic embryo germination of date palm cv. Bouskri

Germination medium Germination and conversion (%)

Callogenesis (%) Necrosis (%)

MS + 1 g/l PVP 12.0 + 2.19 a 64.0 + 4.89 b 24.0 + 3.79 a

MS + 1 g/l PVP+ 1 mg/l GA3

34.4 + 0.97 b 44.8 + 4.27 a 20.8 + 4.96 a

MS + 1 g/l AC 14.4 + 0.97 a 65.6 + 5.30 b 20.0 + 5.79 a

MS + 1 g/l AC + 1 mg/l GA3

33.6 + 2.71 b 52.0 + 3.34 ab 14.4 + 5.15 a

Data are means + standard error. Data in the same column followed by the same letter are not significantly different at the 5% significance level. MS, Murashige and Skoog medium; GA3, gibberellic acid; PVP, polyvinylpyrrolidone; AC, activated charcoal. After transferring the developed plantlets to the glasshouse, a survival rate of 82.35% was observed. In date palm cv. Mejhoul, the survival rate of the plantlets obtained through somatic embryogenesis was 76% (Mazri et al., 2018). In cv. Najda, a survival rate of 80% was reported, while the survival rate of embryo-derived plantlets of cv. Al-Fayda was 77.02% (Mazri et al., 2019 a,b). The results of this investigation showed that date palm cv. Bouskri can be propagated through somatic embryogenesis. The protocol reported here can be used for genetic improvement purposes.

Conclusions

An efficient regeneration system through somatic embryogenesis was developed for date palm cv. Bouskri. Callus formation can be induced on either MS or MS/2 media supplemented with different concentrations of 2,4-D and NAA. However, the optimal culture medium for callus induction was MS containing 20 mg/l 2,4-D. Somatic embryo expression was performed on PGR-free medium while the maturation of somatic embryos was higher on MS medium containing 30 g/l PEG. Somatic embryo germination and conversion into plantlets was achieved on MS medium containing 1 mg/l GA3 and either PVP or activated charcoal for 3 months, followed by 2 months of culture on PGR-free MS medium. After transferring the plantlets to the glasshouse, a high survival rate of 82.35% was observed. The above-described protocol can be used for mass propagation and genetic improvement of date palm cv. Bouskri. Further studies will be carried out to assess the genetic conformity of regenerants.


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