EFFECT OF CULTIVARS, GROWTH REGULATOR AND SUB-CULTURE ON THE PLANTLET SHOOTS FORMATION AND RAPD ANALYSIS OF STRAWBERRY CULTURES IN VITRO

This experiment amid to study the effect of cultivars, growth regulator and number of sub-cultures on the shoots formation of strawberry plantlets during multiplication stage and Random amplified polymorphic DNA (RAPD) analysis of strawberry cultures in vitro during the fourth sub-culture. This experiment included 40 treatments, which were the combination between two strawberry cultivars (Festival and Sweet Charlie), five treatments of growth regulator (BA and GA3) and four number of sub-cultures during shoots formation (multiplication stage). The obtained results showed that, the maximum increment of growth measurement of strawberry plantlets were recorded by Sweet Charlie cultivar .In addition , using 1⁄2 MS-medium without supplemented with any growth regulators (BA and GA3) being the superior treatment for increasing both number of leaves per shoot and shoot length. On the other hand, generally, the fourth sub-culture being the most effective treatment on the growth measurement of strawberry plantlets during multiplication stage. Furthermore, Random amplified polymorphic DNA (RAPD) analysis varied according to the two tested cultivars and the type of for production of disease resistant plants and in plant breeding and crop improvement programs (Mohamed, 2003).


INTRODUCTION
Strawberry (Fragaria X ananassa Duch.) is a natural hybrid of Fragaria chilonsis and Fragaria virginiana is a perennial herb belonging to the Rosaceae Family. The strawberry fruits is delicate in flavor, texture, shape, and rich in some vitamins particularly A, B1, B2, B6, C, E, and some minerals such as calcium, potassium, copper and iron (Glampieri et al., 2015). In addition, fruits are a good source of phytochemical compounds, mainly ellagic acids which have a wide range of biological activity.
Tissue culture technique has been successful on the large scale multiplication of strawberry plants in many countries. This technique can produced millions of plants can be produced in short time from a few mother plants. Beside  and organs depends on some factors such as cultivars (Passey et al.,(2003), Sutan et al.,2010;Mozafari and Gerdakanch, 2012), growth regulators (such as BA and GA 3 ) and their concentration (Sachs et al.,1959;Haber and luippold, 1960;Gamborg et al.1976;Lal et al., 2003, Negi et al.,2008Harugade et al.,2014); number of sub-cultures (Nowere et al., 2011;EL-Zeiny et al.,2013).
Moreover, genetic stability during micro propagation is controlled by various type including genotype, presence of chimera tissue, origin and explant type, medium components, type and concentration of growth regulators. In this connection, EL-Tarras et al. (2001) came to similar conclusion.
There for, the aim of this work was to study the effect of cultivars, growth regulators and number of sub-cultures on the formation of plantlet shoots, as well as Random amplified polymorphic DNA (RAPD) analysis of strawberry plantlets cultures in vitro during forth sub-culture.

MATERIALS AND METHODS
This experiment included 40 treatments, which were the interaction between two strawberry cultivars, five growth regulators and four number of subcultures, as follows: A) Strawberry cultivars 1) Festival. 2) Sweet Charlie.

B) Growth regulators 1) ½ MS-medium (Half salts strength) without
applications of antioxidant (the control treatment).

C) Number of sub-cultures
1) First sub-culture.
These treatments were arranged in a splitsplit plots design with four replicates. Each replicate contained of five glass jars (12.0 × 6.0 cm contained of 50 ml of the culture medium), and each one contained of four explants. The cultivars were arranged in the main plots, while growth regulator treatments were assigned randomly in the sub-plots and number sub-cultures were arranged randomly in the sub sub-plots.
The selected shoots are shown in Fig. (1) were used as a plant material in this stage and subcultured on the half salts strength of basal nutrient (½ MS-medium) which supplemented with the previously growth regulator combinations of BA and GA 3 .

B) Polymerase chain reaction (PCR) analysis (Molecular analysis)
Deoxyribo nucleic acid (DNA) was extracted from in vitro plantlets after the fourth subculture of both cultivars (Festival and Sweet Charlie) and mother plants, ex vitro. Briefly, new and fresh leave samples were collected separately from each cultivar, and the Deoxyribo nucleic acid (DNA) extraction was performed using DNeasy plant Mini Kit (QIAGEN).The method of Williams et al. (1990) used in this connection.
RAPD -PCR reactions were conducted using six primers for two strawberry cultivars i.e. Festival and Sweet Charli to determine the polymorphism that could be associated with differences among five treatments Table (1).

Statistical analysis
All collected data were subjected to proper statistical analysis using Co-Stat software program version 3. The least significant difference (L.S.D.) test at 0.05 level of probability was used to determine statistically the significance of differences among the compared means of various treatments according to Snedecor and Cochran (1980).

A) Growth measurements of plantlets 1) Effect of cultivars
The effect of the two tested strawberry cultivars (Festival and Sweet Charlie) on the shoots formation of plantlets during multiplication stage in vitro are illustrated in Table (2). It is quite clear from such table that, Sweet Charlie being the superior one in number of both shoots formation per explant and leaves per shoot, as well as shoot length as compared with Festival cultivar.
On the other hand, it is also evident from such results that, no obvious differences were detected between the two cultivars on the fresh and dry weight of shoots formation, which were mostly similar in this connection. In this regard, Passey et al. (2003) tested the regeneration ability of seven commercial strawberry cultivars using a range of the explants, they mentioned that, two genotypes showed a limited ability to regenerated shoots in all explants tested. In addition, Sutan et al. (2010) reported that, genotypes was proven to be a critical factor for indirect differences in callus formation ability and shoots regeneration frequency between the two investigated strawberry cultivars. Mozafari and Gerdakanch (2012) found that, shoots multiplication occurred in strawberry cultivars, i.e. Kucrdislun and Merck. The highest number of shoots/culture (3.44) were recorded from Kurdistan cultivar.

2) Effect of growth regulators
Concerning the effect of some growth regulators (BA and GA 3 ) on explant shoots formation of strawberry cultures in vitro, the obtained results in Table (3) and ISSN:2572-3006(Print)2572-3111(Online) http://www.futurejournals.org treatment) being the superior one in respect of number of leaves formation per shoot and shoot length with no significant differences between this treatment and the treatment of ½ MS-medium + 0.01 mg / l BA + 0.01 mg / l GA 3 for shoot length only. In addition using ½ MS-medium which supplemented with 0.5 mg/l BA + 0.3 mg/l GA 3 or 0.01mg/l BA increased the fresh weight of shoots formation, while the treatments of growth regulators (BA and GA 3 ) did not reflect any significant effect on the dry weight of the obtained shoots of strawberry cultures in vitro.
From the previously mentioned results, it could be suggested that, the promotion effect of BA and GA 3 on the formation of both number of shoots per plantlet and leaves per shoot, as well as shoot length and the fresh weight of shoots per plantlet is due to its simulative effect on both cell division and cell enlargement (

3) Effect of number of sub-cultures
It is evident from the obtained results in Table  (4) and Fig. (3) that, number of sub-cultures exerted a marked and significant effect on the shoots formation per plantlets. In addition, the fourth sub-culture recorded the highest number of shoots per plantlet and number of leaves per shoot, as well as shoot length, followed by third sub-culture, respectively. In this connection, Nower et al. (2011) found that, the third sub-culture significantly recorded the highest response in increasing both number of shoots (14.0 shoots per explant) and number of leaves (8.30 per explant) as compared to the first and the second sub-cultures. While, the number of sub-cultures did not reflect any differences in shoot length. Moreover, El-Zeiny et al. (2013) reported that there are relationship between number of sub-cultures and rates of shoots production of global artichoke plantlets. Increasing the number of sub-cultures till fifth times increased gradually the number of shoots production and decreased shoot length.

4) Effect of the interaction between cultivars and growth regulators
The obtained results in Table (5) showed clearly that, the maximum increase in number of shoots per plantlet were more achieved via the interaction treatment between Sweet Charlie cultivar and using ½MS-medium which supplemented with 0.5 mg/l BA + 0.3 mg/l GA 3 . Moreover, the highest values of both number of leaves per shoot and shoot length were recorded by the interaction treatment between such cultivar and using ½ MS-medium without application of any growth regulators (the control treatment), followed by the interaction between the same cultivar and using ½ MS-medium which contained of 0.01 mg/l BA + 0.01 mg/l GA 3 . On the other hand, the maximum increase in the fresh weight of soot were more distinct by the interaction treatment between Sweet Charlie cultivar and using ½ MS-medium+0.01 mg/l BA.
On the contrary, all interaction treatments did not reflect any significant effect on the dry weight of shoots per plantlet. In this regard, Zebrowska and Hortynski (2002) studied the effect of various concentrations of BA (0, 1.6, 3.2 and 6.4 mg /l) in MS-medium in clone B-302 and Kama strawberry cultivar. They found that, leaf explants regenerated only at a concentrations of 3.2 mg and 6.4 mg/l BA in the medium. The higher shoots formation was found to be in clone B-302 than in Kama cultivar and obtained at 3.2 mg/l of BA level (average 6 and 8 shoots /explant). However, at the same concentration of BA in MS-medium did not formation any shoots in Kama cultivar, which regenerated only at concentration of 6.4 mg/l BA. Moreover

5) Effect of the interaction between cultivars and number of sub-cultures
The obtained results in Table ( On the other hand, the interaction treatment between Festival cultivar and the first sub-culture being the inferior one and recorded the lowest values of all parameters which were studied.

6) Effect of the interaction between growth regulators and number of sub-cultures
It is quite clear from the obtained results in Table (7) that, the fourth sub-culture treatment being the most effective as compared with the other sub-culture treatments.
In this connection, the interaction treatment between using ½ MS-medium+ 0.5 mg /l BA + 0.

7) Effect of the interaction between cultivars, growth regulators and number of sub-cultures on the plantlet shoots formation of strawberry cultures in vitro
It is evident from the presented results in Table (8) that, the maximum increase in number of shoots per plantlet were recorded via the interaction treatment between Festival cultivar, using ½ MS-medium which supplemented with 0.5 mg/l BA + 0.3 mg/l GA 3 and the fourth sub-culture. Moreover, the interaction treatment between Sweet Charlis cultivar, the same medium and the fourth sub-culture came in the second rank in this respect .
Furthermore, the maximum, increase in number of leaves per shoot were more achieved via the interaction treatment between Sweet Charlie cultivar, using ½ MS-medium without application any growth regulators and the second sub-culture.
With regard to shoot length, it is quite clear from such results in Table (8) that, the maximum value in this respect was recorded by the interaction treatment between Sweet Charlie cultivar, using ½ MS-medium without application any growth regulators (the control treatment) and the fourth subculture.
From the for going results, it could be suggested that ,all parameters of the plantlets ,i.e. number of both shoots per plantlet and leaves per shoot, as well as shoot length varied greatly according to the tested cultivars, the contained of ½ MS-medium and number of sub-cultures.

Molecular Markers
Identification of six primers for two strawberry varieties, i.e., Festival and Sweet Charlie which generated polymorphic markers were used to Identification of six primer for two strawberry varieties, i.e., Festival and Sweet Charlie which generated polymorphic markers were used to determine the polymorphism that could be associated with differences among five treatments.

1) Festival cultivar
Identification of six primers which generated polymorphic bands was used to determine the polymorphism that could be found among five treatments. RAPD analysis of Festival cv using six primers are illustrated in Table (9). Primer OP-B09 generated two monomorphic bands and the two polymorphism bands with 71.429% polymorphism. 283.196 to 1074.240 b.p.
Whereas, the highest number of bands (12) was generated from Primer OP-C09, and generated three monomorphic bands with 80% polymorphism. The size of bands ranged between 334.001 to 1118.946 b.p.
Primer OP-K01 generated five monomorphic bands and the five polymorphism bands with 50% polymorphism. The size of bands ranged between 297.625 to 1186.595 b.p.
Primer OP-K03 generated four monomorphic bands and three polymorphism bands with 42.857% polymorphism. The size of bands ranged between 204.064 to 629.310 b.p.
Primer OP-O05 generated seven monomorphic bands and four polymorphism bands with 36.346% polymorphism. The size of bands ranged between 245.693 to 1154.764 b.p. Fig. (4) Showed the pattern of amplification product of primer OP-O05.
It is clear that Primer OP-O10 generated five monomorphic bands and nine polymorphism bands with 64.286% polymorphism. The size of bands ranged between 233.292 to 989.581 b.p.
It could be concluded that, the six primers produced 64 bands among them 38 were found polymorphic with 59.375% polymorphism. The number of polymorphic bands per locus ranged from three (OP-K03) to 12 (OP-C09) with an average number of 6.0 bands per locus. DNA markers are proved powerful tools to evaluate the polymorphism (Selim et al., 2010, Hussein et al., 2013 and Suprasanna and Jain 2017).

2) Sweet Charlie cultivar
Identification of six primers which generated polymorphic bands was used to determine the polymorphism that could be found among five treatments. RAPD analysis of Sweet Charlie cv. using six primers are illustrated in Table (     N.S.: Not significant at 0.05 level of probability  N.S.: Not significant at 0.05 level of probability