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Abstract
This investigation was conducted in the Tissue Culture Unit, Horticulture Dept. Fac. of Agric. Moshtohor, Benha university during the period from 2004 to 2007 to study the best way for maximizing protoplast yield and increasing number of viability protoplast as well as enhancing protoplast development of pear rootstocks (Pyrus betulaefolia and Pyrus communis) .The following procedures were studied :
I- Pre-protoplast isolation:
In vivo leaves of both pear rootstocks (betulaefolia and communis pear) were subjected to levels evaluation phenolic compound during the year round. Then sterilized the leaves and treated with different anti-oxidant treatments.
New leaves from in vitro and sterilized in vivo pear rootstocks were divided into small sections and soaked in different plasmolysis treatments in combination treats with different protoplast sources (in vitro and in vivo) .
II- Protoplasts isolation:
Small sections from in vitro were treated by different enzyme combination (mixtures) in combination with protoplast sources. Then different digestive enzyme media and osmotic pressure factor .as well as different incubation periods and different shaking speeds and periods.
III- Protoplast purification:
Different pore sizes, as well as centrifugation speeds and periods were used during purification stage ,
IV- Protoplast development:
Different medium types and different protoplasts densities and hormonal balances were employed to find out the best procedures for protoplast development.
The obtained results can be summarized as follow :
5.1. Pyrus betulaefolia :
5.1.a. Pre-protoplast isolation :
1-It proved that March sample showed the lowest phenolic compounds either totals, free or conjugated contents which assured that the best time for taking explants.
2-It is clear that using of anti-oxidant solution (0.1% ascorbic acid + 0.15 citric acid) was effective in reducing phenolic compounds and reducing their harmful effect.
3- It is recommended to use plasmolysis treatment 9 g/100 ml mannitol for half hour then 13 g/100 ml mannitol for furthermore half hour to enhance successful protoplast isolation.
4- In vitro protoplast source was surpassed in vivo protoplast source as it maximize protoplast yield.
5.1. b.Protoplast isolation :
5- Immersing in vitro explants in enzyme solution supplemented (1.5% cellulase + 1.5% Macerozyme + 0.5% pectinase) was more effective in improving the protoplast yield.
6- The highest protoplast yield was obtained when CPW digestive medium was used.
7- Addition of mannitol to the digestive enzyme medium was preferred in increasing the protoplast yield.
8- Incubation of the explants for 20 hours enhance the highest protoplast yield.
9- Shaking the incubated explants in enzyme solution at 75rpm for 30 min. encourage the best protoplast yield.
5.1.c. Purification:
10- Using sieve at pore size 25m encourage the highest number of viable protoplasts.
11- Centrifugation protoplast with speed rate 1000 rpm for 7.5min. inducted the greatest number of viable protoplasts.
5.1.d. Protoplast development:
12-Murashig & Skoog medium was superior for the best protoplast development than both Gamborg and KM medium types.
13-Using protoplast at density rate 2 X 105 in the culture medium enhanced an improvement in protoplast development.
14- Supplementation of the culture medium with the combination of 1.00 mg/L NAA and 0.3mg/L BAP induced the highest protoplast development.
5.2. Pyrus communis :
5.2.a. Pre-protoplast isolation :
1-The lowest phenolic compounds level was detected at December sample when the leaves was taken.
2- Antioxidant solution (0.1% ascorbic acid + 0.15 citric acid) succeeded in reducing the accumulation of phenolic compounds.
3- using of plasmolyized treatment 9 g/100 ml mannitol for half hour then 13 g/100 ml mannitol for another half hour is required for maximizing protoplast yield.
4- In vitro protoplast source was superior in increasing protoplast yield than in vivo source.
5.2.b.Protoplast isolation :
5- The best results of protoplast isolation was appeared when using enzyme mixture consists 1.5% celtulase + 1.5% Macerozyme + 0.5% pectinase compared with the other enzyme mixtures .
6- CPW medium showed the most superior digestive enzyme medium for increasing protoplast yield .
7- Addition of mannitol to enzyme solution was the more effective in improving protoplast yield and viability.
8- The highest viable protoplast yield were produced the explants were incubated for 20 hours in enzyme mixture .
9- Shaking speed at 75rpm for 30 min maximized protoplast yield .
5.2.c. Purification:
10- Increasing viable protoplast number occurred when using sieve pore size 25m.
11- The best number of viable protoplast was noticed when centrifugation speed rate 1000 rpm for 7.5minutes was used.
5.2.d. Protoplast development:
12- KM proved to be the most suitable medium maximize protoplast division but MS medium provide to be the most suitable medium maximize protoplast development.
13- The highest development of protoplast was obtained when culturing the protoplast with density 2 X 105 .
14- Addition of 1.0 mg/L NAA with either 0.2 or 0.3mg/L BAP to the culture medium resulted in maximized protoplast development.