Pre-isolation , isolation and regeneration protoplasts from leaf mesophyll of in vivo

2Coconut research institute of Chinese academy of tropical agricultural sciences Hainan, China.E-mail: karimalayani@yahoo.com 3Horticulture Department Ghazi University, Dera Ghazi Khan, Punjab Pakistan E-mail: mh.baiea@nrc.sci.eg AbstractTo isolate protoplast, a pre-treatment was completed with the order to reduce and identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5% Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation period, sieve pore size, centrifugation period and hormonal balance was estimated using the techniques for isolation. The quantity of cells was computed as the quantity of cells per square on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW medium using a pore size of 25 μm with using incubation period for 20 hours. Moreover, the best protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant recovery using in apple somatic hybridization programs.


Introduction
The genus Malus has a place with the Rosaceae family and forms with its closely related fruits (Pyrus and Cydonia) and ornaments (Amelanchier, Aronia, Chaenomeles, Cotoneaster, Crateagus, Pyracantha, Sorbus) genera, the subfamily Maloideae (CORNILLE et al., 2012). The apple tree (Malus domestica Borkh) is a standout amongst the most financially imperative fruit grown from the berry crops in world agrarian practice. The world production of the way of culture this fruit depends just on 15-20 varieties (GROSS et al., 2014(, regardless of the world collection of at least 7000 varieties. The traditional method for apple improvement by choosing the best phenotypes from seedlings become from open-pollinated seeds were supplanted by purposeful hybridization around 200 years prior. However, at first little advancement was made in improvement of apple cultivars through controlled crossing, since their initial domestication, apples have been transported around the world and the cultivated species is now grown in all temperate biomes on Earth (HANCOCK et al., 2008). The accomplishment of the moderately ongoing acquaintances must be credited with the determination of parents with great fruit quality. Illustrious Gala, Fuji, and Jonagold were chosen in the original from the best commercial cultivars, notably Golden Delicious and Delicious, available at the time of crossing.
Breeding of new apple cultivars is challenging. The fundamental technique for conventional apple breeding has experienced the intersection and determination of prevalent individuals from a huge number of seedlings. The lengthy juvenile period of the tree and its vast size, requiring a significant lot of time for assessment and an extensive field space have imposed confinements on apple breeding programs.
In the selection procedure and viable usage of the characteristics of specific varieties under particular developing conditions and ensuring practical productivity. Several generations of backcrossing are typically required before parental material suitable for cultivar growing is available. For quite a long time, response to address them has been easing back because of the nature of fruit tree breeding and reproducing: long term; low effectiveness; and consequently the high expense. For the past 20 years, global systems of fruit geneticists have been working to develop research on fruit hereditary qualities went to improving fruit quality characteristics and also protection from biotic anxieties. Protoplast fusion gives chances to combine the genomes of systematically unique species that can't be consolidated explicitly because of incompatibility hindrances (LIU et al., 2007). The generation of gamete-substantial hybrid and hybrid plants likewise has significant potential as far as quality integration (Davey et al., 2005). This innovation has gotten a misfortune and restricted consideration. In the meantime, recent studies identified with the generation of physical hybrid breeds have uncovered the potential role of this strategy in enriching plant germplasm, and for the creation of novel cultivars (ANANTHAKRISHNAN et al., 2006;TROJAK-GOLUCH and BERBE' C, 2007;YERMISHIN et al., 2008).
Fast improvements in biotechnological reproducing have abbreviated the timeframe required for fruit tree rearing, and such systems are presently being connected to apples. Translating of the apple genome (VELASCO et al., 2010) has given knowledge into the development of this species.
The application of somatic hybridization may augment the quality pool accessible for apple breeding and improvement, and might be especially imperative for a standout amongst the most vital dwarfing apple rootstocks used around the world, the cultivar 'M9' (Malus pumila Mill.;HÖHNLE and WEBER, 2007). 'M9' rootstock introduces a long reproductive cycles, a high level of heterozygosity, and frequent self-incompatibility (ZHANG and LESPINASSE, 2006), which put impediments on the utilization of regular breeding techniques. However, the establishment of a protocol for haploid protoplast isolation and culture is an essential for the efficient somatic hybridization program.
Malus domestica 'Anna' cv. which got from the hybrid of Golden Delicious and Red Hadassiya has a decent fruit quality and successfully grown under Egypt conditions. Chilling requirements of such apple cv. is low. Particularly suited to warm winter gardens produces large fresh apples with light green-yellow skin and a slight red blush. Sweet, slightly tart flesh is perfect for crisp eating, cooking and baking. Recommended chilling 200-300 hours, produces at an early age, self-fruitful, anyway a pollinizer, for example, Dorsett Golden will increase production. An apple trees require a period of low temperatures (4 to 10°C) in the fall and winter to allow bud break and growth in the spring. If this period of chilly is inadequate with regards to, the percent of bud that grow will be low and bud break and blossom bud opening will be very sporadic (PUTTI et al., 2003).
Accordingly, the aim of this investigation was to build up a reliable protocol for the isolation and culture of mesophyll protoplasts from the leaves of Malus domestica 'Anna' cv. as an initial step to create another cultivar of apple has great quality and low chilling requirements suitable for Egypt natural conditions.

Materials and methods
This investigation was done at Tissue Culture Unit, NRC, Dokki, Giza, Egypt during the period from 2017 to 2018. All the experimental studies conducted on In vivo Malus domestica 'Anna' cv.

Plant material
All the experimental studies directed on in vivo Malus domestica 'Anna' cv. which derived from the combination of Golden Delicious and Red Hadassiya. The mother plants grown in Research and Creation Station plantation at El-Nobaria area, Behera Governorate, Egypt. Different experimental studies were took place as follow:

Pre-protoplast isolation a. Evaluation of phenolic compounds level
To choose the best anti-oxidant treatment viable in overcoming phenolic issue, recorded the levels of phenolic compounds, various periods and concentrate the seasonal fluctuation of phenolic. For this, Five leaves were collected from different three mother plants at various periods ( Fig  5A) during all the year, i.e., March, June, September and December (at first seven days of every month) record the levels of phenolic compounds (free, conjugated and total).

Protoplast isolation
Leaf disinfestation -1. Collected the new emerged leaves of in vivo from the mother plants ( Fig 5A); 2) Transferred directly to the tissue culture laboratory; 3) Subjected to the running water for 15 minutes to get rid of dirt's and germs; 4) Immersing in soap solution for 5 minutes; 5) Then immersing in 10% Clorox solution (0.5 % NaOCl) commercial bleach with two drops of Tween -20 for 10 minutes; 6) And finally immersed in sterilized distilled water 3 times for 5 minutes, each.
Effect of osmotic pressure factor -Sucrose, mannitol and glucose were the principle osmotic pressure factors applied at a rate 90, 130 and 210 g L -1 individually whereas every source added to CPW salts medium for identifying the ideal osmotic pressure factor, prevailing with regards to succeed in optimizing the osmotic pressure inside and outside (medium osmotic pressure) protoplast valuable to produce rounded protoplast (suitable protoplasts) without occurrence plasmolysis or burst.
Effect of incubation period -In vivo leaf strips ( Fig 5B) immersed in the suitable enzyme mixture were incubated for various periods i.e., 12, 16, 20 and 24 hours to affirm the best incubated time frame actuated the highest viable protoplast yield.

Effect of shaking (Shaking speed and Shaking period):
Different shaking speed i.e., 50, 100, 75 and 125 rpm with different periods i. e., 15, 30, 45 and 60 min. were tested to detect the suitable speed and period which encouraged the highest protoplast yield ( Fig 5C).

Purification
Effect of sieve pore size -The simple experiment, trial was intended to consider the impact of various sieve pore sizes on the rate of purging levels through disposing of debris and processed cell divider buildups. Sieve with various pore sizes were tried, i.e., 25, 50 and 75 µm to choose the best pore size encouraged the highest purification without any hazard on protoplast yield.

Effect of centrifugation (speed and period):
Different centrifugation speeds i.e., 500, 1000 and 1500 rpm speeds were used to confirm the best speed augmented protoplast cleaning and reducing protoplasts damage. Moreover, different periods i.e., 5, 7.5 and 10 minutes were tested to detect the suitable period encouraged the highest protoplast purification with reducing protoplasts damage.

Protoplast culture:
Effect of medium type -In this experiment the isolated protoplasts of In vivo Malus domestica 'Anna' cv. were refined on various media types i.e., MS (Murashige and Skoog, 1962), KM (Kao and Michayluk, 1975) and B5 (Gamborge, 1968) to choose the best culture medium type gave the most elevated protoplast development.
Effect of hormonal balance -Hormones were refined on MS medium supplemented with NAA (1, 2 and 3 mg L -1 ) was added to the development culture media at three levels, each level was combined with each one of the following BAP concentrations (0.1, 0.2 and 0.3 mg L -1 ) as follow: Each experimental unit consisted of Petri dishes (15mm×150mm) which containing protoplast yield. Moreover, Petri dishes were organized as a factorial completely randomized design with three replicates.
Data and calculation -Counting of protoplasts was according to strategy for (BLACKHALL et al., 2002). Also, number of cells was figured as the quantity of cells per each square on haemocytometer. The final count of protoplasts per 1 ml was conveyed by the following equation conditions. Total number of cells = 5n × 10 4 . Where: n = the average of number of cells per each square on haemocytometer. Also, scores were connected for protoplast development which ascertained as the rate of cell division and microcallii as pursue: 1) No cell division or microcalli formed; 2) Below average of cell division and microcalli formed; 3) Average number of cell division and microcalli formed; 4) Above average of cell division and microcalli formed; 5) Excellent (the highest) cell division and microcalli formed.
Statistical analysis -All treatments used in this study were arranged as factorial experiment in a complete randomized design according to SAS and Statistica 9.0 (StatSoft. Inc. 2009). The obtained data were subjected to analysis of variance and statistically analyzed using standard division (SD).

Results
Evaluation of phenolic compounds level -There was a persistent increment altogether, free, and conjugated phenolic compounds determined during March, June, September, and December periods respectively (Fig. 1). Hence, the lowest level of phenolic compound showed up at the March sample, which demonstrated that the best time for taken the explants for protoplast isolation. Additionally, this information reflects the ideal antioxidant treatment expected to lessen or take out the phenolic compounds exist during taking the explants.

Anti-oxidant treatment
The combination of 0.1% ascorbic acid, 0.15% citric acid and 0.5% PVP treatment brought about the noteworthy decrease of phenolic compounds as compared and other treatments (Table 1). Meanwhile, the combination between antioxidant solution arrangement and P.V.P. took the second rank in reducing the phenolic compounds pursued by P.V.P. treatment. However, the reverse was true when combination treatment of ascorbic acid, citric acid and P.V.P was used. Means within a column followed by the same letter/s are not significantly different at p=0.05 Note: "AA" means Ascorbic Acid, "CA" Citric Acid, "PVP" Polyvinylpyrolliden, "EM" Enzyme Mixture, "M" Mannitol, and "S" Sucrose.

Plasmolysis
Results for plasmolysis demonstrated that soaking source explants in plasmolysis treatment containing 90 g L -1 mannitol for half hour, then 130 g L -1 mannitol expanded the quantity of plasmolized cells pursued by plasmolysis arrangement (3) containing 130 g L -1 mannitol +0 sucrose. In any case, the most reduced numbers of plasmolized cells were delivered from plasmolysis (1) (Control: 0 mannitol + 0 sucrose) (Table 1). Concerning the interaction, between protoplasts source and Plasmolysis treatments it is very apparent that in vitro explant treated with the plasmolysis 5 (Plasmolysis for 30 min in CPW 9M + 30 min in CPW 13M) boosted the quantity of plasmolized cells, trailed by in vivo explant combination with the equivalent plasmolysis. However, the mix of in vivo explant with plasmolysis (4) and plasmolysis (1) incited the least qualities.

Effect of digestive enzyme medium -
The results presented in ( Fig. 2A) revealed for the effect of digestive enzyme medium on protoplast yield (Fig.5 D1). It is noticed that CPW medium was superior in increasing the protoplast yield compared with other media used. However, the lowest result was obtained when using MS culture medium.

Effect of osmotic pressure factor -
The adding of mannitol to the way of culture medium created the highest viable protoplast yield as compared with the other osmotic pressure factors (Fig. 2B). The lowest protoplast yield was acquired when glucose was used as its clear form.

Effect of hormonal balance -
In general, above results summarized that using of 1.0 mg L -1 NAA and concentrations of BAP under investigation (0.2 and 0.3 mg L -1 ) was the best hormonal equalization used as suitable for maximized protoplast development and expanded cell division (Fig. 2C). The supplementation the culture medium with 3.0 mg L -1 NAA was favored as the increase of protoplast development. In any case, using free NAA medium gave the most exceedingly high protoplast development. In addition, 0.2 mg L -1 of BAP was suggested as it augmented protoplast development compared and the other BAP concentrations under investigation. In any case, the supplementation of 1.0 mg L -1 NAA and 0.3 mg L -1 BAP prevailing with regards to enhancing protoplast development. Value similar concentration of NAA and 0.2 mg L -1 BAP took the second rank in delivering the best protoplast development. While, the culture medium free from hormones prompted no impact on protoplast development (Fig.5 D1-5).

Effect of incubation period
It is seen that the using incubation period of 20 hours was viable in improving the protoplast yield treatment with the other incubation periods (Fig. 3). The results of manage the impact of incubation periods on protoplast yield incubation periods for 16 h took the second rank in enhancing the protoplast yield pursued with 24 h incubation period. In any case, the lowest protoplast yield was seen when incubated for 12 h.

Shaking speed -
The best shaking rate that boosted the quantity of protoplast with less damage was 75rpm (Fig. 3). Similarly, expanding shaking speed up to 100rpm decreased protoplasts yield because of expanding damage protoplasts yet the shaking velocity of 50rpm took the second rank in enhancing protoplast yield. Fig.  3 portrays the impact of shaking period on protoplast yield. It is clear that greatest protoplast yield (2.07x 10 5 ) was actuated when the increment-shaking period from 0.0 min. to 30 min. pursued by shaking period for 15 min. and, 45 min respectively. However, the minimal protoplasts yield acquired when shaker was not utilized (control) this might be because shaking encouraged enzyme mixture digestion of cell walls and free protoplasts appeared.

Effect of protoplast density
It is clear that increasing cultured protoplast density from 0.5 x 10 5 to 2.0 x 10 5 resulted in enhancing in protoplast development as (3.33) was obtained when cultured density was 2 x 10 5 . Meanwhile, continuous increase of protoplast density up to 2.5 x10 5 inducted an adverse effect on protoplast development. The above results reflect the importance of using protoplast density 2.0 x 10 5 in maximizing protoplast development (Fig. 3).
Effect of different treatments on yield and quality of protoplast 1. Effect of sieve pore size -The increased sieve pore size results in a decreasing numbers of viable protoplast (Fig. 4A). In the interim, the most elevated of the number of viable protoplast was incited by using a pore size of 25 µm pursued by 50 µm lastly the least protoplasts instigated when 75 µm pore size was used. The previously mentioned outcomes suggested using sieve pore size 25 µm incited the most astounding protoplast number. These outcomes might be because of expanding the sieve pore size generated higher quantities of protoplasts cell residues, clumps of undigested tissues and debris to pass through the filter and in turn affect badly on protoplast yield.

Effect of centrifugation speed -
The use of centrifugation speed at 1000 rpm was more effective in increasing the number of protoplast followed by 500rpm (Fig. 4B) reflecting on a number of viable protoplast. However, continuous increase in centrifugation speed up to 1500rpm induced the lowest protoplast yield.

Centrifugation period -Expanding
the centrifugation period up to 7.5 minutes (Fig. 4C) incited appositive impact on the expanding number of viable protoplast in correlation with other periods under investigation. In the meantime, expanding centrifugation period from 7.5 to 10 minutes brought about reducing protoplast viability. In any case, using centrifugation period 5.0 minutes took the second rank in enhancing protoplast viability.

Effect of medium type -
The effect of different media types on protoplast development can be 345 seen in Fig. 4D. It appears that the superiority of MS medium over both KM and B5 media. However, B5 medium showed the worst effect on protoplast development. The above results indicate the suitability of MS medium for the best protoplast development.

Discussions
The present protocol was the initial step to check the potential of mesophyll leaf tissue using new techniques for in vivo isolation in apple protoplast cultures. Strategies like pre isolation and using of antioxidants use in vivo to enhance protoplast generation were rarely used for apple. Numerous endeavors have been made for significant harvests protoplast isolation, including sugarcane, maize, potato, citrus, carrot, cotton, and rice and so on. (RAI et al., 2011) Exposure of protoplasts was likewise published for different species, for example, grape vine, orange tree, lemon, tomato, and tobacco (SˇVA'BOVA' AND LEBEDA 2005). Physiologically uniform stages, Protoplasts is an elective that utilizes quite a bit of the extensive cell populace compared to various types of explants, thus, leading towards an opportunity to present new protocols both in vivo and in vitro conditions for efficient isolation regeneration and selection using less space.
However, a limited number of species capable of rebuilding cell walls and under-going mitotic cell divisions to produce callus and normal plant formation. Often, only genotypes carefully selected in many species undergo production of protoplast-derived plants (DAVEY et al., 2005).
In this exploration, we could control essentially polyphenols that restrain mitotic cell division in protoplast isolation following the treatments with varying level of antioxidants, which was correlated with the density of protoplasts. The increase in protoplast efficiency observed for both types of particular treatment application. However, a stronger response of cells was observed when the protoplast from the antioxidant treated cell was extracted. This may be explained by the cells able to reconstruct cell walls infect negligible or reduce phenolic compounds in comparison to the non-treated cells. Similar observations have been accounted for in the protoplast of In vitro culture of unfertilized ovules in carrot (KIEŁKOWSKA AND ADAMUS 2010).
Analysis of the effect of different factors, including time of explant taking, using of anti-oxidant solution on protoplast isolation and culture all the factors have significantly positive role, such as plasmolysis treatments, enzyme mixtures, different media and other than these factors such as shaking, centrifugation and hormonal balance were the most important.
Taking explant in the March sample demonstrated the most reduced phenolic compounds use of antioxidant solution 0.1% ascorbic acid + 0.15% citric acid+ 0.5% P.V.P, these outcomes are in accordance with the discoveries of (SAVITA et. al., 2011). They declared that citrus acid and ascorbic acid controlled browning of coconut explant, yet polyvinyl-pyrrolidone was insufficient at the rate of 1 mg L -1 in controlling oxidation of the phenolic compounds. In addition, introduction to 90 g L -1 ml mannitol for half hour give preferred results over 130 g L -1 ml mannitol which is in concurrence with the discoveries of (SHRESTHA et al., 2007) reported that explant regeneration from cell suspension-derived protoplasts of Phalaenopsis plasmolyzed for 30 min in CPW medium with 0.5 M mannitol pursued by 30 min in CPW medium with M mannitol (CPW 13 medium) plasmolyzed protoplast increment respect 4.5 × 10 6 g/ FW protoplasts with 60% viability. (HISAMOTO AND KOBAYASHI, 2010) likewise washed in the mesophyll tissues in a solution of 6% mannitol and was plasmolyzed in CPW medium with 0.5 M mannitol pursued by 30 min in CPW medium 0.7 M mannitol. Using 1.5% cellulase + 1.5% Macerozyme + 0.5% pectinase with CPW digestive related medium delivered high density protoplast, these outcomes adapted to the discoveries of (LIN et al., 2014). They discovered that CPW 13M was the best protein medium used for protoplast detachment from leaves of stone fruit (Prunus spp. ). Medium supplemented with 1% PVP and 0.5 mM MES helped in the prevention of browning and protoplast cell wall regeneration. (MEHRI 2003) who discovered that CPW 13M was a profoundly proficient medium for digestion related enzymes mixture to isolate protoplast from prunus carasus L. cv" Montmorency".
Meanwhile, adding mannitol as osmotic pressure factor to the media empowered generation of the highest protoplast numbers as revealed that adding 0.7% mannitol to the incubation medium expanded protoplast viability got from cell suspension of apple cultivars (Malus domestica cultivars fuji and Jonagold) and Malus prunifolia var ringo. In addition, (MEHRI 2003) got the best yield and viability of protoplast of Prunus cerasus L. isolated from leaf mesophyll and leaf callus using enzyme solution containing 13% mannitol and 5mM MES. However, (SEGUI et al., 2006) discovered that the best viability of protoplast from apple (Malus domestica var. fuji ) when used 0.8% M mannitol as osmaticum . Besides, Incubation of the explants for 20 hours additionally encouraged in isolation of protoplast, incubated in an enzyme solution at 25°C in the dark for 18 hours' apple (Malus domestica Borkh) explant. Likewise, with (SHRESTHA et al., 2007) who obtained a high amount of protoplast from Phalaenopsis when incubated in an enzyme solution in the dark under 28°C for 18 hours. Shaking with incubating of explants in an enzyme solution at 75rpm for 30 min delivered a good yield of protoplasts.
Using a sieve at pore size 25 µm give the best filtrate that the isolation of protoplast of citrus was best with filtration through 50 µm and 30 µm nylon screens. Centrifugation protoplast with speed rate 1000rpm for 7.5min creates a clear layer pattern (QINGHUA ZHANG et al., 2006). They isolated protoplasts of Citrus unshiu in the filtrate was additionally cleansed by centrifugation in a 25% sucrose/13% mannitol gradient for 6 min at 88 g. Murashig and Skoog medium was prevalent for the best protoplast with density rate 2.5 X 10 5 in the way of culture medium, best cell division with high protoplast thickness 0.5-2.5 x 10 5 protoplasts for each ml of pear Pyrus spp when refined on MS Medium. (YOO et. al., 2007) also get best results get best results of cell division from protoplasts of apple (Malus X domestica) cv. "fuji" when refined on MS medium enriched with 2 mg 2,4-D and 1mg benzyladenine (BA/liter) and 0.8% agar. Other than MS medium, addition to the mix of 1.00 mg L -1 NAA and 0.3 mg L -1 BAP were most appropriate for protoplast recovery. (XIANG et. al., 2004) Who effectively refined the protoplasts of wheat on NN medium enriched with 2 mg L -1 NAA and 0.2 mg L -1 BAP at 28 o C in the dark and same were reported with high recurrence of cell division of Vitis thunbergii protoplast, when culture medium was enhanced with 2 mg L -1 NAA and 0.2 mg L -1 benzyl adenine.
Regarding, planting efficacy and ability protoplast to regenerate plants from pre-medicated explants showed a smooth response, likely to be correlated with different levels of antioxidants. The maximum recovery efficiency that allowed us to regenerate plant cells was higher and fall in a range of 20 to 85 % premeditated depending on the treatments and to that of time and season of application. Similar results were recorded by (RAI et al., 2011). Successful regeneration and selection with different treatments was also reported on protoplast  (5) protoplast development (1=protoplasts, 2=buding stage, 3= protoplast division, 4=microcalli formation, 5=more microcalli formation). isolation process of chrysanthemum (KUMAR et al., 2008), carnation (THAKUR et al., 2002) and that citrus (SAVITA et al., 2011). However, exposure to toxins like polyphenols can result in a decrease and recovery capacity and can create undesired freaks. Despite what might be expected, using procured and techniques may yield higher recovery rate and additionally healthier plants (SHARMA et al., 2010). Subsequently, in this experiment, we applied a pre-medication system with seasonal variation in phenolic level and anti-oxidation to in a mixture of various media enriched with various hormonal levels inserted higher density of protoplasts. These conditions used in this process ensured sufficient favorable conditions to each cell present in the culture. These conditions were additionally used with protoplast isolation of sugar cane (MAHLANZA et al., 2013). Loss of viability is regular in protoplast cultures of several species. (BABAOG˘LU, 2000), (ADITYA AND BAKER 2003), (SRISAWAT AND KANCHANAPOOM 2005), (KIM et al., 2006), and(CHABANE et al., 2007) reported challenges in protoplast cultures. (DAVEY et al., 2005) attributed the decline in viability to be due to the sensitivity of the plasma membrane, due to an inadequate reduction in the osmolarity of the culture medium.
Along these lines, the achieved above protocol can give a premise to the isolation and development of protoplast and to plant recovery framework, beginning from leaves, for the improvement and breeding program of apple cultivars.

Conclusion
The protocol developed and embraced in this manuscript is proficient for protoplast isolation and recovery of cultivar studied. The results of this exploration likewise recommended that this protocol could be utilized for fusion experiments for somatic hybridization using other cultivars to produce new cultivar suitable for Egypt environmental conditions.