Egypt. J. Plant Breed. 21(1):1 – 12 (2017)
EFFECT OF BENZYLAMINOPURINE
CONCENTRATION AND NUMBER OF SUBCULTURES ON BEHAVIOR OF SOME STRAWBERRY CULTIVARS
IN VITRO
S.F. EL-Sayed1, A.M. EL-Sawy2, Sahar S. Taha1, Manal Sh. Gomah3
1. Vegetable Crops, Fac. Agric., Cairo University, Giza, Egypt-
2. Plant Biotechnology Dept., National Res. Center, El-Behoos St, Dokki, Giza, Egypt 3. PICO Company, Giza, Egypt
ABSTRACT
This study was conducted was the Plant Tissue Culture Laboratory of PICO Comp., Giza, Egypt, during the period of 2010-2014. Meristem tips of three strawberry (Fragaria x ananassa) cultivars, namely Sweet Charlie, Festival and Camarosa were excised and cultured on multiplication MS medium containing 0.50, 0.25or 0.10 mg/l BAP. Subculture was carried every four week intervals till sixth subculture. Camarosa cv. exhibited significant higher multiplication rate than the other two cultivars; i.e. Sweet Charlie and Festival. The sixth subculture showed the highest multiplication rate (3.6, 3.4 and 3.26 in cvs Sweet Charlie, Festival and Comarosa, respectively). The number of shoots per explant at different concentration of BAP was varied depending on the cultivar and number of subcultures. Molecular analysis by using RAPD-PCR indicated that mass propagation via meristem tip culture till the sixth subculture is reliable in producing genetically similar plants to the mother ones.
Key words: Strawberry, BAP, Micropropagation, Subculture, Molecular analysis, RAPD-PCR.
INTRODUCTION
Strawberry has been becoming one of the major vegetable crops in Egypt for fresh fruit consumption, processing and export. The cultivated area was doubled through the past years; reaching 13888.1 Feddan in 2011 with production of 242297 tons (FAO 2012).This expansion required the availability of pathogen-free transplants depending on the introduction of modern micropropagation systems. Traditionally, strawberry plants are propagated by runners derived from unclear stock materials via shoot tip culture (Boxus 1974, 1977 and Reinetrt and Bujaj 1977).
Micropropagation of strawberry offers a number of advantages.
Many researchers have reported increased vigor and runner production in tissue-culture derived strawberry plants. This response in temporary, usually observed only in the tissue culture propagated plants and the first runners, but is very advantageous to the nursery producer (Swartz et al 1981 and Cameron and Hancock 1986). The second advantage is the ability to rapidly multiply new cultivars. More than one million plants can be micropropagated from a single meristem tip in a year. In addition, micropropagation allows rapid and flexible production of healthy, high- quality strawberry plants which are usually true-to-type (Hughes 2002). In vitro techniques also are important tools for modern plant improvement
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programs to introduce new traits into selected plants, to multiply elite selections and to develop suitable cultivars in the minimum time (Taji et al 2001). Used in conjunction with classical breeding methods, an efficient in vitro shoot proliferation and regeneration system could accelerate cultivar development programs. The ability to regenerate plants is crucial to the successful application of In vitro methods (Cao and Hammerschlag 2000).
A shoot regeneration system can be used to develop transgenic plants following genetic transformation of plant cells and to identify and/or induce somaclonal variants. In vitro propagation systems also allows the production of variant plants, of which, some might show unique characteristics as new somaclones.
Shoot regeneration in strawberry using MS medium containing BAP was reported with some workers (Ashrafuzzaman et al 2013, Murti and Yeoung 2013, Diengngan and Murthy2014 and Danial et al 2016). Different concentrations of auxins and cytokinins were used by several workers (Mir et al 2010, Hasan et al 2010,Danial et al 2016 and Kumar et al 2016) who reported that the best results in strawberry culture media are obtained by 0.25-2.5 mg/l cytokinin and 0.25-1.0 mg/l auxin.
The performance of micropropagated plants is also varied according to their multiplication subculture. The economics of strawberry tissue culture industry is directly depending on the number of multiplication subcultures. As number of subcultures is increased (i.e. more than 8) then number of plantlets obviously increases which gives more profit.
The present study was designed to investigate the role of concentration of BAP in multiplication medium and number of subcultures on the behavior of three strawberry cultivars in vitro.
MATERIALS AND METHODS
This study was conducted in the Plant Tissue Culture Laboratory of PICO Comp., Giza, Egypt, during the period of 2010-2014.Shoot tips of strawberry (Fragaria x ananassa) cvs Sweet Charlie, Festival and Camarosa were isolated from runners of greenhouse grown plants. The shoot tips were washed with running tap water several times, and surface sterilized by immersion in 70% (v/v) ethanol for a few seconds, then soaked in a 20% Clorox (Commercial bleach 2.5% sodium hypochlorite) for 15 min with two drops of tween 20, which was used as a wetting agent. Thereafter, shoot tips were rinsed five times in sterile distilled water. Meristem tips (5mm long) were isolated under binuclear and cultured on solidified MS medium (Murashige and Skoog 1962) free-hormone and incubated until plantlets were obtained.
To study the effect of BAP concentration on the multiplication rate of meristem tips, derived shoots obtained from the previous stage were exited and subcultured on solidified multiplication MS medium containing 0.5, 0.25 or 0.1 mg/l BAP. The pH of the medium was adjusted at 5.6- 5.8.
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Meristem tips were cultured into glass culture jars (300 ml) containing 50 ml of the multiplication medium. Each treatment consisted of three replicates, each replicate consisted of 5 jars, which arranged in complete randomized design (RCP). Subcultures were done every four weeks into fresh medium of the same composition till the sixth subculture. All cultures of each treatment were incubated at 25±2°C under a 16 h day length with illumination of 100 µmol/m-²/S-¹ Osram cool white18 W fluorescent lamps.
Shoots produced roots on the same medium.
Percentage of proliferation, 4 weeks after meristem culture and multiplication rate at the end of every subculture were recorded.
Genomic DNA extraction: Genomic DNA was isolated on a mini- prep scale as mentioned by Murray and Thomposon (1980). Small pieces (0.5 g) of leaf tissue of in vitro-derived plantlets and greenhouse derived via traditional propagation plants were frozen in liquid nitrogen in Eppendorf tubes and homogenized in 500 µ l of extraction buffer ( 2
% CTAB, 1.4 M NaCl, 20 mM EDTA pH 8.0, 100 mMTris-HCl, pH 8.0, 0.1 M ß-Mercatoethanol). The extract was incubated at 60 οC for 20 min. To this 500µ l of phenol: chloroform: isoamyl alcohol (24:24:1) were added and mixed by vortexing for 30 sec followed by centrifugation at 10,000 xg for 5 min at room temperature. The aqueous phase was transferred to another tube. This was once again extracted with 500 µ l of chloroform: isoamylalcohol (24:1) in Eppendorf tube. To the aqueous phase, 0.6 volume of isopropanol were added, precipitated the genomic DNA and spooled the fibrous genomic DNA. Genomic DNA was then washed three times with 70% ethanol, dried in vacuum , dissolved in TE containing 10 mg/ml RNase and incubated at 37ºC for 30 min., followed by extraction with phenol: chlorform: isoamylalchol and the aqueous phase was transferred to a fresh tube. Thereafter, the genomic DNA was precipitaed by adding 0.3 M sodium acetate, pH 5.2 (final concentration) and 2.5 vol of ethanol and collected by centrifugation at 10,000 x g for 20 min at 4οC. The pellet was washed with 70% ethanol, vacuum dried and dissolved in TE.
PCR conditions: Five random oligonucleotide (10 mer) primers (Operon technologies Inc., Alameda, California) were designed for use in RAPD analysis. The primers are OPK01(5′ TGC CGA GCT G 3′), OPK02 (5′ GTG AGG CGT C 3′), OPK03 ( 5′ CCC TAC CGA C 3′ ), OPK04 (5′ TCG TTC CGC A 3′), and OPK05 (5′ CAC CTT TCC C 3′).The PCR reactions were carried out in 50 µl volumes containing 100 ng of genomic DNA , 1.0 µ M of each primer , 200 µ M of dATP, dDTP, dCTP, dGTP, 10 mMtris-HCl, pH 8.3, 50 mMKCl, 1.5 mM MgCl2 and 0.001 % gelatine. The Taq DNA polymerase concentration was 1.5 units per assay. PCR reaction was conducted using a Perkin Elmer 2400 (Germany) thermocycler programmed for 45 cycles as follows : 94 ºC
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/5 min (1 cycle) , 94 ºC / 30 sec, 36 ºC / 30 sec, 72 ºC /2 min ( 45 cycle ) and 72 ºC/7 min (1 cycle ), then held at 4ºC. The amplification products of PCR were size-separated by gel electrophoresis in 1% agrose gels with 1 x TBE buffer using a Pharmacia G N. 100 submarine gel electrophoresis apparatus and stained with ethidium bromide and visualized with UV transilluminator and photographed. A 50bp DNA ladder (Promega) was used as a standard with molecular sizes of 1000, 800, 750,700,650, 600,550, 500,450, 400,350, 300, 250,200,150,100 and 50bp. DICE computer package was used for the analysis of RAPD-PCR products (Yang and Quiros 1993).
Statistical Analysis
Factorial experiment with three factors arranged in complete randomized design was used for analysis all data with three replications for each parameter. The treatment means were compared by least significant difference (L.S.D.) test as given by Snedecor and Cochran (1994) by used Assistant program.
RESULTS AND DISCUSSION Proliferation percentage
Data presented in Table 1, show that the most of the excised meristem proliferated gradually in all cultivars. After one week of culturing, the lowest proliferation percentage was produced in Camarosa cv., but after 2 and 3 weeks cv. Festival showed the lowest proliferated percentage.
Meanwhile, Sweet Charlie cv. had the highest proliferated percentage after 1, 2 and 3 weeks. The proliferated percentage in each cv. reached 100%
after 4 week.
Table 1. Percentage of proliferation, one, two, three and four weeks after meristem culture.
Period (week)
Cultivar
Mean Sweet Charlie Festival Camarosa
1 97.80 95.30 87.70 93.60
2 97.80 90.10 93.70 93.87
3 100.00 90.10 93.70 94.60
4 100.00 100.00 100.00 100.00
Mean 98.90 93.88 93.78
LSD value at 0.05:
Cultivar: 0.91, Period: 1.05 and Interaction: 1.81
These results are in agreement with Gubis et al (2003), Feyissa et al (2005), Landi and Mezzetti (2006) and Kumar and Reddy (2010) who reported that genotype is one of the most important factors affecting regeneration. Ara et al (2013) cultured runner tips of seven strawberry genotypes on MS medium. They noted some differences in shoot
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proliferation among them. Also, Murti and Yeoung (2013) reported that in MS medium with IBA, cv. Camarosa shoot grew better than cv. Redpearl with higher plantlet and leaf number. Also, Sutan et al (2010) observed distinct differences in shoot regeneration frequency between the two ornamental strawberry varieties investigated.
Multiplication rate
As shown in Table 2, Camarosa cv. exhibited significantly higher multiplication rate than the other two cultivars; i.e. Sweet Charlie and Festival, which revealed non-significant differences in multiplication rate between them.
Table 2. Effect of BAP concentrations, and number of subcultures on multiplication rate in three strawberry cultivars.
Cultivar BAP con.
(mg/l)
Number of subcultures
1th 2nd 3rd 4th 5th 6th Mean
Sweet Charlie
0.5 2.8 2.8 1.5 2.5 3.1 3.7 2.73 0.25 2.7 2.7 1.9 3.0 3.3 3.6 2.87 0.1 2.2 2.2 1.3 2.2 3.0 3.5 2.4 Mean 2.57 2.57 1.57 2.57 3.13 3.6 2.67
Festival
0.5 2.1 2.1 2.0 3.0 3.2 3.5 2.65 0.25 2.1 2.1 2.0 2.5 3.0 3.3 2.5
0.1 2.3 2.3 2.0 3.3 3.0 3.4 2.72 Mean 2.17 2.17 2.0 2.93 3.07 3.4 2.62
Camarosa
0.5 2.3 2.3 3.5 2.5 3.2 3.5 2.88 0.25 2.2 2.2 3.5 2.2 3.4 3.0 2.75 0.1 2.1 2.1 2.9 3.0 3.5 3.3 2.82 Mean 2.2 2.2 3.3 2.57 3.37 3.26 2.82 0.5 2.57 2.57 1.57 2.57 3.13 3.6 2.67 0.25 2.17 2.17 2.0 2.93 3.07 3.4 2.62
0.1 2.2 2.2 3.3 2.57 3.37 3.26 2.82
Mean 2.31 2.31 2.29 2.69 3.19 3.42 LSD value at 0.05:
Cultivars (cv.): 0.15 BAP Concentrations (BAP): 0.05 Number of Subcultures (Sub): 0.22 cv x BAP: 0.27 cv x Sub: 0.38 BAP x Sub: 0.38 cv x BAP x Sub: 0.66
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The variation in multiplication stage among cultivars appeared as differences in number of shoots (Chien-Ying et al 2009 and Mozafari and Gerdakaneh 2012), frequency of shoot formation (Ara et al 2013), plant height and leaf number (Murti and Yeoung 2013). Recently, El-Sayed (2016) indicated that Sweet Charlie cultivar produced more shoot numbers than Florida cultivar.
Concerning effect of BAP concentration, the highest multiplication rate was obtained when BAP was used at 0.1 mg/l, followed by BAP at 0.5 mg/l then at 0.25 mg/l. The importance of BAP for regeneration of strawberry has been emphasized by different researchers (El-Sawy 2007, Biswas et al 2007 and Sakila et al 2007) who showed that BAP for regeneration of strawberry is important. BAP was added to the multiplication medium at concentrations ranged from 0.1 to 4.0 mg/l (Biswas et al 2007 and 2008, Ashrafuzzaman et al 2013, Mohamed 2014, Diengngan and Murthy 2014 and Danial et al 2016). The proper BAP concentration for the highest multiplication rate varied from a study to another one. This differences may be attributed to the medium used, where some investigators used Knoppheller (K-H) medium (Abramenko, 1983), others used Knop medium (Biswas et al (2007 and 2008), but most of them used Murashige and Skoog medium (Adak 2011, Ara et al 2012,Mohamed 2014 and Kumar et al 2016).
Boxus (1981) reported that decreasing the BAP concentration from 1 mg/l to 0.5 or 0.25 mg/l reduced occurrence of somaclonal variation. Lopez- Aranda et al (1994) indicated that the use of low BAP concentrations on strawberry has been recommended by several authors, since it decreases the risk of phenotypic abnormalities after the field establishment of micropropagated plants. The multiplication rate was constant during the first four subcultures, and then it was significantly increased at the fifth subculture and reached its maximum rate at the sixth sub culture. The sixth subculture showed the highest multiplication rate(3.6, 3.4 and3.26 in cvs Sweet Charlie, Festival and Comarosa, respectively). Boxus (1981) confirmed that number of subcultures had great influence on in vitro shoot formation of strawberry. In this regard, the number of subcultures is generally limited to a maximum of 10-12, as a high number of subcultures was associated with some physiological problems in the field. Isac and Coman (2012) reported that up to 10 multiplication steps can be achieved in strawberry culture in vitro. Data of El-Sayed (2016) confirmed that number of subculture had a significant effect on number of shoots and cluster weight. The third subcultures scored higher values of shoot number/explant and cluster weight than the first and second subcultures.
Generally, Lopes- Aranda et al (1994) indicated that the number of subcultures (3-8) in strawberry did not affect productivity; in accordance
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with another observation they pointed out that problems arise when using material with over 10 subcultures.
As for interaction effect, results in Table 2, show that the highest multiplication rate was obtained in the sixth subculture with BAP at 0.5mg/l for cvs Sweet Charlie and Festival, which was 3.7 and 3.5 for both cultivars, respectively. For ‘Camarosa’, the highest multiplication rate was obtained in the third and sixth subculture with BAP at 0.1mg/l.
Finally, from the previously mentioned results, it could be suggested that number of shoots per explant at different concentrations of BAP were varied depending on the cultivars and number of subcultures. This finding is similar to that published by EL-Sawy (2007) who stated that the average number of shoots/jar depended on the number of the subcultures and cultivars. Also Debnath (2006) and Sonmez and Kafkas (2012) reported that explant types and cultivars of different genetic background may give different regeneration responses.
Mozafari and Gerdakaneh (2012) found that shoot multiplication occurred in Kurdistan and Merck strawberry cultivars. The highest number of shoots per culture (3.44) was recorded from Kurdistan cultivar. There was a strong and intricate interaction between plant growth regulators, culture conditions and cultivar.
Molecular analysis:
Two kinds of plant materials were used, in vitro derived plantlets and greenhouse-plants derived via traditional propagation by runners.
Genomic DNAs of both materials were extracted and compared by RAPD-PCR, using random oligonucleotide primers. Of the five primers, one of them (OPK01) successfully yielded 47 bands across the cultivars tested. The other primers were not as efficient as the mentioned primer (OPK01) in generating successive PCR products. Size ranged from 230 to 950 bp depending on the cultivar and the plant material. For computer analysis to detect the pairwise differences between the two materials, intensive bands were considered as present (1), while weak or absent bands were considered as absent (0). Table 3 and Fig. 1, show the reaction of primerOPK01 with the two different materials in the three cultivars Sweet Charlie, Festival and Camarosa. Forty seven (47) different bands with size ranged from 230 to 950 bp were yielded. Three bands out of them were polymorphic. Their sizes were 950 bp in case of Camarosa and 230 and 250 bp in case of sweet Charlie of OPK01 ,but in case of Festival no polymorphic band was obtained . The percentage of polymorphism was 7.7, 12.5 and 0.0% in Camarosa, Sweet Charlie and Festival, respectively. In case of cultivar Camarosa primer OPK01 produced 13 different bands with sizes ranging from 320 to 950 bp. Out of them one band was polymorphic. Its size was 950 bp of OPK01.
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Table 3. Primer with arbitary sequence tested for its effectiveness in the RAPD - PCR analysis that produced polymorphic bands of strawberry.
Cultivar
Number of bands in the gel
Size of the polymorphic
bands(bp)
% of polymorphism Total polymorhic
Camarosa 13 1 950 7.7
Sweet
Charlie 16 2 230,250 12.5
Festival 18 - - 0.0
Total 47 3 230,250,950 6.4
Fig. 1. RAPD-PCR polymorphasim of DNA for three cultivars of strawberry using OPK01.
1 - In vitro derived plantlet.
2- Traditionally propagated plant.
M- 1kb DNA Ladder (promega).
1 2 1 2 2
Camarosaa
Sweet Charlie 1
Festival
800bp
50bp 1000bp
M
Camarosa
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On the other hand, the succeeded primer OPK01 in case of cultivar Sweet Charlie produced 18 different DNA bands ranging in size from 230 to 850 bp. Out of them, two were polymorphic, their sizes were 230 and 250 bp of OPK01. But OPK01 in case of Festival produced 18 different DNA bands with size range from 230 to 850 bp, and no polymorphic band was obtained. Table 3, summarizes the relevant results of the primer used.
Furthermore, most of the obtained bands from in vitro-derived plantlets in primer used with the three cultivars are confirmed to be present in the fingerprints of cold greenhouse-plants and the percentage of polymorphism is 6.4. It means in all cultivars with OPK01, that the RAPD fingerprints produced were almost identical. The results of the present finding are in agreement with Castiglione et al (1993), who found no RAPD fingerprint variation when different plants of the same clone of Populus species.
Among the various methods developed to micropropagated plants, enhanced axially branching culture has become the most important propagation method. This method is especially advantageous because it is simple and the propagation rate is relatively high (Pierik, 1991). More importantly, it is generally considered to be an in vitro culture system with low risk of genetic instability (Pierik 1991 and Schoofs 1992), because the organized meristems are generally more resistant to genetic changes that might occur during cell division or differentiation under in vitro conditions (Shenoy and Vasil 1992). Notwithstanding this consideration, there are numerous reports on the incidence of somaclonal variation among micropropagated plants.
For example, reports have indicated the occurrence of somaclonal variation in micropropagated bananas and plantains (Schoofs 1992) raised through meristem culture. Various kinds of leaf chlorosis coupled with multiple apexing and dwarfing in strawberry (Martinelli 1992).
In conclusion, the present investigation indicated that mass propagation via tissue culture (meristem tip culture) is reliable in producing clones genetically similar to the mother plants, till sixth subculture with 0.5 mg/l BAP. It was also concluded that RAPD approach is convenient, fast and reproducible to detect the presence of genetic variation associated with tissue culture of strawberry
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ريثأت زي رت كو س ي ع ةعارز ا ةداعإ تارم ددعو نيرويبو يما ليز ب ا
لمعم ا لخاد وارف ا فا صأ ضعب
ديس يحتف ديس ا
،1
دمحم يواص ا لداع و2
رحس ط حيمس عمج نابعش لا مو 1
3
1 . رضخ ا مسق -
ةعارز ا ةي - ةرهاق ا ةعماج -
ةزيج ا - رصم
2 . ةيثارو ا ةسدنه ا مسق -
ثوحب يموق ا ز رم ا -
يج ا ةز – رصم
3 . و يب ه رش –
ةزيج ا - رصم
و يب ة رشب ةجس اا ةعارز لمعم يف ةسارد ا ذ تيرجإ –
ةزيج ا - نم ةرتف ا يف رصم 2212
ي ا
2214 نم يمق ا ميتسرم ا لزع مت.
3 ة وارف ا نم فا صا ازورام و لافيتسيف و يراش تيوس)
ةئيب ي ع تعرزو(
ي ع يوتحت جو سوجيشاروم 2.5
و 2.25 و 2.1 ل ةعارز ا ةداعا تيرجإ. نيرويب و يما ليز ب رت /مج م 4
يراش تيوس نيف ص ا ا ب ةراقم اب فعاضت ةبس ي عا ازورام ف ص ا يطعا. سداس ا رم ا يتح عيباسا ( ا ردق فعاضت ا ةبس يف ةدايز ي ا تدا ةسداس ا ةرم ةعارز ا ةداعإ . لافيتسيفو 3.6
– 3.4 - 3.26 ) يف
فا صاا لافيتسيفو يراش تيوس و يما ليز ب ا تازي رت ي ع يتاب ءزج ل عرفاا ددع. ي اوت ا ي ع ازورام و
(لس ستم ا ةرم ب ا لعافت مادختساب ئيزج ا لي حت ا. ةعارز ا تارم ددعو ف ص ا فاتخأب تف تخا نيرويب RAPD-
ارز ا ةداعإ يتح ةيميتسرم ا ةمق ا مادختسأب راث إا نا حضوا )PCR جات إ ةقوثوم قيرط ةسداس ا ةرم ةع
.مأا تاب ايثارو ة باشتم تاتاب
ابنلا يبرتل يرصملا لجملا (
: ) - ( 1 2 )