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Abstract

In vitro shoot formation and cost of multiplication of pineapple cultured on liquid MS medium of different strengths (0.5x, 1.0x, 1.5x, 2.0x) enriched with sucrose at 20 g/L and Benzylaminopurine (BAP) at 2.0 mg/L were compared at different pH adjustments (5.0, 5.5, 6.5) and different volumes of medium dispensed per culture (3, 4, 5, 6, 7, 8 ml) after two months of incubation. Highest shoot formation (10 shoots) obtained in doubled strength MS (2.0x) adjusted to pH 5.0 and dispensed at 7 ml per culture. Lowest rate (2 shoots) obtained in half strength MS (0.5x) adjusted to pH 5.5 and 6.5 and dispensed at volume larger than 4 ml per culture. Doubled strength MS adjusted to pH 5.0 and dispensed at 4 ml resulted in the highest total shoot per liter (6750 shoots) at total cost of $64.85 per liter of medium and lowest cost per shoot (0.92 cents) while adjusting the same medium to pH 5.5 and pH 6.5 resulted in low total shoots (4500 and 3750 shoots) and doubled the cost per shoot, (1.54 and 1.85 cents) respectively. Half strength MS (0.5x) adjusted to pH 5.0 and dispensed at 7 ml per culture resulted in lowest total shoots (429 shoots) and highest cost per shoot (8.3 cents). Assessment of different treatment based on total shoots and cost per liter of medium and cost per shoot is very practical for management of commercial multiplication protocol than shoot formation per explant. Selection of pH adjustment in accordance with the medium strength in hand and the intended volume to be dispensed per culture was very crucial for higher rate and lowest cost of in vitro multiplication.

Keywords: Medium strength; Medium volume; pH adjustment; Total shoots; Cost per shoots; Ananas comosus

Introduction

Solid and liquid full-strength MS (1.0x) adjusted to pH 5.7 and dispensed at 20 ml per culture is the most common practice for testing of pineapple in vitro culture. In many cases, full strength MS medium was favored over SH, White and B5 media [1,2], and B5 and AZ medium [3] for both of establishment and multiplication stages. [4,5] on the other hand, used MT medium for both stages. Using of different medium for each stage and modified MS medium for both stage was also reported. MS medium was suggested for establishment and B5 for multiplication [6]. On contrary, either N basal salt [2,7,8] or MT medium [9] was suggested for establishment while MS for multiplication. Modification of some MS medium components was also tried for pineapple in vitro culture. For instance, half strength MS [10,11], half strength of MS salt but full vitamins [12], MS full salt but the MS vitamin components was replaced by vitamins of White medium [13], B5 medium [14] and vitamins of Morel medium [15]. MS full macronutrient and vitamins but its micronutrient components was replaced by that of B5 [1] and with that of Knudson medium [16,17] found that suitability of medium type depended on length of incubation period and hormone treatments. After 30 days of incubation in 6 different media (CH, DW, LS, MS, MT, and N) best shoot multiplication was obtained in MT and LS while after 60 days the best multiplication obtained in MT, CH and MS and after 75 days obtained in CH medium. In most cases of pineapple culture, both of establishment and multiplication media were usually adjusted to 5.7 and dispensed at 20 to 25 ml per culture. However, in few cases pH was adjusted to 5.0 [18,19,5], 5.5 [15] and to pH 6.5 [20].

Best pH adjustment was found to depend on sucrose enrichments and medium states [21] and sucrose enrichment and cultivars [22]. In agar solidified MS, best pH for Smooth cayenne was pH 6.5 and for Moris was pH 5.0. In vitro culture of pineapple were carried in different volumes of medium per culture ranged from 3 [18], 7 [5], 10 [19], 12.5 [23], 20 [24,20,2,5], 25 [25,26], 30 [27,14,28], 40 [23] to 50 ml [29,30] per culture. Different medium types differ in either type or concentrations of their components or both and on ratio of one of their component to another's. Any one, some or all of these differences could be the cause of different rate of shoot formation obtained in different media type and media modifications. Furthermore, since, the amount of nutrient available per culture depends not only on the type of medium or its modifications but also on medium strength, pH adjustment and volume of medium dispensed per culture, certain medium strength, medium volume and pH combinations could be better than others for explant growth and multiplication. The objective of this study was to compare the effect of four different MS strength (0.5x, 1.0x, 1.5x and 2.0x) on Moris pineapple in vitro shoot formation and how that effect could be manipulated by adjusting the medium to different pH (5.0, 5.5, 6.0 and 6.5) and by dispensing medium at different volumes per culture (3, 4, 5, 6, 7 and 8 ml).

Materials and Methods

Factorial experiment consist of 72 combination of 4 MS medium strength (0.5x, 1.0x, 1.5x, 2.0x), 3 pH adjustments (pH 5.0, 5.5 and 6.5) and 6 medium volumes (3, 4, 5, 6, 7 and 8 ml) dispensed per culture were tested for their effect on shoot formation of pineapple after 60 days of incubation under constant temperature and photoperiod of 16 hours light provided by white cool fluorescent lamps and 8 hours of darkness.

Media preparation and explants source

Double strength (2.0x) MS medium [31] was prepared by dissolving of 5.89 grams of MS powder in 750 ml of distilled water. The medium was then divided in 4 beakers each received 75, 150, 225 and 300 ml and marked H, F, O and D. The medium in each beaker was converted respectively to half (0.5x), full (1.0x), one and half (1.5x) strength by completing the volume of each beaker to 300 ml using distilled water. All media irrespective of their strength were supplemented with sucrose at 20 g/ L and BAP at 2.0 mg/ L. The beaker containing half strength medium (marked H) was divided equally into three glass jars (100 ml each) and jars were marked H1, H2 and H3. Beaker containing full strength (F), one and half strength (O) and double strength (D) medium were divided also into three glass jars (100 ml per jar) and marked F1, F2 and F3; O1, O2 and O3; D1, D2 and D3. Medium on all jars marked with number 1, 2 and 3 were adjusted to pH 5.0, 5.5 and 6.5 respectively. The jars were closed with autoclavable plastic lids and the media were sterilized by autoclaving at 121 0C and 1.5 kg/ cm2 for 20 minutes. After that the content of jar marked H1 was dispensed under laminar into 18 culture tubes each three tubes received 3, 4, 5, 6, 7 and 8 ml and numbered 1 to 6 respectively. The same procedure repeated for other jars marked H2, and tubes were numbered 7 to 12 and jars marked H3 and tubes were numbered 13 to 18. Same procedure was repeated for jars marked F1, F2 and F3, O1, O2 and O3, D1, D2 and D3 and the culture tubes with equal volume of medium obtained from jar F1, F2 F3, jars O1, O2, O3 and jars D1, D2 and D3 were numbered 19 to 24, 25 to 30 and 31 to 36; 37 to 42, 43 to 48 and 49 to 54; 55 to 60, 61 to 66 and 67 to 72 respectively. Three shoots from Moris stock cultures were placed under laminar flow cabinet in each culture tube and incubated under constant temperature of 25 0C and 16 hour of light provided by fluorescent lamps for two months.

Data collection and statistical analysis

After two months of incubation, the multiple shoots complex was picked out of the culture tubes and separated into individual shoot for counting the shoots and measuring their lengths. Each culture tube was considered as replicate and table with three replicates for total shoots per culture tube was constructed. The data of each replicate was either divided by 3 (explants per culture) or multiplied by 1000 and dividing the result by the medium volume dispensed per culture tube (number of cultures per liter) to establish two tables each with three replicates one for average shoot formation per explant and one for total shoots per one liter of medium respectively. The data were subjected to analysis of variance and mean separation by Duncan Multiple Range Test at p ≤ 0.05 using SAS statistical package 6.12.

Results

The highest shoot formation per explant (10 shoots) was obtained in combination of double strength (2.0x) medium, adjusted to pH 5.0 and dispensed at 7 ml per culture. The second best (9 shoots) obtained in five combinations. Three of these five combinations consisted also of double strength (2.0x) adjusted to pH 5.0 but dispensed at 4 and 8 ml and adjusted to pH 5.5 and dispensed at 8 ml per culture. The other two consisted of one and half strength (1.5x) adjusted to pH 5.0 and to pH 5.5 and both dispensed at 7 ml (Table, 1). The highest shoot formation at each fixed medium strength of 0.5x, 1.0x, 1.5x and 2.0x was 4, 8, 9 and 10 shoots, all obtained in medium adjusted to pH 5.0 but dispensed at different volumes per culture (4, 6, 7 and 7 ml respectively). The lowest shoot formation in medium strength 0.5x, 1.0x and 2.0x, on the other hand, was 2 shoots obtained in medium adjusted to pH 5.5 and dispensed at 6 ml, adjusted to pH 5.5 and dispensed at 4 ml and to pH 6.5 and dispensed at 8 ml while the lowest shoot formation in medium strength 1.5x was 3 shoots obtained in medium adjusted to pH 6.5 and dispensed at 3 ml per culture. At any combination of pH adjustment and medium volume dispensed per culture, medium strength (2.0x) resulted in higher or at least equal rate of shoot formation than any other medium strength (Table, 1). When media were dispensed at 4 and 7 ml per culture, the highest shoot formation (9 and 10 shoots) obtained in medium strength 2.0x adjusted to pH 5.0. When dispensed at 8 ml, the highest shoot formation (9 shoots) obtained also in medium strength 2.0x adjusted to pH 5.0 and to pH 5.5. When media were dispended at 3 ml per culture, the highest shoot formation (6 shoots) obtained in medium strength 2.0x adjusted to pH 5.0 and to pH 6.5 and in medium strength 1.5x adjusted to pH 5.0. At fixed volume of 5 ml per culture, the highest shoot formation (7 shoots) obtained in medium strength 2.0x adjusted to pH 5.0 and medium strength 1.5x adjusted to pH 6.5 while at fixed volume of 6 ml the highest shoot formation (8 shoots) obtained in medium strength 2.0x and medium strength 1.0x both adjusted to pH 5.0.

At all medium volumes per culture except 8 ml, adjusting medium strength (1.0x) to pH 5.0 resulted in higher shoot formation than other pH adjustments. Comparing medium adjusted to pH 5.5 and 6.5, on the other hand, showed that the shoot formation in cultures containing 6 and 8 ml of medium adjusted to pH 6.5 was two times (6 and 8 shoots) that of medium adjusted to pH 5.5 (3 and 4 shoots) and equal shoot formation obtained in both pH adjustments (3, 3 and 4 shoots) in cultures containing 3, 5 and 7 ml. At all medium volumes per culture except 5 ml, adjusting the medium strength (1.5x) to pH 5.0 resulted in higher shoot formation than other pH adjustments. When the medium dispensed at 5 ml per culture, medium which was adjusted to pH to 6.5 resulted on contrary in more shoots (7 shoots) than those adjusted to pH 5.0 (6 shoots) and pH 5.5 (5 shoots). Adjusting medium to pH 6.5 resulted also in more shoots (5 shoots) than adjusting to pH 5.5 (4 shoots) if the medium dispensed at 4 ml. However, if the medium dispensed at 7 ml per culture, adjusting the medium to pH 5.5 resulted in contrary in more shoots (9 shoots) than adjusting to pH 6.5 (6 shoots). No different in shoot formation between medium adjusted to pH 5.5 and 6.5 and dispensed at 3, 6 and 8 ml (3, 5 and 6 shoots each) and between medium adjusted to pH 5.0 and 5.5 and dispensed at 7 ml per culture (9 shoots each). At all medium volumes per culture adjusting medium strength (2.0x) to pH 5.0 resulted in equal or more shoots than adjusting to pH 5.5 and 6.5. Medium adjusted to pH 5.0 resulted in more shoots than those adjusted to pH 5.5 and pH 6.5 if medium dispensed at 4, 5 and 7 ml per culture. No different in shoot formation, on the other hand, between medium adjusted to pH 5.5 and 6.5 and dispensed at 6 ml (5 shoots) and between medium adjusted to pH 5.0 and 5.5 and dispensed at 3 (6 shoots) and 8 ml (9 shoots) per culture. Medium adjusted to pH 5.5 resulted in more shoots (9 shoots) than adjusting to pH 6.5 if the medium dispensed at 7 ml per culture (6 shoots).

Highest total shoots per liter of medium among all of the combinations was 6750 shoots obtained in double strength (2.0x) medium adjusted to pH 5.0 and dispensed at 4 ml per culture and the lowest total was 429 shoots obtained in half strength medium (0.5x) adjusted to pH 5.0 and dispensed at 7 ml per culture (Table, 2). The highest total shoots per liter of medium at medium strength 0.5x, 1.0x and 1.5x was 3000, 4000 and 6000 shoots respectively all obtained in medium adjusted to pH 5.0 and dispensed at 3 ml. The lowest total shoots per liter of each medium strength (0.5x, 1.0x, 1.5x and 2.0x), on the other hand, obtained at different combinations of pH adjustments and medium volume per culture. The lowest total shoots of medium strength 0.5x (429 shoots) obtained in medium adjusted to pH 5.0 and dispensed at 7 ml per culture while the lowest total shoots of medium strength 1.0x (1500 shoots) obtained in medium adjusted to pH 5.5 and dispensed at three different volumes (4, 6 and 8 ml) and the lowest total shoots of medium strength 1.5x (2250 shoots) obtained in medium adjusted to pH 5.5 and dispensed at 8 ml and in medium adjusted to pH 6.5 and dispensed at two different volumes (6 and 8 ml) per culture. The lowest total shoots of medium strength 2.0x (750 shoots) obtained in medium adjusted to pH 6.5 and dispensed at 8 ml per culture.

Highest total cost per liter of medium using medium strength 0.5x, 1.0x, 1.5x and 2.0x was $80.59, $81.82, $83.05 and $84.28 obtained when medium was dispensed at 3 ml per culture while the lowest total cost was $32.02, $33.25, $34.48 and $35.71 obtained when medium was dispensed at 8 ml per culture respectively (Table 3). The cost per shoot, on the other hand, ranged from 0.92 to 8.3 cents. Nine combinations resulted in the lowest cost (0.92 cents) per shoot; (medium strength 2.0x adjusted to pH 5.0 and dispensed at 4, 6, 7 and 8 ml per culture and adjusted to pH 5.5 and dispensed at 8 ml, medium strength 1.5x adjusted to pH 5.0 and dispensed at 7 and 8 ml and adjusted to pH 5.5 and dispensed at 7 ml per culture and medium strength 1.0x adjusted to pH 5.0 and dispensed at 6 ml). On seven of these nine combinations of lowest cost per shoot, the media were adjusted to pH 5.0 and the media of the other two were adjusted to pH 5.5. In five of these combinations the medium strength was 2.0x, in three was 1.5x and in one was 1.0x. However, these nine combinations of lowest cost per shoot resulted in different total shoots ranged from 2250 to 6750 shoots and different total cost ranged from $34.48 to $64.85 per liter of medium (Table, 3). Four combinations of medium strength, pH and volume of medium per culture, on the other hand, resulted in highest cost per shoot (8.3 cents). In all of these four combinations of highest cost per shoot, the medium strength was 0.5x but at different pH adjustments and medium volumes per culture (pH 5.0 and 7 ml; pH 5.5 and 3 and 4 ml; pH 6.5 and 4 ml.). Assessment of different treatment based on total shoots and cost per liter of medium and cost per shoot is very practical for management of commercial multiplication protocol than shoot formation per explant. Selection of pH adjustment in accordance with the medium strength in hand and the intended volume to be dispensed per culture was very crucial for higher rate and lowest cost of in vitro multiplication.

Discussion

The results of this study (Table, 1 and 2) demonstrated that double strength (2.0x) MS medium was the best medium strength and half strength (0.5x) MS was not an option for in vitro multiplication of pineapple. Double strength MS medium resulted in the highest shoot formation per explant (10 shoots), highest total shoots per liter (6750 shoots) and lowest cost per shoot (0.92 cents), but at high total cost per liter of medium ($64.85). On contrary, half strength medium resulted in loss of 50 % of explant capability of shoot formation (4 shoots) and total shoots production per liter of medium (3000 shoots) and tripled the cost per shoot (2.8 cents). Full strength (1.0x) MS medium adjusted to pH 5.7 is the most common medium for pineapple in vitro multiplication. However, the shoot formation in medium strength 1.5x and 2.0x adjusted to pH 5.5 was two times higher than that at medium strength 1.0x adjusted to pH 5.5. To be as better as medium strength 1.5x and 2.0x, full strength (1.0x) MS medium have to be adjusted to pH 6.5 and dispensed at 6 and 8 ml per culture and adjusted to pH 5.0 and dispensed at 6 ml (Table, 1). Adjusting full strength (1.0x) MS medium to pH 6.5 [20] and pH 5.0 [5] were used for pineapple and resulted in formation of 13 and 7 shoots per explant respectively but the medium per culture was three time more (20 ml) than the medium volume used in this study (6 and 8 ml). [22] Found that at 10 ml per culture of agar solidified full strength MS medium, different pineapple cultivars required different sucrose enrichment and pH adjustment; sucrose at 20 g/ l and pH 6.5 for Smooth cayenne and sucrose at 30 g/l and pH 5.0 for Moris cultivar. However, in liquid MS, the best pH adjustment for Moris remained the same as in solid media (pH 5.0) but the sucrose enrichment reduced to 20 g/l [21].

This study showed that adjusting the medium to a very specific pH was very crucial for shoot formation and cost of multiplication and its cruciality depended on the medium strength and volume dispensed per culture (Table 1 and 3). Assessment of different treatment based on total shoots and cost per liter of medium and cost per shoot would be more practical for management of commercial multiplication protocol than shoot formation per explant. Selection of pH adjustment in accordance with the medium strength in hand and the intended volume to be dispensed per culture was very crucial for higher rate and lowest cost of in vitro multiplication. At all medium volumes per culture except 8 ml, adjusting medium strength (1.0x) to pH 5.0 and at all medium volume per culture except 5 ml adjusting medium strength (1.5x) to pH 5.0 resulted in higher shoot formation than other pH adjustments. Medium strength (1.0x) and (1.5x) adjusted to pH 6.5 and dispensed at 8 and 5 ml per culture resulted in contrary in more shoot formation than adjusting to pH 5.0. In both media strength, comparing medium adjustments to pH 5.5 and pH 6.5, on the other hand, showed that whether adjusting to pH 6.5 would result in more, equal or less shoot formation than adjusting to pH 5.0 depended on how much of the medium was dispensed per culture (Table, 1). Similar, at all volumes dispensed per culture medium strength (2.0x) adjusted to pH 5.0 resulted in equal or more shoot formation than the other pH adjustments. However, whether adjusting to pH 5.0 would result in equal or more and whether pH 5.5 would result in more or less shoot formation than adjusting to pH 6.5 depended on the medium volume dispensed per culture.

These variations indicated that using of improper pH adjustments of medium or improper volume of medium per culture could cause great loss of in vitro shoot formation capability. Comparing the highest and lowest shoot formation obtained at different pH adjustments showed that adjusting of medium strength 0.5x, 1.0x, 1.5x and 2.0x to improper pH could lead, depending on medium volume dispensed per culture, to loss of up to 2, 4, 2 and 5 shoots which otherwise was possibly obtainable per explant respectively. Similar, comparing of highest and lowest shoot formation obtained at different volumes of medium per culture showed that dispensing of medium strength 0.5x, 1.0x, 1.5x and 2.0x at improper volume per culture could lead, depending on the medium pH adjustment, to loss of up to 3, 5, 6 and 4 shoots of otherwise possibly obtainable per explant respectively. Medium pH adjustment affected also the total shoots per liter of medium (Table 2). Comparing of highest total and lowest shoots per liter of medium showed that improper pH adjustment of medium strength 1.0x, 1.5x and 2.0x that did not fit the intended medium volume planned to be dispensed per culture could lead to loss of 1000 to 2500, 1800 to 2143 and 750 to 2750 of potentially obtainable total shoots per liter of medium respectively.

Shoot formation rate is the most commonly used parameter for assessment of different in vitro treatments. It is very important parameter to compare treatments effectiveness and as indicator of potential mass production. However, it serves physiologist more than propagators. For propagator, particularly those with low budget and small market, total shoots, total cost per liter of medium and cost per shoot are the most important three bases for decision making. Using of total shoots per liter of medium for assessment of different treatments combine evaluation of the shoot formation effecting factors with management of facilities used for in vitro tissue culture, facilitate estimation of cost per shoot and serve the purpose of commercial mass production more than shoot formation rate per explant. Dispensing the medium at smaller volume per culture increased the possibly obtainable total shoots per liter, but required more culture tubes, shelving space and working hours than dispensing the medium at larger volume per culture. Medium strength 2.0x adjusted to pH 5.0 and dispensed at 7 ml per culture resulted in highest rate per explant (10 shoots) but low total shoots (4286 shoots) and low total cost ($39.87) per liter while dispensing the medium at 4 ml per culture resulted in second best rate per explant (9 shoots) but highest total shoots (6750 shoots) and high total cost per liter ($64.85) and both treatments (two medium volumes) resulted in equal (0.92 cents) cost per shoot (Table, 1, 2 and 3). Selection between these two treatments (two volumes) which resulted in equal cost per shoot (0.92 cents) would be a compromise between saving $24.98 and production of 2464 extra shoots per liter and would depend on budget and facilities available and time set for production and delivery of specific amount of propagules according to client request. Another seven combinations of media strength, pH adjustments and medium volume per culture resulted in lowest cost per shoot (0.92 cents) at different total shoots per liter ranged from 2250 to 6750 shoots and different total cost ranged from $34.48 to $64.85 (Table, 2 and 3). Compromise would be between even larger saving ($30.37) and larger extra shoot production (4500 shoots) per liter of medium. In commercial laboratory where several liters of medium are used, saving of $24.98 to $30.37 per each liter of medium is very critical for management of the available budget and maintaining competitive market price for the product. In conclusion, combinations of pH adjustment and volume of medium dispensed per culture determined the shoot formation rate and total shoots per liter of medium and consequently have great impact on total cost per liter and cost per shoot. Hence, previous reports in which multiplication factors were optimized at fixed pH need to be reconsidered and using of fixed pH as a common procedure in tissue culture should no longer be adopted. In any future experiment, for optimizing of multiplication and reduction of cost per shoot, the medium pH should be adjusted in accordance with volume of medium intended to be dispensed per culture, cultivars, sucrose enrichments and medium states.

Cost is a major obstacle of micropropagation. Different approaches were followed to reduce the cost of pineapple multiplication. First approach is to reduce the cost of medium by using of liquid medium and cheap sucrose alternatives [32-34]. Second approach is to reduce the cost of labors, vessels and shelving space by using of bioreactor immersion system [14,35]. However, in both approaches of cost reduction, the cost per shoot which is the most valid cost comparison was not reported and the total cost was not itemized into different cost items. In addition, in all of these approaches pH adjustment was fixed at 5.7 and large volume of medium was dispensed per culture. pH adjustment is not a cost item, but the results of this study showed that pH adjustment could be considered as possible mean of cost reduction. pH adjustment affected the cost per shoots through its effect on shoot formation rate per explant and total shoots per liter of medium. MS strength 2.0x adjusted to pH 5.0 and dispensed at 4 ml per culture resulted in high total shoots (6750 shoots) and lowest cost per shoot (0.92 cents) while adjusting the same medium to pH 5.5 and pH 6.5 resulted in low total shoots (4500 and 3750 shoots) and doubled the cost per shoot, 1.54 and 1.85 cents respectively (Table 2 and 3). [21] reported that the cost per shoot at the second, third and fourth cycle of multiplication of 3 explants incubated in 8 ml of full strength (1.0x) MS medium adjusted to pH 5.0 for 60 days was 1.8 cents. Table 3 showed that using of 3 explants in double strength (2.0x) MS medium adjusted to pH 5.0 and dispensed at 4 ml reduced the shoot cost to half (0.92 cents). Generally, cost of any product could not be effectively reduced unless the cost factors itemized. [36,37] reported that the major cost factor of pineapple in vitro multiplication was the electricity cost for operating of air conditions and illumination of incubation room. In countries where outdoor incubation is possible [27], focus on investigation and optimization of combination of pH adjustments, medium volumes per culture and incubation under outdoor conditions would exploit the geographically inherited advantages of these countries and provide them with strong competitive edge on the world market.

Conclusion

Optimization of multiplication factors at fixed pH adjustment which is a common practice in tissue culture need to be reconsidered. The pH adjustment should be selected in accordance with medium strength and the intended volume of medium to be dispensed per culture. Optimization of pH adjustment is not only important for optimization of multiplication but it could also be a possible significant approach for reduction of in vitro multiplication cost. Using of multiplication rate for assessment of different treatments did serve the goals of physiologist, but, it may not so in point view of propagators. Use of total shoots per liter of medium and comparing of cost per shoot is more appropriate for selection of best compromise between treatment with highest multiplication rate, highest total shoots and lowest total cost per liter of medium and lowest cost per each single shoot. Testing of double strength medium (2.0x) enriched with table sugar at wide range of pH adjustments (4.0 to 7.5), medium volumes (3 to 10 ml) and explants density (3 to 10 explants) per culture over different incubation periods (30 to 75 days) may lead to a combination that would result in the most lowest cost per shoot, lowest total cost and highest total shoots per liter and proposed for investigation.

Acknowledgment

This study was supported by Ministry of higher Education, Libya.

Abbreviation

MS (Murashiege and Skoog Medium); BAP (Benzyleamino Purine); SH (Schenk and Hildebrandt Medium); B5 (Gamborg Medium); AZ (Abo El-Nil and Zettler Medium); CH (Cheng Medium); DP (Dutcher and Powel Medium); LS (Linsmair and Skoog Medium); MT (Murashige and Tucker Medium); N (Nitch Medium).

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