In Vitro Induction and Identification of Polyploid Amorphophallus muelleri Blume Plants by Colchicine Treatment
Wahyu Widoretno1*), Rodliyati Azriningsih2), Deden Sukmadjaja3) and Mufidatur Rosyidah4)
1) Faculty of ..., Universitas Brawijaya, Malang, East Java, Indonesia
2) Faculty of ..., Universitas Brawijaya, Malang, East Java, Indonesia
3) Research Center of Horticulture and Estate Crops, Research Organization Agriculture and Food, National Research and Innovation Agency, Indonesia
4) Faculty of ..., Universitas Brawijaya, Malang, East Java, Indonesia
*) Corresponding author E-mail: [email protected] Received: November 28, 2022 /Accepted: January 23, 2023
ABSTRACT
This research aimed to induce in vitro and identify polyploidy plantlets of Porang (Amorphophallus muelleri Blume) after colchicine treatment. In vitro shoot base explants of porang were cultured on MS medium containing different colchicine concentrations of 30, 60, 90, and 120 mg/l for four weeks while the control was medium without colchicine. After four weeks, all treated and untreated explants were multiplied and regenerated into plantlets. Flow cytometry and chromosome count were employed to determine the plantlet’s ploidy level and chromosome number, respectively. The result showed that colchicine inhibited explant growth, shoot formation, and plantlet regeneration. Increasing colchicine concentration reduces the capacity of explant in shoot formation, the number of shoots and plantlet regeneration. Of 19 plantlets regenerated from colchicine-treated explants, nine plantlets were mixoploid. Percentage of mixoploid plantlet regenerated from treated explant with 30 mg/l colchicine was higher than 60 mg/l colchicine.
Mixoploid plants are mixed diploid, tetraploid, and octoploid cells. Chromosome count confirmed that the diploid cell of A. muelleri has 26 chromosomes and a colchicine-induced triploid and tetraploid cell has 39 and 54 chromosomes, respectively.
Keywords: Amorphophallus muelleri Blume; Colchicine; Flow cytometry; Polyploidy INTRODUCTION
Porang (Amorphophallus muelleri Blume) is a tuber plant belonging to the family Araceae that grows in forest. Tubers of porang contain about 15-65% glucomannan, higher than their relative plants (Saleh et al., 2015; Wahyuni et al., 2020). Glucomannan has high economic potential, which is used in various food, chemical and pharmaceutical industries. Exports of porang tuber continued to increase, during 2019 it was 11,702 tons and in 2020 it was 16,002 tons (BPS, 2021). However, National porang production is still unable to meet export needs. One of the problems in porang cultivation is the limited availability of superior seeds (Santosa, 2014).
Porang is usually propagated using tubers. Although it can reproduce by seed, the seeds are apomixes (Poerba et al., 2009). Vegetative propagation with tubers or apomixes does not occur genetic recombination; as a result, genetic improvement of plants through conventional breeding is not effective.
Tissue culture techniques have great potential to produce superior plants, but this potential has yet to be fully exploited in developing countries. The quality of porang seeds will affect crop yields thus, it is necessary to develop superior porang seeds and supply them on an industrial scale.
The results of research on various plants showed that chromosomal duplication made it possible to obtain plants that had superior characteristics, such as larger size, higher phytochemical content compared their diploid. The tetraploid plant Atropa belladonna had a higher alkaloid content (Huang et al., 2010), Salvia miltiorrhiza produced higher levels of flavonoid and terpenoid (Gao et al., 1996), and the polyploid cells of Panax ginseng, Datura stramonium and Scutallaria biacalensis had a higher content of bioactive compounds than their diploids (Kim et al., 2004; Berkov & Philipov. 2002, Gao et al., 2002).
Technique of in vitro polyploidy induction using colchicine has been successfully carried out on patchouli, peanut and tobacco plants (Widoretno, 2016, Omidbaigia et al., 2010, Tulay and Unal. 2010}.
Colchicine is able to produce cell polyploidy because it inhibits the addition of α- and ß- tubulin molecules
to microtubules ends. Treatment with colchicine can impede the synthesis of microtubules in dividing cells, which prevents the separation of chromosomes, and lead to cell polyploidy (Tulay and Unal 2010).
In order to support development program of the superior product through biotechnology-based plant breeding, this research activity is directed at developing polyploidy porang in vitro to improve of tuber raw materials and glucomannan content. The availability of superior porang seed will support the increase in porang production in Indonesia.
MATERIALS AND METHODS Plant Material and In Vitro Propagation
A diploid of A. muelleri Blume (2n = 26) was used for chromosome duplication. In vitro shoots were regenerated on MS medium + 0.1 mg/l NAA + 3 mg/l BA and sub cultured at intervals of approximately 6 weeks. The culture was kept at 25 ± 1oC under a light of 16/8 h (light/dark) cycle. The study was conducted from January to November 2022 at Plant Tissue Culture Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang and Cell & Tissue Biology Laboratory, Center for Research and Development of Agricultural Biotechnology and Genetic Resources (BB Biogen), Bogor.
Polyploidy Induction and Shoot Multiplication
The 1-2 cm shoots after six weeks in vitro culture were used as explant for the polyploidy experiment (Fig 1). Shoot base explant was isolated from in vitro shoot and then inoculated on solid MS medium containing different concentration of colchicine 30, 60, 90, and 120 mg/l. As a control, the shoot base was cultured on a medium without colchicine. The cultures were incubated at 25 ± 1oC in the dark for four weeks.
For each treatment was composed of ten replicates (bottles), And in a bottle, 5 explants were cultured. At the end of the exposure time, the response of explant and the number of shoot per explant were evaluated.
After four weeks all colchicine treated explants were cultured on induction media without colchicine for shoot formation. The number of total shoots and number of shoots per explant were counted. The formed shoots from colchicine treated and untreated explants were regenerated into plantlet.
Determination of Ploidy Level
Flow cytometry and chromosome counting from root tip cells were used to estimate the ploidy level of all plantlets regenerated from untreated and treated shoot base.
Flow Cytometry Analysis
The ploidy level of the regenerated plantlet was estimated by flow cytometry. Twenty-five milligrams shoot tissue from colchicine-treated and untreated explants were chopped in plastic clip containing 500 µl of cold ice buffer solution (LB01) for 60 seconds. The crude extract was then filtered through CellTrics 30 µm to obtain filtrate. The filtrate was stained with propidium iodide 175 µl. Samples were analyzed using flow cytometry. Leaves from a diploid plant were used as the control.
Fig. 1. Culture of porang shoots and shoot base explants used for in vitro polyploidy induction: A. Small shoots started to form from callus; B. The 1-2 cm shoots after four weeks of culture. C. Shoot bases as explants for polyploidy induction.
A B C
Counting of Chromosome
Chromosome counting and observation was performed using squashing method. Root tips of 1 month-old plantlet were soaked in 0.004 M hydroxyquinoline for four hours. The treated root tips were rinsed with water and then fixed in a mixture of 1 part glacial acetic acid and 3 parts ethanol for 24 h. The fixed root tips were rinsed with water and then hydrolyzed in 1 N HCl for 2 minutes at 65 oC. The root tip was stained with 2 % aceto orcein for 30 minutes. The root tips were carefully cut off and then squashed on the microscope slide. The chromosomes were observed and counted using a light microscope (Olympus CX 31) at 1000x magnification. The chromosomes number was used to classify the plants. The plants, which have chromosome number 28, were classified in diploid plant (2n = 28), the plants with double chromosome 56 were classified in tetraploid plant (4n = 56) and the plant with mixed ploidy were regarded as mixoploid plants.
Statistical Analysis
A Complete Randomized Design (CRD) with ten replications was used in the experiment. Analysis of Variance (ANOVA) followed by Duncan Multiple Range Test (DMRT) at 5% significance level was used to identify the mean differences among the treatments.
RESULTS AND DISCUSSION Influence of Colchicine on Growth of Shoot Base Explants
The porang shoots used as explants were shoot base (Fig. 1) and these were from 1-2 cm in vitro shoots. The explants were cultured on MS medium supplemented with BA 3 mg/land colchicine at various concentrations (0, 30, 60, 90 and 120 mg/l) for four weeks.
The addition of colchicine on culture medium affected the growth response of explants and shoot formation in explants. After four weeks, explants cultured on colchicine-free medium and medium with the addition of colchicine at a low concentration of 30-60 mg/l seemed to be larger than those cultured on medium with the addition of higher colchicine concentration (90-120 mg/l). In addition, some small shoots emerged from those explants. Explants cultured on medium with higher concentrations of colchicine formed less shoots and even some of them were blackish brown and unable to form shoots at all (Fig. 2).
Furthermore some explants died when cultured on the medium with 120 mg/l colchicine.
Colchicine-induced polyploidy inhibited the shoot formation of shoot base explants. The higher colchicine concentration contained in medium, induced higher inhibition on shoot formation (Fig. 3). The inhibition of shoot formation in explants cultured on medium with low colchicine concentrations of 30-60 mg/l was 20-40%, while inhibition caused by the higher concentrations of colchicine was approximately 60%. The total number of shoots formed from explants cultured on colchicine-free medium was 29.2 with the average shoot number per explant of 5.8. While, the number of shoots formed from explants cultured on medium supplemented with higher concentrations of colchicine (90-120 mg/l) was only about 11-12 shoots and 1-3 shoots per explant in average.
Fig. 2. Growth response and shoot formation of shoot base explants cultured on medium with colchicine:
A. Control; B-E. Colchicine treatment: B. 30 mg/l; C. 60 mg/l; D. 90 mg/l; E. 120 mg/l.
A B C D E
The in vitro colchicine-induced polyploidy in shoot base explants inhibited explant growth and shoot formation. The effect of colchicine on inhibition of explant growth and shoot formation in porang was due to the fact that colchicine hindered mitosis. It is related to the binding of colchicine to tubulin thereby preventing the formation of microtubules during cell division. Colchicine is believed to interfere with cell division through its disruptive action on the mitotic spindle. Inhibition mechanism by colchicine occurs through direct or indirect influence of colchicine on the spindle. Indirect action is by the activation of enzymes that attack the spindle while direct action involves binding of colchicine to the spindle fibers causing the spindle fibers to dissociate into protein subunits (Taylor, 1965).
The higher the concentration of colchicine was the greater growth inhibition of explant and shoot formation would be. In addition to inhibition of shoot formation, shoots formed from high concentration colchicine-treated explants seemed to be stunted and thick, yellowish in color with abnormal structures. It indicates that high colchicine concentrations are toxic to explants. According to Leung et al., (2015), at low concentrations, colchicine inhibits microtubule growth and at high concentrations, colchicine promotes microtubule depolymerization and it causes high toxicity in normal tissues.
The Influence of Colchicine on Shoot Multiplication and Plantlet Regeneration
After 4 weeks cultured on medium containing colchicine, the shoots were then sub cultured on MS medium + BA 3 mg/l without colchicine addition for shoot multiplication. During the culture, the shoots began to enlarge and multiplied, but some differences occurred regarding the growth and multiplication of the shoots on the explants previously treated with colchicine. The explants without colchicine treatment or low colchicine concentrations (30 mg/l) showed better shoot multiplication compared to those higher conchicine concentrations. The former explants produced more and greener shoots, while the later formed fewer, yellow, and seemed to be abnormal shoots. Browning even occurred in explants that formerly treated with the highest concentration of colchicine, 120 mg/l (Fig. 4).
The 4-weeks colchicine treatment greatly influenced the explants ability in the shoots formation and multiplication on colchicine-free medium. The higher colchicine concentrations, induced greater inhibition on greater shoot formation (Fig. 5). Around 30% inhibition occurred in 30-60 mg/l colchicine-treated explants, while the explants that were previously treated with 90-120 mg/l colchicine underwent about 45- 60% inhibition. The total number and average of shoots formed in non-treated explants, 30-60 mg/l colchicine-treated explants, and 90-120 mg/l colchicine-treated explants were 39.3 and 7.9, 27-28 and 5- 6, 16-21 and 3-4 shoots respectively.
Remarks: The same notation on observation parameter shows no significant difference based on DMRT at 5%
significance level.
Fig. 3. The effect of colchicine addition in the medium on the shoot formation after 4 weeks culture: A. Total number of shoots; B. Shoot number per explant.
a ab
bc c
c
a
a
ab bc
c
Aside from affecting explant growth and shoot formation, colchicine treatment on shoot basal explants also hindered plantlet regeneration. Explants treated with high concentrations of colchicine had low plantlet regeneration ability. Control explant and colchicine-treated explants up to 60 mg/l were able to regenerate to form plantlets well. In contrast, the plantlet regeneration was completely blocked in 90 and 120 mg/l colchicine-treated explants. In these colchicine concetrations, the shoots formed failed to develop further and no plantlet was formed (Fig. 6).
Fig. 4. Response of shoot formation and growth of colchicine-treated shoot base explants after four weeks sub culture on colchicine-free medium. A. Control; B-E. After colchicine treatment: B. 30 mg/l; C. 60 mg/l ; D. 90 mg/l; E. 120 mg/l.
Remarks: The same notation on the observation parameter shows no significant difference based on DMRT test at 5%
significance level.
Fig. 5. The effects of colchicine treatments on shoot formation after four weeks cultured on colchicine-free medium: A. Total number of shoot; B. Shoot number/explant.
Fig. 6. Plantlets regeneration from shoot base explants treated using colchicine at various concentrations.A. Control; B-E. After colchicine treatment: B. 30 mg/l; C. 60 mg/l; D. 90 mg/l; E. 120 mg/l concentrations.A. Control; B-E. After colchicine treatment: B. 30 mg/l; C. 60 mg/l; D. 90 mg/l; E. 120 mg/l.
A B C D E
a
b bc
bc c
a
b
bc c bc
B
A C D E
Inhibition of shoot formation from colchicine-treated explants was due to inhibition of cell division and decrease in morphogenesis. Ye et al. (2010) showed that the inhibition of shoot growth after colchicine treatment was caused by a decreasing rate of cell division. Meanwhile, according to Bennici et al. (2006), high concentrations of colchicine reduced morphogenesis. A decrease in the ability to form shoots by colchicine was also observed in Petunia hybrida. The percentage of shoot regeneration in 2 cultivars decreased from 78 and 90% in control to 13.3 and 5.2% when 0.025% colchicine was added for 2 weeks.
In addition, plantlets regenerated from colchicine-treated explants were short and grew slowly, and had abnormally dark leaves (Abu-Qaoud and Shtaya, 2014). Colchicine added to the culture medium also significantly inhibited the shoot regeneration of Echinacea purpurea L. (Nilanthi et al., 2009) and plantlet regeneration of orchid Vanda hybrid (Vanda limbata Blume X Vanda tricolor Lindl. var.suavis (Tuwo and Indrianto, 2016); The higher colchicine concentration induced lower regeneration rate. Tuwo and Indriato (2016) reported that colchicine treatment decreased the number and length of roots, number and length of leaves. Colchicine also delayed the shoot development and induced more callus on the surface of the explants (Nilanthi et al., 2009).
The study on in vitro culture of Digitalis lanata indicated that the percentage of explant survival and the potential for shoot regeneration were affected by the duration of exposure and the concentration of colchicine (Bhusare et al., 2021). The higher the concentration of colchicine and the longer the treatment duration induced lower percentage of successful seed germination of Bletilla striata (Li et al., 2018), Eriobotrya japonica (Blasco et al., 2015) and Sesamum indicum (Anbarasan et al., 2014) under in vitro conditions. Administration of colchicine also significantly reduced the percentage of seed germination of Cadonopsis lanceolata, Hordeum vulgare and Vicia narbonensis (Kwon et al., 2016; Sourour et al., 2014;
Bakry et al., 2007).
Influence of Colchicine on Induction of Polyploidy
Polyploidy induction by colchicine is a well-known approach for chromosome doubling that can be used to produce polyploid plants. Determination of ploidy level of plantlet regenerated from without and with colchicine treated explant was identified based on flow cytometry analysis and chromosome number observation. The result of flow cytometry analysis showed that all polyploid plantlet were mixoploid with no tetraploid plantlet was observed. Mixoploids plantlets are made up of mixed ploidy and the mixoploid observed in this study was diploid, tetraploid and octaploid cells. The histogram of flow cytometry results in Fig. 7 was a representative fluorescence profile nuclei from plantlet of diploid and mixoploid from colchicine- treated explant. The ploidy level of flow cytometry analysis showed the G1 peak of diploid 2n (R2) was on channel 104 (10,000) of relative fluorencent intensity, whereas mixoploid plantlets with diploid, tetraploid and octaploid showed a peak at the channel 10,000, 20,000, and 40,000 respectively (Fig. 7). Percentage of mixoploid plantlet regenerated from the explants treated with 30 mg/l colchicine were higher than those treated with 60 mg/l colchicine (Table 1). The higher colchicine concetrations (90 and 120 mg/l) inhibited the shoot regeneration and then shoot became necrotic so no plantlet was obtained.
Mixoploid plantlets of A. muelleri have been obtained by in vitro induction using colchicine on shoot base explants. Observation and counting of chromosomes were carried out to confirm the result of flow cytometry. The chromosome count of the initial diploid plant of A. Muelleri was 2n = 2x = 26, according to a mitotic examination of the apical meristems of the roots. Most of plantlet regenerated from treated explant was mixoploid with tetraploid cells 2n = 4n = 52 and/or triploid cells 2n = 3x = 39 (Fig. 8B-D) in addition to diploid cells 2n = 2x = 26, as shown in Fig. 8. Among the 19 regenerated plantlets from explants treated with colchicine, 9 plantlets were confirmed mixoploid by chromosome count and flow cytometry analysis.
The effective concentration of colchicine to induce polyploidy of chromosome was 30 mg/l (Table 1).
Colchicine is commonly used in plant breeding to induce polyploidy since it is an antimitotic drug.
The substance prevents the microtubule formation, thus inhibits the segregation chromosomes during mitosis and induces polyploidy (Manzoor et al., 2019). Colchicine affects microtubule depolymerization through the formation of colchicine-tubulin complex. The intensity of the complex should be proportional to the polyploidy inducing capacity of the colchicine. The ability of colchicine to induce polyploidy was affected by concentration and duration of exposure of this mutagen (Udensi and Ontui, 2013). Polyploid have been successfully induced by colchicine treatment in some species, such as Watsonia lepida N.E. Brown
(Ascough et al., 2008), Capsicum frutescens L. (Pliankong et al., 2017), Rhododendron fortune Lindl (Mo et al., 2020), Trigonella foenum-graecum L. (Omezzine et al., 2012), Populous hopeiensis (Wu et al., 2022).
.
Fig. 7. Flow cytometry histogram of colchicine treated and control porang (A. muelleri Blume). A. Histogram of original porang plant (in vivo), the diploid peak (R2) close to channel 104 (10000); B. Histogram untreated plantlet, the diploid peak (R2) is set at channel 10000 and the tetraploid peak (R3) (because of a small amount of cell division) is observed at channel 20000; C and D. Histogram for mixoploid plantlet from treated explant with 30 and 60 mg/l colchicine showing a small number of tetraploid (R3, peak at channel 20,000) and octaploid nuclei (R4, peak at channel 40000) in addition to the diploid peak (R2) (channel 10,000); X-axis is number of nucleic counted; Y-axis is relative fluorescence intensity.
Table 1. Ploidy level of A. muelleri plantlets regenerated from basal shoot treated with colchicine Colchicine Concentration
(mg/l)
Number of Regenerated Plantlet
Percentage of Diploid (%)
Percentage of Mixoploid (%)
0 10 100 0
30 11 45 55
60 8 50 50
90 0 - -
120 0 - -
A B
C D
Fig. 8. Metaphase chromosome number in root tip of A. muelleri plantlet. In the control plantlet, diploid cell, 2n = 26 chromosomes; B-D. In the colchicine treated plantlets; B. Triploid cell, 2n = 3x = 39 chromosomes and tetraploid cell 2n=3x=52 chromosomes, C and D. Tetraploid cell 2n = 4x = 52 chromosomes.
In this study, mixoploid were induced but no tetraploid was observed in all of the treatments. Similarly, in Trigonella foenum-graecum L., colchicine produced mixoploid (Omezzine et al., 2012), whereas in Watsonia lepida, Rhododendron, colchicine produced more plants of mixed ploidy than stable tetraploid (Ascough et al., 2008; Vainola and Repo, 2001). Previous studies indicated that the presence of mixoploids is related to process of in vitro polyploidy induction (Ewald et al., 2009). According to Ascough et al (2008), colchicine treatment on multicellular tissue as starting material can cause more plant of mixed ploidy than stable tetraploid.
Mixoploids are generally through to be unstable because of the asynchronies among different cell type in the mixoploid cell cycle (Nilanthi et al., 2009). However, several stable mixoploid in Populus have been reported in previous studies (Xu et al., 2016; Liu et al., 2018). According to Ascough et al (2008), for producing a plant of only one ploidy level, it is necessary to use individual cells as explant. Meanwhile, Vainola and Repo (2001) stated that concentration and duration of colchicine treatment that is highly lethal (low survival) can reduces the number of diploid and mixoploid plants.
CONCLUSION AND SUGGESTION
In vitro induction of polyploidy A. muelleri Blume had effects on growth explant, shoot formation and plantlet regeneration. Higher colchicine concentration is very toxic and may lead to the onset of necrosis in shoot base explant and reduced shoot regeneration of explant. Through ploidy level analysis, mixoploid plantlets of A. muelleri Blume produced using colchicine and in vitro technique.
ACKNOWLEDGEMENT
We are grateful to Universitas Brawijaya and Ministry of Education and Culture, Research and Technology for supporting of this research through Penelitian Dasar Unggulan Perguruan Tinggi grant for 2022.
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