الفهرس 
Establishment of date palm somatic embryogenesisOnly 14 pages are availabe for public view
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Abstract
This study had been carried out at the Horticulture Department, Faculty of Agriculture, Minoufiya University, Shebin El-Kom, Egypt; the experiments had been conducted in the Central Laboratory for Date Palm Researches and Development (CLDPRD), Plant Biotechnology Department, Agricultural Research Centre, Ministry of Agriculture, Giza, Egypt; during the period from 2009 to 2013. The study aimed to investigate the ability to produce plantlets by micropropagation through tissue culture technique from date palm Sewi cultivar. In order to efficiently maximizing the induction of plants via indirect somatic embryogenesis, it is important to study the influence of different culture systems, physical and chemical factors on the initiation, maturation and germination of somatic embryos to plantlets and growth in vitro than during the acclimatization stages.
1. Establishment stage:
Plant material, sterilized and formation of callus cultures (Establishment stage).The propagation process was started with the selection of healthy offshoots from mother date palm trees of Sewi cv. (Egyptian semi-dry date palm cultivar),were removed from their mother plants having a weight of 5-7 kg and height of 50-80 cm. Shoot tips and leaves primordial as explants of the offshoots were excised, after removing all outer leaves, fibers and roots, and then surface-sterilized, and then surface-sterilized, where Explants were surface sterilized under aseptic conditions by using ethyl alcohol (70%) for 1min followed by immersion in (0.5g/l) mercuric chloride (HgCl2) for 5min and then rinsed one-time with sterile distilled water and transferred to double surface sterilization by commercial Clorox (5.25%) sodium hypochlorite (NaOCl) supplemented with two drops of Tween-20 per 100 ml solution, the first one by 40% Clorox for 15 min and thoroughly washed with sterilized distilled water for one time and the second one by 60% Clorox for 25 min and then washed with sterilized distilled water for three times. 1.1. Callus formation:
Sterilized shoot tips and leaf primordial were longitudinally divided and cultured on Murashige and Skoog (1962) medium supplemented with 3 mg/l 2iP + different concentration of 2,4-D at 3.0, 5.0, 10.0 or 20.0 mg/l plus 1.5g/l activated charcoal.
Experiment 1: Effect of different combinations between N6-(2-iso-pentenyl adenine) (2iP) and different concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D) on swelling, browning and callus formation percentage from shoot tip and leaf primordial explants of date palm Sewi cv. after 24 weeks.
The most results of this experiment are summarized as follows:
1.1.1. Swelling percentage: Leaf primordial explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 20.0 mg/l 2,4-D recorded the highest swelling percentage (80%); while, shoot tip explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 3.0 mg/l 2,4-D showed the lowest significant value of swelling (20%)
1.1.2. Browning percentage: Leaf primordial explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 20.0 mg/l 2,4-D recorded the highest browning percentage (5%); while, shoot tip explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 10.0 mg/l 2,4-D showed the lowest significant value of browning (0.5%).
1.1.3. Callus formation percentage:Shoot tip explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 10.0 mg/l 2,4-D recorded the highest callus formation percentage (64%); while, leaf primordial explants cultured on MS medium supplement with 2iP at 3.0 mg/l + 3.0 mg/l 2,4-D showed the lowest significant value of swelling (10%).
2. Somatic embryos of maturation (maturation stage):
2.1. Embryogenic callus formation and development to somatic embryos:
Friable callus resulted from establishment stage was divided into 0.5 g/jar and cultured on medium by different culture systems.
2.1.1. Experiment 1: The effect of culture systems and MS medium on fresh weight value, number of pro-embryos, number of completely somatic embryos, remain embryos and total embryos of date palm Sewi cv. after 12 weeks.
The most results of this experiment are summarized as follows:The highest mean value of callus fresh weight, number pro-embryos, number of completely somatic embryos, remain embryos and total embryos (4.17, 43, 32.67, 190 and 222.67, respectively) has been obtained from application of mixing system culture. While, the lowest mean value of callus fresh weight, number pro-embryos, number of completely somatic embryos, remain embryos and total embryos (1.29, 9, 2.33, 0.0 and 2.33 respectively) were obtained at Solid cultures.
2.1.2. Experiment 2: Effect of MS salts strength, 2,4-Dichlorophenoxy acetic acid (2,4-D) and activated charcoal (A.C) concentrations on fresh weight value, number of pro-embryos, number of somatic embryos, browning degree and vitrification degree of friable callus of date palm Sewi cv. cultured in mixing system after 12 weeks.
friable calls (0.5g/jar) was chopped into small pieces on filter paper, transferred aseptically into 30 ml solid medium in 350 ml jars and cultured on full MS and Half strength medium supplemented with 0.0, 0.5 and 1.0mg/l 2,4-D in addition of activated charcoal at 0.0, 0.5, 1.0 and 2.0 g /l, 30g/l, sucrose200 mg/L KH2PO4-2H2O and 100 mg/l glutamine, decanted 5 ml/jar liquid medium same composition.
The most results of this experiment are summarized as follows:
2.1.2.1. Growth value (fresh weight): Full MS medium salt strength with 0.5 mg /l 2,4-D and 1.0 g/l activated charcoal recorded the highest significant value of callus fresh weight (4.73), while half MS medium salt strength without 2,4-D and activated charcoal showed the lowest significant value of callus fresh weight (0.99). 2.1.2.2. Number of pro-embryos: Full MS medium salt strength supplemented with 0.5 mg /l 2,4-D and 1.0 g/l activated charcoal gave the highest significant value of pro-embryo number (60); in addition full MS medium salt strength without 2,4-D and activated charcoal showed the lowest significant value of pro-embryo number(8).
2.1.2.3. Number of completely somatic embryos: Full MS medium salt strength supplemented with 0.5 mg /l 2,4-D and 1.0 g/l activated charcoal gave the highest significant value of mature somatic embryos number (52), compared with half MS medium salt strength without 2,4-D and activated charcoal showed the lowest significant value of mature somatic embryos number (0.0).
2.1.2.4. Browning degree:
Full MS medium salt strength with 0.5 mg /l 2,4-D and 0.5 g/l activated charcoal recorded the highest significant value of browning degree (2.3). While, half MS medium salt strength without and with 0.5, 1.0 mg/l 2,4-D and 1.0 or 2.0 g/l activated charcoal showed the lowest significant value of browning degree (1).
2.1.2.5. Vitrification degree:
Half MS medium salt strength with 0.5 mg /l 2,4-D and 0.5 g/l activated charcoal recorded the highest significant value of vitrification degree (2.3), whereas full MS medium salt strength with 0.5 mg /l 2,4-D and 2.0 g/l activated charcoal recorded the lowest significant value of vitrification degree (1.0), also half MS medium salt strength without 2,4-D and 2.0 g/l activated charcoal gave the same vale (1.0).
2.1.3. Experiment 3: Effect of different concentrations of sucrose on growth value and development of date palm Sewi cv. cultured in mixing system after 9 weeks.
This experiment was conducted to record the effects of sucroseat 30, 45, 60 and 75g/l on callus fresh weight value, number of pro-embryos, number of somaticembryos, browning and vitrificaion degree during maturation.
The obtained results may be summarized as follows: The addition of maturation medium supplemented with 45 g/l sucrose significantly increased the value of callus fresh weight, number of pro embryos and number of somatic embryos formation (4.89 g/culture, 66.7 pro embryos and 51.7 embryos, respectively), while the lowest value of callus fresh weight resulted from using 30 g/l sucrose (4.22). Whereas the lowest value of number of pro embryos and number of somatic embryos formation (33.3 pro embryos and 21.9 embryos, respectively) were obtained from using 75 g/l sucrose. The highest significant value of browning degree and vitrificaion degree (1.8 and 2.0,respectively) were obtained from using 75 g/l sucrose. While the lowest value of browning degree (1.3) resulted from using 30 g/l sucrose, but the lowest value of vitrification degree (1.0) resulted from using 45 g/l sucrose.
2.1.4. Experiment 4: Effect of thidizuron (TDZ) concentrations on growth value and development of date palm Sewi cv. cultured in mixing system after 9 weeks.
This experiment was conducted to record the effects of TDZ at 0.0, 0.05, 0.1, 0.2 and 0.4mg/l on callus fresh weight value, number of pro-embryos, number of somatic embryos, browning and vitrificaion degree during maturation.
The most results of this experiment are summarized as follows: The highest value of callus fresh weight (4.84)was obtained on culture medium supplemented with TDZ at 0.2 mg/l, whereas the highest value of number of pro embryos and number of somatic embryos formation (70 pro embryos and 55 embryos, respectively)were obtained on culture medium supplemented with TDZ at 0.1 mg/l. the lowest value of callus fresh weight, number of pro embryos and number of somatic embryos formation (2.96, 24 and 32.3, respectively) were obtained from using 0.40 mg/l TDZ. The highest significant value of browning degree and vitrificaion degree (2.0 lowest value of browning degree and vitrificaion degree (1.3 and 0.3, respectively) resulted from using 0.1 mg/l TDZ
2.1.5. Experiment 5: Effect of abscisic acid (ABA) concentrations on growth value and development of date palm Sewi cv. cultured in mixing system after 9 weeks.
This experiment was conducted to record the effects of ABA at 0.0, 0.25, 0.5, 1.0, 2.0 and 4.0 mg/l on callus fresh weight value, number of pro-embryos, number of somatic embryos, browning and vitrificaion degree during maturation.
The most results of this experiment are summarized as follows:The highest value of callus fresh weight (4.49)was obtained on culture medium free ABA, whereas the lowest value of callus fresh weight(2.11) was obtained from using 2.0 mg/l ABA. The highest value of number of pro embryos and number of somatic embryos formation (66.7 and 59, respectively)were obtained on culture medium supplemented with ABA at 0.5 mg/l. While the lowest value of number of pro embryos and number of somatic embryos formation (35 and 29.3, respectively) were obtained from using 4.0 mg/l ABA. The highest browning degree had been obtained on culture medium supplemented with ABA at 2.0, 0.25 and 0.0 mg/l (2.0), while, the lowest value was recorded on media supplemented with ABA at 1.0 or 4.0 mg/l (1.3). The highest vitrification degree had been obtained from cultures containing ABA at 2.0 mg/l (1.7),the lowest vitrification degree had been obtained from cultures containing ABA at 0.5 mg/l (0.7).
2.1.6. Experiment 6: Effect of polyethylene glycol (PEG 4000) concentrations on growth value and development of date palm Sewi cv. cultured in mixing system after 9 weeks.
This experiment was conducted to record the effects of (PEG 4000) at 0.0, 5, 10, 15 and 20 g/l on callus fresh weight value, number of pro-embryos, number of somatic embryos, browning and vitrificaion degree during maturation.
The most results of this experiment are summarized as follows:The highest value of callus fresh weight, number of pro embryos, number of somatic embryos formation and browning degree (4.49, 68, 54 and 2.0, respectively)were obtained on culture medium free PEG. While the lowest value (2.96, 24, 32.3 and 0.7, respectively) were obtained from using 20 g/l PEG. The highest vitrification degree had been obtained from cultures containing PEG at 20or 0.0 g/l (1.0), the lowest vitrification degree had been obtained from cultures containing PEG at 5.0, 10.0 or 15.0 g/l (0.7).
2.1.7. Experiment 7: Better effect of different concentrations of ABA, PEG, TDZ and interaction between them on the maturation somatic embryo of date palm Sewi cv. cultured in mixing system after 9 weeks.
a. Better effect of different concentrations of TDZ and ABA and interaction between them.
Using different concentrations of TDZ at 0.1 and 0.2mg/l combined with ABA at 0.25 and 0.5 mg/l, resulted in: The highest significant value of callus fresh weight, number of pro embryos and number of somatic embryos formation (4.60, 73.3 and 65, respectively) were observed by the addition of 0.2 mg/l TDZ and 0.5 mg/l ABA to culture medium. Whilst, the lowest significant value of callus fresh weight and number of completely somatic embryos formation (3.67 and 38.7, respectively) were recorded by using medium containing 0.1 mg/l TDZ + 0.25 mg/l ABA, but using maturation medium containing 0.2 mg/l TDZ + 0.25 mg/l ABA gave the lowest value of pro embryos number.
Using medium containing 0.2 mg/l TDZ and 0.25 or 0.5 mg/l ABA recorded the highest value of vitrification degree (1); while, using maturation medium supplemented with 0.1 mg/l TDZ + 0.25 or 0.5 mg/l ABA recorded the lowest value of vitrification degree(0.7).
No browning had been obtained from cultures containing TDZ 0.1 mg/l and ABA 0.25 mg/l (0.0). while, using medium containing 0.2 mg/l TDZ and 0.25 or using medium supplemented with 0.1 mg/l TDZ + 0.5 mg/l ABA recorded the highest value of browning degree(0.7).
b. Better effect of different concentrations of TDZ and PEG4000 and interaction between them.
Using different concentrations of TDZ at 0.1 and 0.2mg/l combined with PEG 5, 10 and 15 g/l, resulted in: The highest significant value of callus fresh weight (4.73) was observed by the addition of 0.2 mg/l TDZ and 5 g/l PEG to culture medium. Whilst, the lowest significant value (3.60) was recorded by using medium containing 15 g/l PEG + 0.1 or 0.2 mg/l TDZ.Using medium containing 0.2 mg/l TDZ and 10 g/l PEG recorded the highest value of pro embryos and mature somatic embryos number (69 and 47.7, respectively); while, using maturation medium supplemented with 0.1 mg/l TDZ + 5 g/l PEG recorded the lowest value (55 and 33, respectively). Using medium containing 0.2 mg/l TDZ and 5g/l PEG recorded the highest value of vitrification degree(1.3); while, using maturation medium supplemented with 15 g/l PEG + 0.1 or 0.2 mg/l TDZ recorded the lowest value (0.3). No browning had been obtained from cultures containing TDZ 0.1 mg/l + PEG 5.0 g/l or medium with 0.2 mg/l TDZ + 10 g/l PEG (0.0). While, using medium containing 0.2 mg/l TDZ and 15g/l PEG recorded the highest value of browning degree (0.7).
c. Better effect of different concentrations of ABA and PEG4000 and interaction between them.
Using different concentrations of ABA at 0.25 and 0.5 mg/lcombined withPEG 5, 10 and 15 g/l, resulted in:
The highest significant value of callus fresh weight (3.93) was observed by the addition of 0.25 mg/l ABA and 15 g/l PEG to culture medium. While, the lowest significant value (2.80) was recorded by using medium containing 5 g/l PEG + 0.5 mg/l ABA.
using medium containing 0.5mg/l ABA and 15 g/l PEG recorded the highest value of pro embryos and mature somatic embryos number (60 and 50, respectively); while, using maturation medium supplemented with .25 mg/l ABA + 10 g/l PEG recorded the lowest significant value (46 and 37.7, respectively).No significant difference could be observed between values of vitrification and browning degree by using ABA, PEG or interaction among them. But using maturation medium supplemented with 15 g/l PEG + 0.25 or 0.5 mg/l ABA and medium supplemented with 5 g/l PEG + 0.5 mg/l ABA were giving the lowest value of vitrification degree(0.3).whereas using maturation medium supplemented with 0.5 mg/l ABA + 10 or 15 g/l PEG were giving free browning (0.0).
2.1.8. Experiment 8: study the effect of amino acid (arginie, proline and glutamine) on maturation somatic embryogenesis of date palm Sewi cv. cultured in liquid media after 9 weeks.
2.1.8.1. The test both amino acids alone and the interaction between them.
This experiment was conducted to record the effects of amino acids (arginie, proline and glutamine) at 0.0, 100, 150, 200 and 250 mg/l on callus fresh weight value, number of pro-embryos, number of somatic embryos, browning and vitrificaion degree during maturation.
The most results of this experiment are summarized as follows:Using 250 mg/l glutamine increased significantly fresh weight value, number of pro embryos and number of somatic embryos (4.75 g/culture, 74 pro-embryo/ culture and 58 embryo/ culture).while the lowest value for them were obtained by using 150 mg/l proline, 150 mg/l arginine and100 mg/l arginine (2.77 g/culture, 17.7 pro-embryo/ culture and 17 embryo/ culture).No browning had been obtained from cultures containing proline at 150 mg/l compering with medium free amino acids gave the highest value.No vitrification was observed from cultures containing arginine at 150 mg/l or glutamine at 200 mg/l compering with medium free amino acids gave the highest value.
2.1.8.2. The effect of different concentrations of acids with each.
This experiment was conducted to record the effects of amino acids (arginine + proline + glutamine) at 0.0, 100, 150, 200 and 250 mg/l on callus fresh weight value, number of pro-embryos, number of somatic embryos, browning and vitrificaion degree during maturation.
The most results of this experiment are summarized as follows: Using 250 mg/l arginine + proline + glutamine increased significantly fresh weight value, number of pro embryos and number of somatic embryos (5.23 g/culture, 72.7 pro-embryo/ culture and 55 embryo/ culture) in addition free browning compared with using medium free amino acids.Using medium containing 200 mg/l arginine + proline + glutamine recorded the lowest value of vitrification degree (0.3) compering with medium free amino acids gave the highest value.
2.1.9. Experiment 9: Effect of desiccation period on callus of date palm Sewi cv. through maturation stage.
This experiment was conducted to record the effects of desiccation period on fresh weight. Callus of 1 g was held in the air current of Laminar flow cabinet for different periods (0.0, 0.5, 1.0, 2.0 and 3 hours), Also calculate the number of pro-embryos, number of somatic embryos during maturation.
The most results of this experiment are summarized as follows Increase desiccation period callus to three hours was cultured medium maturation gave the highest values of fresh weight, number of pro embryos and number of somatic embryos (3.21 g/culture, 71 pro-embryo/ culture and 39 embryo/ culture, respectively). While the lowest significant values were recorded with undesiccated callus cultures (1.50 g/culture, 35 pro-embryo/ culture and 5 embryo/ culture, respectively).
2.2. Germination stage:
2.2.1. Experiment 1: Effect of different concentration gebirillic acid and 0.1mg/l
TDZ with 0.1 mg/l NAA on germination of somatic embryos.
In this stage, somatic embryos formed on previous stage were cultured on media containing GA3 0.0, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/l and TDZ at 0.1 mg/l with 0.1 mg/l NAA. The results revealed that:Using germination medium containing 0.4 mg/l GA3 and 0.1 mg/l TDZ + 0.1 mg/l NAA recorded the highest values of number of secondary somatic embryos and percentage of germinated embryos (26 and 75, respectively) compared with using free medium of GA3.
Using germination medium containing 0.8 mg/l GA3 + 0.1 mg/l TDZ + 0.1 mg/l NAA or medium free GA3 + 0.1mg/l TDZ + 0.1 mg/l NAA recorded the lowest value of vitrification degree (0.33) compering with medium containing 0.1 mg/l GA3 + 0.1 mg/l TDZ +0.1 mg/l NAA or 0.2 mg/l GA3 + 0.1mg/l TDZ + 0.1 mg/l NAA gave the highest value (1.33).
2.2.2. Experiment 2: Effect of desiccation period on embryos of date palm through germination stage.
This experiment was conducted to record the effects of desiccation period on fresh weight. Somatic embryos un-germinated of 1 g was held in the air current of Laminar flow cabinet for different periods (0.0, 1.0, 2.0, 3.0 and 4.0 hours), also calculate the number of secondary somatic embryos, percentage of germinated embryos and vitrification degree during germination. The results revealed that:Increase desiccation period embryos to four hours was cultured medium germination gave the highest values of embryos fresh weight and germinated embryos percentage (3.20 g/culture and 80%, respectively), also recorded no vitrification of embryos and lowest number of secondary somatic embryos (17).While the lowest significant value of germinated embryos percentage was recorded with undesiccated embryos cultures (62 %).
3. Multiplication stage:
3.1. Effect of different types and concentrations of cytokinins on shoots multiplication.
A small cluster containing 3 - 4 embryos were used as the explant materials in this stage. Were cultured on media containing different types and concentrations of cytokinins. The results revealed that:The highest significant value for number of secondary somatic embryos and growth value were recorded with using medium supplemented with 0.5 mg/l Kin + 0.50 mg/l 2iP (24.67 and 3.67). In addition to appeared the lowest browning degree.The highest value for number of shoots, shoot length and number of root were recorded with using medium supplemented with 0.25 mg/l Kin + 0.25mg/l 2iP (12 shoots, 3.6 cm and 3.3 root).
3.2. Effect of different types and concentrations of cytokinins and auxin on shoots multiplication.
A small cluster containing 3 - 4 embryos were used as the explant materials in this stage. Were cultured on media containing different types and concentrations of cytokinins with auxin (0.1 mg/l NAA). The results revealed that:The highest significant value for number of secondary somatic embryos, shoots length and growth value were recorded with using medium supplemented with 0.5 mg/l Kin + 0.50 mg/l 2iP + 0.1 mg/l NAA (33.67 secondary embryos,3.7 cm and 4). In addition to appeared the lowest browning degree.The highest value for number of shoots and number of root were recorded with using medium supplemented with 0.5 mg/l BA + 0.5mg/l 2iP + 0.1 mg/l NAA (11.67 shoots and 4 root).
4. Rooting stage:
Effect of auxin and potassium silicate on rooting of date palm Sewi cv. recorded after 3 months.
Shoot explants (9 – 12 cm) were cultured on media supplemented with NAA at 0.5 mg/l+ IBA at 1 mg/l and potassium silicate different concentration at 0.05, 0.1, 0.2, 0.4 and 0.5 mg/L each were added to half MS basal nutrient medium.The most results of this experiment are summarized as follows:The highest significant value of rooting percentage, root number, shoot length (cm), leaves number and growth value were observed when culture medium supplemented with 0.5 mg/l NAA + 1.0 mg/l IBA, without potassium silicate (100%,7.67 roots, 24 cm, 5 leaves and 4, respectively). The highest value of root length (cm) was recorded with using medium supplemented with 0.5 mg/l potassium silicate without auxins (4.3 cm).
The highest value of leaf width (cm) was recorded with using medium supplemented with 0.05 mg/l potassium silicate + 0.5 mg/l NAA + 1.0 mg/l IBA (2.0 cm).5. Acclimatization stage:Effect of different soil type on survival percentage of date palm Sewi cultivar during acclimatization stage (4 months).Plantlets cultivated in different Soil culture type (sand, peat, vermiculite, perlite and compost) and during different acclimatization period (after 1, 2, 3 and 4 months). Calculate the percentage of survival plantlets through acclimatization.The most results of this experiment are summarized as follows:Plantlets cultivated in soil culture containing Compost and Perlite (1:1, v/v) after one month had higher percentage values (76%) of survival of acclimatization as compared to those produced under the other treatments. While Plantlets cultivated in soil culture containing sand and peat after four month recorded the lowest percentage values (44%) of plantlets survival of acclimatization.SDS-PAGE Protein electrophoresis in callus and somatic embryos:Appeared several the new banding patterns with comparative control this was result of the addition ABA only or combination with TDZ concentrations suitable to culture media, during maturation stage. Lead to stimulation inactive genes for the production of new proteins. Further demonstrating the maturity and quality of embryos.Recommendation:1. To obtain the highest and greatest value of embryogenic callus, cultivation of shoot tip on MS medium supplemented with 2,4-D at 10 mg/l, 2iP at 3 mg/l and 1.5 g/l activated charcoal.2. To increase the fresh weight value of the callus during the maturation stage, using mixing system of solid and liquid medium containing full MS salt strength with reducing the ammonium nitrate to the half and added 0.5 mg/l 2,4-D, 1 g/l activated charcoal and 45 g/l sucrose with 0.2 mg/l thidiazuron.3. To get the best and highest number of pro embryos and number of completely somatic embryos during the maturation stage, cultivation of embryogenic callus on medium M1 supplemented with thidiazuron at 0.2 mg/l and ABA at 0.5 mg/l.4. To obtain the highest number of secondary somatic embryos during germination stage, embryos must cultivate embryos on the MS medium containing 0.4 mg / l GA3, 0.1 mg / l TDZ and 0.1 mg / l NAA. While to obtain highest percentage of embryos germinate should be partial desiccation of embryos for 4 hours and reculture on the same medium previously.5. To increase multiplication of embryos and growth values of shoots through multiplication stage using MS supplement with Kin and 2iP concentration at 0.5 mg / l with 0.1 mg / l NAA.6. To get the highest percentage of rooting and growth value of the plantlets should be planted on medium containing half MS salts strength plus 0.5 mg / l NAA and 1.0 mg / l IBA.7. To obtain a higher percentage value of survival during acclimation stage, cultivation plantlets in mixture of compost with Perlite (1: 1 v / v). media should be used.