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Abstract

Three experiments were used to investigate relationship between in vitro rooting treatments (medium states; solid, liquid, IBA concentrations; 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l, incubation periods; 30, 45, 60 and 75 days), rooting parameters (plantlet height, root number), polyethylene cover and pineapple plantlets survival of acclimatization. Data analysis showed no significant difference between survival of plantlets rooted in solid versus liquid (t≤0.331), IBA contained versus IBA devoid (t≤0.241) medium, rooted versus rootless shoots (t≤0.453) and between plantlets rooted in different concentration of IBA (p≤0.828). The survival was mainly affected by plantlets height (p≤0.0003), incubation period (p≤0.0001) and polyethylene cover (t≤0.015). All plantlets that were taller than 45 mm survived acclimatization irrespective of medium states that were rooted in and number of roots per plantlet. In vitro rooting for 75 days resulted in survival of 94% of the plantlets while only 71% of the plantlets that were in vitro rooted for 30 days survived acclimatization. Intermediate in vitro rooting period of 45 and 60 days resulted in intermediate survival percentage (80% and 85%). The survival of covered plantlets (83%) was significantly higher than uncovered plantlets (59%).

Keywords: Acclimatization; Plantlets survival; Ananas comosus; In vito rooting

Introduction

In vitro rooting of pineapple could be obtained in agar solidified full strength [1-3] and half strength MS medium [4-7] and in liquid full strength [8-11] and half strength MS medium [12-13]. In vitro rooting was also obtained in solid [14] and liquid White medium [15] and solid MT medium [16]. However, while some of the researchers used medium free of hormone [17], others used medium enriched with singly applied IBA [1,3,4,6,8,12], NAA [7,16,18], IAA [19,20] and others used combination of IBA and NAA [5,14,21,22], IBA and IAA [23,24], IBA and KN [11]. In vitro rooting of pineapple was also obtained in MS medium enriched with BAP in combination with activated charcoal [9] and in combination with 2, 4-D [25]. The most commonly used incubation period for rooting of pineapple is 30 days. However, 45 [3,21,26], 60 [1,27] and 75 days [18,28] of incubation in solid and 60 [8,10,11] and 70 days [15] of incubation in liquid was also reported. Previous in vitro rooting studies focus on the effect of hormone types and concentrations on in vitro root induction and the recommended rooting treatments was selected based on rooting percentage, root number and length [5,7,15,21,26,29]. In addition to the above rooting pararmeters, plantlets height was also used for assessment of different rooting treatments [1,8,10,12,20,28]. In vitro rooting is usually carried out based on assumption that in vitro root formation as measured in number and length will increase the plantlets survival during the following acclimatization stages. Acclimatization, on the other hand, is carried out by subjecting the plantlets to gradual changes from the in vitro conditions of high humidity, low light and stable temperature to ex vitro conditions to increase also the survival and growth of plantlets on open nusery bed and field. Rooting substrate types and mixing ratio [3,7,30,31,32], light intensity [33], hydroponic unit [19] and diazotrophic bacteria [34] were tested for plantlets survival during acclimatization stage. In many cases, over 88% of plantlets survival was reported.

However, it is not clear if the survival was mainly a function of the factors and conditions tested during acclimatization or it may a result of more complicated interrelation between quality of the obtained in vitro rooted plantlets (root numbers, length and plantlets height) and the conditions and factors tested during both in vitro rooting and acclimatization stages. Although, both of the in vitro rooting and acclimatization stages are conducted for the same purpose which is increasing survival of plantlets, the relation between in vitro rooting treatments (medium states, hormone types and concentrations, length of incubation period), parameters used for assessment of rooting treatments and survival of acclimatization (main goal of in vitro rooting) were rarely reported [4,35,36]. For valid recommended rooting and acclimatization treatments, these two stages need to be studied in connection with each other. Assessment of rooting treatments and selection of best rooting parameter for evaluation of rooting treatments should be based on the effect of that rooting parameter on plantlets survival of acclimatization stage. Separate evaluation of rooting and acclimatization stages would highly likely come up with improper recommended treatment for one or both of the two stages. Evalaution of in vitro rooting and acclimatization treatments in connection with each other would not only help in selecting the best rooting parameter for evaluation and selection of the most optimum rooting and acclimatization treatment, but also result in better management and lower cost of rooting and acclimatization stages. Using either solid or liquid medium, hormone or hormone free medium, shorter or longer incubation for in vitro rooting is expected to have different effect on the different rooting parameters and on the in vitro hardening process. Consequently, plantlets of different rooting parameters is expected to have different plantlets growth and survival percentage under the tested factors of acclimatization. The objective of this study is to investigate the relationship between rooting treatments (IBA at 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l, medium states (solid and liquid), rooting periods (30, 45, 60 and 75 days), rooting parameters (plantlets height, roots per plantlet) and pineapple plantlets survival percentage after two months of acclimatization under shade house.

Materials and Methods

Three different rooting experiments were conducted to test the effect of rooting treatments (IBA concentrations, medium states, and incubation period length), rooting parameters (plantlets height, number of roots) and polyethylene cover on the plantlets survival of acclimatization stage.

IBA concentrations and medium states experiment

One liter of full strength MS medium [37] was prepared and divided into six glass jars (150 ml each) marked with numbers one to six. No hormone was added to jar number one and IBA at 0.5, 1.0, 1.5, 2.0 and 2.5 mg/l was added to jars numbers two - six respectively. The pH adjusted to 5.7 and the content of each of the six jars divided into two jars to one of them agar were added at concentration of 7 g/l. The twelve glass jars placed in autoclave and subjected to 1200C and pressure of 1.5 kg/cm for 20 minutes. The content of each of the twelve jars divided under laminar floor into nine culture tubes and numbers 1- 9, 10 - 18, 19 -27, 28 - 36, 37- 45 and 46- 54 were given to culture tubes containing liquid medium and numbers 55- 63, 64- 72, 73- 81, 82- 90, 91- 99 and 100 - 108 were given to culture tubes containing solid medium. One Moris pineapple shoot was placed per each of the 108 culture tubes and incubated under 16 hours of light and constant temperature of 250C. After 60 days of incubation, plantlets were removed out of each culture tube for counting roots number per plantlet and measuring roots length and plantlets height. After that, each plantlet was placed back on the same culture tube it was taken from. Hence each plantlet could be traced back to the type of the rooting treatment it received and the kind of rooting parameters it had developed (root number, root length, plantlet height). The collected data used to establish tables for each rooting parameter, rooting treatment and tested factor and were grouped as rootless versus rooted, hormone treated versus hormone free, liquid versus solid media. The plantlets from solid and from liquid media each were assorted also into four categories according to plantlet height, 25 (20- 30 mm), 35 (31- 40 mm), 45 (41- 50 mm) and 55 (51- 60 mm) mm tall and four categories according to roots number, 0 (rootless), 2 (1- 2 roots), 4 (3- 4 roots), 6 (5- 6 roots) roots per plantlet. The plantlets were transferred to 72- cells trays filled with garden soil mix and laid out starting from a marked cell on the tray following predetermine direction in accordance with a drown sketch. Hence, each plantlet on the trays could be traced back to type of rooting treatments and rooting parameters it developed. The trays were enclosed within polyethylene sheets and placed under normal room conditions for 21 days. After that each three plantlets were transferred to pre-marked pots (10 x 5 cm.) filled with compost and placed under shade house. Plantlets were watered every four to six days and after 60 days the numbers of survival plantlets were counted. The data of survival were presented in table for each rooting parameter and each rooting treatment versus other and were analysis using t test and the data of the plantlets which were assorted into different groups according to height and root number and IBA concentratios were subjected to analysis of variance and Duncan Multiple Range Test using SPSS statistical package No. 11.

Table 1: Anova analysis of rooting treatments (IBA concentrations and medium states) and rooting parameters (plantlets height and root number per plantlets) effect on pineapple survival of acclimatization.

Table 2: Effect of rooting treatments (IBA concentrations, medium states, incubation periods) and rooting parameters (plantlets height, roots per plantlet) on pineapple survival of acclimatization stage.

Means followed by same letters were not significantly different at probability of 0.05 according to

Duncan Multiple Range Test.

Nt (Not tested) solid medium was not used for testing of incubation period effect on the plantlets survival of acclimatization

Table 3: Effect of polyethylene cover, medium states, IBA and in vitro root formation on pineapple survival of acclimatization.

Incubation period experiment

After four multiplication cycles, 360 shoots were individually in vitro rooted in culture tubes containing 8 ml of liquid half strength MS medium enriched with IBA at 2.0 mg/l and sucrose at 30 g/ l. After 30, 45, 60 and 75 days of incubation, 90 plantlets were removed from the culture tubes and their height, root number and length were recorded and transferred (irrespective of the plantlets height, root number and length) to six pots filled with sand (15 plantlets per pot), saturated with water, enclosed within polyethylene sheet and kept under room conditions. The polyethylene cover was partially opened after 10 days and completely removed after 21 days and the pot moved to shade house. The plantlets were watered every four to six days and the numbers of survived plantlets were counted after 60 days under shade house. The number of survived plantlets were arranged in table of four incubations periods and six replicates (15 plantlets per replicate) and subjected to analysis of variance and Duncan Multiple Range Test at p ≤ 0.05 using SPSS statistical package No.11.

Polyethylene cover experiments

After four multiplication cycles, 150 pineapple shoots were rooted in agar solidified full strength MS medium enriched with sucrose at 30 g/l and IBA at 0.5 mg/l for 75 days under 16 hour of light and 8 hours of darkness and constant temperature of 250C. After 75 days of incubation, the shoots were removed from cultures, roots cleaned from agar and transferred to pots filled with sands (15 plantlets per pot). Half of the shoots (75 plantlets) were covered with polyethylene sheets and the other half was not covered and both kept indoor under normal room conditions for 21 days. Uncovered plantlets were watered each three days. The cover were partially opened after 15 days and completely removed after 21 days and the pot moved to shade house. The plantlets were watered every four to six days and the numbers of survived plantlets were counted after 60 days under shade house. The plantlets survival data were arranged in table of covered vs uncovered and analyzed using t-test at p≤0.05.

Results

Rooting treatments, rooting parameters and survival

Anova analysis of data (Table 1) showed that the plantlets survival of acclimatization was neither affected by the IBA concentrations (p≤0.83) or medium states (p≤0.229 ) nor by the interaction of IBA with medium states (p≤0.205) or interaction of medium states with plantlets height (p≤0.247). IBA concentrations and medium states during in vitro rooting stage did not have any direct (independent) or indirect (dependent) affect via interaction with each other or with plantlet height on the survival of acclimatization. On contrary, the plantlets survival of acclimatization was mainly affected by direct (independent) affect of plantlet height (p≤0,0003 and to some extent by interation of the medium states and number of roots per plantlet (p≤0.0618). Table 2 supported also that acclimatization survival was not effected by the IBA concentrations but by plantlets height. About 90% of the plantlets that were taller than 45 mm at the end of rooting stage survived acclimatization under shade house irrespective of medium states that were rooted in. The survival of those shorter than 45 mm, on the other hand, varied depending on medium states and the root number per plantlets. The survival of the plantlets that were rooted in liquid and were 25 and 35 mm tall had higher survival percentage (57 and 78% respectively) than those rooted in solid (33 and 57% respectively). The survival of plantlets rooted on solid increased and the survival of those rooted in liquid medium decreased as the root per plantlet increased from two to six roots. All plantlets (100%) which rooted in solid medium and produced six roots survived acclimatization while the survival of the plantlets which produced four, two roots and rootless ones were 78, 67 and 56% respectively. On contrary, only about 50% of the plantlets that produced six roots in liquid medium passed the acclimatization stage. For plantlets rooted in liquid medium, there were no significant different in the survival percentage between rootless plantlets and those which form less than four roots. About 78% of rootless and those with less than four roots survived acclimatization and each had higher survival percentage than plantlets with six roots per plantlet (Table 2). Furthermore, table 3 showed also that the difference in plantlets survival between those rooted in solid versus those rooted in liquid medium (t≤0.333), rooted in medium with versus medium without IBA (t≤0.241) and rooted plantlets versus rootless (t≤0.453) were not significant.

Incubation periods, polyethylene cover and survival

The differences in the plantlets survival between plantlets rooted in liquid medium for different incubation incubation periods were significant (p≤0.00001) and the plantlets survival increased from 71 to 94% as the incubation increased from 30 to 75 days (Table 1 and 2). Intermediate incubation period of 45 and 60 days resulted in intermediate percentage of survival (81% to 85%). In addition, enclosing of the plantlets within polyethylene sheets for the first 21 days before being transferred to shade house resulted in significant increase in plantlets survival of acclimatization (t≤0.015) compared to those that were not covered with polyethylene (Table 3). About 83% of the plantlets that were enclosed within polyethylene sheet survived acclimatization compared to 59% of the uncovered plantlets. In summary, the plantlets survival of acclimatization was mainly affected by plantlets height at the end of rooting stage (p≤0.0003), interaction between roots number per plantlet and medium state (p≤0.061) in which the plantlets were rooted in (Table 1), length of incubation period of rooting stage (Table 2) and by enclosing of the plantlets within polyethylene sheets (t≤0.015 ) for the first 21 days before being transferred to shade house (Table 3), For pineapple, it seemed that in vitro rooting is not required and rooting percentage, root number and length is not an indicator of expected capability of acclimatization survival. Plantlet height and other criteria such as stomata quick response might be more important than root number and length for assessment of in vitro rooting treatments and expectation of acclimatization survival.

Discussion

In all in vitro rooting studies, rooting percentage, root number and length are usually recorded and used for assessment of treatments. However, no justification was provided that these parameters of assessment will help achieving the goal of in vitro rooting (survival of acclimatization). This study demonstrated that pineapple plantlets survival of acclimatization was neither affected by in vitro rooting treatments (medium states and IBA concentrations) nor rooting parameters (rooting%, number, and length). The pineapple plantlets survival of acclimatization was mainly affected by plantlets height at the end of rooting stage (Table 1 and 2), length of incubation periods (Table 2) and by enclosing of the plantlets within polyethylene sheets for the first 21 days before being transferred to shade house (Table 3). Plantlets height was more important than other rooting parameters (rooting percentage, root number and length) for survival of acclimatization as well as for determination of proper length of in vitro rooting stage. Whether the plantlets were rooted in solid or liquid medium, in vitro rooting of pineapple should not be terminated before the plantlet reached 45 mm in tall. Over 90% of the pineapple plantlets that were taller than 45 mm survived acclimatization irrespective of medium states and whether it form roots or were rootless (Table 2). A 45 mm tall plantlets is a reachable plantlet height during in vitro rooting stage. Smooth cayenne shoots developed into 70 mm [20], 90 mm [10] tall plantlets after 75 days of incubation in half strength MS medium enriched with NAA at 1.0 mg/l and Moris shoots developed into 56 mm [20] tall plantlets after 60 days of incubation in liquid quarter strength MS enriched with IAA at 2.5 mg/l. If the in vitro rooting has to be ended before plantlets reached the 45 mm tall, termination of incubation period of those rooted in liquid should be done before plantlets form more than four roots and termination of those rooted in solid after the plantlets form more than four roots. The best rooting treatment is that in which either one or both of these two requirements (plantlet height and root number) obtained in shortest incubation period. [38] reported that longer pineapple shoots has higher acclimatization survival. The percentage of acclimatization survival increased from 20 to 100% as the shoots size increased from 40 to 80 mm in length. However, [39] reported if the shoot fresh weight was 0.05 g, shorter shoot of just only 20 mm long would survive acclimatization under greenhouse. Plantlets height was also found to be crucial for survival of Euphorbia fulgens [40], woody plants [41] and oil palm plantlets [36]. Previous studies, in which different rooting treatments and incubation period were used, reported pineapple plantlets of different height, 30 [16], 50 [2], 70 [42] and 90 mm [10] but did not elaborate about the relation between plantlets height and length of incubation period and acclimatization survival.

Little attention was paid to the effect of rooting treatments and rooting parameters on the plantlets survival of acclimatization. [4] found that pineapple shoots which rooted in hormone contained MS medium enriched with sucrose had higher percentage of acclimatization survival (96%) than those rooted in MS medium devoid of sucrose (49.5%). Similar, [35] reported that pineapple shoots rooted in liquid medium with luffa matrix as support had higher survival percentage (95%) than those rooted in agar solidified medium (78%). However, they did not elaborate about the number of roots per plantlet in both media or the height of the plantlets used for acclimatization. Using oil palm, [36] reported equal percentage of acclimatization survival (about 82%) of embryo shoots rooted in stationary liquid, semi solid and double phase medium. However, the survival of those that were treated with IBA was higher (78%) than those treated with NAA (30%) and those rooted in hormone free medium (39%). Rooted embryo shoots of oil palm had higher survival percentage (75%) than rootless shoots (47%). They found also that the percentage of survival increased from 22% when plantlets height was 25 mm to 100% when height was 75 mm and decreased from 85% when root length was 10 mm to 69% when the root length exceeded 15 mm.

Extending the incubation periods (in vitro rooting stage) from 30 to 75 days increased the pineapple plantlets survival from 71% to 94% (Table 2). The higher survival percentage of plantlets that were left in rooting medium for longer period could be related to some sort of in vitro hardening process and particular plantlet quality. Depletion of nutrients of the medium and changes in the physical and chemical conditions inside the vessel as a result of plantlets growth may lead to some sort of in vitro hardening that increased and improved as the incubation period increased in length and consequently increased the survival of acclimatization. Whether in vitro hardening does take place over longer incubation period could be verified by measuring of wax and cuticle layer, chloroplast shape, stomata quick response, increase in weight and starch accumulation within plantlets tissues and photosynthetic ability. The plantlets which used in this study for comparing the effect of different rooting incubation periods on the acclimatization survival were not grouped according to root number per plantlets and plantlets height. So new experiments in which the plantlets assorted into different group of plantlet height and roots per plantlet before being subjected to acclimatization conditions is needed to identify the criteria that enable longer incubation to improve acclimatization survival. Longer incubation period of rooting did result in higher plantlets survival, but on the other hand it would increase the electricity bill of the in vitro rooting stage [12]. Future search should be directed for compromise between lowest cost of shortest incubation period and highest acclimatization survival of longer one. It is very important that rooting treatments and incubation periods should be assessed and terminated based on plantlets quality (rooting parameters) which expected to improve the acclimatization survival potential such as plantlet height, wax and cuticle layer and quick stomata response rather than the common used parameters of rooting percentage, root number and length.

Using of polyethylene cover for 21 days befor the plantlets moved to shade house resulted in survival of 83% of the plantlets compared to 59% of the uncovered plantlets (Table 3). The plantlets used to test the effect of polyethylene cover were rooted in solid medium for 75 days and were not assorted according to their quality before covering. The 59% survival of the uncovered plantlets of different qualities indicated a possibility that plantlet of certain quality, (height for instance), could survive in open pots under room condition and shade house without cover or misting requirement. Future experiment in which plantlets assortment into groups of different quality before being subject to acclimatization is needed to identify which is the plantlet quality that enables the uncovered plantlets to survive acclimatization. Acclimatization are usually done under greenhouse with misting and controlled light intensity and humidity [33] and inside growth chamber [23,43]. Polyethylene cover is cheaper and simpler alternative to installing, operating and maintenance of misting system and greenhouse and growth chamber with controlled environmental factors. For small busniss with small budget and low production target in developing countries, polyethylene cover would maintain high survival and low cost of acclimatization.

Since rooting treatments and in vitro root formation was not as crucial as the plantlets height for pineapple survival of acclimatization, it would be better to replace in vitro rooting by elongation stage. Elongation stage with different procedures [13,38,44,45] was suggested to facilitate separation of shoots, enhance growth, increase shoots length and to improve ex vitro survival and acclimatization. On the other hand, [39,43,46] reported respectively that 20, 30 and 50 mm long shoots could directly transferred from multiplication medium to acclimatization. Shoots of 40 - 75 mm long [7] were reported during multiplication in liquid and shoots of 70 - 95 [3], 57 - 63 [22], 49 - 53 mm long [5] were reported during multiplication in solid medium. Other researchers using different multiplication treatments reported average shoot length of 30 [18], 37 [10] and 55 mm long [47] per multiplication cycle [48]. Reported that the shoot length during multiplication depended on explant size and density and about 70% of shoots were shorter than 20 mm. The shoot length increased over subculture, by the fourth subculture the shoot length reached 31 mm [18, 49]. The above reports indicated that it is possible that shoots longer than the minimum 45 mm required for survival of acclimatization (as demonstrated in this study) could be produced during multiplication stage. So, instead of elongation and rooting , it is seems possible and would be even better approach to search for multiplication treatment in which reasonable multiplication rate and shoots taller than 45 mm are obtained simultaneously. If such multiplication treatment developed and optimized, it will save the cost and time of rooting and elongation stages and would result in substantial reduction in cost of micropropagation. [39] reported that pineapple shoots with average fresh weight of 0.05 g survived acclimatization condition under greenhouse. Using Spathipuyllum floribundum, [50] reported that shoots of heavy weight obtained during multiplication stage showed higher acclimatization survival than shoots of light weight. [51] observed that the changes in average shoot weight and shoot formation rate of pineapple during a single multiplation cycle occurred in alternative pulses of high and low increase of weight and rate of shoot formation. Using of shoot weight during multiplication or in vitro rooting stage as criteria for pineapple capability of acclimatization survival and as a parameter for in vitro rooting treatments assessment is suggested for future investigation. In summary, for better micropropagation system, rooting and acclimatization stage should be studied and evaluated in connection with each other. After rooting stage, the plantlets should be separated into groups according to root number, root length, plantlet height and weight before being subject to acclimatization treatments. Future studies of in vitro rooting should focus in determining the relation between rooting parameters and percentage of survival during acclimatization. Population size and levels of tested factors of both of rooting and acclimatization stage should be large enough to establish correlation, t test and regression analysis rather than just Anova analysis and means separation test. Survival of acclimatization is a key step for successful micropropagation system. If none or only few plantlets survived acclimatization stage, it means failure of the project and higher cost of production irrespective of how optimal the rooting or multiplication stage was.

Conclusion

Pineapple plantlets survival of acclimatization was neither affected by in vitro rooting treatments (medium states and IBA concentrations) nor rooting parameters (rooting, number, and length). The pineapple plantlets survival of acclimatization was mainly affected by plantlets height at the end of rooting stage, length of incubation periods and by enclosing of the plantlets within polyethylene sheets for the first 21 days before being transferred to shade house. For pineapple, it seemed that in vitro rooting is not required and rooting percentage, root number and length is not an indication of capability of acclimatization survival. For valid recommended rooting and acclimatization treatments, these two stages need to be studied in connection with each other. Assessment of rooting treatments and selection of best rooting parameter for evaluation of rooting treatments should be based on the effect of that rooting parameter on plantlets survival of acclimatization stage. Plantlet height was more important than root number and length for assessment of in vitro rooting treatments. About 90% of the plantlets that were taller than 45 mm at the end of rooting stage survived acclimatization under shade house irrespective of medium states, IBA concentrations that were rooted in and whether it form roots or were rootless.

Funding

This study was supported by Ministry of higher Education, Libya

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