MORPHO-PHYSIOLOGICAL CHANGES IN PINEAPPLE PLANTLETS [ Ananas comosus ( L . ) Merr . ] DURING ACCLIMATIZATION

Pineapple producing countries lack good quality propagation material to expand cultivars into new areas. Previously, research protocols may increase the offer of high quality plantlets and speed the introduction of new pineapple cultivars. The present work is to evaluate the morpho-physiological changes in plantlets of pineapple [Ananas comosus (L.) Merr. ‘MD-2’] during the acclimatization phase. Plantlets were acclimatized under 80% relative humidity, 25.5 °C temperature and photosynthetic photon flux of 400-500 μmol m-2 s-1 as average for 45 d under natural photoperiods. All measurements (plant length, number of leaves and roots, fresh weight, width and length of leaf ‘D’, net photosynthesis and total transpiration rate) were carried out at the end of in vitro rooting phase coincident with 0 d of acclimatization and at 15, 30 and 45 d thereafter. Photosynthetic activity of in vitro plantlets did not increase during the first 30 d of the acclimatization phase. After 30 d, photosynthetic activity ranged from 5.72 to 9.36 μmol CO2 m-2 s-1 while total transpiration ranged from 6.0 to 1.42 mmol H2O m -2 s-1. During the first 30 days there were no significant differences in number of leaves, length or width of the longest (‘D’) leaf (cm) or plant length (cm). However, after 45 days plant fresh weight (g), length and width of the ‘D’ leaf (cm) and root number all increased significantly, while transpiration (mmol H2O m -2 s-1) declined. There were small but significant decreases in chlorophyll a and b (μg g-1 mf.). Increased photosynthetic activity after 30 d shows that the increase in light intensity and the reduction of relative humidity during acclimatization did not constitute inhibitory factors.


INTRODUCTION
Pineapple [Ananas comosus (L.) Merr.] is vegetatively propagated so expanding the available supply of planting material required to expand production in new areas or introduce a new cultivar is an expensive and time consuming process.Based on typical planting densities for various cultivars, one hectare of the crop requires from 35 000 to 75 000 propagules (BARTHOLOMEW; PAULL; ROHRBACK, 2003).
The MD-2 cultivar, developed by Del Monte Fresh Produce International Inc., has almost completely taken over the international fresh fruit market (LOEILLET; DAWSON;PAQUI, 2011).In order for the pineapple industries to remain competitive, it is imperative that they expand plantings of 'MD-2' pineapple on a commercial scale (BARTHOLOMEW et al., 2009).Rapid expansion of planting material requires the implementation of a meristem bank to establish plantlets production schemes with the use of biotechnology techniques (LEAL; COPPENS D 'EECKENBRUGGE, 1996).
The key means of regeneration is the use of meristematic tips (BOTELLA; FAIRBAIN, 2005).All these protocols have disadvantages related with the low coefficients of multiplication and the high cost and the low possibility of automation (WATT, 2012).That is why the explosive expansion of this industry depends on the existence of new technologies to facilitate automation and improved protocols to optimize the acclimatization of plants ( KITTO, 1999).
However, the potential use of these procedures is limited by the low survival rates and plant growth during the acclimatization stage.Plants cultured in vitro are exposed to high levels of organic and inorganic nutrients, high relative humidity, low light intensity, and limited exchange of CO 2 and O 2 .As a result, in vitro cultivated pla nts h ave morphol ogica l and physi ologi cal abnormaliti es (BARBOZA et al., 2006).Those modifications make them very sensitive to the abrupt environmental changes experienced when they are removed from culture containers and placed under ex vitro conditions (KELLER; LUTTGE, 2005;BRAGA et al., 2011).As a result, a gradual acclimatization period is normally required after ex vitro transplantation to reduce stress as in vitro plants progressively acclimatize to the new growing condition (YANES; GONZÁLEZ; RODRÍGUEZ, 2000).
Bioplant Center developed a novel protocol for the production of in vitro cultivated plants based on the use of liquid medium and temporary immersion technology coupled with the implementation of a semiautomatic system.The system reduces the time required to generate sufficient quantities of plantlets for the creation of high quality pineapple plantlets banks.Using this technology, a pilot plant produced about one million 'MD-2' plantlets in a small growth chamber (ESCALONA et al., 1999).However, little is known about the morpho-physiological changes undergone by 'MD-2' plantlets during the acclimatization phase of the process.The objectives of this research were to characterize the morphological and physiological changes that occurred in plantlets during the acclimatization process.It is expected that the informat ion would guide furth er research on improvements in acclimatization that would speed the process or improve rates of establishment under field conditions.

Plant material and growth conditions.
Pineapple plantlets (Ananas comosus 'MD-2') were micropropagated according to the protocol proposed by Daquinta and Benegas (1997) using the crown buds as explants.

In vitro culture conditions.
All in vitro manipulations were performed in aseptic conditions.The culture media were sterilized by autoclaving at 121° C at 118 kPa pressure.The plantlets were grown in transparent magenta plastic containers (7.2 x 8.6 x 9.2 (H) cm) with 40 mL of liquid medium.The photoperiod was of 16 h at a photosynthetic photon flux (PPF) of 75 µmol m -2 s -1 and 26±2° C of temperature during all phases.

Environmental conditions in acclimatization phase.
Uniform micropropagated plantlets of  4.0 cm height, 7.0-8.0g fresh weight and containing 5-6 true leaves were selected from the plastic culture vessels (ESCALONA et al., 1999), dipped in 3.0 mL L -1 Previcur Energy ® (Bayer Cropscience) for 5 minutes and planted in 1:1 (v:v) red ferralytic soil and filter cake (derived from sugarcane bagasse) in polyethylene bags (396.26cm 3 ) as containers for acclimatization (VILLALOBOS et al., 2012).Plantlets were acclimatized under 80% relative humidity (RH), 25.5°C temperature and photosynthetic photon flux (PPF) of 400-500 µmol m -2 s -1 as average for 45 d under natural photoperiods.During the first week, plants were misted for 15 seconds every 45 minutes beginning at 9:00 am each day.A foliar fertilizer mix containing 16.0 g of crystalline N-P-K and 1.0 g of Multimicro Combi (Haifa Chemicals Ltd., Haifa Bay 26120, Israel) in 16 L of water was applied every ten days.
Morpho-physiological measurements were carried out at the end of in vitro rooting phase coincident with 0 d of acclimatization and at 15, 30 and 45 d thereafter.

Morphological and physiological analysis.
Forty plants were collected at each time interval and data were taken on plant length (cm), number of leaves and roots; fresh weight (g), width and length of leaf 'D', defined as a leaf forming a 45 degrees with the vertical axis.
Chlorophyll was extracted from five leaf disks (0.28 cm 2 ) taken from the 'D' leaves of five plantlets.
Disks were incubated in 3 mL DMSO at 65º C for 1 hour, and absorbance was measured at 645 and 663 nm.The chlorophyll concentration of the extracts was calculated using the equations described by Porra et al. (2002).

Photosynthesis measurements.
Net photosynthesis (A, µmol m -2 s -1 ) was measured at the ambient relative humidity and air temperature.The analyzer and cuvette (PP Systems CIRAS-2 Portable Photosynthesis System and PLC6 2.5 cm 2 cuvette, http:// www.ppsystems.com/)were automatically calibrated before every measurement.Light was fixed at 600 µmol m -2 s -1 for the determination of the A/Ci curve, while the CO 2 was fixed at 400 µmol mol -1 , these values were obtained from saturation curve of light and CO 2 in pineapple plants.
Measurement of A and total transpiration rate (T, mmol H 2 O m -2 s -1 ) were always made on the youngest fully expanded leaves and between 9:00 am and 10:00 am.

Statistical analysis.
At each sampling day, six plants were randomly selected to measure photosynthesis and transpiration, 40 for counting leaves, width and length of leaf 'D' (cm), plant length, root number, 20 for measuring fresh weight and 10 for measuring chlorophyll content.
Analysis of variance was conducted using SPSS Program.Duncan's multiple range tests and Student's tdistribution were used for mean separation at p< 0.05 level.

RESULTS AND DISCUSSION
Total transpiration of in vitro leaves was highest at 0 d and declined significantly by 15 d after transfer to ex vitro conditions, after which it remained constant until the end of the experiment (Figure 1 A).Leaves initially became shorter and wider when plantlets were subjected to the higher light levels of the acclimatizing environment (Table 1).The low light levels during in vitro culture likely caused some etiolation of the leaves.
It appears that in only 15 d, the stomata become functional to control water loss since transpiration was stable from that point on.This observation is consistent with the fact that during acclimatization, the transpiration is gradually reduced as stomata become functional and cuticular water loss declines (HERRERA, 2009).When stomatal and cuticular transpiration rates in in vitro leaves are very high it is difficult to maintain plant turgidity after transplanting ex vitro (SEON et al., 2000).
Photosynthesis at 0 d was low at the end of in vitro phase and remained for the first 30 days after transfer to ex vitro conditions (Figure 1 B).The significantly higher level Cote, Folliot and Andre (1993) found that in vitro pineapple plants required a few months and a plant fresh weight of about 30 g to complete the transition from C3dependent photosynthesis to predominantly CAM assimilation.Thus, it seems unlikely that plants in this study, which weighed only about 11 g at 45 days after transfer to ex vitro conditions (Table 2), would demonstrate significant CAM activity.
In the evaluation at 'D' leaf width, it was unchanged at 15 d after plant transference to acclimatization after which width increased progressively with time to day 45 (Table 1).Plant length was significantly less after 15 and 30 d than it was at day 0, but by day 45 had regained the height at day 0. Aragon et al. (2012) found that stress imposed by water deficit and higher photosynthetic photon flux switched pineapple plants from C3 to CAM behavior.In our results we can find more number of roots, higher fresh weight and photosynthetic activity at 45 d in acclimatization period to report for this author at 56 d in CAM induced pineapple.Maybe, the environmental conditions used in aclimatización process (increase in light intensity and quality of natural light) were decisive factors for it results.
The effects of increment in light intensity and the gradual reduction of relative humidity at early stage were demonstrated in pineapple plantlets (Ananas comosus) by Yanes, González and Rodríguez (2000) and Silva et al. (2008).Batagin et al. (2009) found an increase on cuticle thickness, on wavy contours of epidermal cells, and on the distribution and quantity of mesophyll fibers were observed during acclimatization process, evidencing the light conditions interference in morphological characteristics of the pineapple plantlets.In vitro propagation of pineapple has demonstrated that light is the factor with more influence on plant quality (GONZALEZ et al., 2005) and provides better agronomic and anatomical change during the acclimatization phase (DAMASCENO; SOUZA; GOMES, 2008).
During the acclimatization phase, plantlet increased 1.41 the root number, while stem diameter was 0.32 cm and the plant fresh weight reached 3.25 g.The main cause of fresh weight increase was found in the new emission of root, stem diameter, width of leaves and length of plants  leaves during the experiment.In pineapple, the major drymatter accumulation is associated with a high leaf area index and the ability to maintain their photosynthetic capacity for long periods of time (BARTHOLOMEW;MALÉZIEUX, 1994).However, in pineapple plantlets the fresh weight increased significant during 45 days, presumably due the new environmental conditions and the photoautotrophic behavior of the plantlets.Generally, it is recognized that environmental change during in vitro to ex vitro conditions cause stress in plants, expressed in terms of accumulation of abscisic acid (ABA), proline and reactive oxygen species (CARVALHO et al., 2006;BATROVA;POSPOSILOVA;SYNKOVA, 2008).However, results on in vitro pineapple have shown that it is possible to affect the carbon metabolism by changing the environmental temperature during the light and the dark period (NIEVOLA et al., 2005).Temperature and plant heights also had influences notably in the morphologycal variable in plants cultivated in a greenhouse (DAMASCENO; SOUZA; GOMES, 2008).In our results, apparently, no prolonged stress was observed during acclimatization phase, demonstrated by the growth of the evaluated variables in all process.
After transfer of plantlets to acclimatization condition a sharp and not significant decrease in chlorophyll a and b was observed during the first 15 days (Table 3) and was constant until 45 d.The chlorophyll a and b content continued to decrease until 45 d (8.1 and 3.8 µg g -1 FW respectively).This higher content in chlorophyll in young in vitro leaves is indicative of adaptation to shade.Under these conditions, the light intercepting units are extensive, and higher chlorophyll are associated with higher investment of the plant into light harvesting complex of photosystem II (LHC II), and antenna pigments.This might be explained by a rapid dismantling of the light harvesting antenna to avoid overreduction of the photosynthetic electron chain, which may lead to photoinhibition (BATROVA; POSPOSILOVA; SYNKOVA, 2008).
Chlorophyll a/b ratio showed no significant reduction (0.02 µg g -1 FW) at the times of transfer to different environmental conditions during the first 30 d.This is not in contradiction with the result found by Fila et al. (2006) that recorded high chlorophyll contents in plantlets growth under in vitro condition compared with ex vitro acclimatized.

Table 1 -
Change of morphological variables of pineapple plantlets (Ananas comosus 'MD-2') during acclimatization.Means followed by different letters in rows are significantly different using ANOVA, Duncan's test, p  0.05.
z Number of days after transplantation.

Table 2 -
Change in morphological variables of pineapple plantlets (Ananas comosus 'MD-2') at the initial and final evaluation during acclimatization.

Table 3 -
Change in chlorophyll a, chlorophyll b, and total chlorophyll content in pineapple plantlets during acclimatization phase.Means followed by different letters in rows are significantly different using ANOVA, Duncan's test, p 0.05.
z Number of days after transplantation.