EFFECT OF SUCROSE, INORGANIC SALTS, INOSITOL, AND THIAMINE ON PROTEASEEXCRETION DURING PINEAPPLE CULTURE IN TEMPORARY IMMERSION BIOREACTORS

A. PEREZ*, L. NAPOLES, C. CARVAJAL, M. HERNANDEZ, AND J. C. LORENZO

Bioplant Centre, University of Ciego de Avila, 69450, Cuba

(Received 9 June 2003; accepted 6 November 2003; editor H. M. Schumacher)

Summary

Although pineapple plants have been found to produce proteases ex vitro, most of the biotechnological investigations on

this crop have been focused on propagation. The procedure involving the use of temporary immersion bioreactors is one of

the most outstanding because of its high multiplication rate. We previously recorded specific protease activity in the

culture medium during the pre-elongation step of this protocol. Therefore, we decided to modify the culture medium

composition of this phase looking for an increase in protease excretion. Four independent experiments were performed to

evaluate the effects of different levels of sucrose (0–350.4 mM), inorganic salts [0–200% Murashige and Skoog (MS) salt

strength], inositol (0–2.20 mM), and thiamine (0–1.2mM). The following indicators were recorded: shoot fresh mass per

bioreactor; and protein concentration, proteolytic activity, and specific protease activity in culture media. Specific protease

activity, the most important indicator recorded, was highest with 262.8 mM sucrose, 100% MS salt strength, 0.3mM

thiamine and no inositol. Results shown here demonstrate that conditions adequate for propagation purposes (87.6 mM

sucrose, 100% MS salt strength, 0.55 mM inositol, 0.3mM thiamine) are not always adequate for protease excretion.

Key words: Ananas comosus (L.) Merr.; micropropagation; proteases; pineapple; bioreactor.

Introduction

Pineapple plants have been described as sources of proteases

(Apte et al., 1979), and they are frequently used in medicine and

the food industry. Proteases have been used for treatment of cancer

(Batkin et al., 1988; Florez, 1995; Targoni et al., 1999), digestion

disorders (Kelly, 1996), viral diseases (Ransberger and Stauder,

1993; Kleef et al., 1996), swelling and immune-modulation

problems (Melis, 1990; Lotti, 1993; McBrige, 1999; Metzig et al.,

1999; Engwerda et al., 2001; Leipner et al., 2001). Moreover,

proteases are regarded as a food complement (Kleef et al., 1996;

Losada, 1999; La Valle et al., 2000) to soften meats and dehydrated

eggs (Miller, 1982; Lawrie, 1985; Bailey and Light, 1989).

Proteases are also frequently used for culture medium formulation

(Headon and Walsh, 1994). However, most of the in vitro research

on pineapple has been focused on propagation.

As one case in point, a procedure by Escalona et al. (1999)

involves the use of temporary immersion bioreactors. Such a

protocol includes three in vitro steps: shoot multiplication (45 d),

pre-elongation (14 d), and elongation (14 d); and seems to be the

most efficient protocol for pineapple shoot proliferation described to

date. Multiplication rates are higher than 1:60 while other protocols

only reach 1:8. We previously found that pineapple shoots excrete

proteases into the culture medium during the pre-elongation phase

of Escalona et al.’s (1999) procedure (Hernandez et al., 1999).

Therefore, we carried out an optimization of growth stage and

hormone content to increase protease excretion (Perez et al., 2003).

However, a variation of levels of other chemical compounds of the

culture medium, such as sucrose, inorganic salts, inositol, and

thiamine, remained to be tested.

Researchers investigating secondary metabolite formation have

shown that these medium components have an influence on

metabolism. Zenk et al. (1984), Igbavboa et al. (1985), Sato et al.

(1991), Zhang et al. (1997), Xu et al. (1998), and Verma et al.

(2002) have tested the effect of carbohydrate type and levels. Do

and Cormier (1991), Hanagata et al. (1992, 1993), Fang et al.

(1998), Gao et al. (2000), Massot et al. (2000), and Trejo-Tapia et al.

(2003) have studied the role of inorganic salts. Sepehr and

Ghorbanli (2002) have experimented with different concentrations

of inositol and vitamins.

Regarding pineapple proteases, we are not aware of any other

research group investigating their production under in vitro

environment. We have only found the report by Apte et al. (1979)

about proteolytic enzymes detected in callus cultures and shoot

leaves of Ananas sativus, a relative of the Ananas comosus (L.) Merr

commercial pineapples. Apte et al.’s (1979) report was limited to

the evaluation of the influence of culture age on production of

proteases, and the effects of variations of the chemical

microenvironment were not studied. Therefore, the present research

was conducted in order to test if different levels of sucrose,

inorganic salts, inositol, and thiamine affect protease excretion

during pineapple plant culture in temporary immersion bioreactors.

To our knowledge, this kind of study has not been reported to date.*Author to whom correspondence should be addressed: Email aperez@

bioplantas.cu

In Vitro Cell. Dev. Biol.—Plant 40:311–316, May–June 2004 DOI: 10.1079/IVP2004529q 2004 Society for In Vitro Biology1054-5476/04 $18.00+0.00

311

Materials and Methods

Plant material. Pineapple buds (cv. MD2) were collected from field-grown plants and cultured following the protocol by Daquinta and Benegas(1997). Explants were placed in conventional plant containers (300 ml) formicropropagation where 25 ml of liquid culture medium fed five explants.The culture medium included MS (Murashige and Skoog, 1962) salts,0.55 mM inositol, 0.3mM thiamine-HCl, 87.6 mM sucrose, 4.4mM 6-benzyladenine (BA), and 5.3mM naphthaleneacetic acid (NAA). Youngpineapple shoots were transferred to the multiplication culture medium (asdescribed above except 9.3mM BA and 1.6mM NAA) after 45 d ofimplantation. Shoots proliferated for 6 mo. and were subcultured at 45-dintervals. Such shoots were placed in temporary immersion bioreactors (inaccordance with Escalona et al., 1999) to carry out the four experimentsshown here.

Effect of sucrose on pineapple shoot fresh mass and protease excretionduring culture in temporary immersion bioreactors. This experimentconsisted of five treatments (0.0, 87.6, 175.2, 262.8, and 350.4 mMsucrose). The basal medium included 4.2mM gibberellic acid (GA), 100%MS salt strength, 0.55 mM inositol, and 0.3mM thiamine-HCl.

Effect of inorganic salts on pineapple shoot fresh mass and proteaseexcretion during culture in temporary immersion bioreactors. Thisexperiment consisted of five treatments (0, 50, 100, 150, and 200% MSsalt strength). The basal medium included 4.2mM GA, 262.8 mM sucrose,0.55 mM inositol, and 0.3mM thiamine-HCl.

Effect of inositol on pineapple shoot fresh mass and protease excretionduring culture in temporary immersion bioreactors. Five treatments werecompared (0.00, 0.55, 1.10, 1.65, and 2.20 mM inositol). The basal mediumincluded 4.2mM GA, 262.8 mM sucrose, 100% MS salt strength, and 0.3mMthiamine-HCl.

Effect of thiamine on pineapple shoot fresh mass and protease excretionduring culture in temporary immersion bioreactors. Five treatments werecompared (0.0, 0.3, 0.6, 0.9, and 1.2mM thiamine-HCl). The basal mediumincluded 4.2mM GA, 262.8 mM sucrose, and 100% MS salt strength.

Experimental conditions. Cultures were maintained at 25 ^ 18C with30mE m22 s21 (white fluorescent light) and an 8 h photoperiod. Shoot freshmass per bioreactor, protein concentration, proteolytic activity, and specificproteolytic activity in the culture media were measured at 21 d of culture.

Determination of protein content and protease activity. Culture mediumwas filtered and concentrated 10 times (dialysis, polyethylene glycol 8000)for biochemical evaluations. Protein content was determined in accordancewith Lowry et al. (1951). Absorbency (650 nm) was recorded and a standardcurve for bovine serum albumin was used. Proteolytic activity was measuredin accordance with Anson (1938). Hemoglobin was used as a substrate (2%,

pH 6.8). The reaction mixture contained 500ml of hemoglobin solution and100ml of concentrated culture medium. Reaction was allowed for 20 min(378C), then stopped with 1.0 ml trichloroacetic acid (5%). Supernatant wastested with Folin–Ciocalteau reagent. Absorbency (650 nm) was measuredand a tyrosine standard curve was used. Enzyme activity was defined as Ug21 plant fresh mass [U ¼ enzyme quantity hydrolyzing 1mmol tyrosine permin (378C)]. Specific activity was calculated as the rate of proteolytic activityrelative to protein content.

Statistical analysis. All experiments were repeated three times. Theexperimental design was completely randomized. The Statistical Package forSocial Sciences (Version 8.0 for Windows, SPSS Inc.) was used. Intervalsaverage ^ standard error (SE) were calculated.

Results and Discussion

Our previous research (Perez et al., 2003) and the present one

were conducted in a consecutive manner. This means, for instance,

that the best treatment in terms of specific protease activity of the

first experiment was used in the rest of the experimental program,

and so on. Therefore, modification of growth stage from 14 to 21 d in

our previous research increased specific protease activity in the

culture medium from 0.049 to 0.064 U mg21 protein (from 0.196 to

0.259 U l21 culture medium). The use of 4.2mM GA instead of

2.8mM elevated specific activity to 0.076 U mg21 protein

(0.307 U l21 culture medium). On the other hand, removal of

2.2mM BA from the culture medium increased specific activity to

0.088 U mg21 protein (0.352 U l21 culture medium).

In the experiments shown here, sucrose at 87.6 and 175.2 mM

increased pineapple shoot fresh mass (g per bioreactor; Fig. 1A).

Protein content increased to the highest sucrose level tested

(350.4 mM sucrose; Fig. 1B). Maximum proteolytic and specific

proteolytic activities were recorded in the culture medium

supplemented with 262.8 mM sucrose (Fig. 1C, D).

Sucrose is used in plant cell, tissue, and organ culture for two

main purposes: as a carbon source and as a regulator of cell osmosis

(George, 1993). Because of low carbon dioxide and light supplies,

most in vitro cultures are autotrophic-incompetent. They are not

able to proliferate properly without an exogenous supply of

FIG. 1. Effect of sucrose on shoot fresh mass (A), protein content (B), proteolytic activity (C), and specific activity (D) in the culturemedium during pineapple culture in temporary immersion bioreactors. Vertical bars represent average ^ SE.

312 PEREZ ET AL.

carbohydrates (Cournac et al., 1991; Debergh and Zimmerman,

1991; Kotvun and Daie, 1995; Desjardins et al., 1996; Pospısilova

et al., 1997; Ticha et al., 1998). On the contrary, very high sucrose

levels cause cell dehydration, increase in endogenous abscisic acid

content (Zeevaart and Creelmann, 1988; Meurs et al., 1992), and

therefore, a reduction of proliferation (Pierik, 1990). In our

experiment, we also observed this effect. The increase in shoot fresh

mass (g per bioreactor) was lower when 0 or 350.4 mM sucrose was

used (Fig. 1A).

The most important indicator recorded in this research was the

specific proteolytic activity, which is typical of investigations on

protease production (Chavez et al., 1990). We observed the

optimum specific protease activity in the culture medium

supplemented with 262.8 mM sucrose (Fig. 1D). This concentration

is much higher than Escalona et al.’s (1999) formulation (87.6 mM

sucrose) and caused a reduction of shoot fresh mass (g per

bioreactor; Fig. 1A). Therefore, pineapple shoots might have been

under osmotic stress with 262.8 mM sucrose. For other plants, a

relationship has been found between secondary metabolites and

stress. Sato et al. (1991) recommended sucrose at 350.4 mM for

anthraquinone production by Rubia tinctorium cultures. Xu et al.

(1998) reported that sucrose at 292.0 mM had a positive effect on

salidroside synthesis by Rhodiola sachalinensis calluses. Gao et al.

(2000) suggested supplying sucrose in a range between 233.6 and

350.4 mM to obtain red and yellow pigments in Carthamus

tinctorius cell cultures. Moreover, enzymes like proteases have been

found to occur in response to stress (Beers et al., 2000) as has been

demonstrated also for secondary metabolites.

However, it is important to note that excessive stress with

350.4 mM sucrose was detrimental for protease production by

pineapple cultures in temporary immersion bioreactors (Fig. 1D).

Sucrose at 350.4 mM caused a dramatic increase in protein levels in

the culture medium (Fig. 1B) but the proteolytic activity was reduced

(Fig. 1C). Many other proteins and enzymes, not proteases, have

been found to occur in response to extreme stress [e.g., glucanases,

peroxidases, invertases (George, 1993), esterases, amylases (Aviles

et al., 1994)].

Pineapple shoot fresh mass (g per bioreactor) and protein content

in the culture medium were not modified by variations in the

strength (0–200%) of MS inorganic salts (Fig. 2A, B). However, a

reduction in proteolytic activity in the culture medium was observed

when salt concentration was reduced (Fig. 2C). The highest specific

protease activity was recorded in the culture medium supplemented

with 100% MS salt strength (Fig. 2D).

Inorganic salts are supplied exogenously in most in vitro culture

systems. It is believed they are necessary, among other functions, to

form proteins (e.g., NO32, NH4

þ), to control water movement (e.g.,

Kþ), to be part of the energetic mechanism (e.g., PO432), to be

included in the cell wall architecture (e.g., Ca2þ), and to activate

enzymes (e.g., Mg2þ, Mn2þ) (Salisbury and Ross, 1992).

Considering this, we did not expect the results shown in Fig. 2A.

A relationship between MS salt strength and pineapple shoot

proliferation (g per bioreactor) was not observed. Even total removal

of inorganic salts did not cause statistically significant differences

with respect to other treatments.

An explanation of this result could be in the culture conditions of

the plant material before the experiment. Pineapple shoots were

always cultured in accordance with Daquinta and Benegas (1997).

This protocol includes an MS inorganic salt composition that is one

of the most enriched formulations for in vitro culture. Therefore,

pineapple shoots used in this experiment might have contained

large amounts of endogenous inorganic salts. Such endogenous

accumulation might have allowed them to survive without any

exogenous supply after establishing the experiment. Results

obtained with 200% MS salt strength were not expected either.

This highly concentrated treatment did not seem to have caused

pineapple shoot osmotic stress because fresh mass (g per bioreactor)

and protein content were not affected (Fig. 2A, B). Pineapple plants

in vitro seem to be as insensitive to salt pressure as in vivo. Field

studies have found that pineapple tolerates large amounts of

FIG. 2. Effect of MS inorganic salts on shoot fresh mass (A), protein content (B), proteolytic activity (C), and specific activity (D) in theculture medium during pineapple culture in temporary immersion bioreactors. Vertical bars represent average ^ SE.

EFFECTS ON PROTEASE EXCRETION IN PINEAPPLE 313

mineral fertilizers without affecting its agronomic performance

(Pena et al., 1998). However, further repetitions of this experiment

might produce significant differences among treatments. In contrast,

specific proteolytic activity depended on the strength of the MS salts

and the maximum value was obtained at 100% (Fig. 2D).

Inositol increased up to 2.20 mM affected pineapple shoot fresh

mass (g per bioreactor; Fig. 3A). Inositol at 1.10 mM increased

protein content (compared to 2.20 mM inositol; Fig. 3B) and

proteolytic activity in the culture medium (Fig. 3C). However,

inositol did not increase specific proteolytic activity (Fig. 3D).

Inositol has been described as a natural constituent of plants

(Balla, 1998) involved in cell membrane permeability (Loewus and

Murthy, 2000; Stevenson et al., 2000). It stimulates cell division

when added at low concentrations to the culture medium (George,

1993). However, calcium–inositol and ferrous inositol complexes

are formed in culture media including high levels of inositol.

Uptake of these complexes by plants is very difficult (McArdle,

2003) and, therefore, osmotic potential of the culture medium is

increased (Drøbak and Watkins, 2000). Such a high osmotic

potential limits plant biosynthesis processes and growth (Zhu,

2001). In our experiment, we also observed a negative effect of very

high levels of inositol (higher than 1.10 mM; Fig. 3).

Thiamine in the range of 0.3–1.2mM reduced pineapple shoot

fresh mass (g per bioreactor; Fig. 4A). The highest protein content in

the culture medium was recorded when 0.6mM thiamine was added

(Fig. 4B). On the contrary, maximum proteolytic and specific

FIG. 3. Effect of inositol on shoot fresh mass (A), protein content (B), proteolytic activity (C), and specific activity (D) in the culturemedium during pineapple culture in temporary immersion bioreactors. Vertical bars represent average ^ SE.

FIG. 4. Effect of thiamine on shoot fresh mass (A), protein content (B), proteolytic activity (C), and specific activity (D) in the culturemedium during pineapple culture in temporary immersion bioreactors. Vertical bars represent average ^ SE.

314 PEREZ ET AL.

proteolytic activities were measured when the culture medium

included 0.3mM thiamine (Fig. 4C, D).

Thiamine has been reported as an essential factor in the

biosynthesis of some amino acids (Miernyk et al., 1987) and in

carbohydrate metabolism (George, 1993). Regarding supplemen-

tation of thiamine to the culture medium, Ikeda et al. (1979) stated

that exogenous supply was not necessary as cells habituated to

thiamine and its precursors. On the contrary, Barwale et al. (1986)

reported that increasing the thiamine level up to 5.04mM was

convenient for some in vitro culture systems. In our experiment, an

exogenous supply of thiamine reduced pineapple shoot fresh mass

(g per bioreactor; Fig. 4A). Shoots cultured without thiamine seemed

to be habituated and, therefore, they may have synthesized the

thiamine level required. Consequently, an exogenous supply (0.3–

1.2mM thiamine) might have resulted in an overdose. In contrast,

an exogenous supply of thiamine increased the protein content, and

proteolytic and specific proteolytic activities in the culture medium

(Fig. 4B–D).

To produce pineapple proteases, in terms of specific proteolytic

activity, results shown here prompted us to modify sucrose and

inositol levels of Escalona et al.’s (1999) pre-elongation phase

culture medium. Sucrose was increased from 87.6 mM to 262.8 mM

because this modification allowed elevation of the specific protease

activity from 0.088 to 0.207 U mg21 protein (from 0.352 to

0.829 U l21 culture medium; Fig. 1D). Inositol was removed as no

effect was observed in the range of 0–1.10 mM (Fig. 3D). On the

contrary, MS inorganic salt strength (100%) and thiamine level

(0.3mM) were not changed as Escalona et al.’s (1999) formulation

caused the highest specific proteolytic activity in the culture

medium (Figs. 2D, 4D; about 0.207 U mg21 protein and 0.827 U l21

culture medium). In brief, experiments shown in our previous paper

(Perez et al., 2003) and in the present one allowed an increase in

specific protease activity in the culture medium from 0.049 to

0.207 U mg21 protein (from 0.196 to 0.829 U l21 culture medium,

422.4%). Other factors, such as genotype and physical micro-

environment, remain to be tested and they are being studied in our

laboratory in continuing investigations.

Acknowledgments

This research was partially supported by the Third World Academy ofSciences and the Cuban Ministry for Science, Technology and Environment.The authors are grateful to Ms. Monifah Telesford (Saint Vincent and theGrenadines) for her critical reading of the manuscript.

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