Crude Fermented Extract Containing Gibberellic Acid Produced by Fusarium moniliforme is an Alternative to Cost Reduction in Biofactories

ABSTRACT

Nidularium procerum and Nidularium innocentii (Bromeliaceae) were cultivated in vitro on media supplemented with different sources and levels of GA₃ (gibberellic acid). These sources were the commercial powder (analytical degree) and fermented extract obtained by Fusarium moniliforme via solid state fermentation. The in vitro elongation and rooting of these plants were evaluated after 50 days of cultivation. The GA₃ present in the fermented extract possess the same effect of purified GA₃ (analytical degree) for the increase of the height of aerial part of shoots of N. innocentii, but not for the N. procerum being the GA₃ fermented extract in a lesser degree. The GA₃ fermented extract influences negatively the rooting in N. innocentii, while GA₃ analytical degree practically does not interfere in the rooting. On the other hand, in N. procerum, both the GA₃ sources reduce the root number and do not interfere in rooting percentage. GA₃ crude fermented extract is an alternative to reduce costs, however, its results can vary depending on the species and parameter evaluated. The fermented extract was stored at temperature during 260 days and its shelf life presented a suitable stability, maintaining 92% of its initial GA₃ amount.

Keywords:
Shelf life; bromeliad; gibberellin; solid state fermentation; micropropagation; biofactory

INTRODUCTION

Plant tissue culture is the only methodology that can produce a large quantity of clonal plants in a short time with high phytosanitary quality ^[1]. Therefore, a great number of plant biofactories (i.e., places where clonal plants are produced in great scale using plant tissue culture) are dispersed around the world producing enormous amount of clonal plants. Although these plants are more expensive than the conventional plant production, some advantages encourage their use, such as production during all year (independent of seasons), selected elite genotypes, plants free of diseases and use of reduced spaces. Moreover, the plant tissue culture is an important tool that allows the production of primary and secondary metabolites in environmental controlled conditions, independent of climatic conditions ^[2]. However, there is a great cost to establish and to maintain the operations in these biofactories.

The development of new cheaper methods for in vitro culture are necessary to the biofactories to become more profitable. In this context, some advances have been done, including: (1) automated methods of in vitro culture, using bioreactors ^[3]; which reduce costs due to the use of a liquid culture medium that eliminates the use of agar, that is the most expensive component in the culture medium ^[4] and decreases the amount of labour (2) New culture media using industrial wastewater such as vinasse are used as source of mineral nutrients ^[1,5] (3) The substitution of the conventional sources of the MS culture medium nutrients ^[6] by locally available fertilizers ^[7] (4) Growth room using natural light to culture in vitro plants and (5) the use of macro and micronutrients as precursors for plant growth regulators (e.g., Zinc as precursor of indole-3-acetic acid); (6) alternative methods to produce plant growth regulators, such as jasmonates, gibberellic acid and indole-3-acetic acid ^{[8, 9, 10]} and (7) chemical sterilization methods of the culture medium ^[11] has contributed to decreased costs of production.

Gibberellic acid (GA₃) is a plant hormone that belongs to the group of gibberellins ^[12]. Plants produce low amounts of GA₃. Therefore, commercially, this hormone is produced by microorganisms (i.e., by fermentation) or by chemical synthesis. Nowadays, it is industrially produced by submerged fermentation, but this process results in low yield with high production costs. One alternative to reduce the costs of GA₃ production is the solid-state fermentation (SSF) that allows the use of agro-industrial residues ^[9]. SSF was carried out using Fusarium moniliforme and citric pulp (CP) extract giving 5.9 g GA₃.kg^-1 dry CP after three days of fermentation ^[13]. Nevertheless, the purification process of this molecule elevates the production costs, consequently studies comparing the purified GA₃ with GA₃ fermented extract not purified must be carried out to evaluate the possibility of use these fermented extracts in the more different ways.

The aim of this research was to evaluate the effects of GA₃ fermented extract obtained by SSF and comparing its effects against the synthetic GA₃ (analytical degree) for in vitro elongation and rooting of shoots of two ornamental bromeliads: Nidularium innocentii and Nidularium procerum. The fermented extract stability at room temperature was also studied to analyze its shelf life.

MATERIAL AND METHODS

Gibberellic acid sources

The synthetic GA₃ was a commercial powder (analytical degree ≥ 90%) obtained from Sigma-Aldrich Chemical (USA) and was dissolved with 1N NaOH and solubilized with distilled water. The GA₃ fermented extract was obtained via SSF by Fusarium moniliforme strain LPB 03 using citric pulp supplemented with sucrose as substrate. The production was carried out in column bioreactors (250 mL volume) with forced aeration (30mL.min^-1) with 70% humidity during 5 days. GA₃ was extracted with sodium phosphate buffer (pH 7.4) and filtered. This material was precipitated with 20% ethanol and the supernatant containing GA₃ was not purified ^[13]. Quantitative determination of GA₃ of the crude fermented extract was performed by spectrophotometry at 254 nm absorbance ^[14].

In vitro establishment of bromeliads

The seed disinfection of Nidularium procerum and Nidularium innocentii was performed as proposed by ^[12]. This process consisted of the seed immersion in 70% ethanol (v/v) during one minute, followed by immersion in commercial bleach (1% active chlorine) for 20 min, and rinsed three times with distilled sterilized water. The germination medium was MS ^[6], with 30 g.L^-1 sucrose and solidified with 6 g.L^-1 agar. Seedlings were multiplicated on MS medium supplemented with 30 g.L^-1 sucrose, 2 μM naphthalene acetic acid (NAA), 4 μM 6-benzylaminopurine (BAP) and solidified with 6 g.L^-1 agar.

In vitro elongation and rooting using different GA ₃ sources and levels

Shoots (2 cm height) from clusters (multiple shoots) propagated in vitro were used as explants. Basal medium was composed by MS with 30 g.L^-1 sucrose and solidified with 7 g.L^-1 agar. This experiment was organized in a two-way ANOVA (2 × 3) in which the factor A consisted of two sources of GA₃ (analytical degree and fermented extract) and factor B consisted of GA₃ levels which were 0, 4.1 and 8.2 μM. The root number, rooting percentage, height of the aerial part (cm), leaf number, lateral shoot number, fresh weight (g) and lateral shoot percentage were evaluated after 50 days of in vitro culture. It was used 1.5 mL.L^-1 and 3.0 mL.L^-1 of the GA₃ fermented extract that corresponds to the levels 4.1 and 8.2 µM GA₃, respectively. All GA₃ solutions were sterilized by microfiltration (0.22 μm) and added to cooled autoclaved media at about 60^oC inside the laminar flowhood.

Stability of the GA ₃ present in fermented extract at room temperature

A sample of GA₃ fermented extract was collected after the fermentation and placed in 5 mL tubes. These tubes were sealed with thread caps and stored at room temperature in the dark. These tubes were used to quantify weekly the GA₃ (present in fermented extract) until 260 days. The GA₃ content was determined according to methodology described by ^[14]. The data were expressed in percentage relative to the GA₃ initial amount, which was considered as 100%.

Culture conditions and statistical analysis

The pH of all media was adjusted to 5.8 and then were autoclaved at 1.1 kg.cm^-2 and 121ºC for 20 min. The cultures were kept at 25 ± 2ºC under white fluorescent light (28 μmol m^-2 s^-1) with a 16 h photoperiod. The experimental design was completely randomized in a factorial arrangement (two-way) with five replicates of five explants. The data was submitted in a normality analysis for the Lilliefors´s test, and submitted to the analysis of variance (ANOVA) followed by Duncan’s test at a P<0.05. All statistical analyses were done following the procedures of the software SOC ^[16]. Variables from counting were transformed to $\sqrt{\text{x}+0.5}$ and variables from percentage were transformed to arcsin$\sqrt{\text{x}/100}$.

RESULTS AND DISCUSSION

Effects on N. innocentii

The best result for the height of the aerial part was obtained in the presence of GA₃, nevertheless, the levels of 4.1 and 8.2 did not differ statistically. However, there were no statistical differences between the type of sources of GA₃ (Table 1). Similar results were observed for the same species, but cultivated in double-phase medium using GA₃ (analytical degree), wherein the best result for this species was 7.3 cm of height obtained at 8.2 µM ^[15]; due to the culture in the double-phase medium promotes a higher growth of the plants. This difference can be due to the higher ability of absorption. This larger absorption can be attributed to the lack of physical barriers in the culture medium, which increases the explant contact with nutrients, plant growth regulators and water whereby it usually increases the in vitro growth ^[17].

Table 1
Characteristics of Nidularium innocenti i cultivated on media supplemented with different sources of GA₃ (analytical degree and fermented extract) and levels of GA₃ after 50 days of in vitro culture.

	Root number				Rooting percentage
GA3	Gibberellic acid source				Gibberellic acid source
(µM)	GA3 (AD)1	GA3 (FE)	Mean		GA3 (AD)	GA3 (FE)	Mean
0	5.4 abA2	5.2 aA	5.3		100 aA	100 aA	100
4.1	5.0 bA	2.7 bB	3.8		96 aA	72 bB	84
8.2	5.7 aA	1.4cB	3.5		100 aA	41.6 cB	70.8
Mean	5.4	3.1			98.6	71.2
	Height of the aerial part (cm)				Leaf number
GA 3	Gibberellic acid source				Gibberellic acid source
(µM)	GA 3 (AD)	GA 3 (FE)	Mean		GA 3 (AD)	GA 3 (FE)	Mean
0	4.4	5.1	4.7 b		16.9 aA	16.5 aA	16.7
4.1	5.1	5.7	5.4 a		10.7 bB	14.3 bA	12.5
8.2	5.6	5.7	5.6 a		11.0 bB	13.3 cA	12.1
Mean	5.0 A	5.5 A			12.8	14.5
	Number of lateral shoots per explant				Fresh weight (g)
GA 3	Gibberellic acid source				Gibberellic acid source
(µM)	GA 3 (AD)	GA 3 (FE)	Mean		GA 3 (AD)	GA 3 (FE)	Mean
0	2.0 aA	2.1 aA	2.0		0.476	0.354	0.415
4.1	0.7 bA	0.9 bA	0.8		0.318	0.437	0.377
8.2	0.6 bA	0.5 cA	0.5		0.340	0.304	0.322
Mean	1.1	1.1			0.378	0.365
	Lateral shoots (%)
GA 3	Gibberellic acid source
(µM)	GA 3 (AD)			GA 3 (FE)		Mean
0	88 aA			88 aA		88
4.1	53 bA			40 bA		46.5
8.2	48 bA			61.2 bA		54.6
Mean	63			63

• ¹
  Source of gibberellic acid: AD = Analytical degree GA₃ and FE = Fermented extract containing GA₃.
• ²
  Treatments followed by same lower case letters in the same column and same capital letters in the line do not differ significantly (P<0.05) by Duncan´s test.

Major rooting percentage of tissues was verified in the absence of GA₃ (0 µM), however there were no statistical differences among the levels using the analytical degree GA₃ source, nevertheless higher levels of GA₃ present in the fermented extract decreased the rooting percentage (Table 1). The best results for the root number were obtained in the absence of GA₃ (0 µM) and 8.2 µM GA₃ (analytical degree). The presence of GA₃ (crude fermented extract) in the media decreased the root number (Table 1). Similar result was found in Lavandula angustifolia cultivated on GA₃ fermented extract; however, this other GA₃ fermented extract was partially purified; this lower rooting was attributed to the presence of impurities that remained in the fermented extract even after the partial purification ^[9]. Different species had presented different behavior related to root number when they were cultured with different levels of GA₃. In potato (Solanum tuberosum), a concentration of 0.248 mg.L^-1 GA₃ had doubled the root number per plantlet (5.6 roots per plant) when compared to control (2.3 roots per plant) ^[18]. But bromeliads, normally root in culture medium free of plant growth regulators, such as in Dyckia macedoi ^[19], D. agudensis ^[20], D. maritima ^[21], Vriesea scalaris ^[22] and Orthophytum mucugense ^[23]. The main use of GA₃ in bromeliads is to promote the shoot elongation to easy the manipulation due to little size of explants in multiple shoots (clusters).

The concentration of the fermented extract containing GA₃ seems also to have an impact over the inhibition of root induction as observed in the reduction of rooting percentage and root number in N. innocentii, it was used 1.5 mL.L^-1 and 3.0 mL.L^-1 of GA₃ fermented extract that corresponds to 4.1 and 8.2 µM GA₃, respectively. The increase in the levels of unknown compounds present in this fermented extract has an inhibitory effect on the rooting, as also was demonstrated in L. angustifolia, which high concentrations inhibited completely the rooting (0%), at level of 31.2 mL.L^-1 that corresponded to 1.0 mg.L^-1 GA₃ ^[9]. However, the GA₃ level (analytical degree) did not influence the rooting percentage (Table 1).

The GA₃ presence on the culture medium, independent of the GA₃ source and level used; decreased the percentage of lateral shoots and number of lateral shoots.The highest value for percentage of lateral shoots and number of lateral shoots per explant was observed in a culture medium free of gibberellic acid (0 µM GA₃) (Table 1). However, when the aim is to promote the elongation and rooting, normally the lateral shoots are undesirable. Thus, the lowest values for percentage of lateral shoots were observed in a culture medium supplemented with 4.1 µM GA₃ (fermented extract) and 8.2 µM GA₃ (analytical degree) (Table 1). The lowest values for number of lateral shoots were observed in a culture medium supplemented with 8.2 µM GA₃ (fermented extract and analytical degree). The lateral shoots produced in this phase of elongation and rooting is a result from prior multiplication phase due to cytokinins presence ^[21], whereas cytokinins are primarily responsible for breaking apical dominance and consequent lateral shoot induction ^[24].

The GA₃ levels of both sources (analytical degree and fermented extract) decreased the leaf number (Table 1). Different results for leaf number were found in potato (Solanum tuberosum) where the concentration of 0.248 mg.L^-1 of GA₃ increased the leaf number (7.3) as compared to control (3.3) ^[18]. These results found in bromeliads possibly are associated with a larger energetic spend used for elongation of the aerial part of the plant, which results in a decrease of roots, leaves and shoots. The best result for leaf number was obtained at 0 µM GA₃ (analytical degree and fermented extract) (Table 1). The leaf number per explant is a characteristic normally correlated to shoot number per explant and a great number of leaf number per explant can be important when leaf explants were used to induce new shoots or calli, as some micropropagation or somatic embryogenesis protocols were established, as is the case of Cryptanthus sp. ^[25], Neoregelia cruenta ^[26], Ananas comosus ^[27] and Vriesea scalaris ^[22].

The GA₃ level and sources (analytical degree and crude fermented extract) did not influence the fresh weight (Table 1). Different results were obtained for Lavandula angustifolia cultivated in vitro with different GA₃ sources (analytical degree or purified partially fermented extract). In this case, there was a decrease in the fresh weight proportionally to the increase of the GA₃ level. Nevertheless, there was no difference between the GA₃ sources ^[9].

Effects on N. procerum

The best result for the height of the aerial part was obtained in the presence of GA₃, nevertheless, the levels of 4.1 and 8.2 did not differ statistically; however, there were statistical differences between the type of source of GA₃, being the GA₃ analytical degree superior than GA₃ fermented extract (Table 2). N. procerum seems to be more sensitive than N. innocentii to certain compounds present in fermented extract, what influences on the height of the aerial part. Nevertheless, according to this result it is possible that different species possess different answers to GA₃ crude fermented extract, presenting results completely or partially suitable.

Table 2
Characteristics of Nidularium proceru m cultivated on media supplemented with different sources of GA₃ (analytical degree and fermented extract) and levels of GA₃ after 50 days of in vitro culture.

	Root number				Rooting percentage
GA3	Gibberellic acid source				Gibberellic acid source
(µM)	GA3 (AD)1	GA3 (FE)	Mean		GA3 (AD)	GA3 (FE)	Mean
0	3.8 aA2	3.8 aA	3.8		92	96	94
4.1	2.4 bA	2.3 bA	2.3		95	96	95.5
8.2	2.4 bA	2.2 bA	2.3		88	88	88
Mean	2.8	2.7			91.6 A	93.3 A
	Height of the aerial part (cm)				Leaf number
GA3	Gibberellic acid source				Gibberellic acid source
(µM)	GA 3 (AD)	GA 3 (FE)	Mean		GA 3 (AD)	GA 3 (FE)	Mean
0	4.9	4.8	4.8 b		16.1	16.9	16.5
4.1	7.7	6.3	7.0 a		10.5	14.9	12.7
8.2	7.8	6.2	7.0 a		11.6	15.9	13.7
Mean	6.8 A	5.7 B			12.7 B	15.9 A
	Number of lateral shoots per explant				Fresh weight (g)
GA3	Gibberellic acid source				Gibberellic acid source
(µM)	GA 3 (AD)	GA 3 (FE)	Mean		GA 3 (AD)	GA 3 (FE)	Mean
0	1.8	2.2	2.0		0.227	0.207	0.217 a
4.1	0.8	2.4	1.6		0.189	0.189	0.189 a
8.2	0.8	2.6	1.7		0.212	0.197	0.204 a
Mean	1.1 B	2.4 A			0.209 A	0.197 B
	Lateral shoots (%)
GA3	Gibberellic acid source
(µM)	GA 3 (AD)			GA 3 (FE)			Mean
0	68			65.5			66.7
4.1	44			77			60.5
8.2	56			65.5			60.7
Mean	56 B			69.3 A

• ¹
  Source of gibberellic acid: AD = Analytical degree GA₃ and FE = Fermented extract containing GA₃.
• ²
  Treatments followed by same lower case letters in the same column and same capital letters in the line do not differ significantly (P<0.05) by Duncan´s test.

The best result for the root number was obtained in the absence of GA₃ (0 µM). The presence of GA₃ in the medium decreased the root number. GA₃ sources did not influence the root number in N. procerum (Table 2). The rooting of N. procerum was not inhibited by impurities present in the fermented extract.

The percentage of lateral shoots varied from 88 to 96% (Table 2). The GA₃ sources presented statistical differences, being the fermented extract superior to analytical degree. The highest value for the leaf number per explant was observed in a culture medium free of gibberellic acid (16.9 leaves per explant). However, there was no statistical difference in GA₃ levels, but there was statistical difference in GA₃ sources, being the fermented extract superior to analytical degree (Table 2).

The fresh weight did not present significant difference for GA₃ levels (Table 2). Similar result was observed in nodular cultures of Vriesea reitzii cultivated on MS medium supplemented with 5 or 10 µM GA₃ ^[28]. However, the GA₃ sources presented statistical difference, being the analytical degree superior to fermented extract (Table 2). Micropropagation has been employed to enhance the mass production, quality, and sterilized condition of the targeted plant ^[29]. Nevertheless, a great yield in biomass is desired when the aim of the work is to produce primary or secondary metabolites; moreover, micropropagated plants with larger biomass can promote higher survival rates during the acclimatization.

The percentage of lateral shoots and number of lateral shoots per explant presented statistical difference in the GA₃ sources, being the fermented extract superior than analytical degree (Table 2). This result can be interesting in the case which the multiplication is desired. Nevertheless, it is known that cytokinins are the the most indicated plant growth regulators for promotion of shoot multiplication as demonstrated in innumerous studies performed in bromeliads, such as Vriesea scalaris ^[22], Nidularium procerum and Nidularium innocentii ^[15], Aechmea blanchetiana and Aechmea distichantha ^[30] and Neoregelia concentrica ^[31]. Moreover, the use of the GA₃ present in the fermented extract to induce multiplication could be a good alternative to cytocynins case the relation cost/benefit be advantageous, considering wheter there is cost reduction.

Stability of the GA ₃ present in fermented extract at room temperature

Stability tests were performed at room temperature with GA₃ fermented extract to test the possibility to produce and commercialize a liquid extract. GA₃ concentration remained constant till the 14^th day of storage. After this time, there was a slight decline in the amount of GA₃ reaching 92% that was stable during 260 days of storage. The GA₃ stability of the fermented extract produced and tested in the present study is considered satisfactory, with a loss of activity of less than 10% after 260 days at room temperature. Furthermore any stabilizers or additives could be added to the extract, which could further enhance the storage life of the product.

CONCLUSIONS

The GA₃ present in the fermented extract possess the same effect of purified GA₃ (analytical degree) for the increase of the height of aerial part of shoots of N. innocentii, but not for the N. procerum. The GA₃ fermented extract influences negatively the rooting in N. innocentii, while GA₃ analytical degree practically does not interfere. On the other hand, in N. procerum, both the GA₃ sources reduce the root number and do not interfere in rooting percentage. GA₃ crude fermented extract is an alternative to reduce costs replacing the GA₃ analytical degree (i.e., most expensive), however, its results can vary depending of the species and parameter evaluated. The fermented extract storage was at room temperature during 260 days and its shelf life presented a suitable stability, maintained 92% GA₃ initial amount.

ACKNOWLEDGEMENTS

Authors thank Capes (Coordination for the Improvement of Higher Level -or- Education - Personnel), CNPq (the National Council for Scientific and Technological Development) and Fundação Araucária for financial support and scholarships, which enabled the development of this research.

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  Martins JPR, Schmildt ER, Alexandre RS, Castro EM, Nani TF, Pires MF, et al. Direct organogenesis and leaf-anatomy modifications in vitro of Neoregelia concentrica (Vellozo) L. B. Smith (Bromeliaceae). Pak. J. Bot. 2014; 46(6): 2179-2187.

Publication Dates

• Publication in this collection
  2018

History

• Received
  16 Mar 2017
• Accepted
  26 June 2018