IN VITRO MICROGRAFTING OF RED GLOBE (VITIS VINIFERA L)

This study was carried out to evaluate the micrografting success of Red globe cultivar (Vitis vinifera L.) on three rootstocks Ramsey, Freedom and SO4 and investigate the effect of some factors on the micrografting success. Furthermore, compatibility in the graft union was studied. The results showed that Red globe on un-rooted Freedom achieved the highest percentages of scion survival (100.00 %), scion bud burst (60.00%), graft union formation (60.00%), and rooting (40.00%), also longest shoot (2.00cm), highest root number (8.00) and root length (4.50cm). Murashige and Skoog (MS) medium Supplemented with Indole Butyric Acid (IBA) at 1mgL -1 achieved the highest percentages of scion survival (95.20%), scion bud burst (65.43%), graft union formation (82.22%) also, longest shoots (1.84 cm) and maximum average leaf number (2.50). Regarding the effect of MS strength, 25% MS, 50% MS and 75% MS achieved positive effects on all parameters compared with full MS. The anatomical study revealed that Red globe on Freedom combinations were the best due to enough callus formation, less necrotic layers, and a wavy continuity of new cambium were determined. Sterilized 1:1:1 (v:v) peatmoss+perlite+sand mix gave the highest survival rate (100.00%) and showed superior vegetative growth. Freedom micrografts achieved the highest survival rate (100.00%), better growth and longer roots compared with those on SO4 and Ramsey (80, 70%) respectively.


INTRODUCTION
Grape is a popular crop and has many cultivars with different purposes in the world around. Red globe is an attractive table grape due to its berry size, color, and favorable for both local market and export. Dormant cuttings are usually used in grapevine propagation besides grafting on phylloxera-resistant rootstocks. However, conventional grafting is slow and does not produce pathogen-free plants perfectly. In vitro micrografting is a new technique applied in aseptic conditions and used on a large scale for plant multiplication as an alternative to the conventional one (32). Micrografting is a rapid process that can produce thousands of healthy plants in a short time as well as improves the yield and grape quality in addition to eliminating the common pathogens (Virus, Phylloxera, Nematodes, root rots..etc) that lead to massive yield and quality losses, and able to overcome some of the anatomical and physiological problems when some species and cultivars merge together (17,32). Selecting the right rootstocks that adapted to the local climate conditions is very important (19) that affect efficiently the cultivars resistance (18). Micrografting technique has been used on several species including grape (5), Cashew (54), Pistachio (43), and cherry (15). Micrografting success can be affected by several factors including scion origin and age, basal medium formulation, sucrose concentration, micrografting procedure, growth regulators, and rootstock type (5). (24) obtained grafting success of 83 % by using in vitro scion (0.5-1cm cm) of Le Conte pear on in vitro decapitated P betulaefolia as rootstock. (47) reported that maximum graft success was observed on Murashige and Skoog liquid medium containing 2% sucrose. (6) stated that the highest graft success (24.55% and 21.89%) was obtained when micrografts were cultured on MS semi-solid media at 3 and 6 % sucrose respectively. (1) used 1 cm of a rootstock and a shoot-tip of a scion and made a good connection of the two pieces then cultured on MS medium solidified with 6gL -1 agar, 3% sucrose. (49) reported that IBA was insignificantly better than NAA in all parameters, while NAA stimulated forming callus masses on rootstocks, and He recommended Thompson seedless on Freedom micrografts planted on B5 medium contained IBA at 2 mg/l, Flame seedless on Ramsey micrografts planted on B5 medium contained IBA at 3 mg/l. After grafting, compatibility between stock and scion tissues is really important to obtain an optimum grafted plant (22). (31) explained in the study of histological structure of compatible and incompatible plants that necrotic cells layer in incompatible plants was thick, and it was noted the presence of callus as small parts around the grafting area, leading to the separation of scion and stock later. (49) illustrated that Freedom rootstock was more active in cell dividing compared with Harmony rootstock. Moreover, vascular connections were detected although the cambia of both scion and rootstock were in some cases not matched. For the successful transfer to the open field, in vitro plantlets have to pass through a gradual change from high relative humidity and low light intensity of the flask stage into low relative humidity and high light intensity of the open nursery and field. After rooting, the plantlets are transferred into pots filled with rooting substrates, left open and placed inside growth chamber (3), or placed inside propagator units (51). It could also either covered with plastic bags or enclosed inside polyethylene sheet and placed under a glasshouse with natural daylight (30). This study aimed to assess the micrografting success of Red globe grape cultivar on different rootstocks under in vitro conditions and investigate the influence of some factors on the grafting success. Furthermore, determining the compatibility via anatomical study in the graft union. Moreover, acclimatization of the obtained micrografts.

MATERIALS AND METHODS Plant material and explants preparation
One-year-old transplants were used as mother plants for Red globe grape cultivar and three rootstocks named Freedom, Ramsey, and SO4 for explants. Nodes 1-2 cm long were used as explants. The leaves were removed, and a short part of the petiole left. The explants were washed with water, and a few drops of soap then put under a slow flow of tap water for an hour. Explants were surface sterilized in 1% sodium hypochlorite with 2-3 drops of Tween-20 for 10, 15, and 20 minutes as a surfactant. Then, the nodal cuttings were rinsed 3 times with sterilized water then divided into separate stem node explants which were cultured (4-5 per jar). The basal medium and glassware preparation Full strength and free hormone MS as a basal medium was used (44) in the establishment stage. Sucrose at 3% was used as a source of energy and the medium was adjusted pH 5.8 ±1 before being solidified with purified Agar (Agar-Agar) at 7 g/L, then autoclaved at 100 psi and 121 ᵒ C for 20 min. Glass jars (400 ml) contained nearly 40 ml medium per jar have been used during microprpagation stage. In addition, glass culture tubes (150×25mm) received 20 ml medium per tube in the micrografting experiments. The glass jars and tubes were washed with detergent then soaked in a 5% solution of sodium hypochlorite (NaOCl) for an hour then rinsed with water before using them.

Scion and rootstock shoots multiplication
In vitro explants from the initiation stage were cultured on a full-strength MS medium supplemented with a plant growth regulator (5 mgL -1 6-furfurylaminopurine Kin, 3% sucrose and 0.7% Agar-Agar). The pH was adjusted to 5.8% before autoclaving (121 ᵒ C, 20 min). Micrografting stage Preparation of scion: 4-week-old In vitro shoots of Red globe from the final subculture with the desired thickness, and internodes have been taken as scions for micrografting. Under aseptic conditions, the selected shoots were cut to 5-10 mm in length and each one had an axillary bud. Preparation of rootstocks 8-10-week-old in vitro shoots of Freedom, Ramsey and SO4 rootstocks were used for micrografting. Shoots with a good diameter and long internodes were selected then shortened to (15-20 mm) in length inside the laminar cabinet under aseptic conditions. Micrografting procedure Cleft-grafting method was applied. A 0.5 cm slit was made in the rootstock shoot and a wedge from a scion shoot was cut and inserted it in the rootstock making a maximum connection of the two pieces. In all cases, the rootstock and scion should be as one union. Grafted combinations were cultured on MS medium solidified with 7gL -1 agar and containing 30gL -1 sucrose. Some experiments were carried out to improve the in vitro micrografting success among the studied cultivar and rootstocks. In addition, the factors involved in scions performance and the rooting of micrografts. Micrografts rooting percentage (adventitious roots forming on the rootstock stem). 8) Number of roots per micrograft. 9) Average root length per micrograft. Effect of rooted and un-rooted rootstocks on grafting success: Red globe scions were grafted on both rooted and un-rooted rootstocks and inoculated in a full strength and hormone-free MS medium. The parameters were recorded 4 weeks after grafting. Effect of IBA concentration on grafting success: A full strength of MS supplemented with IBA at 3 concentrations (0.1, 0.5, and 1 mgL -1 ) was studied on the rooting percentage and other grafting parameters in order to select the best concentration. Effect of medium strength 4 different strengths of free hormone MS (25,50,75, and full-strength) had been studied to understand their influence on the micrografts performance. Parameters were recorded 4 weeks after grafting. Anatomical study: Transverse sections were made across the successful graft union of well rooted and growing micrografts eight weeks after the culture date in order to study the compatibility and incompatibility limits among the studied cultivars and rootstocks. Samples were cut and fixed in formalin-acetic acidethanol 70% (FAA) for 48 h, then dehydrated in ascending ethanol. Paraffin wax was infiltrated and samples were embedded (28). Cross-sections were microtomed (12-16 µm) mounted to glass slides and stained with safranin-fast green schedule (50). Sections were examined and photographed with a light microscope (Leica DM 2500).

Incubation conditions
The cultures were incubated at 25±2ᵒC with 16 hours of light using fluorescent lamps (2 lamps per shelf) and 8 hours dark of 2000-2500 lux light intensity at cultures level. Acclimatization 6-week-old micrografted plantlets had been removed from the culture tubes then washed with running tap water to remove any medium residual then treated with a fungicide solution (Prochloraz 45% EC) 1gL -1 , then potted into a proper sterile medium and kept inside a glass chamber covered with transparent plastic in the incubation room for 4 weeks and watered constantly before being transferred to the glasshouse. Effect of soil mixture on the acclimatization success: Several soil mixtures of peatmoss:perlite 1:1(v/v), peatmoss: sand 1:1 (v/v) and peatmoss: perlite: sand 1:1:1(v/v) were tested. Effect of rootstock type on acclimatization success: The objective of this experiment was to investigate the effect of rootstock type on the survival percentage. Micrografted plantlets of Red globe combined with Freedom, Ramsey and SO4 rootstocks were planted in a soil mixture of peatmoss: perlite: sand 1:

Data analysis
A completely randomized design (CRD) was used and data were analyzed using SASS 9.1 and means were compared by Duncan's multiple range test at p≤0.05 level of confidence. (14).

RESULTS AND DISCUSSION
Effect of rooted and un-rooted rootstocks on grafting success : Data in Table 1 exhibits the effect of rooted and un-rooted rootstocks on some grafting characteristics of Red globe micrografts. It is plain that grafts of Red globe on Freedom rootstock significantly achieved the highest mean scion survival percentage (SS%) compared to those grafted on other rootstocks (100.00%). On the other hand, insignificant differences were recorded between the rooted and un-rooted rootstocks in this parameter. Interaction between the two studied factors was significant as Red globe scions grafted on rooted and un-rooted freedom gave the highest SS% (100.00%), whereas the lowest values were on un-rooted Ramsey and rooted SO4 (60.00%) for each. On the same side, Red globe scions grafted on Freedom gave significantly the highest mean bud burst percentage (BB%), whereas the lowest BB% was in those grafted on SO4(50, 29.5%) respectively. Also, insignificant difference was observed between the rooted and un-rooted rootstocks in BB%. Regarding the interaction, micrografts on un-rooted Freedom significantly gave the highest BB% (60.00%), while the lowest value was in micrografts on un-rooted SO4 (25.00%). It is obviously shown that insignificant differences were observed among means of rootstocks or rooted and un-rooted rootstocks or the interactions between the two factors in mean average shoot number per scion (ASN). Concerning the average shoot length (ASL), insignificant differences were observed among rootstocks regardless of roots presence or between rooted and un-rooted rootstocks regardless of rootstocks. Interaction between the studied factors was significant as the highest values were in the grafts of Red globe on un-rooted Freedom and SO4 (2.00cm) for each one, while the lowest values were in rooted Freedom and SO4 (0.50 cm) for each one. The results showed the mean average leaf number (ALN) in the grafts of Red globe on SO4 rootstock (3.35). In terms of roots effects, the grafts of Red globe on un-rooted rootstocks gave the highest mean ALN compared to the rooted one (3.34). Regarding the interactions, there were significant differences between the two factors that appear clearly in grafts of Red globe on un-rooted SO4 (4.40). It was clear that grafts of Red globe on Freedom rootstock showed the highest mean of graft union formation percentage (GUF%), compared to other rootstocks (50.00%), while the lowest GUF% was on SO4 (25.00%). Also, insignificant difference was recorded between rooted and un-rooted rootstocks regardless of rootstock types. Concerning the interactions, Red globe grafts on un-rooted Freedom showed the highest GUF% (60.00%), while the lowest GUF% was in un-rooted SO4 (25.00%). Concerning micrografts rooting percentage (MR%), Red globe grafts on both Freedom and Ramsey achieved the highest mean of MR% (20.00, 16.50%) respectively with insignificant difference between them . On the other hand, Red globe grafts on un-rooted rootstocks gave the highest MR%, regardless of the rootstocks (24.33%). Regarding the interaction, Red globe grafts on un-rooted Freedom achieved the highest MR%,while the rooted rootstocks did not appear any The results showed that Red globe grafts on Freedom significantly gave the highest mean of average root number (ARN) per micrograft (4.00) followed by the grafts on Ramsey (0.50) then on SO4 (0.00). Regarding the effect of the roots, un-rooted rootstocks were better significantly as gave the highest ARN regardless of the rootstocks (3.00). Concerning the interaction, there were significant differences between the two studied factors which appeared clearly in Red globe grafts on un-rooted freedom (8.00), while the grafts on all rooted rootstocks did not give any new roots (0.00). Red globe grafts on Freedom gave the highest mean of average root length (ARL) followed by the grafts on Ramsey then on SO4 (2.25, 0.50, 0.00 cm) respectively with significant differences among them. On the other side, Red globe grafts on un-rooted rootstocks achieved the highest mean of ARL significantly (1.83 cm) regardless of rootstocks. Interaction between the studied factors revealed that grafts of Red globe on unrooted Freedom gave the highest ARL (4.50 cm) significantly, while the roots of the rooted rootstocks did not respond or continue their growth.

un-rooted SO4
it is clear that Red globe micrografts achieved the best results on Freedom rootstock cultured on hormone-free and full-strength MS medium as their micrografts gave the highest percentages of scion survival (100.00%), scion bud burst (50.00%), graft union formation (50.00%), rooting (20.00%). These findings went in the same line with those obtained by (49) who reported that Freedom proved to be the best rootstock for Superior scions. On the contrary, (16) stated that Ramsey rootstock gave the highest rate of success of Thompson seedless and Superior, if compared to Dog Ridge for both scions. Moreover, the shoot length of Thompson seedless scion on Ramsey rootstock was higher than Thompson seedless scion on Dog Ridge rootstock. In addition, superior cultivar on both Ramey and Dog Ridge showed the same trend. (52) reported that the highest grafting success rate was obtained from Early Cardinal cultivar grafted on 41B followed by Early Cardinal on Ramsey with the rate of 71.4%. Many researchers found that micrografting success varies with the rootstocks because of the compatibility reaction between the grafting partners. (5) found that using Kober 5BB rootstock, the micrografting success rates reached 40.9 % and 68.2% for Hafizali and Emir varieties respectively. On the other hand, the obtained results showed that Red globe micrografts regardless of the rootstocks were remarkable on un-rooted rootstocks as they gave the best results in all studied parameters compared to the rooted one. It was observed that after the roots had shortened, they turned into brown color and stopped completing their growth which may be attributed to phenolic compounds from the cut surfaces and their oxidation by polyphenol oxidase and peroxidase enzymes caused discoloration of the tissues which resulted in poor micrografting (36). The phenolic compounds present in the cut surfaces inhibit the development of new cells and lead to graft union failure (26). These findings went in parallel with those obtained by (62) who stated that the physiological state of roots extremely influences the survival rate. Effect of IBA concentration on grafting success: The effect of IBA concentration on some grafting characteristics of Red globe grape micrografts is illustrated in Table 2. The micrografts of Red globe on different rootstocks varied in their response to IBA concentrations and that appeared clearly on Freedom rootstock which significantly achieved the highest mean scion survival percentage (SS%) compared to those grafted on other rootstocks (95.20%). On the same trend, significant differences were recorded among IBA concentrations in this parameter, so it was recorded that 1mgL -1 IBA scored the highest SS% significantly (95.20%) and the lowest SS% was at 0.1mgL -1 IBA (71.47%). Interaction between the two studied factors was significant as Red globe scions grafted on freedom gave the highest SS% at 0.1 and 0.5 mgL -1 (100.00%), Ramsey and SO4 at 1mgl -1 IBA (100.00%), whereas the lowest values were on Ramsey and SO4 at 0.1 mgL -1 (57.20%) for each one. On the same side, Red globe scions grafts on Freedom gave significantly the highest mean bud burst percentage (BB%), whereas the lowest BB% was in those grafted on Ramsey (78.83, 30.67%) respectively. Also, significant differences were observed among IBA concentrations in means BB%, which appeared significantly in 1 mgL -1 (65.43%), while 0.5 mgL -1 gave the lowest mean BB% (50.00%). Regarding the interactions, micrografts on Freedom significantly gave the highest BB% at 0.1 and 1 mgL -1 (87.38, 82.60%) respectively and SO4 at 0.5 mgL -1 (83.20%), while the lowest values were in micrografts on Ramsey at 0.5 mgL -1 (0.00%) and on SO4 at 0.1mgL -1 (0.00%). It was shown that insignificant differences were observed among means of rootstocks or IBA concentrations or the interactions between the two factors in mean average shoot number per scion (ASN). Significant differences in means of average shoot length (ASL) were observed among rootstocks, Freedom significantly scored the maximum ASL compared to other rootstocks (2.46 cm) regardless of IBA concentrations. On the other side, Red globe micrografts cultured on MS supplemented with 1 mgL -1 IBA achieved the highest ASL (1.84 cm). Interaction between the studied factors was significant as the highest value was obtained in the grafts of Red globe on Freedom at 1 mgL -1 (3.50 cm), while the lowest values were on Ramsey at 0.5 mgL -1 and on SO4 (0.00 cm) for each one. The results revealed that significant differences were recorded in the mean of average leaf number (ALN) which appeared significantly in the grafts of Red globe on Freedom rootstock (3.60). In terms of IBA concentration effects, insignificant differences were observed among them although 1 mgL -1 gave the highest mean ALN compared to other concentrations (2.50). Regarding the interactions, there were significant differences between the two factors that appear clearly in grafts of Red globe on Freedom at all IBA concentrations but it was better at 0.1 mgL -1 (3.80), while the lowest values were on Ramsey at 0.5 mgL -1 and on SO4 at 0.1 mgL -1 (0.00) for each one. It was obvious that grafts of Red globe on Freedom rootstock showed the highest mean of graft union formation percentage (GUF%), compared to other rootstocks (84.80%), while the lowest GUF% was on Ramsey (55.55%). Also, significant differences were recorded among IBA concentrations regardless of rootstock types, therefore 1 mgL -1 gave the highest GUF% (82.22%). Concerning the interactions, the highest GUF% was in Red globe grafts on Freedom at 1 mgL -1 (100.00%), on Ramsey at 0.1mgL -1 , and on SO4 at 0.5mgL -1 (100.00%), while the lowest GUF% was on Ramsey at 0.5 mgL -1 and on SO4 at 0.1 mgL -1 (0.00%). Concerning micrografts rooting percentage (MR%), Red globe grafts on Freedom significantly achieved the highest mean of MR% (84.12%). On the other hand, 0.1 mgL -1 gave the highest mean MR% regardless of the rootstocks (47.60%). Regarding the interaction, Red globe grafts on Freedom at 0.1 mgL -1 achieved the highest MR% (100.00%), while the lowest MR% was on Ramsey at 0.5 and 1 mgL -1 (0.00%) and on SO4 at 0.1 mgL -1 (0.00%). The results showed that Red globe grafts on Freedom significantly gave the highest mean of average root number (ARN) per micrograft (11.20). Regarding the IBA concentrations effect, insignificant differences were observed in the mean ARN regardless of the rootstocks. Concerning the interaction, there were significant differences between the two studied factors which appeared clearly in Red globe grafts on freedom at 0.1 and 1 mgL -1 (12.60, 13.00) respectively, while the grafts on Ramsey at 0.5 and 1 mgL -1 or on SO4 at 0.1 mgL -1 did not give any roots (0.00). Red globe grafts on Freedom and SO4 gave the highest mean of average root length (ARL) significantly (3.74, 4.00 cm) respectively with insignificant differences among them. On the other side, insignificant differences were recorded among IBA concentrations in the mean of ARL regardless of rootstocks. Interaction between the studied factors revealed that grafts of red globe on SO4 gave the highest ARL at 0.5 mgL -1 (7.00 cm) significantly, whereas Freedom and Ramsey gave the highest ARL at 0.1 mgL -1 (4.69, 3.71 cm) respectively.   (55). Some workers used plant growth regulators to improve graft success through improving the callus formation, and vascular tissue differentiation. Auxins are the first of the major plant hormones to be discovered. (37). In the present study IBA addition to MS medium had been found effective for improving the percentages of scion survival, scion budburst, graft union formation, and rooting of the Red globe micrografts regardless of the rootstocks. These findings show the correlation between the successful graft unions and the cell activity and growth that improve callus formation (37). Auxin plays a vital role in the development of compatible unions and enhances the differentiation of vascular tissues (55). Auxin enhances many developmental effects by regulating gene expression (60). (58) Found that NAA was effective in improving the micrograft success in walnut. (49) Reported that IBA was insignificantly better than NAA in all parameters, while NAA stimulated forming callus masses on rootstocks. The obtained results indicated that the effects of auxin, IBA, on the measured parameters were dependent on applied concentrations where treatment of 1.0 mgL -1 had significantly enhancing effects. The highest percentages of scion survival, scion budburst, graft union formation and shoot length were observed in 1.0 mgL -1 treatment, whereas 0.1mgL -1 had a positive effect on rooting%, the average number of roots and root length. This might be related to the fact that higher concentrations of IBA are inhibitory both to root induction and elongation. (2) Indicated that the high concentrations of auxin enhances ethylene production which inhibits the rooting. Auxin has a great role in many developmental activities, and can impact cell division, cell growth, or cell differentiation (60). (25) Indicated that IBA at 0.5 ppm decreased the rate of establishment, shoot, and root values in the grafts with Rupestris du Lot, but had a favorable effect on the grafts with 140 Ruggeri at doses up to 4 ppm. (49) Recommended Thompson seedless on Freedom micrografts planted on B5 medium contained IBA at 2 mgL -1 , Flame seedless on Ramsey micrografts planted on B5 medium contained IBA at 3 mgL -1 . On the other hand, the obtained results illustrated that Red globe on Freedom micrografts gave significantly the best results compared to other rootstocks in all studied parameters. These findings went in parallel with the results obtained by (49). Table 3 exhibit the effect of medium strength on some grafting characteristics of Red globe grape micrografts. It is distinct that Freedom significantly achieved the highest mean scion survival percentage (SS%) compared to those grafted on other rootstocks (93.75%) regardless of medium strength. On the other side, significant differences were observed among medium-strength levels in this parameter, so it was recorded that full MS scored the highest SS% significantly (86.67%) and the lowest SS% was at 50% MS (58.20%). Interaction between the two studied factors was significant as Red globe scions grafted on freedom gave the highest SS% at 25% MS and full MS (100.00%), also on Ramsey at 25% MS (100.00%) and on SO4 at full MS(80%). On the other hand, Red globe scions grafts on Freedom gave significantly the highest mean scion bud burst percentage (SBB%), whereas the lowest SBB% was in those grafted on Ramsey (73.88, 33.33%) respectively. Also, significant differences were recorded among medium strength levels in mean SBB%, which appeared significantly in 75% MS (71.97%), while Full strength gave the lowest mean SBB% (30.00%). Regarding the interaction, micrografts on SO4 significantly gave the highest SBB% at 75% strength MS (100.00%), while at 50% MS and 75% MS on Freedom without significant difference between them (85, 7, 83.30 %) respectively and at 25% MS on Ramsey (50.00 %). The results indicated that insignificant differences were observed among means of rootstocks or medium strength or the interactions between the two factors in the mean average shoot number per scion (ASN).

Effect of medium strength Data in
It is clear that significant differences were observed among rootstocks in mean of average shoot length (ASL). Superior on SO4 and on Freedom micrografts significantly scored the maximum ASL compared to Ramsey (2.15, 2.03 cm) regardless of medium strength. On the other side, Red globe micrografts cultured on 25% MS achieved the highest ASL (2.67 cm) regardless of rootstocks. Interaction between the studied factors was significant as the highest value was obtained in the grafts of Red globe on SO4 at 25% MS (3.50 cm) and the lowest values were on Freedom and on SO4 at full MS (0.50 cm) for each one. It was revealed that insignificant differences were recorded among the rootstock in mean of average leaf number (ALN). In terms of medium strength effect, significant differences were observed among them as micrografts of Red globe at 25% MS, 50% MS, and 75% MS gave the highest mean ALN without significant difference among them. Regarding the interactions, there were obvious differences between the two factors that appear clearly in grafts of Red globe on Freedom at 75% MS (4.2), on SO4 and on Ramsey at 25% MS (4.80, 3.80) respectively, while the lowest values were on SO4 at full MS (2.00). Obviously found that grafts of Red globe on Freedom and Ramsey rootstocks showed the highest mean of graft union formation percentage (GUF%) (88.75, 93.75%) respectively, while the lowest GUF% was on SO4 (66.63%). Also significant differences were recorded among medium-strength levels regardless of the rootstock types which were clearly shown at 50% MS as gave the highest GUF% (100.00%). Concerning the interaction, the highest GUF% was on Freedom at 50% MS and full MS (100.00%), on Ramsey at all strength MS (100.00%) except 75% MS (75.00%) and on SO4 at 25% and 50% MS (100.00%) while the lowest GUF% was at full MS (0.00%). Concerning micrografts rooting percentage (MR%), the grafts on Freedom significantly achieved the highest mean of MR% (70.83%). On the other hand, 50% MS gave the highest mean MR% regardless of the rootstocks (91.73%). Regarding the interaction, the grafts on Freedom at 25% and 50% MS, on SO4 at 50% MS and on Ramsey at 25% MS achieved the highest MR% (100.00, 100.00, 100.00, 87.38%) respectively, while the lowest MR% was on all rootstocks at full MS (0.00%). Red globe grafts on Freedom significantly gave the highest mean of average root number (ARN) per micrograft (10.50). Regarding the medium strength effect, significant differences were observed in mean ARN regardless of the rootstocks which shown that 25%, 50 and 75% MS gave the highest ARN without significant differences among them (6.50, 8.00, 7.13) respectively. Concerning the interaction, there were significant differences between the two studied factors which appeared clearly on freedom at 50% and 75% MS (15.20, 14.80) respectively, the grafts on Ramsey at 25% and 50% MS (4.20, 5.20) respectively, while SO4 at 25%, 50%, and 75% MS have high ARN without any significant differences among them, whereas the lowest value was observed on all rootstocks at full MS which did not give any new roots (0.00). The results indicated that insignificant differences were recorded among the rootstocks in the mean of average root length (ARL). On the other side, significant differences were recorded among mediumstrength levels in the mean of ARL regardless of rootstocks and it appeared significantly at 25% MS (2.26 cm). Interaction between the studied factors revealed that the highest values of ARL were on Freedom at 75% MS, (2.76 cm), on Ramsey, and SO4 at 25% MS.  The graft success as a means of plant propagation is greatly influenced by the nature of the culture medium used. The purpose of the culture medium, is to provide optimum conditions for the growth of the grafts. The most widely used culture medium is MS medium, because most plant cell cultures react to it favorably. It is classified as a high salt medium in comparison to many other formulations, with high levels of nitrogen, potassium, and some of the micronutrients, particularly boron and manganese (11). The findings of this experiment showed that the strength of the medium had a significant effect on all parameters measured. The highest values for all parameters measured were obtained from the micrografts cultured on 25%, 50% and 75% MS medium showing a significant difference compared to full MS which obtained the lowest values except scion survival percentage. It was clear from the obtained results that the graft growth was better using lower strength of MS. The highest percentage of scion budburst was obtained on 75% MS, while the highest percentages of graft union formation and rooting were obtained on 50% MS, whereas the longest shoot and root were obtained on 25% MS. On the contrast, full MS produced the lowest values in all studied parameters and prevented the rooting in all micrografts on the studied rootstocks. These findings are in agreement with those obtained by (7) who stated That the highest root length and root number in micro cuttings of tea were obtained with application redundancy MS medium (25% macro element). The lowest root length and root number in micro cuttings of tea were obtained with application full MS medium. (53) stated that the reduction in the strength of MS medium resulted in the increase of in vitro shoot and root formation from blue berry. (44) reported that root number and root length of Stevia rebaudiana plant cultures were significantly influenced by the strength of MS medium (25%MS, 50% MS and full strength MS). Several researchers in the tissue culture of grapes used MS medium (52,1,49). (35) reported that rooting 'Marechal Foch'grapevine in half-strength MS salts was superior to rooting in full-strength MS salts. The effect of the medium strength could be possibly associated with particular components of the culture medium. For example, even minor changes in the concentration of trace elements can affect plant organogenesis in vitro. (10) observed a radical reduction in the number and length of roots induced from white poplar (Populus alba) cultures when zinc concentration in the culture medium was increased. Anatomical study Red globe grafted on freedom rootstock new xylem Figure 10 shows two stages in connection cambium development. ʺAʺ an early stage, the cambium is well developed (arrows). In ʺB The development of vascular tissues (xylem and phloem) from the activity of the connection cambium. Although the graft is apparently successful but there is a weak zone lacking the vascular tissues indicated with arrow heads in ʺBʺ. Figure 11 illustrates that there was an advanced stage similar to figure ʺBʺ, the two partners of cambium produced considerable amounts of vascular tissues, these tissues were not connected (the zone facing the two arrows).

Figure 11. Cross section in the graft union of Red globe/Freedom shows an advanced stage
Red globe grafted on Ramsey rootstock Figure 12 reveals the initial cells of the callus. It initiated from the ray parenchyma of the xylem, from the cambial derivative cells and from the phloem parenchyma.  In Figure 14, B shows an early stage of connection cambium differentiation (arrows). A and C , somewhat later stage of cambium development connecting the two partners of the graft (arrows in C). Compatibility is extremely important for The success of grafting on the of the graft union to make fast development of vascular match between the stock and the scion (42), which will allow continuing the growth of both the root and the canopy (23,34). The callus is a tissue produced to cover wound surfaces of the plant and formed by parancimatic cells differentiating from the phloem zone near the cambium. In further periods there are cellular differentiation in callus, some cells transform to produce cambium and cambium produces xylem and phloem. Thus vascular tissue between stock and scion is formed and water and food transfer is provided (37,59,8). Researchers reported that after grafting, callus continues development thus the gaps between stock and scion filled and necrotic layers break and disappear (12,61) as small pale-colored necrotic masses while other parts of it remained as very dark-colored tissue. According to (9) in situations where necrotic layers have not broken, cambial differentiation took a long time. In this study, Freedom rootstock activity was clearly observed in cell dividing when grafted compared to Ramsey and SO4 which were producing limited callus cells in grafting union. However, the anatomical observations in this study went harmonically with the present results that the highest bud burst and graft union formation percentage of the studied cultivars scions were obtained when grafted on Freedom rootstock. These observations were similar to those obtained by (49). In this situation, the anatomical observations indicated that successful grafting was observed when Red globe grafted on Freedom rootstock as both stock and scion were sharing together the formation of callus cells to fill the gaps between them in parallel with breaking the necrotic layers and a new cambium produced perceptible amounts of vascular tissues (xylem and phloem) these observations in agreement with that stated by (42, 49). Regarding Red globe on Ramsey rootstock combinations, graft union formation was more difficult because of less callus formation and dense necrotic layers caused more wavy cambial contact and relatively weak vascular tissues (4,23). On the same side, the combinations of Red globe on SO4 showed that the vascular tissues of the stock and scion were unmatched although the stock and scion contributed to the formation of callus and intermingled, no connecting cambium was established and dense necrotic layers were observed. (9,13,45). It is observed that Red globe is quite compatible with Freedom rootstock supported by the anatomical observations that declared enough callus formation, less necrotic layers, and a wavy cambial continuity of new cambium. Whereas on Ramsey and SO4 combinations where has relatively less callus formation, dense necrotic layers caused quite wavy cambial continuity result to from the weak and late formation of vascular tissues. These findings also revealed that different stock scion combinations gave different levels of callus formation and rooting compatible with the previous studies. Acclimatization of micrografted plantlets Effect of soil mixture : The survival rates of micrografted Red globe on Freedom transplants after acclimatization planted on different artificial soil mixtures were presented in Figure 15. It is clear that the plantlets affected by the characteristics of the mixture, which the sterilized 1:1:1 (v:v) peatmoss+perlite+sand mix were found to be effective of in vitro plantlets hardening which significantly gave the highest survival (100%) and showed superior vegetative growth compared to other soil mixtures, whereas the lowest rate was in 1:1 (v:v) peatmoss+perlite (50%). It seemed that organic matter represented with peatomoss strongly affected the plantlets survival, whereas the presence of sand with peatmoss as 1:1 (v:v) may modify the concentration in the mixture leading to improvement in survival rate (80%) Immediately after transplantation, visible wilting was observed. However, the water status of plants could still stable after some days or weeks.  (19), soil (33), and sand-peatmoss (21). Among various potting mixtures tried, the mixture containing sand+peatmoss+perlite (1:1:1) was found to be the most suitable. Physical, chemical and biological properties of the potting mixture display a vital role in the establishment of in vitro produced plantlets (27). The better performance of peatmoss might be attributed to its ability to improve the biological properties of the mixture. Sand may be responsible for providing sufficient aeration and high water holding capacity besides satisfied water drainage and good ventilation providing by perlite. Hence mixing sand, peatmoss, and perlite in equal volumes might have helped in giving a better grip for the roots, ample aeration, and sufficient organic matter. Similar results have also been obtained by (48,41,21). The results presented in Figure 21 show that Freedom rootstock is considered more effective for Red globe micrografts than SO4, as it achieved the highest survival rate (100%) compared to those on SO4 (80%). Moreover, a difference in vegetative growth was recorded which was vigorous and higher on Freedom than on SO4. Furthermore, Freedom was obviously had the highest number of roots and longer than in SO4 rootstock. . Plantlets of Red globe/SO4 4 weeks after the acclimatization Acclimatization success is greatly associated with the plantlets survival and considered the best indicator for the selection of suitable genotype and soil medium. On the other hand, the low growth rate during the acclimatization stage could be handled by proper fertilization and irrigation schedule after transferring the survived plants to nursery beds or bigger pots (21). The remarkable growth and roots of the survival plantlets could be explained by the successful acclimatization and these results is in agreement with those obtained by (37) who reported that rooting of grafted plants may happen when the roots first initiated in vitro and after that grown outdoor on root growing media and those recorded by (46,29) who stated that the more successful transplantation of plantlets, the high growth can be achieved. Furthermore, different stages were noted during the acclimatization firstly, an adaptation stage with slow shoot growth and root formation, secondly a fast growth of roots and shoot (56). These present results went in parallel with (49) who reported that Freedom micrografts achieved a satisfied response to transplant in sterile soil mixture through Superior on Freedom micrografts gave the highest survival rate (70%) followed by Flame seedless and Thompson seedless (60%).

CONCLUSION
The findings of this study showed that Red globe on un-rooted Freedom rootstock significantly achieved the highest percentages of scion survival (100.00%), scion bud burst (60.00%), graft union formation (60.00%), rooting (40.00%), shoot length (2.00 cm), root number (8.00) and root length (4.50 cm). Regarding the response to IBA concentrations, Red globe on Freedom was better than other studied rootstocks which achieved the highest values in all studied parameters. It is obvious that the micrografts on 1mgL -1 IBA achieved the highest percentages of scion survival (95.20%), scion bud burst (65.43%), graft union formation (82.22%) also, longest shoots (1.84 cm), and maximum average leaf number (2.50), while on 0.1mgL -1 IBA gave the highest rooting percentage (47.60%). Red globe on Freedom micrografts on MS medium supplemented with 0.1 mgL -1 IBA recorded the highest percentages of scion survival (100.00%), scion bud burst (87.38%), rooting (100.00%), and the highest average root number (12.60). on the other side, the results of the medium strength experiment showed that 25% MS, 50% MS, and 75% MS achieved positive effects on all parameters compared with full MS. The results of the anatomical study have shown that the callusing level in graft union was higher in Freedom compared to Ramsey, and SO4. It was clearly noticed that Red globe on Freedom combinations was Successfully survived as enough callus formation, less necrotic layers, and a wavy cambial continuity of new cambium were identified. Acclimatization results revealed that 1:1:1 (v:v) peatmoss+perlite+sand mix significantly gave the highest survival (100.00%) and showed superior vegetative growth. Red globe on Freedom micrografts achieved the highest survival rate (100.00%), better vegetative growth, and longer roots compared with those on SO4 and Ramsey (80.00, 70.00%) respectively.