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Agronomía Colombiana

Print version ISSN 0120-9965

Agron. colomb. vol.38 no.3 Bogotá Dec. 2020  Epub June 01, 2021

https://doi.org/10.15446/agron.colomb.v38n3.86430 

Scientific note

Ginkgo biloba L. mini-cuttings: indole butyric acid, substrates, and biochemical composition of the mother plants

Miniestacas de Ginkgo biloba L.: ácido indolbutírico, substratos y composición bioquímica de las plantas madre

Renata de Almeida Maggioni1 

Leandro Porto Latoh1 

Leandro Marcolino Vieira1  * 

Emilio Romanini Netto2 

Katia Christina Zuffellato-Ribas1 

1 Programa de Pós-graduação em Agronomia (Produção Vegetal), Universidade Federal do Paraná (UFPR), Curitiba, Parana (Brazil).

2 Programa de Pós-graduação em Meio Ambiente e Desenvolvimento, Universidade Federal do Paraná (UFPR), Curitiba, Parana (Brazil).


ABSTRACT

The objective of this study was to evaluate the viability of the Ginkgo biloba mini-cutting technique, as well as the influence of substrates and different concentrations of indole butyric acid (IBA) on adventitious rooting in addition to the protein and sugar content in the mini-cutting. Mini-cuttings were 4 ± 1 cm in length, with the bases immersed in solutions of 0, 1000, 2000, and 3000 mg L-1 IBA. They were then planted in polypropylene tubes using two substrates (vermiculite and Tropstrato®) and maintained under greenhouse conditions for 60 d. The experiment was carried out with a 2 x 4 factorial scheme (substrates x IBA). There was no influence of IBA application on the promotion of rhizogenesis in Ginkgo biloba mini-cuttings. The rooting percentages were higher than 55% regardless of the treatment used. The vermiculite substrate showed a higher number of roots (4.94) and lower mortality (11.60) of mini-cuttings than Tropstrato®. We conclude that the mini-cutting technique is feasible for Ginkgo biloba, and the use of IBA is not necessary. We found that the induction of adventitious rooting depended on the biochemical composition of the mother plants, due to the translocation of non-reducing sugars and leaf proteins for root formation.

Key words: vegetative propagation; medicinal plants; plant growth regulators; medicinal plant cloning; rooting

RESUMEN

El objetivo de este estudio fue evaluar la viabilidad de la técnica de miniestacas de Ginkgo biloba, así como la influencia de diferentes sustratos y diferentes concentraciones de ácido indolbutírico (IBA) en la formación de raíces adventicias además del contenido de proteínas y azúcares en las miniestacas. Las miniestacas tenían 4 ± 1 cm de longitud, y sus bases se sumergieron en soluciones de 0, 1000, 2000 y 3000 mg L-1 IBA. Luego las miniestacas se plantaron en tubos de polipropileno usando dos sustratos (vermiculita y Tropstrato®), bajo condiciones de invernadero durante 60 d. El experimento se llevó a cabo en un esquema factorial 2 x 4 (sustratos x IBA). No hubo influencia de la aplicación de IBA en la promoción de la rizogénesis de las miniestacas de Ginkgo biloba. Los porcentajes de enraizamiento fueron superiores al 55%, independientemente del tratamiento utilizado. El sustrato de vermiculita mostró un mayor número de raíces (4.94) y menor mortalidad (11.60) de miniestacas que el Tropstrato®. Por lo tanto, se puede concluir que la técnica de miniestaca es factible para Ginkgo biloba,y que el uso de IBA no es necesario. Se encontró que la inducción de raíces adventicias depende de la composición bioquímica de las plantas madre, debido a la translocación de azúcares no reductores y proteínas de la hoja para la formación de las raíces.

Palabras clave: propagación vegetativa; plantas medicinales; reguladores del crecimiento; clonación de plantas medicinales; enraizamiento

Introduction

Ginkgo biloba L. (Ginkgoaceae) is a deciduous species from Asia whose extracts, leaves, and fruits have been used in the treatment of mental illnesses for more than 2000 years (Zhang et al., 2011). It is one of the most studied and popular medicinal plants with contents of gincolids and flavonoids, free radical scavenging properties, and a proven effect on the human cardiovascular system, particularly in cerebral circulation (Lin et al., 2008; Van Beek & Montoro, 2009; Song et al., 2010; El-Ghazaly et al, 2015). Besides its medicinal potential, gingko also has a high ornamental potential, with fan-shaped leaves, lush color that turns greenish-brown in autumn and yellowish in winter (Tommasi & Scaramuzzi, 2004; Bitencourt et al., 2010).

Because it is a dioecious species, seedling production requires plants of both sexes, and this hinders species propagation (Singh et al., 2008). In addition, vegetative propagation of the species requires plant growth regulator application and, even so, the produced plants have low vigor of (Acharya et al., 2001; Valmorbida & Lessa, 2008; Bitencourt et al., 2010). Based on this, it is necessary to develop and evaluate vegetative rescue techniques to optimize asexual propagation.

Vegetative propagation of plants has been one of the most important methods of clonal forestry (Martins et al., 2011) in recent decades, representing significant gains for the industry and society (Steffens & Rasmussen, 2016) and ensuring uniformity in planting, higher productivity, and low cost and allowing the selection of desirable characteristics (Xavier et al., 2009; Wendling et al., 2016). Mini-cutting is a vegetative propagation technique that can be considered a specialization of conventional cutting. This technique basically consists of the use of seedlings propagated by seeds or rooted cuttings that will constitute a mini garden (Alfenas et al., 2009). The mini-cutting success is mainly related to cell and tissue rejuvenation that favors meristem differentiation to rooting formation (Ferriani et al., 2010).

The rooting and survival of mini-cuttings can be influenced by several factors such as physiological conditions, type and position of propagules, collection season, juvenility, substrate, and the use of plant growth regulators for rooting induction (Carpanezzi et al., 2001; Rezende, 2007). The level of endogenous auxin is considered a critical factor in adventitious rooting in many tree species, requiring the use of exogenous auxins to induce rooting (Wendling et al., 2015).

The objective of this study was to evaluate the viability of the Ginkgo biloba mini-cutting technique, testing different substrates and concentrations of indole butyric acid (IBA). In addition, the contents of sugars and proteins related to the rhizogenesis of the species will verify the relationship between adventitious rooting and the biochemical composition of cuttings.

Materials and methods

Experiment I - Gingko biloba mini-cuttings

Gingko biloba shoots were collected on October 16, 2017 from ~30 mini-stumps conducted in a mini-garden system (Fig. 1A) at the Forest species propagation laboratory (Embrapa Forests, Colombo-PR, 25°19' S and 49°09' W, 950 m a.s.l.). After collection, the plant material was kept in styrofoam boxes with water to avoid dehydration. The material was then transported to the Macropropagation Laboratory at the research and study group on cuttings (GEPE), located at the Federal University of Paraná (UFPR), in Curitiba - PR (25°44' S and 49°23' W, 920 m a.s.l.). From the shoots of the mini-stumps, stem mini-cuttings were obtained with 4 ± 1 cm length, with a bevel cut in the base and straight cut at the tip, maintaining a leaf in the apical portion (Fig. 1B).

FIGURE 1 A) Mini-stumps, B) mini-cutting, and C) experiment installed in the greenhouse. 

After preparation, mini-cutting bases were immersed in the indole butyric acid (IBA) plant growth regulator in 50% hydroalcoholic solution for 10 sec (Bitencourt et al., 2010), according to the treatments 0 mg L-1, 1000 mg L-1, 2000 mg L-1 and 3000 mg L-1. The planting was carried out in polypropylene tubes with 53 cm3 capacity, using two substrates separately: vermiculite of fine granulometry and the commercial substrate Tropstrato®. The cuttings were planted at about % the depth of the base and maintained for 60 d in a greenhouse with intermittent mist (temperature of 24°C ± 2°C and 80% relative humidity) (Fig. 1C), located in the Biological Sciences Department of the Federal University of Paraná.

The experiment was arranged in a completely randomized design with a 2 x 4 factorial scheme (substrates x IBA concentrations) for a total of 8 treatments with 4 replicates and 14 mini-cuttings per experimental unit, totaling 448 mini-cuttings.

After 60 d, the following variables were evaluated: percentage of rooted mini-cuttings (mini-cuttings that emitted roots of at least 1 mm in length) (Fig. 2A), number of roots per mini-cutting, average length of three major roots per mini-cutting (cm), percentage of mini-cuttings with calluses (mini-cuttings alive, without roots, with formation of undifferentiated cell mass in the base) (Fig. 2B), percentage of live mini-cuttings (mini-cutting without calluses and without roots) (Fig. 2C), percentage of dead mini-cuttings (mini-cuttings with necrotic tissues); percentage of mini-cutting shoots (live mini-cuttings with or without roots and callus, showing shoots of new leaves) (Fig. 2A), and percentage of mini-cuttings that retained their original leaves (live mini-cuttings, with or without roots and callus, which retained the original leaves at the time of evaluation) (Fig. 2A).

FIGURE 2 A) Rooted mini-cutting, B) mini-cutting with callus and C) live mini-cutting. LM - leaf maintenance; Sh - shoots of new leaves. 

Experiment II - Biochemical composition of Gingko biloba mini-cuttings

For the biochemical analysis, ten Ginkgo biloba mini-cuttings were used at the time of the rooting and ten mini-cuttings at the time of evaluation at 60 d. For the quantification of the total protein content, the method described by Bradford (1976) was used and the total soluble sugar contents were determined by the phenol-sulfuric method described by Dubois et al. (1956).

Statistical analysis

For both experiments, the treatment variances were tested for homogeneity using the Bartlett's test (P<0.05) and normality test [Shapiro-Wilk (W)]. Subsequently, the variables were subjected to analysis of variance (ANOVA) and those with significant differences in relation to the F test had their means compared with the Tukey test (P<0.01 and P<0.05). Only the variables live mini-cuttings, percentage of mortality, callus formation, and percentage of mini-cuttings that retained their original leaves did not show normality but were homogeneous with the Bartlett test.

Results and discussion

Experiment I - Gingko biloba mini-cuttings

There was no significant interaction (P<0.05) between the substrate x IBA concentrations for all the evaluated variables, indicating that these factors can act alone in the adventitious rooting of the species. The concentrations of IBA did not influence the induction and development of Ginkgo biloba roots (Tab. 1). In a previous conventional cutting study of the species, Valmorbida and Lessa (2008) observed that the IBA influenced adventitious rooting formation, when compared to the treatment without the use of this plant growth regulator, with rooting percentage results up to 80.55%. Bitencourt et al. (2010) found statistical differences with higher concentrations of IBA (4000 and 8000 mg L-1) bound in talc on ginkgo cutting rooting (45.00 and 46.25% rooting percentages, respectively).

TABLE 1 Rooting percentage (R), number of roots per mini-cutting (NR), average length of three major roots per mini-cutting (LR), live mini-cuttings (A), percentage of mortality (M), callus formation (C), percentage of mini-cutting shoots (Sh), percentage of mini-cuttings that retained their original leaves (LM), subjected to substrates and concentrations of indole butyric acid (IBA) in Ginkgo biloba mini-cuttings. 

Means followed by the same letter In each variable do not differ according to the Tukey test at 5% probability. CV: coefficient of variation.

Thus, since the effect of IBA on the rooting of the species has not been observed, the results obtained in this study are promising considering the high rooting rates (mean of 62.05%). This result shows a positive effect of the rejuvenation of the plant material in the adventitious rooting of Ginkgo biloba and indicates the potential of the minicutting technique for the species, resulting in a reduction in the cost of production since plant regulators are not used for root induction.

One of the advantages of the mini-cutting technique is precisely the reduced need for exogenous auxin application due to the reinvigoration of parent plants (Stuepp et al., 2015; Stuepp et al., 2017b). Similarly, Stuepp et al. (2017a) confirm the efficiency of the mini-cutting technique for the species, resulting in a high rooting index (92.5%). The low percentages of live and dead mini-cuttings in all treatments are a consequence of the high levels of rooting with fast formation, demonstrating the adaptation of the material to greenhouse conditions. The high potential of rhizogenesis of Ginkgo biloba mini-cuttings validates the efficiency of the mini-cutting technique for the species.

Regarding the variable mini-cuttings with callus, the Trop-strato® substrate promoted a higher percentage of callus that differed statistically from the vermiculite substrate. The treatment without IBA application and with 1000 mg L-1 showed a higher percentage of callus when compared to the others; however, these treatments did not have an effect on the rooting of cuttings. The treatments that showed the highest percentage of calluses may be promising, indicating that a longer permanence of the mini-cuttings in the greenhouse could result in root induction. This fact is justified since the treatments with higher percentages of calluses were the same with lower percentages of rooting (Tab. 1). This inverse relationship between the percentage of callus and adventitious root formation has already been reported by other authors who observed that the formation of adventitious roots in Ginkgo biloba can occur from the callus tissue formed at the base of cuttings or mini-cuttings. This suggests that root induction could result from a longer permanence of mini-cuttings in the greenhouse (Valmorbida & Lessa, 2008; Bitencourt et al., 2010; Stuepp et al., 2017a).

There was a significant difference between the concentrations of IBA in the percentage of shoots, with the highest percentages observed in the treatments without IBA application and in the concentration of 3000 mg L-1 (Tab. 1). According to Moubayidin et al. (2010), root growth occurs when, in the apical meristem, cell division prevails over differentiation. This occurs when there is a greater concentration of auxins promoting cell division, in contrast to the cytokinin concentrations that promote cellular differentiation.

The auxin: cytokinin ratio regulates tissue morphogenesis. While a high auxin:cytokinin ratio stimulates the formation of adventitious roots, a low ratio leads to higher growth of the shoot (Taiz et al, 2017). According to Agullo-Anton et al. (2011) and Agullo-Anton et al. (2014), exogenous auxin supplementation in cuttings affect the endogenous cytokinin concentration, influencing adventitious rooting. Souza and Miranda (2006) found in an in vitro study that the proliferation of shoots and root formation in Gerbera jamesonni depends on the balance between auxin and cytokinin. Although a greater number of shoots was observed in the treatment without synthetic auxin application, adventitious rooting was not influenced, indicating that the presence of leaves induced an auxin-favorable balance.

It is possible to verify a close relationship between the rooting percentage and adventitious root development by maintaining mini-cutting leaves in all treatments (Tab. 1). The importance of leaf maintenance for these two variables has been reported in the literature, especially related to the presence of certain leaf compounds, such as carbohydrates, auxins and rooting cofactors, which can be translocated by phloem to the base of the cuttings, thus stimulating root formation (Bona & Biasi, 2010; Fragoso et al, 2015).

Experiment II - Biochemical composition of Gingko biloba mini-cuttings

Adventitious rooting is influenced by the biochemical composition of propagules (Yan et al, 2017). Some studies have demonstrated the correlation between rooting and carbohydrate content (Aslmoshtaghi & Reza-Shahsavar, 2010; Ragonezi et al, 2010; Denaxa, et al, 2012).

The carbohydrates translocated by phloem are non-reducing sugars such as sucrose, because they are less reactive than reducing sugars (Taiz et al, 2017). This fact can be seen in Figure 3, where it is possible to verify a decrease in the non-reducing sugar concentration at the time of mini-cutting evaluation (60 d after experimental set up). Additionally, it is possible to observe a high level of non-reducing sugars at the moment of root evaluation.

FIGURE 3 Biochemical composition of different organs of Ginkgo biloba and collection time. I - Installation; E - evaluation. Means followed by the same letter do not differ according to the Tukey test (P<0.05). 

These carbohydrates translocated by phloem can be used as an energetic resource in root induction and development (Aslmoshtaghi & Reza-Shahsavar, 2010; Souza et al, 2015) and as a carbon source for biosynthesis of amino acids and nucleic acids (Fachinello et al, 2005). They can also act in the regulation of gene expression (Wang & Ruan, 2013). Thus, the reduction of endogenous carbohydrate levels indicates that these sugars were used during root emission and growth (Husen & Pal, 2007), as demonstrated in the present study.

The quantity of protein in the mini-cuttings observed at the moment of evaluation was also lower than that observed at the moment of setting up the experiment (Fig. 3). Proteins are biochemical compounds associated with rooting, that can help in the induction, formation and development of adventitious roots (Taiz et al, 2017), and maybe involved in the signaling and biosynthesis of auxins (Franklin et al, 2011; Hornitschek et al, 2012; Zhang et al, 2017).

When evaluated separately from cuttings, the reduction in the levels of non-reducing sugars and proteins during rooting that is associated with the high contents of these compounds in the roots (Fig. 3), indicates an ideal redirection of the source of drain energy. This fact reassures the importance of the nutritional status of the stock plant since it is directly related to the formation of the root system (Hartmann et al, 2011), as confirmed by the high rooting indexes in the present study. Furthermore, these results reinforce the importance of the nutritional status of the stock plant that directly affects the mini-cutting rooting and survival, and that is justified by the high rooting results observed in the present study.

Conclusion

The mini-cutting technique is feasible for the production of Ginkgo biloba seedlings regardless of the substrate used. Additionally, we concluded it is not necessary to use a plant growth regulator to induce rooting.

The induction of adventitious roots is dependent on the biochemical composition of the mother plants.

Acknowledgments

The authors would like to acknowledge the GEPE (Research and Study Group on Cuttings) and UFPR (Federal University of Paraná) for providing the infrastructure used in this research and the Coordination for the Improvement of Higher Education Personnel (CAPES), by financial support of the research and scholarships granted.

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Author's contributions RDAM, LPL, LMV, and KCZ formulated the research goals and aims, RDAM, LPL, LMV, and ERN developed the methodology, RDAM, LMV, and KCZ conducted the research and investigation process, RDAM prepared the initial draft and created the visualization/data presentation, LPL implemented the software, LPL and LMV applied the statistical techniques to analyze study data, ERN and KCZ carried out the critical review, commentary, and revision of the manuscript, KCZ verified the overall replication/ reproducibility of the results/experiments, provided the study materials, oversaw, managed and coordinated the research activity planning and execution, and acquired the financial support for the project.

Received: May 15, 2020; Accepted: December 11, 2020

*Corresponding author: leandro.marcolino@ufpr.br

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