Effects of sorghum (sorghum bicolor L.) root exudates on the cell cycle of the bean plant (phaseolus vulgaris L.) root

Effects of sorghum (sorghum bicolor L.) root exudates on the cell cycle of the bean plant (Phaseolus vulgaris L.) root*

Angela Maria Gattás Hallak¹, Lisete Chamma Davide² and Itamar Ferreira Souza³

^*Part of a dissertation presented by A.M.G.H. to the Universidade Federal de Lavras (UFLA), in partial fulfillment of the requirements for the Master's degree.

¹Departamento de Ciências Naturais da Fundação de Ensino Superior de São João Del Rei, Caixa Postal 110, 36300-000 São João del-Rei, MG, Brasil. Send correspondence to A.M.G.H.

²Departamento de Biologia and ³Agricultura da Universidade Federal de Lavras, Caixa Postal 37, 37200-000 Lavras, MG, Brasil.

ABSTRACT

Two experiments were conducted to test the allelopathic effect of sorghum (Sorghum bicolor L.) root exudates on bean (Phaseolus vulgaris L.) cell division. Research was conducted in the greenhouse of the Wistock Agricultural Research Institute of Minas Gerais State (EPAMIG) and in a laboratory of the Federal University of Lavras (UFLA). Sorghum variety BR-601 and bean variety Carioca MG were used. The exudate, called sorgoleone (SGL), was obtained by methylene chloride and acetic acid extraction from sorghum roots seven days after sowing on Petri dishes, and refrigerated until use. Solutions of 0.01, 0.05, 0.10, and 0.15 mM were prepared using Johanson solution as the SGL solvent. Seven-day-old bean seedlings grown in vermiculite in a greenhouse were transplanted to the solution. Seven days after transplantation, the beans did not show any sign of phytotoxicity; however, cytogenetic observations showed that SGL reduced the number of cells in prophase, metaphase, and anaphase stages. Colchicine effects were observed among cells in metaphase on the third and fifth days after treatments and varied with SGL concentrations. By the seventh day, the colchicine effects were inversely proportional to concentration, which varied from 34.3% for 0.01 mM to 6.6% for 0.15 mM. SGL acts as a mitotic inhibitor. It probably depolymerizes the microtubular proteins and induces the formation of colchicine metaphases causing polyploid nuclei. A largest period of SGL treatment also induced chromosome breaks and bridge formation in anaphase and telophase. Although SGL cannot be used as a herbicide for bean cultures, its allelochemical effects on other cultures are the factors that will define the use of sorghum as a natural herbicide.

INTRODUCTION

Vegetable substances that inhibit germination or growth of other plants could eventually become efficient substitutes for synthetic herbicides, depending on their selectivity and toxicity (Muller, quoted by Dionello-Batista and Basta, 1984). Hydroquinones are substances produced by and exudated from sorghum (Sorghum bicolor L.) roots. Reduced and oxidated forms of hydroquinones either stimulate or inhibit plant germination (Netzly et al., 1988). Sorghum hydroquinones have been found to influence other plants (Breazeale, 1924; Guenzi et al., 1967; Lehle and Putnam, 1983 and Panasiuk et al., 1986). Netzly and Butler (1986) used chemical tests and found proteins, lipids, free amino acids, phenols, and quinones in sorghum root exudates. It was not until l988; however, that Netzly et al. (1988) using mass spectrometry identified a new compound which was denominated sorgoleone (SGL).

Sorghum produces a factor that inhibits weed development and growth. This affects wide-leafed weeds more than narrow-leafed weeds. Whether sorghum inhibits, stimulates, or is indifferent depends on the environment, species, and allelochemical quantity released (Einhellig and Rasmussen, 1989). The aim of our research was to analyze the effect of sorghum root exudates on the cellular cycle of bean (Phaseolus vulgaris L.) plants.

MATERIAL AND METHODS

The Carioca MG bean was used to test the allelopathic effect of SGL. The experiments were conducted in the greenhouse of the Agricultural Research Institute of Minas Gerais State (EPAMIG) and in the Vegetable Anatomy and Cytology laboratory at the Federal University of Lavras (UFLA).

Bean seeds were germinated on trays with vermiculite for 7 days. The young plants were transferred to 200-ml glass flagons with Johanson solution (Pizzolato and Regehr, 1978) and stayed in it for 24 h, in order to adapt to the solution and recover from the stress caused by transplantation. Subsequently, the plants were transferred to flagons with 0.01, 0.05, 0.10, or 0.15 mM SGL solutions.

SGL was extracted from the sorghum roots germinated on Petri dishes at room temperature. The roots had been immersed in methyl chloride and acetic acid (20 ml:50 ml) for one second. After the 3rd, 5th and 7th days of treatment, bean root tips of approximately 5 mm were collected for cytogenetic analysis. The roots were fixed in a solution of ethyl alcohol and acetic acid (3:1) and, after hydrolysis in 1 N HCl at 60ºC, they were stained with 2% Giemsa in phosphated buffer, pH 6.8. The blades were set with Entellan (Guerra, 1983). Each treatment (concentrations of SGL) had four replications. Two thousand cells per treatment were considered for cytological analysis on the 3rd, 5th and 7th days of treatment.

We observed the following aspects of cellular division:mitotic index (number of cells in division x 100/number of meristematic cells), colchicine metaphase index (number of colchicine metaphases x 100/number of cells in metaphase), presence of bridges, fragments, late chromosomes, and micronuclei in anaphase and telophase. Regression and variance analyses were performed.

RESULTS

Daily observations of the young bean plants did not detect growth or morphological alterations. The bean plants did not show any visual symptoms of phytotoxicity under these experimental conditions.

All SGL concentrations induced reduced mitosis after three days of treatment when compared with the control (Table I). The dose that had the greatest effect on cellular division was 0.10 mM SGL. In the second collection (5th day of treatment), however, the lowest dose, 0.01 mM, had the greatest effect on cellular division. The mitotic index gradually increased after the third dose (0.10 mM SGL), but did not equal the control. The third collection (7th day of treatment) showed that as the dose concentration increased the mitotic index gradually decreased.

Table I
- Effects of SGL dosage and length of treatment on mitotic index, metaphases, and colchicine metaphases (%) in the bean plant.

SGL concentrations (mM)	3rd day			5th day			7th day
	MI	MEI	CMI	MI	MEI	CMI	MI	MEI	CMI
0.00	21.90	12.25	0.00	23.00	9.00	0.00	30.25	32.00	0.00
0.01	17.40	3.50	0.00	17.55	10.00	12.50	30.45	11.00	34.3*
0.05	12.25	3.50	0.00	18.20	14.00	41.0*	27.30	12.50	31.50
0.10	10.80	5.00	9.00	21.40	11.00	18.20	24.15	11.25	19.60
0.15	13.25	4.50	37.9*	21.60	12.50	26.00	23.25	14.00	6.60

MI - Mitotic index; MEI - metaphase index; CMI - colchicine metaphase index.

*Higher percentage of colchicine metaphases in each treatment.

There was a significant difference in the number of colchicine metaphases among treatments. All SGL concentrations, except for 0.01 and 0.05 mM on the 3rd day of treatment, led to colchicine metaphase formation (Table I).

A large dose of SGL was necessary to induce colchicine metaphase on the third day of treatment (Table I). After the 5th day, even the lowest concentrations caused a higher percentage of colchicine metaphases. On the last day of treatment, the percentage of metaphases decreased with the increase of the dosage.

The effects of concentration and time of collection upon the cellular cycle of the bean plant root were determined (Figure 1). The total number of cells observed in each phase of the control was considered equal to 100 and correlated with the number of cells observed in the treatments. The number of cells in interphase, at all concentrations, was always greater than in the control.

Figure 1
- Effect of sorgoleone (SGL) dosage and length of treatment on the cellular cycle of the bean plant.

Treatment with 0.01 mM SGL causeal a small increase in interphases from the 3rd to the 5th day of treatment, and a decrease on the 7th day of treatment. The 0.05 mM treatment on the 3rd day had a greater number of interphases than the 0.01 mM treatment. There was a decrease in the number of interphases on the 5th and 7th days. The largest number of interphases was observed on the 3rd day of treatment with 0.10 mM SGL. This number decreased when a concentration of 0.15 mM was used.

The number of prophases of the treated plants was generally inferior to that of the control. The 0.01 mM treatment showed a decrease from the 3rd to the 5th day and then an increase on the 7th day, which was larger than the 3rd and the control. Treatments with 0.05, 0.10 and 0.15 mM SGL decreased the number of prophases. In all treatments, the number of cells in metaphase had an abrupt increase from the 3rd day to the 5th day and an abrupt decrease from the 5th to the 7th day. Anaphase and metaphase were the phases which changed the most, and had the greatest values. Late chromosomes, bridges, and fragments in anaphase and telophase on the 5th and 7th days of treatment with 0.0l, 0.05 and 0.10 mM SGL were also observed ( Figure 2).

Figure 2
- Effect of sorgoleone (SGL) on bean species plants: A - normal metaphase (control); B - colchicine metaphase (0.05 mM SGL - 7th day of treatment); C - bridge in telophase (0.10 mM SGL - 7th day of treatment); D - anaphase with late chromosomes (0.05 mM SGL - 7th day of treatment); E - polyploid metaphase.

Regression and variance analyses of the cellular cycle of the young bean plants demonstrated significant differences in dosage and length of treatment. Regarding the doses administered, the coefficient of determination was significant at 1% level (R² = 0.91). Considering the length of treatment, it was found R² = 0.99 for interphase level and R² = 0.99 for mitotic index.

DISCUSSION

The effects of SGL on the bean plant included reduction of the mitotic index as well as induction of colchicine metaphase, late chromosomes, bridges, and chromosome fragments. The increase in the number of colchicine metaphases shows that SGL probably has an effect similar to colchicine, depolymerizing the microtubular proteins and blocking fiber formation during the mitotic state. This hypothesis was reinforced by the appearance of polyploid nuclei 2n = 4X = 44 (Figure 2) with 0.10 mM doses after seven days of treatment. Other studies on the effect of SGL on stem development of bean plants showed deformations in the cellulose walls of xylem cells, which also demonstrates the depolymerizing effect of SGL on microtubule proteins (Hallak, 1996). The observation of bridges and fragments in the anaphases and telophases of bean plantules treated with SGL suggested probable allelochemical effects, causing chromosome breaks.

There are some indications that chromosome malformations decrease and vanish during the development of plants from seeds kept in storage (Murata et al., 1984). Previous studies suggest that old Datura seeds retained malformations causing changes in meiosis and abortion in pollen grains, which was transmitted to the next generations (Gerassimova, 1935; Navashin and Gerassimova, 1936; Gunthardt et al., 1953).

CONCLUSIONS

SGL acts as a mitotic inhibitor. It probably depolymerizes the microtubular proteins and induces the formation of colchicine metaphases causing polyploid nuclei. A larger period of SGL treatment also induced chromosome breaks and bridge formation in anaphase and telophase.

Although SGL cannot be used as a herbicide for bean cultures, its allelochemical effects on other cultures are the factors that will define the use of sorghum as a natural herbicide.

ACKNOWLEDGMENTS

The authors thank laboratory technician Ana Hortência, for her cooperation. A.M.G.H. was the recipient of a CAPES fellowship.

RESUMO

Foram instalados experimentos em casa de vegetação da Empresa Agropecuária de MG (EPAMIG) e laboratório da Universidade Federal de Lavras (UFLA) em 1994, com o objetivo de testar a ação alelopática de exsudados de raiz de sorgo (sorgoleone - SGL), variedade BR-601, sobre plantas jovens do feijoeiro, variedade Carioca MG. Sorgoleone foi extraído das raízes de sorgo 7 dias após a semeadura em placas de Petri. Soluções de 0,01, 0,05, 0,10 e 0,15 mM de SGL foram preparadas usando solução de Johanson como solvente. Plantas de feijão com 7 dias de idade, germinadas em vermiculita, em casa de vegetação, foram transplantadas para estas soluções. O feijoeiro não mostrou sinais de fitotoxicidade durante o período de tratamento; porém, observações citogenéticas mostraram que concentrações de 0,01 mM e acima reduziram o número de células nas fases de prófase, metáfase e anáfase em relação à testemunha. Entre as células em metáfase, observaram-se metáfases colchicínicas. As percentagens de metáfases colchicínicas no 3^o e 5^o dias após o transplante variaram com as concentrações de SGL; no 7^o dia, estes valores foram inversamente proporcionais às concentrações, variando de 34,3% para 0,01 mM a 6,6% para 0,15 mM de SGL. Pode-se observar ainda nas células em anáfase e telófase cromossomos formando pontes e atrasados, bem como a ocorrência de metáfases poliplóides.

(Received July 1, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  01 July 1997