Moderately strong allelopathic activities were found in four bamboo species, <i> Bambusa multiplex </i> cv. Houraichiku; <i> Phyllostachys bambusoides </i> cv. Madake; <i> P. nigra </i> cv. Hachiku; <i> Sasa kurilensis </i> cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, <i> B. multiplex </i> and <i> P. bambusoides </i> stimulated the recipient lettuce growth, <i> i.e. </i> , non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of <i> P. nigra </i> and <i> S. kurilensis </i> were less stimulatory or inhibitory. Inhibitory effect of <i> S. kurilensis </i> was the strongest among four bamboo species. Furthermore, highly inhibitory effects of <i> S. kurilensis </i> protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.
For application of bamboo allelopathy as natural herbicide in agriculture, allelopathic activities of 80 species of bamboo were investigated using in vitro bioassay method of allelopathy, “sandwich method”, using lettuce as a recipient plant [
Recently, a new in vitro bioassay method of allelopathy, “protoplast co-culture method” was developed [
In this report, we selected four bamboo species of different classification, e.g., monopodial or sympodial, and of different ecological distributions, i.e., temperate, tropics, and subarctic. Suspension cultures and isolation of their protoplasts had been developed in Phyllostachys bambsoides Sieb. Et Zucc. (Pb, Madake) [
Plants of four bamboo species, i.e., Phyllostachys bambusoides, Madake (Pb); Bambusa multiplex Raeush, Houraichiku (Bm); Phyllostachys nigra Munro var. Henonis Hachiku (Pn); Sasa kurilensis Makino et Shibata, Chishimazasa (Sk) (
Suspension cultures of Bm was generated from the node and Pb and Pn were from the young shoot as described previously [
Lettuce seedlings were prepared as described previously [
The sandwich method was performed as reported [
Scientific name | Classification | a (2n) | cultivar (Common name) | Distribution | b |
---|---|---|---|---|---|
Phyllostachys bambusoides Sieb. Et Zucc | Bamboo Monopoidal | 48 | Madake | Temperate (10˚C - 30˚C) c | Pb |
Phyllostachys nigra Munro var. Henonis | Bamboo Monopoidal | 48 | Hachiku | Temperate (10˚C - 30˚C) | Pn |
Bambusa multiplex Raeush | Banboo Sympoidal | 72 | Houraichiku | Tropics (20˚C - 40˚C) | Bm |
Sasa kurilensis Makino et Shibata | Sasa Monopoidal | 48 | Chishimazasa | Subarctic (<15˚C) | Sk |
a. Chromosome number, b. Abbrebiations, c. an optimal temperature range for growth.
of dried bamboo leaves were sandwiched between two layers of 5 ml of 0.5% agar (powder, gelling temp. 30˚C - 31˚C, Nacalaitesque Co. Ltd. Kyoto, Japan) in six multi-well plates (Nunc). Length of hypocotyls and roots of germinated seeds of lettuce was measured after three days of incubation at 20˚C in the dark. The control treatment consisted of seeds germinated in the absence of dried leaves. Data were recorded as % growth of the control and averaged with standard error (SE).
Bamboo protoplasts were isolated by enzyme combination of 2% of Cellulase RS (Yakult) and 0.5% of Pectolyase Y-23 (Kyowa Kasei) in 0.6 M mannitol solution for 3.5 to 9 hours incubation. Protoplasts were passed through 42, 63 and 80 µm sized meshes depending on the diameters of protoplasts, and washed with mannitol solution three times by centrifugation at 100 g for 4 min.
Lettuce cotyledons protoplasts were isolated as described previously [
Viability of protoplasts were determined using fluorescein diacetate [
Protoplasts numbers were counted using a hemocytometer. Five μL each of protoplasts in 0.6 M mannitol solution were put in the 50 μL of liquid MS medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 μM benzyladenine (BA), or 10 μM of 2,4-D as plant hormones, and 3% sucrose and 0.6 M mannitol, in a well of 96-well culture plate (Falcon 3075). Final protoplast densities of bamboo were 4 × 103/mL to 105/mL, and of lettuce were 6 × 103/mL to 105/mL. The protoplasts were cultured at 28˚C in a humid incubator (CO2-in- cubator without the supply of CO2, APC-30DR, ASTEC Co. Ltd.). The numbers of non-spherically enlarged lettuce protoplasts and small numbers of divided cells were counted under an inverted microscope (Olympus CK40) after 4 days of culture. The numbers of divided cells including colonies composed from four or more cells were counted after 8 days of culture. Percentages of control without addition of bamboo protoplasts were calculated and averaged with SE at lettuce protoplast densities of 6 - 25 × 103/mL (6, 12, 25 × 103/mL).
Image analysis of yellow color accumulation of lettuce protoplasts after protoplast co-culture was performed as described previously [
Pb and Sk and different cultivars of Pn and Bm were in the big list of sandwich method of bamboo [
Difference between young leaves and mature leaves was reported in Mucuna gigantea, whose putative allelochemical was L-DOPA, though the inhibition was higher in young than mature leaves [
Protoplasts were well isolated by using 2% Cellulase RS and 0.5% Pectolyase Y-23 in 0.6 M mannitol, from two-week old suspension cultures of all four species tested. They were very dense culture maintained at high phosphate-con- taining MS basal medium.
Protoplasts of Pb and Bm could not be isolated from one-week-old suspension cells even after 24 hours incubation (data not shown). Two week-old culture is good growing stage for all four species.
from the protoplasts of bamboo suspension cells under an inverted microscope. In the control without bamboo protoplasts, 91% of lettuce protoplasts were non- spherically enlarged cells, which shows cell wall formation, and 9% were divided cells. Stimulation effects with low protoplast density (1.2 × 104/mL) of Pb and Bm were prominent. In contrast, Pn and Sk clearly inhibited the growth of lettuce at the low protoplast density and at higher densities. Almost no growth was obtained with 2.5 × 104/mL of Sk. Thus differences in inhibition on lettuce growth at cell wall formation stage were found among four bamboo species. Sk protoplasts were most inhibitory on recipient lettuce protoplasts.
This report is the first report that 50% or more stimulation effect was observed by protoplasts of test plants on lettuce protoplasts at cell wall formation stage. In other woody plants, which had very inhibitory allelopathic activities in both sandwich method and protoplast co-culture method, e.g., Derris indica [
Effects of bamboo protoplasts on the growth of lettuce protoplasts after 8 days of culture were shown in
protoplasts densities of bamboo up to 2.5 × 104/mL, Pb and Bm were stimulatory, while Pn and SK were similarly inhibitory as of 4 day pattern in
No difference between cell enlargement stage and cell division stage was clearly reported in one of tree mangrove species, Derris indica. And the effect of a putative allelochemical, rotenone showed the same pattern of inhibition at both stages [
scanning of 96-well culture plate using an inexpensive scanner, analysis with free software Image J [
In this report, at 17 day of culture, 6 × 103/mL data which yellow color intensity was low, were deleted and the data at lettuce protoplast densities of 1.2 × 104/mL to 105/mL were averaged.
Calculated yellow values at high bamboo densities gave sometimes minus value because we deducted the values of bamboo themselves without lettuce protoplasts [
In the sandwich method, not very strong inhibitory allelopathic activities were repeatedly found in four bamboo species tested. However, in the protoplast co- culture method, with digital image analysis, Sk was most inhibitory among four bamboo species. In all three stages of recipient lettuce protoplast growth, i.e., non-spherical cell enlargement, cell divisions and yellow color accumulation, Sk had the strongest inhibitory effect. At stages of non-spherical cell enlargement and cell divisions, Bm and Pb were much stimulatory at lower protoplast densities of bamboo. Pn was in between of Bm and Pb, and SK. No stimulation on yellow color accumulation was observed in four bamboo species tested.
Such a difference of activities among assay methods of allelopathy was also reported. Sasa veitchii (Sasa glabra), Kokumazasa, was very inhibitory (2% radicle growth) in another assay method, plant box method, which measure the effects of living root exudates on lettuce seedlings growth, however, only 33% growth of control was reported in sandwich method [
SK plants are subarctic which naturally grow in northern Hokkaido island, Japan and were planted in highway slopes, while Pb and Pn are temperate and Bm is tropics. In this report, protoplast co-culture method was performed at 28˚C. Test at lower temperature can be considered. However, though 20˚C is the best for lettuce seedlings growth in the sandwich method, lettuce protoplasts could be well cultured up to 30˚C in the co-culture method with mangrove species which grow in the tropical area [
Different inhibition patterns in protoplast co-culture assay and differences among different bioassay methods might be caused by different concentrations of the same allelochemical or difference of allelochemicals among four bamboo species. Secondary metabolites synthesized in the late growth stage of plant development are thought to be allelochemicals in leaf litter. Leaf leachate and aqueous extracts of a tropical bamboo, Phyllostachys edulis was reported to be inhibitory to the growth of recipient plants including lettuce [
Bamboo is well known as a source of plant hormones, e.g., gibberellins, for rapid growth of shoots. However, in the lettuce protoplast co-culture assay system, gibberellic acid was inhibitory, while antagonistic hormone, abscisic acid (ABA), stimulated the lettuce protoplast growth at 0.1 - 10 μM range [
A study is underway to investigate chemical constituents in protoplasts of bamboo species by using microscopy image analysis of flavonoid and lipid [
Ogita, S. and Sasamoto, H. (2017) In Vitro Bioassay of Allelopathy in Four Bamboo Species; Bam- busa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis. American Journal of Plant Sciences, 8, 1699-1710. https://doi.org/10.4236/ajps.2017.87117