FACTORS AFFECTING PROPAGATION OF STRAWBERRY (Fragaria spp.) THROUGH TISSUE CULTURE TECHNIQUES.

FACTORS AFFECTING PROPAGATION OF STRAWBERRY (Fragaria spp.) THROUGH TISSUE CULTURE TECHNIQUES.

 

Hamdy Ahmed Emara

Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City,  Minufiya Province, Egypt.

 

ABSTRACT

An efficient method for shoot regeneration, root formation from runner tips and acclimatization of strawberry plantlets was developed. Runner tips of 1-2 cm long were used as source of explnts. After surface sterilization apical meristems of 3-5mm long were isolated and used as explnts. At multiplication stage, results indicated that the highest vegetative parameters (shoot number, shoot length and leaf number) were observed when MS medium supplemented with 1 mg/l BA followed by the medium contained 1 mg/l BA and 0.1 mg/l IBA. However control treatment showed a significant similar result in shoot length only. Results of this study indicated that, BA was more effective in enhancement the growth of strawberry in vitro compared to Kin and TDZ. 

At rooting stage, it was clear that MS medium at full strength containing 30 g/l sucrose significantly surpassed all other combinations of MS strengths and sucrose concentrations in increasing root number and length per plantlet and fresh weight/plantlet. The same treatment enhanced the shoot length but without significant difference compared to some other combinations. The treatment contained 3 g/l agar with 6 g/l perlite significantly enhanced root formation (number and length) as well as shoot length, fresh weight/plantlet, and leaf number/plantlets. Finally, plantlets were successfully acclimatized and the soil mixture contained peatmoss : perlite (2 : !, V/V) observed high percentage of survival of plants ( 80%) with enhancing both root number and length/plantlet, plantlet height and leaf number/plantlet.

Keywords: Propagation, strawberry, fragaria spp., tissue culture techniques.

 

INTRODUCTION

The strawberry is a herbaceous perennial member of the family rosaceae. The garden strawberry (Fragaria x ananassa Duch), originated in Europe around 1750 as a hybrid between the pistillate South American Fragaria chiloensis Duch and a North American Fragaria virginiana Duch.

 For many centuries before, strawberries had been a favorite among the fruits of the temperate world. They were valued for delicious flavor and fragrance, for health-restoring qualities and as harbinger of spring (Wilhelm and Sagen, 1974). Following further hybridizations, especially since 1850, Fragaria x ananassa has developed into the large, fragrant, tasty red fruit that is now cultivated worldwide. The high degree of genetic heterozygosity present in Fragaria spp. enabled the development of strawberry cultivars adapted to widely varying environment conditions and resistant to several diseases and pests. Not only the genetic variability, but also a high adaptability and plasticity of the strawberry plant itself give this crop such a remarkable range of adaptation (Darrow, 1966). That heterozygosity was explained by Gaafar and Saker (2006) as there are more than 20 Fragaria species worldwide, there are seven basic types of chromosomes that they all have in common. However, they exhibit different polyploidy. Some species are diploid, having two sets of the seven chromosomes (14 chromosomes total). Others are tetraploid (4x = 28), hexaploid (6x = 42), octoploid (8x = 56) or decaploid (10x = 70). Strawberry is cultivated all around the world, not only for its digestive and tonic properties, but because of the nutritional value of its fruits, important source of folate, vitamin C, fiber, potassium, flavonoids, autocianidin, phytochemicals and antioxidants. Prior experiences with strawberry micropropagation indicate that vitroplants are more uniform, produce higher number of runners, have better survival in the field, and the fruit yield increases in 24% than plants propagated by the traditional method (Kikas et al., 2006).                                      

The aim of this study was to establish a protocol for in vitro production of virus free strawberry plants through examination of the effect of: first, different factors (different growth regulators, sucrose concentration and type of gelling agent) on in vitro shoot proliferation and root formation from meristematic explants. Second, the effect of different soil mixtures on acclimatization of in vitro-produced strawberry plantlets.

 

MATERIALS AND METHODS

 

This work was carried out at Tissue Culture Lab., Dep. of Plant Biotechnology, Genetic Engineering and Biotechnology Research Institute, Minufiya University, Sadat City, Egypt, during the years of 2005 and 2007.

 

Source of explants:

Mother plants of strawberry (Fragaria x ananassa Duch.) grown in the farm of Minufiya university, Sadat city, were used as source of explants during this study. Runner tips of 1-2 cm long were taken from mother plants as that long was suitable for sterilization procedures.

 

Surface sterilization of explants:

Explants were rinsed under running tap water for 30 minutes. For surface sterilization, explants were immersed in sodium hypochlorite solution (2%) containing two drops of Tween 20 per 100ml for 20 minutes. The plant materials were then, rinsed three times with sterile distilled water.

After sterilization, explants were shortened to remove the surfaces of explants and meristems of 3-5mm long were isolated as final explants. These explants were cultured with good contact on the surface of the medium.

 

Culture medium:

Murashige and Skoog (MS) medium (1962) was used in all experiments of this study including 6 g/l agar, 100 mg/l myoinositol and 30 g/l sucrose.

 

Sterilization of the nutrient media:

            Following preparation of the medium and prior to addition of agar, the pH was adjusted to 5.8. The medium was poured into culture jars of size 350ml where each jar contained 30ml of the medium and capped with polypropylene closures. The culture jars were autoclaved at 121 ^oC and 1.2 kg/cm² air pressure for 20 minutes.

 

Experiments of this study were designed as following:

Establishment stage (Establishment of strawberry cultures in vitro):

            Meristem tips were cultured on hormone-free MS solid medium to establish clean and healthy materials for next experiments. Cultures were incubated for 4 weeks in the growth room.

 

Incubation conditions

            All cultures of this study were incubated at 25 ^oC ± 2 day and night. Light was provided by fluorescent tubes giving an intensity of 1500 lux at the level of culture vessels for 16 hours per day.

 

Multiplication stage:

The effect of different growth regulators on shoot proliferation from strawberry explants was studied in this stage, as three different experiments:

 

A)- Effect of different concentrations of benzyl adenine (BA) and indole butyric acid (IBA) on the growth of strawberry in vitro.

Shoots obtained from the growth of the explants (meristems), during the previous stage, were cultured in MS medium supplemented with BA at different concentrations (0.0, 0.5, 1.0, and 2.0 mg/l) in combinations with IBA at different concentrations (0.0, 0.1, 0.5 and 1.0 mg/l). Cultures were incubated in the growth room (the same incubation conditions mentioned before) for 4 weeks. Then, data were recorded as shoot number, shoot length and leaf number.

 

(B)- Effect of different concentrations of kinetin (Kin) and IBA on the growth of strawberry in vitro:

In vitro obtained shoots of strawberry were cultured in MS medium contained Kin and IBA at the same concentrations of cytokinin and auxin and the same combinations as mentioned in step, A. After 4 weeks, the same parameters (shoot number, shoot length and leaf number) were recorded.

 

(C)- Effect of different concentrations of thiodiazron TDZ and IBA on the growth of strawberry in vitro.

In vitro obtained shoots of strawberry were cultured in MS medium contained TDZ and IBA at the same concentrations of cytokinin and auxin and the same combinations as mentioned in step, A. After 4 weeks, the same parameters (shoot number, shoot length and leaf number) were recorded.

 

Rooting stage:

      According to the evaluation and results of the previous stage, recommendation can be raised as to use MS medium contained 1 mg/l BA for strawberry shoot multiplication in vitro. These shoots were used to examine the effect of different factors on in vitro root formation as following:

 

A- Effect of different strengths of MS medium in combinations with different sucrose concentrations on root formation and the growth of strawberry after 4 weeks in vitro:

Shoots of strawberry obtained from multiplication stage were cultured on different strengths of MS, ⅛, ¼, 1/, and full strength of MS medium in combinations with various concentrations of sucrose, 10, 15, 20, 25 and 30 g/l. All treatments of this experiment contained 1 mg/l IBA. After 4 weeks, data were recorded as, root number/plant, root length, shoot length and fresh weight/plant

 

B- Effect of different gelling agents of the nutrient medium and perlite (each alone and in some combinations) on root formation and the growth of strawberry after 4 weeks in vitro.

All treatments of this experiment contained 1 mg/l IBA. The experiment contained the following treatments:

a- Agar    (6 g/l)                                   d- Gelrite (1 g/l) +   perlite (6 g/l)

b-Gelrite (2 g/l)                                    e- Agar      (3 g/l) + perlite (6 g/l)

c- Perlite (6 g/l)

 Data were recorded after 4 weeks as, root number/explant, root length, leaf number/explant, shoot length and fresh weight/plant

 

Acclimatization stage:

Effect of different soil mixtures on the growth of strawberry in the greenhouse:

            According to the results of the previous experiments, plantlets were grown during the previous rooting stage on MS medium supplemented with 1 mg/l IBA, 30 g/l sucrose and solidified with 3 g/l agar with 2 g/l perlite. Then, plantlets were transferred to the greenhouse and planted individually in pots of 6 cm diameter and filled with different soil mixtures as following:

                                            

(1)-1 Peatmoss:1 Perlite         (2)-1 Peatmoss:2 Perlite      

(3)- 1 Peatmoss:3 Perlite        (4)- 2 Peatmoss:1 Perlite       

(5)- 2 Peatmoss:2 Perlite        (6) - 2 Peatmoss:3 Perlite

(7)- 1 Peat.:1Sand                   (8)-1Peat.:2Sand                 

(9)- 1Peat.:1Sand                    (10)-2 Peat.:1Sand                 

(11)-2Peat.:2Sand                   (12)- 2Peat.:3Sand  
          Plantlets in pots of all treatments were covered with transparent polyethylene bags (for two week) to maintain a high humidity around them. Data were recorded after 1 month of transplanting as survival percentage, leaf number/plantlet and plantlet height (cm), and root number and length/plantlet.

All experiments were repeated twice and the represented data were averages. Results of these experiments were analyzed by analysis of variance (ANOVA) according to Gomez and Gomez (1984).

 

RESULTS AND DISCUSSION

 

Multiplication stage:

A-Effect of different concentrations of BA and IBA on the growth of strawberry in vitro.

1- Shoot number:

In Table 1, data on the main effect of BA on shoot formation indicate that the medium contained 1.0 mg/l BA significantly proved to be the best for shoot formation compared to other concentrations. Control (hormone-free medium) showed the lowest shoot number. As for the main effect of IBA, data reveal that the presence of IBA in the medium significantly decreased the shoot number compared to control that observed the highest record in that concern. Concerning the interaction, results clear that BA alone at 1.0 mg/l significantly showed the highest record of shoot number {14.70, (Fig. 1)} followed by the treatment contained 1.0 mg/l BA and 0.1 IBA that a significant similar response. In that concern, the use of 0.5 mg/l BA and 0.5 mg/l IBA was reported for shoot proliferation of strawberry (Navatel et al., 1989), while Marcotrigiano et al. (1984) found that BA at 0.3 mg/l was as effective for shoot proliferation as 1 and 3 mg/l. Boxus (1999) revealed that when shoot tips of strawberry were cultured on a medium supplemented with 0.5 mg dm^-3 BA, 0.1 mg dm^-3 GA₃and 6.4 g dm^-3 agar, shoots were multiplied. Singh and Pandey (2004) reported that half strength MS medium supplemented with 1 mg / l BA and 1 mg/l IBA produced the highest number of shoots (13 per explant). Moreover, Kaushal et al. (2006) found that callus of strawberry differentiated into shoots after transferring to MS medium supplemented with 0.5 Kin + 2 mg/l BA + 0.25 mg/l NAA. Well developed shoots were transferred to multiplication medium containing 0.5 mg/l Kin + 0.5 mg/l BA and 1.0 mg/l GA_3. The importance of BA, in strawberry shoot formation, was also reported by Lal et al. (2003) who found that the maximum number of shoots per explant was observed in MS medium supplemented with BAP at 4.0 mg/l.

 

Table 1: Effect of different concentrations of BA and IBA on shoot number/explant after 4 weeks.

 

 2- Shoot length:

            Data on the main effect of BA on shoot length (Table  2) observe that the addition of BA to the medium did not stimulate the shoot length, and highest shoot length was significantly obtained with control medium. Regarding the main effect of IBA on shoot length, the same trend as that of BA was observed, and control surpassed all IBA concentration in that concern.

          Concerning the interaction, the same two treatments that mentioned above (1.0 mg/l BA alone, and 1.0 mg/l BA + 0.1 mg/l IBA) significantly showed the highest shoot length. However, control treatment significantly observed similar response in shoot length compared to those two treatments.

Table (2): Effect of different concentrations of BA and IBA on shoot length after 4 weeks in vitro.

 

Damiano (1978) found that the addition of 1 or 2 g of activated charcoal per liter of MS medium promotes elongation of both the shoots and roots of strawberry.

 

3- Leaf number:

            In Table 3, data on the main effect of BA on leaf number/explant indicate that the treatment contained 1.0 mg/l BA resulted in the highest value of leaf number compared to other treatment including control. However, data on the main effect of IBA on leaf number/explant show different response, as the control and the medium contained 0.1 mg/l IBA significantly showed similar higher response and surpassed all treatments in increasing the leaf number. Concerning the interaction, mostly the same trend that observed in shoot number was obtained here, as the same two treatments contained 1.0 mg/l BA alone or 1.0 mg/l BA + 0.1 mg/l IBA showed the highest significant increase in leaf number/explant compared to all other treatments including control. The effect of tissue culture on leaf formation was studied by Zebrowska et al. (2003) who revealed that number of leaves per microplant in the autumn after planting and in the next year of cultivation was significantly higher in comparison to the standard propagate seedlings

 

B- Effect of different concentrations of Kin and IBA on the growth of strawberry in vitro.

1- Shoot number:

            In Table 4, data on the main effect of Kin on shoot number, reveal that the presence of Kin did not show any positive effect in that concern, as all treatments of Kin significantly showed similar response to that obtained with control treatment. Results of the main effect of IBA in shoot number showed the same trend as mentioned before with Kin. Regarding the interaction, most

Table 3: Effect of different concentrations of BA and IBA on leaf number/explant after 4 weeks in vitro.

 

of treatments including control, significantly observed similar increase in shoot number (Figure 1) compared to two treatments (0.5 mg/l Kin + 0.5 mg/l IBA or 0.5 mg/l Kin + 1.0 mg/l IBA) that significantly showed lower effect in that concern. However, some investigations recommended the presence of BA with Kin in the multiplication medium, Kaushal et al. (2006) found that callus of strawberry differentiated into shoots after transferring to MS medium supplemented with 0.5 Kin + 2 mg/l BA + 0.25 mg/l NAA. Well developed shoots were transferred to multiplication medium containing 0.5 mg/l Kin + 0.5 mg/l BA and 1.0 mg/l GA_3. Weifeng et al. (2004) revealed that Kin used together with BA for adventitious bud induction gave better results than Kin alone, but, Waithaka et al. (1980) revealed that axillary dormancy was released by including a large level (50 µM) of Kin in the culture medium.

 

Table 4: Effect of different concentrations of Kin and IBA on shoot formation of strawberry in vitro, after 4 weeks.

 

2- Shoot length:

      Data in Table 5 on the main effect Kin on shoot length indicate that, although Kin at 0.5 mg/l observed higher value of shoot length followed by the medium contained 2.0 mg/l Kin without significant difference, but control treatment showed a significant similar increase when compared with the treatment of 0.5 mg/l Kin. Concerning the main effect of IBA, data indicate that the addition of IBA, at any of the used levels, decreased the shoot length compared to the control that significantly showed the highest record in that concern (3.36). As for the interaction, both treatments of 0.5 mg/l Kin alone and control significantly showed similar higher responses and surpassed all other treatments in enhancement the shoot length.

 

  Table 5: Effect of different concentrations of Kin and IBA on shoot length of strawberry in vitro, after 4 weeks.

kin mg/l	Shoot length
	IBA mg/l
	0.0	0.1	0.5	1.0	Mean (A)
0.0	3.95 a	2.90 bc	2.85 bc	3.10 bc	3.20 (a)
0.5	3.90 a	2.95 bc	2.95 bc	2.80 bc	3.15 (ab)
1.0	3.05 bc	2.80 bc	2.90 bc	2.65 c	2.85 (c)
2.0	2.55 c	3.05 bc	3.30 b	2.60 c	2.87 (bc)
Mean (B)	3.36 (a)	2.93 (b)	3.00 (b)	2.79 (b)

 

       3- Leaf number:

 Data on the main effectof Kin on leaf number (Table 6) show that control surpassed all treatments of Kin in that concern. Regarding the main effect of IBA on leaf number, the two levels 0.1 and 0.5 mg/l IBA observed higher records in leaf number. However, control showed a significant similar increase in that concern. The highest level of IBA (1.0 mg/l) showed the lowest response. As for interaction, interestingly, control treatment and all treatments of IBA alone significantly increased the leaf number and surpassed all other treatments.

 

Table (6): Effect of different concentrations of Kin and IBA on leaf number/explant of strawberry in vitro, after 4 weeks.

C- Effect of different concentrations of TDZ and IBA on the growth of strawberry in vitro after 4 weeks.

 1- Shoot number:

            In Table 7 data on the main effect of TDZ clear that the treatment contained 0.5 mg/l TDZ significantly showed the highest shoot number/explant (2.43) compared to all other records. Control showed the lowest record in that concern. Regarding the main effect of IBA, data illustrate that a significant similar increase was resulted with all levels of IBA and control, except the level 0.1 mg/l that showed lower effect in shoot formation. As for the interaction, significant increase in shoot number was obtained when 0.5 mg/l TDZ was combined with either 0.5 mg/l or 1 mg/l IBA (Figure 1). These two treatments surpassed all other treatments including control (hormone-free medium). In that concern, Yonghua et al. (2005) reported that highest regeneration and number of shoots per explant were achieved in shoot regeneration medium containing 1.5 mg/l TDZ + 0.4 mg/l IBA + 1.0 mg/l AgNO₃

 

Table (7): Effect of different concentrations of TDZ and IBA on shoot number/explant of strawberry in vitro, after 4 weeks.

 

 

2- Shoot length:

            Data on the main effect of TDZ on shoot length (Table 8) reveal that control treatment showed a significant increase in shoot length compared to all treatments of TDZ that observed lower records in that concern. Concerning the main effect of IBA, results clear that IBA at all used levels resulted in a significant decrease in shoot length compared to the control that showed a higher response. As for the interaction, interestingly, control treatment significantly increased the shoot length compared to all other treatments, either contained TDZ or IBA each alone or in combinations.

 

 

 Table 8: Effect of different concentrations of TDZ and IBA on shoot length/explant of strawberry in vitro, after 4 weeks.

3- Leaf number:

            In the same Table 9, data on the main effect of TDZ show that, the presence of TDZ affected negatively the leaf formation, and the control surpassed all used levels of TDZ. As for the main effect of IBA, all used levels of IBA significantly showed similar increase in leaf formation, but control treatment observed the same effect without significant difference. Concerning the interaction, a significant similar response was observed with some sporadic treatments including all levels of IBA each alone, control and the treatment contained 5.0 mg/l TDZ alone or combined with 0.5 or 1.0 mg/l IBA. Lower responses were obtained with the other treatments. The increase in TDZ concentration from 0.5 to 2.0 mg/l either alone or in combinations with IBA showed a significant decrease in leaf number.    

 

Table 9: Effect of different concentrations of TDZ and IBA on leaf number/explant of strawberry in vitro, after 4 weeks.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: Effect of different growth regulators on the growth of strawberry after 4 weeks in vitro.

 

Rooting stage:

A- Effect of different MS strengths and sucrose concentrations on root formation and the growth of strawberry plantlet after 4 weeks in vitro.

1- Root number:

In Table 10 data on the main effect of MS strengths on root number show that, highest response was significantly obtained with full MS or ¼ MS, but 1/8 and ½ MS came in the second order. As for the main effect of sucrose, results indicate that the gradual increase in sucrose concentration up to 30 g/l was combined with a gradual increase in root number. Both levels (10 and 15 g/l) significantly showed the lowest records in shoot number without significant difference between each other. Concerning the interaction, it was clear that the highest record of roots was resulted with the medium consisted of full MS with 30 g/l sucrose, all other treatments observed lower responses in that concern (Fig. 2). In that concern, some investigations did not recommend full MS for strawberry rooting, Kaushal et al. (2006) found that rooting of strawberry was done in MS half strength + 1.0 mg/l IBA and 0.2 mg/l activated charcoal. The same strength was recommended for strawberry rooting by Yonghua et al. (2005) who reported that half strength MS containing 1.0 mg/l AgNo₃ was optimum medium for rooting. Kikas et al. (2006) found that high salt concentration of MS promoted active proliferation but the shoots remained too short for rooting, but with low salt concentration of MS, the proliferation rate was lower but shoots elongated enough. Gautam et al. (2001) indicated that the highest root induction frequency obtained was 95.23% on ¼ MS medium with IBA at 1.0 mg/l and charcoal (200 mg/l). However, Lal et al. (2003) found that maximum rooting was obtained in both full and half strength MS medium supplemented with IBA at 1.0 mg/l. Mereti et al. (2003) found that highest percentages of rooting were achieved in MS medium contained 10 µM IBA (92%) and 10 µM IAA (82%).

 

Table 10: Effect of different MS strengths and sucrose concentrations on root number/plantlet of strawberry after 4 weeks in vitro.

MS salt strength	Number of root
	Sucrose g/l
	10	15	20	25	30	Mean (A)
1/8 MS	3.20 fg	2.20 gh	4.40 bcde	4.60 bcd	3.60 DEF	3.60 (b)
¼ MS	3.40 EF	3.80 cdef	4.80 BC	5.40 B	4.40 bcde	4.36(a)
½ MS	1.60 h	3.60 def	4.80 bc	4.40bcde	4.80 bc	3.84(b)
MS	3.20 fg	4.00 cde	3.00 defg	3.20 fg	8.60 a	4.40(a)
Mean (B)	2.85 (c)	3.40 (c)	4.25(b)	4.40(b)	5.35 (a)

 

2- Root length:

            In Table 11, data on the main effect of MS strengths on root length reveal that the highest significant record was obtained with full MS, all other strengths showed lower effect. Results of the main effect of sucrose on root length show that, the highest record of root length was obtained with the medium contained 30 g/l sucrose followed by the medium of 20 g/l sucrose without significant difference. Lower responses were obtained with the other levels of sucrose and the lowest response was resulted with the medium contained 10 g/l. Concerning the interaction, data indicate that full strength of

Table 11: Effect of different MS strengths and sucrose concentrations on root length/plantlet of strawberry after 4 weeks in vitro.     

MS salt strength	Root length
	Sucrose g/l
	10	15	20	25	30	Mean (A)
1/8 MS	2.00 ghi	1.50 ij	2.90 bcd	2.40 defg	2.10 fgh	2.18 (b)
¼ MS	3.00 bc	1.20 j	2.40 defg	2.10 fgh	2.60 cdef	2.26(b)
½ MS	2.30 efg	1.70 hij	3.30 b	2.60 cdef	2.60 efg	2.40 (b)
MS	1.30 j	2.50 cdefg	2.10 fgh	2.70 cde	4.80 a	2.68(a)
Mean (B)	2.15 (c)	1.73 (d)	2.68 (ab)	2.45 (b)	2.90 (a)

 

MS medium and supplemented with 30 g/l sucrose significantly surpassed all other treatments in increasing the root length (Figure 2).

 

3- Shoot length:

            Results in Table 12 on the main effect of MS strengths on shoot length show that no significant difference was observed between all strengths in that concern. Regarding sucrose concentration, data on the main effect indicate that the level 10 g/l sucrose significantly resulted in the lowest shoot length (3.35), significantly higher responses were resulted with the other sucrose levels without significant difference between them. As for the interaction, sporadic treatments showed high responses in shoot length, including ¼ MS contained 30 g/lsucrose, ½ MS contained 15, 20, 25 or 30 g/l sucrose and full MS with 20, 25, or 30 g / l sucrose without significant difference. In that concern, Kikas et al (2006) found that high salt concentration of MS promoted active proliferation, but the shoots remained too short for rooting, but with low salt concentration of MS, the proliferation rate was lower but shoots elongated enough. Damiano (1978) found that the addition of 1 or 2 g of activated charcoal per liter of MS medium promotes elongation of both the shoots and roots of strawberry.

 

Table 12: Effect of different MS strengths and sucrose concentrations on shoot length of strawberry after 4 weeks in vitro.  

 

Figure 2: Effect of different MS strengths on root formation and the growth of strawberry after 4 weeks in vitro.

 

4- Fresh weight/plant:

            Data in Table 13 on the main effect of MS strengths on fresh weight/plant show that a significant gradual increase of fresh weight/plant was resulted from the increase in MS strength, and the highest value was recorded with full MS (0.378 g). Regarding the main effect of sucrose, data show that the level 30 g/l significantly observed the highest fresh weight followed by 25 g/l. control showed the lowest record in that concern. As for the interaction, results indicate that full MS medium with 30 g / l sucrose observed the highest fresh weight / plant compared to all other treatments including control. The level 10 g/l sucrose showed the lowest records with all MS strengths.

 

Table 13: Effect of different MS strengths and sucrose levels on fresh weight/plantlet of strawberry after 4 weeks in vitro.     

MS salt strength	Fresh weight/plant (g)
	Sucrose g/l
	10	15	20	25	30	Mean (A)
1/8 MS	0.214 hi	0.256 efgh	0.292 def	0.348 cd	0.240 fgh	0.270   (c)
¼ MS	0.214 hi	0.150 j	0.308 de	0.260 efgh	0.232 gh	0.233 (d)
½ MS	0.212 hi	0.374 c	0.292 def	0.278 efg	0.404 c	0.312 (b)
MS	0.162 ij	0.280 efg	0.298 de	0.464 b	0.464 a	0.378 (a)
Mean (B)	0.200 (e)	0.265 (d)	0.298 (c)	0.338 (b)	0.391   (a)

B- Effect of different gelling agents of the nutrient medium and perlite on root formation and the growth of strawberry plantlets after 4 weeks in vitro:.

1-Root number:

Data in Table 14 show that the highest root number was significantly observed with the medium contained 3 g/l agar with 6 g/l perlite (8.10), that was followed by different media, 6 g/l perlite only, 6 g/l perlite with 1 g/l gelrite, or 6 g/l agar only. The lowest value in that concern was recorded with the medium supplemented with 2 g/l gelrite only (Figure 3). Boxus (1999) revealed that when shoot tips of strawberry were cultured on a medium supplemented with 0.5 mg dm^-3 BA, 0.1 mg dm^-3 and 6.4 g dm^-3 agar, shoots were multiplied and subsequently rooted in vitro on the same medium without BA and GA_3.

 

Table 14: Effect of different gelling agents of the nutrient medium and perlite on root formation and the growth of strawberry plantlets after 4 weeks in vitro.

 

2-Root length:

     In the same Table 14 indicate that the same medium (3 g/l agar with 6 g/l perlite) significantly increased the root length compared to all other t

 

3-Shoot length and leaf number:

      Results of both parameters showed no significant difference between all used treatments.

 

4-Fresh weight/plant

       Data in Table 14 indicate that a higher significant increase in fresh weight/plant was recorded with the medium contained either 6 g/l perlite with 3g/l agar or 6 g/l perlite alone compared to other treatments. The treatment of 2 g/l gelrite significantly came in the second order. The lowest values were significantly recorded with both treatments contained 6 g/l agar alone or 6 g/l perlite with 1 g/l gelrite. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3: Effect of different gelling agents of the nutrient medium and perlite on root formation and the growth of strawberry plant after 4 weeks in vitro

Acclimatization stage:

Effect of different soil mixtures on acclimatization of strawberry:         

            Data in Table 15 clear that soil mixture contained peatmoss and perlite (2:1, v/v) or 2:2 (v/v) resulted in the highest survival of plantlets (80%) compared to all other soil mixtures. However, the only soil mixture that showed the highest records in all other measured parameters (plant length, leaf number/plant, root number/plant and root length) contained 2 peatmoss: 1 perlite  (Figure 4), followed by the mixture contained 2 peatmoss : 2 perlite that showed similar high responses in the same parameters except its lower effect in leaf number. Some other mixtures significantly observed sporadic similar results but in one or two parameters only. The mixtures contained either 1 peatmoss: 3 sand or 2 peatmoss: 3 sand showed no survived plantlets. Mereti   et al. (2003) found that highest percentage of rooting were achieved in 10 µM IBA (92%) and 10 µM IAA (82%), although the respective survival rates during acclimatization were only 28 and 55%, respectively.

 

      Table 15: Effect of different soil mixtures on acclimatization of strawberry after one month in the greenhouse. 

Soil mixtures (v/v)	No. of leaves	Plant height	No. of root	Root length	Survival %
Peat. 1:Perlite 1	5.33 bc	6.33 a	4.67 c	3.00 ab	66.6
Peat. 1:Perlite 2	5.00bcd	4.33 b	5.67 bc	3.17 a	40.0
Peat. 1:Perlite 3	5.67ab	4.17 b	5.33 c	2.50 cd	20.0
Peat. 2:Perlite 1	6.67a	6.00a	7.00 ab	2.83 abc	80.0
Peat. 2:Perlite 2	4.33cde	5.67 a	7.67 a	3.17 a	80.0
Peat. 2:Perlite 3	4.00def	4.16 b	4.67 c	3.00ab	50.0
Peat. 1:Sand 1	4.00def	2.60 c	4.33 c	3.00ab	21.00
Peat. 1:Sand 2	4.00def	2.83 c	4.33 c	2.67 bc	16.6
Peat. 1:Sand 3	0.00g	0.00 d	0.00 e	0.00 f	0.00
Peat. 2:Sand 2	3.67ef	2.67 c	2.00 d	2.00e	25.00
Peat. 2:Sand 2	3.00f	3.67 bc	1.67 d	2.17 de	33.33
Peat. 2:Sand 3	0.00g	0.00 d	0.00 e	0.00 f	0.00

 

Figure  4: One month old plantlets of strawberry grown in soil mixture consists of peatmoss : perlite (2:1, v/v).

 

REFENCES

 

Boxus, P. (1999). Micropropagation of strawberry via axillary shoot proliferation. Plant Cell Culture Protocols. Methods in Molecular Biology. Part 111. Plant Propagation In Vitro. Hall R. D. (ed.) Humana Press Inc. Totowa N.J., Vol. 3: 103-114.

Damiano, C. (1978). Il carbone attivo nella coltura in vitro della fragola. Frutticoltura, 40: 49-50.

Darrow G. M. (1966). The strawberry history, breeding and physiology. Holt, Rinehart Winston, New York.

Gaafar, R. M. and M. M. Saker (2006). Monitoring of cultivars identity and genetic stability in strawberry varieties grown in Egypt. World Journal of Agricultural Sciences, 2 (1): 29-36.

Gomez, K. A. and A. A. Gomez (1984): Statistical Procedures for the Agricultural Researches. John Wiley and Son, Inc., New York.

 

Guatam, H.; R. Kaur; D. R. Shama and T. Neetu (2001). A comparative study on in vitro and ex vitro rooting of micropropagated shoots of strawberry (Fragaria × ananassa Duch.). Plant Cell Biotechnology and Molecular Biology, 2(3 / 4): 149-152.

Kaushal, K.; A. K. Nath, and D. R. Sharma (2006). Establishment of callus culturesnand plant regeneration in strawberry (Fragaria × Ananassa Duch.) cv. Chandler. Indian Journal of Plant Physiology., 11 (2): 136-144.

Kikas, A.; A. Libek and V. Vasar (2006). Influence of micropropagation on the production of strawberry runner plants, yield and quality. Acta Horticulturae, 708: 241-244.

Lal, M.; S. Sharma and M. V. Hegde (2003). Micropropgation of strawberry (Fragaria × ananassa Duch). Indian Journal of Horticultural Research, 37 (3): 231-234.

Marcotrigiano, M.; H. J. Swartz; S. E. Gray; D. Tokarcik and J. Popenoe (1984). The effect of benzylamino purine on the in vitro multiplication rate and subsequent field performance of tissue culture-propagated strawberry plants. Adv.Strawberry Prod., 3: 23-25.

Mereti, M.; K. Grigoriadou and N. Leventakis (2003). In vitro rooting of strawberry tree. (Arbutus unedo L.) in medium solidified by peat – perlite mixture in combination with agar. Acta Hort., 616: 607-609.

Murashige, T. and F. A. Skoog (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473-479

Navatel, J. C.; J. G. Rancillac and M. Rancillac (1989). Field behaviour of strawberry plants issued from micropropagation: 7 years of experimental studies (1981-1987). In: Proc COST 87 Worksh Strawberry Micropopagation, May 19, Forli EEC.

Singh, A. K. and S. N. Pandey (2004). Genotypic variation among strawberry cultivars for shoot organogenesis. Acta Horticulturae, 662: 277-280.

Waithaka, K.; A. C. Hildebrandt and M. N. Dana (1980). Hormonal control of strawberry axillary bud development in vitro. Journal of Am. Soc. Hortic. Sci., 105: 428-430.

Weifeng, Y.; J. Wanmei and Y. Shupin (2004). Effect of plant growth regulators on adventitious shoot regeneration from strawberry (Fragaria  ananassa) leaf discs. Plant Physiology Communications., 40 (4): 447-449.

Wilhelm, S. and J. E. Sagen (1974). A history of the strawberry, from ancient gardens to modern markets., Div. Agric. Sci., Univ. Cal. Berkeley, pp 3-10.

Yonghua, Q.; Z. Shanglong; Z. Lingxiao; Z. Daoyu and A. Syed (2005). Response of in vitro strawberry to silver nitrate (AgNo₃). Horti. Science, 40 (3): 747-751.

Zebrowska, J. I.; J. Czernas; J. Gawronski and J. A. Hortynski (2003). Suitability of strawberry (Fragaria x  ananassa Duch.) microplants to the field cultivation. Food, Agriculture and Evironment., Vol. 1 (3&4): 190-193.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   العوامل المؤثرة على إکثار نبات الفراولة  باستخدام

تقنیات زراعة الأنسجة

 

 

حمدی أحمد عمارة

قسم بیوتکنولوجیا النبات – معهد الهندسة الوراثیة – جامعة المنوفیة- ج.م.ع.

 

 

        لبدء تلک الدراسة تم استخدام القمم الخضریة للمدادات بطول 1 – 2سم کمصدر للأجزاء النباتیة (المرستیمات القمیة) المستخدمة للزراعة بالمعمل. وبعد التعقیم السطحی تم فصل تلک المرستیمات القمیة بطول 3-5ملم والتى استخدمت کمنفصلات نباتیة لبدء الزراعة بالمعمل، حیث تم زراعتها على بیئة موراشیج وسکوج الخالیة من منظمات النمو لمدة 4 أسابیع " مرحلة البدایة أو التأسیس".

        وفى مرحلة التضاعف: والتى أجریت لدراسة تضاعف الأفرع الناتجة من المرحلة السابقة، أثبتت النتائج أن بیئة موراشیج وسکوج المحتویة على 1ملجم/لتر بنزیل أدینین أظهرت أفضل الصفات الخضریة التى تم قیاسها "عدد الأفرع ، طول الأفرع ، عدد الأوراق" ، ویأتى بعدها من ناحیة التأثیر على تلک الصفات ، البیئة المحتویة على 1 ملجم/لتر بنزیل أدینین مع 0.1 ملجم/لتر أندول حمض البیوتیریک وذلک بدون فرق معنوى وقد أظهرت معاملة الکنترول "موراشیج وسکوج بدون منظمات نمو " نتائج مشابهة للبیئات السابق ذکرها ولکن من ناحیة طول الأفرع فقط حیث أظهرت نتائج أقل معنویاً مع باقى الصفات. هذا وقد أظهرت تلک الدراسة تفوق البنزیل أدینین على نمو الفراولة معملیاً مقارنة بالکینیتین والثیودیازرون.

        وفى مرحلة التجذیر: کان واضحاً أن البیئة التی تضمنت قوى أملاح موراشیج وسکوج الکاملة مع 30جم/لتر سکروز تفوقت بفارق معنوى على جمیع المعاملات الأخرى (تضمنت قوى مختلفة لأملاح موراشیج وسکوج ، کاملة ،½ ، ¼ ،⅛ مع ترکیزات سکروز مختلفة ، 30، 25، 20، 15، 10جم/لتر) وذلک من حیث معظم صفات الدراسة فی تلک المرحلة (عدد الجذور للنبات ، طول الجذور للنبات والوزن الطازج للنبات) وقد أظهرت نفس المعاملة زیادة فی طول الأفرع ولکن بدون فرق معنوى مقارنة ببعض المعاملات الأخرى.

        وعند دراسة استخدام مواد مختلفة لتصلب البیئة على التخدیر سواء باستخدامها کل على حدة أو مع بیرلیت، أظهرت النتائج أن البیئة المحتویة على 3جم/لتر آجار مع 6جم/لتر بیرلیت أظهرت تفوقاً واضحاً فی تکوین الجذور سواء من ناحیة العدد أو الطول، وکذلک بالنسبة للصفات الأخرى (طول الأفرع للنبات ، والوزن الطازج للنبات ، عدد الأوراق للنبات) وأخیراً فی مرحلة الأقلمة: تم أقلمة النباتات الناتجة من المعمل بنجاح فی خلیط من التربة أحتوى على بیتموس مع بیرلیت (1:2بالجم ) حیث أظهر هذا الخلیط تفوقاً فی کل صفات الدراسة وهى نسبة النبات الحیة  (80%) وکذلک طول النبات وعدد وطول الجذور وعدد الأوراق. ورغم أن الخلیط بیتموس مع بیرلیت (2:2 بالجم) أظهر تفوقاً مشابهاً بدون فرق معنوى إلا انه کان تأثیره اقل من السابق على عدد الأوراق الناتج للنبات.

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