Induction of polyploids in cayenne pepper (capsicum frutescens L.) through various concentrations of colchicine

Polyploidy was declared as an attempt to obtain a new variety with the hope of increasing the quality of diploid (2n) plants by changing the chromosome arrangement to become tetraploid (4n) using the 2n chili seed soaking method for 30 days at a concentration of colchicine, namely K0 = 0.0% (control); K1 = 0.1%; K2 = 0.2%; and K3 = 0.3%. Data from the analysis of variance (ANOVA) stated that there was a very significant effect (**) on all observed variables. The results of the analysis on the variable percentage of seed germination showed that the treatment with a concentration of 0.0% was 92.20% with the number of chromosomes 24 (2n) and 0.1% as much as 26.20% with the number of chromosomes 48 (4n), while the increase in colchicine concentration (0.2% to 0.3%) has a lethal effect so that no chili seeds germinate (0.00). The average concentration of 0.1% treatment was higher with a large stem diameter, a large number of branches, a small number of leaves, more chlorophyll content with a dark green dun color, a larger number of stomata, a smaller leaf area, shorter flowering, more number of stomata, smaller leaf area, age shorter flowering, more glossy fruit color, shorter physiological maturity, longer fruit stalk size, longer fruit size, larger fruit diameter, heavier fruit, a higher number of harvested fruits, a heavier harvested fruit, number of seeds per fruit, and the weight per 100 seeds is heavier when compared to the control treatment (K0 = 0.0), which is very different.

Illustrations explain the paper; they do not show study data.

AI-generated illustration

↗ Enlarge

Colchicine Concentration vs. GerminationClose ×

AI-generated conceptual illustration (Cayenne pepper seeds (Capsicum frutescens L.)). It explains the idea and does not show study data.

Open full-size image ↗

AI-generated illustration

↗ Enlarge

Chromosome Doubling ConceptClose ×

AI-generated conceptual illustration (Cayenne pepper (Capsicum frutescens) plant cells). It explains the idea and does not show study data.

Open full-size image ↗

Introduction

Cayenne pepper (Capsicum frutescens L.) is one of the horticultural commodities needed by the community as a flavor enhancer for various types of food menus (Murni, 2010) [43] . The distinctively spicy taste of cayenne pepper is caused by the presence of capsaicin (C18H27NO3) contained in the fruit (Silvia et al., 2016) [35] . Differences in capsaicin content are often used as a differentiator between types of characters in cayenne pepper plants (Colney et al., 2018) [5] . Cayenne pepper productivity has been increased in various ways, including through fertilization (Hapsoh at al., 2017) [11] pest and disease control (Herison et al., 2014) [13] , mechanization (Setyanto et al., 2020) [33] , irrigation (Zaitun et al., 2021) [42] , and the use of superior cayenne pepper varieties (seeds) (Andriani et al., 2020) [2] . The intensification program using superior cayenne pepper seeds is the first step to increasing production (Murni, 2010) [43] . According to Effendi et al. (2018) [6] , efforts to improve productivity through the use of superior cayenne pepper seeds require several stages of breeding, including expanding genetic diversity, analyzing character inheritance, and selecting and releasing varieties. Superior cayenne pepper seeds to date are generally produced through breeding activities by crossing cayenne pepper elders who have superior characters so that hybrids are obtained that carry a combination of superior characters from the two crossed elders (Herison et al., 2014) [13] . In addition, superior cayenne pepper seeds can also be obtained through genetic material engineering, such as by carrying out polyploid operations, either triploid (Bakhtiar et al., 2002) [4] or tetraploid (Lelanga and Seran, 2020) [17] . Polyploidy in plants can occur naturally or artificially. Natural polyploids occur through exposure to lightning in plants or through allogamy (crossing) between similar plants, whereas artificial polyploids can be carried out through electrical shock or exposure to mutagens, particularly antimitotic compounds such as colchicine and oryzalins (Sukamto et al., 2010) [38] . Colchicine is a mutagen that is often used for polyploid purposes in plants. The effectiveness of colchicine in manipulating the number of chromosomes is largely determined by the concentration and method of exposure to colchicine and the part or type of plant used. The concentration of colchicine that has a polyploid effect on plants varies between 0.01% to 1% (Suminah et al., 2002) [39] , while the methods of exposure to colchicine can be through soaking, dipping, dripping, greasing, injecting, and spraying on seeds, sprouts, stems, plantlets, or flowers (Mahyuni et al., 2015) 19] . The application of colchicine to diploid plants, that have two sets of chromosomes (2n), can lead to polyploidy, such as the production of tetrapolyd plants, that have four sets of chromosomes (4n). Murni (2010) [43] , stated that the polyploid effect can be observed in morphological, anatomical, physiological, and cytological aspects; as shown by changes in the roots, stems, leaves, flowers, fruit, and composition of proteins, carbohydrates, and vitamins in plants (Sofia, 2008) [37] . This study aims to examine the effect of colchicine concentration on polyploid induction in cayenne pepper plants.

Materials and Methods

This research will be conducted in Lasoani Sub-District, Mantikolore District, Palu City, Central Sulawesi Province, Indonesia (Fig 1) at a geographical position of 0054'15" South Latitude and 119054'1" East Longitude with an altitude of ±135 m above sea level (Habbachi, 2022) [9] . This research will take place from August to December 2022. The tools used were digital scales, analytical balances, a light microscope (1000x magnification), a UV VIS spectrophotometer (ME-UV1100), centrifuges, Eppendorf tubes, glass cuvettes, refrigerators, Petri dishes, spatulas, porcelain cups, vials, tweezers, pipettes, object glass, deglass, a razor, a hotplate, a measuring cup (100 ml, 500 ml, and 1000 ml), a thermometer, a tray (5.5 cm x 3.

↗ Enlarge

Figure 1Close ×

Research Location, Palu City, Central Sulawesi, Indonesia

Open full-size image ↗

Original paper figureSource PDF · page 2 ↗

Each treatment was repeated five times, so that there were 20 experimental units. Each experimental unit used 25 cayenne pepper seeds; thus, the total number of seeds used was 500 seeds.

The data obtained from this study will be analyzed using analysis of variance (ANOVA). The results of the analysis of variance that show a significant or very significant effect will be tested further using the Honest Significant Difference Test (5% HSD) to compare the average values of all treatments tried (Nuryadi et al. 2017) [24] .

Results

The results of the analysis of varian (ANOVA) in the treatment of colchicine concentrations from various observational variables are shown in Table 1.

↗ Enlarge

Table 1Close ×

Results of the Analysis of Variance (ANOVA) in the treatment of colchicine concentrations from various observational variables

Open full-size image ↗

Original paper figureSource PDF · page 3 ↗

Discussion

Discussion

Effect of Colchicine Concentration on Polyploid Induction in Cayenne Pepper Plants

Colchicine is an antimitotic substance that is used in manipulating genetic material, especially in engineering at the chromosomal level. According to Rochmat et al. (2017) [30] , administration of antimitotic compounds can cause mutations in all parts of the plant, from the growing point to the generative organs. The administration of colchicine concentrations showed differences in morphology that could be directly observed during the vegetative phase when compared to diploid plants (Aili et al., 2016) [1] . Based on the results of the analysis of diversity, it was stated that the treatment of cayenne pepper seeds, which were soaked at concentrations of 0.0% colchicine, 0.1%, 0.2%, and 0.3% for 30 days, had a very significant effect on all observed variables. This is in accordance with the opinion of Gultom (2016) [8] , namely that giving the right concentration will result in a change in the vegetative character. The higher the concentration given in polyploid induction, the more cells are exposed and damaged, which can cause morphological changes in the endoplasmic reticulum and Golgi bodies so that cell development becomes abnormal and takes time to grow back to normal (Langhans et al., 2009) [16] . Plants obtained from 0.1% coccine immersion showed a tetraploid effect with a doubling of the number of chromosomes and an increase in coccine concentration to 0.2% and 0.3%, causing inhibition (a lethal effect) on cayenne pepper seed germination. According to Sartika et al. (2020) [32] , the higher the concentration of colchicine, the higher the percentage of tetraploid cells, but the higher the percentage of sprout death. Data analysis of the percentage of seeds germinating stated that the 0.0% concentration treatment averaged 92.20% more than the 0.1% treatment, which only averaged 26.20%, while the increase in colchicine concentration (0.2% to 0.3%) gives a lethal effect so that no cayenne pepper seeds germinate "0.00" (Fig 3). According to Murni (2010) [43] , high concentrations of colchicine and long soaking times can reduce the ability of seeds to germinate, this is due to the toxicity contained in them, causing cell death. According to Miguel and Leonhardt (2011) [21] , effective polyploid induction is a treatment that produces a lot of polyploidy but does not kill plant cells. Nofitahesti and Daryono (2016) [23] , reported that soybean seeds (glycine max (L.) Merr.) at colchicine concentrations of 0.025% -0.05% -0.075% -0.1% -0.15% -0.2%, and 0.25% with soaking times of 6-8-12-16-18 and 24 hours failed to germinate, whereas in a study conducted by Murni (2010) [43] on curly chilli (Capsicum annuum L.) sprouts at a concentration of 0.05% for 24 hours stated that as many as 13% of the sprouts had dead root tips. From the results of this analysis, it can be seen that an increase in colchicine concentration and soaking time results in a decrease in the ability of seeds to germinate. This is in accordance with the statement of Omidbaigi et al. (2010) [25] , concerning the toxicity of colchicine, which can cause lethal effects, namely reactions that occur when substances or chemicals interfere with cell or sub-cell processes in living things, which can result in death, so that further observations, including analysis of the number of chromosomes, were only carried out on treatments with 0.0% and 0.1% colchicine concentrations. Saadah (2021) [31] , stated that soaking seeds in concentrations of colchicine for a long time can cause the chromosome structure to agglomerate and shrink, which is caused by the absorption of too much colchicine fluid (absorption). The results of chromosome analysis (Fig 4), in the treatment with a concentration of 0.0%, it was found that there were 24 (2n) with short spindle thread forms, while in the 0.1% colchicine concentration treatment, there were 48 (4n) with long and wrinkled spindle shapes. [25] , polyploid engineering aims to produce plants with larger morphological sizes and that are resistant to the environment, so from plant height data from various treatments, the concentration of colchicine is 0.1% at 4 WAP, an average of 35.00 cm, 8 WAP, an average of 58.90 cm and 12 WAP, an average of 62.82 cm, higher than the 0% treatment at 4 WAP, an average of 22.20 cm; at 8 WAP, the average was 38.52 and at 12 WAP, the average was 57.90 cm. Zuyasna et al. (2023) [42] , stated that the application of the right concentration of colchicine can have a positive impact on plant tissues and does not cause a decline in growth, resulting in tetraploid plants that are able to grow and develop faster than diploid plants.

↗ Enlarge

Figure 4Close ×

Shape and Number of Chromosomes Treatment 0.0% and Colchicine Concentration 0.1%. Cayenne pepper sprouts without colchicine

Open full-size image ↗

Original paper figureSource PDF · page 4 ↗

↗ Enlarge

Figure 4Close ×

Shape and Number of Chromosomes Treatment 0.0% and Colchicine Concentration 0.1%. Cayenne pepper sprouts without colchicine

Open full-size image ↗

Original paper figureSource PDF · page 4 ↗

↗ Enlarge

Figure 3Close ×

Germinated seeds of chili plants from various concentrations of colchicine

Open full-size image ↗

Original paper figureSource PDF · page 4 ↗

Polyploid plants have large xylem and phloem transport bundles, so the transport of assimilation products becomes more numerous, which has an impact on the very fast growth and development of stem diameter, so tetraploid plants have a larger volume of wood compared to diploid plants (Zaitun et al., 2021) [41] . Based on stem diameter data, it was stated that in the 0.1% colchicine treatment, the average age of Leaves are the organ that determines the assimilation produced for the purposes of plant growth and development.

Based on data on the number of leaves in the 0.0% concentration treatment, the average was 34.40, which was 34.40 more compared to the 0.1% colchicine concentration treatment, where the average was 28.00. This is in accordance with the opinion of Mahyuni et al. (2015) [19] , which states that the application of colchicine can affect leaf formation, the higher the dose given, the fewer leaves are formed.

Polyploid plants contain more chlorophyll than diploid plants, this is indicated by the greener leaf color (Liu et al., 2011) [18] . Based on data on leaf chlorophyll content from the treatment of 0.1% colchicine concentration in plant 1, the average was 3.72 g/l, plant 2 averaged 4.66 g/l and plant 3 averaged 5.64 g/l, more than the 0.0% treatment, which in plant 1 averaged 2.30 g/l, plant 2 averaged 3.28 g/l and plant 3 averaged 4.76 g/l.

The success of colchicine in producing polyploid plants can be determined by observing the shape, size, and number of stomata, which have increased in number compared to diploid plants (Rocmat et al., 2017) [30] . Based on data on the number of stomata, the average 0.1% concentration of colchicine treatment was 98.00 more than the 0.0% treatment average of 69.33 (Fig 5). The existence of stomata is related to the plant's ability to adapt to the environment in terms of transpiration. The more number of stomata, the more parts of the leaf that produce Co2 for the photosynthesis process so that the more chlorophyll (green substance in leaves) is produced (Ere, 2018) [7] .

↗ Enlarge

Figure 5Close ×

Observation of leaf structure with a magnification of 1000x at a concentration of 0.0%, the number of stomata is small, the shape

Open full-size image ↗

Original paper figureSource PDF · page 5 ↗

↗ Enlarge

Figure 5Close ×

Observation of leaf structure with a magnification of 1000x at a concentration of 0.0%, the number of stomata is small, the shape

Open full-size image ↗

Original paper figureSource PDF · page 5 ↗

Fig 5:

Observation of leaf structure with a magnification of 1000x at a concentration of 0.0%, the number of stomata is small, the shape looks large, the distance is far apart and the concentration of colchicine is 0.1%, the number of stomata is large, the shape looks small, the distance is close together Maulana et al. (2021) [20] , stated that polyploids from several types of plants have larger leaf sizes compared to diploid plants, but in this study, the concentration of colchicine on leaf area stated that the 0.0% treatment on the second leaf averaged 8.92 mm 2 and the 3rd leaf averaged 12.47 mm 2 wider than the 0.1% treatment, which had the 2nd leaf area an average of 5.97 mm 2 and the 3rd leaf had an average of 8.45 mm 2 . This is in accordance with the statement of Sartika et al. (2020) [32] , Mutations due to colchicine cause changes in shape, number, and leaf area compared to controls, with a greener color due to increased chlorophyll content (Fig 6).

↗ Enlarge

Figure 6Close ×

Plant Morphology at a concentration of 0.0%, has a large leaf area with a wavy surface, shiny green color, begins to flower at 66 days

Open full-size image ↗

Original paper figureSource PDF · page 6 ↗

Assess the effect of polyploidy (diploid [2n] and tetraploid [4n]) on the yield of cayenne pepper

According to Sa'adah (2021) [31] , plants that experience changes in the arrangement of chromosomes will experience changes in chemical content, one of which is growth hormones such as auxins, cytokinins, and gibberellins, which can affect flowering rates. Syaifudin et al. (2013) [40] , stated that polyploid plants have cells with large transport bundle sizes, causing the process of transporting assimilation products and water to run better at 0.1% on average, 36.60 days faster flowering than the 0.0% concentration treatment on average, 66.00 days longer (Fig 6).

Hasimi et al. (2016) [12] , stated that the natural color change of fruit is influenced by ethylene gas, temperature, fruit cultivars, and chlorophyll content, which undergoes degradation so that carotenoid pigments emerge, which enzymatically appear little by little. Pigment groups contained in fruit, such as chlorophyll, carotenoids, and flavonoids (Antocyanins and anthoxantins), can affect fruit color, which is bright and fades (Pardede, 2013) [26] . Based on the results of visual observations of changes in fruit color (Fig 7) in the 0.0% treatment, when the ovary comes out of the calyx, it is light green; when it starts to develop,it is yellowish green; when it starts to ripen, it is faded yellowish red; and when it is ripe or ready to be picked, it is faded dark red. While for the treatment the concentration of colchicine is 0.1% when the ovary comes out of the dark green calyx, when it starts to develop it is shiny green, when it starts to ripen it is shiny dark orange, and when it is ripe or ready to be picked it is shiny dark red. [10] , polyploid plants contain more secondary metabolites, a faster metabolism, and greater biomass, resulting in brighter fruit colors and faster physiological maturity. This is in accordance with physiologically ripe fruit age data in the 0.0% treatment, it takes an average of 45 days longer than the 0.1% colchicine concentration treatment, which takes an average of 35 days. According to Ramadhani et al. (2013) [28] , the morphological shape of the fruit in each variety varies, this is due to genetic factors, causing differences in traits or genotypes. The fruit morphology of polyploid plants can be used as an indicator of the success of colchicine application (Anggraito, 2004) [3] . Based on data on fruit stalk length in the 0.1% colchicine concentration treatment, the average was 26.43mm longer than the 0.0% treatment, which was an average of 21.91mm. Simonsvsks et al. (2016) [36] , stated that the fruit stalk serves as a place for attachment and release of the fruit from the flower petals, besides that, the fruit stalk is closely related to fruit quality, and the short length of the fruit stalk will affect 36 velotyl compounds, which function as fruit aroma enhancers, and storage time.

↗ Enlarge

Figure 7Close ×

Observation results changes in fruit color from formation to physiological maturity at concentrations of 0.0% and 0.1% Colchicine

Open full-size image ↗

Original paper figureSource PDF · page 6 ↗

↗ Enlarge

Figure 7Close ×

Observation results changes in fruit color from formation to physiological maturity at concentrations of 0.0% and 0.1% Colchicine

Open full-size image ↗

Original paper figureSource PDF · page 6 ↗

According to Kosmiatin and Husni (2018) [15] , fruit shape and size are influenced by the genotype of each variety as well as environmental factors that support fertilization. Data on fruit length in the 0.1% colchicine concentration treatment averaged 34.56mm longer compared to the 0.0% treatment with an average of 28.20mm (Fig 8). Data on fruit diameter in the 0.1% colchicine concentration treatment averaged 8.99 mm larger than the 0.0% treatment with an average of 8.75 mm (Fig 9). Fruit weight data in the 0.1% colchicine concentration treatment averaged 1.26 g heavier than the 0.0% treatment with an average of 0.66 g (Fig 10). Data on the number of fruits harvested in the treatment with 0.1% colchicine concentration averaged 37.00 more than the yield in the 0.0% treatment, with an average of 28.00.

↗ Enlarge

Figure 10Close ×

Cayenne pepper plant fruit weight from treatment concentrations of 0.0% and 0.1% Colchicine

Open full-size image ↗

Original paper figureSource PDF · page 7 ↗

↗ Enlarge

Figure 9Close ×

Fruit Diameter of cayenne pepper plants from treatment with 0.0% and 0.1% Colchicine Concentrations

Open full-size image ↗

Original paper figureSource PDF · page 7 ↗

↗ Enlarge

Figure 8Close ×

Length of cayenne pepper plant fruit from treatment with concentrations of 0.0% and 0.1% Colchicine

Open full-size image ↗

Original paper figureSource PDF · page 7 ↗

Harvest fruit weight data in the 0.1% colchicine concentration treatment averaged 38.35g heavier than the 0.0% treatment with an average of 20.94 g. According to Rahajeng and Rahayuningsih (2013) [27] , seed formation in tetraploid plants is influenced by two factors, namely genetic incompatibility and ineffective fertilization, so that it will produce fruit with a large or small number of seeds. Setyawan et al. (2018) [34] , stated that pollination carried out at cultivation sites with an altitude of ±200 masl with an average temperature of 19-32 °C produced tetraploid fruit with a large number of seeds, while according to Riandoni et al. (2020) [29] , if it was carried out at an altitude of ± 610 masl with a temperature average of 16-29 °C, it would produce tetraploid fruit with a small number of seeds. From the data on the number of seeds per pod, with the height of the study site ± 135 masl, the highest number of seeds was obtained on average at 29.73 in the 0.1% colchicine concentration treatment, while for the 0.0% treatment the average was 23.67. According to Iqbal et al.

(2015) [14] , the shape and size of seeds can affect seed viability in general. Large seed sizes have food reserves (endosperm) such as carbohydrates, proteins, fats, and minerals that function as energy for the embryo during germination, while small seeds and shrivels will experience difficulties during germination. Based on data on weight per 100 seeds, in the 0.1% colchicine concentration treatment, the average was 0.38g heavier than in the 0.0% treatment, where the average was 0.26g (Fig 11). According to Setyanto (2020) [33] , after fertilization, the seeds will experience an increase in size and weight until they reach physiological maturity and then become lighter as the water content decreases due to the increased level of fruit maturity.

↗ Enlarge

Figure 11Close ×

Weight of 100 cayenne pepper seeds from 0.0% and 0.1% colchicine concentration treatments

Open full-size image ↗

Original paper figureSource PDF · page 8 ↗

Conclusion

 Results of analysis of cayenne pepper seeds soaked in 0.0% colchicine concentration; 0.1%; 0.2% and 0.3% for 30 days indicated that all observation variables had a very significant effect.  Cayenne pepper seeds that managed to germinate in the colchicine concentration treatment for 30 days of immersion, stated that the 0.0% treatment germinated as much as 92.20% and the 0.1% treatment as much as 26.20%, while the increase in colchicine concentration (0.2% to 0.3%) gives a lethal effect so that no chili seeds germinate "0.00".  The results of chromosome analysis in the 0.0% treatment amounted to 24 = diploid (2n) with short spindle thread shapes, and the 0.1% colchicine concentration treatment totaled 48 = tetraploid (4n) with spindle thread shapes experiencing clumping and shrinkage caused by absorption of too much colchicine fluid (absorption).  A greener leaf color has more chlorophyll content (0.1%) compared to a leaf that has control (0.0%). The number of stomata is more (0.1%) than in the control treatment (0.0%).  The 0.1% colchicine concentration treatment had morphological forms, the plants were taller with a larger stem diameter than the control (0.0%), the number of branches formed was higher (0.1%) when compared to the control treatment (0, 0%), the number of leaves was higher (0.1%) than the control (0.0%), and the leaf area was smaller (0.1%) when compared to the control (0.0%), which is larger. The age of flowering treatment was 0.1% shorter with an average of 36,60 days compared to the control treatment (0.0%) with an average of 66.00.  Based on fruit morphology, the 0.1% colchicine treatment had longer fruit stalks, longer fruit sizes, larger diameters, and had more fruit weight than the control (0.0%). The 0.0% treatment required a long time to achieve the criteria for physiological ripeness, while the fruit treated with a concentration of 0.1% colchicine required a short average time of 34.87 to meet the criteria for physiological maturity with a shiny red color.  Based on data on the number of fruits harvested, the 0.1% treatment was more effective than the 0% treatment.  Based on the results of the analysis, the average weight of the harvested fruit stated that the 0.1% treatment was heavier than the 0% treatment.  The results of the calculation of the number of seeds in the 0.1% colchicine treated fruit had a higher number than the control treated fruit (0.0%).

 The weight per 100 seeds in the 0.1% colchicine treatment stated that the average was heavier than the control treatment (0.0%).

Acknowledgement

AcknowledgmentsAlhamdulillah, an expression of my gratitude to all parties who have helped in this lecture and research. All the knowledge that researchers receive will be charity for all of you. Data will be available upon request. This information will become part of the advancement of world civilization and be useful for mankind. May Allah forgive all our sins and mistakes and accept our deeds.

Figures and tables from the paper

↗ Enlarge

Figure 2Close ×

Layout Plan of the Experimental Unit.

Open full-size image ↗

Original paper figureSource PDF · page 3 ↗

1. Aili EN. Effect of colchicine administration on phenotypic appearance of forage maize inbred lines (Zea mays L.) in the vegetative growth phase. J Plant Production. 2016 Jun;4(5):370-377.
2. Andriani I, Yuniarsih ET. Technological innovation in chili cultivation in West Sulawesi. J Agercolere. 2020 Sep;2(2):37-44.
3. Anggraito YU. Identify the weight, diameter, and thickness of the fruit flesh melon (Cucumis Melo, l) cultivar action 434 tetraploid as a result colchicine treatment. Berk. Biological Panel. 2004 Dec;10:37-42.
4. Bakhtiar, Nurzuhairawaty. Change some great chili (Capsicum annum L.) due to administration of colchicine. J Agrosains. 2002 Jul;4(1):1-6.
5. Colney L, Tyagi W, Rai M. Morphological and molecular characterization of two distinct chilli cultivars from North Eastern India with special reference to pungency related genes. Scientia Horticulturae. 2018 May;240:1-10.
6. Gultom T. Effect of colchicine on chromosome number of garlic (Allium sativum) local cultivars doulu. J. Biosains. 2016 Dec;2(3):223-231.
7. Handayani T, Witjaksono, Nugraheni KUT. In vitro Induction of Tetraploid in Guava (Psidium guajava L.). J Biologi Indonesia. 2017 Aug;13(2):271-278.
8. Hapsoh, Gusmawartati, Amri AI, Diansyah A. Response of growth and production of curly chili (Capsicum annuum L.) to the application of compost and inorganic fertilizers in polybags. J. Hortikultura Indonesia. 2017 Apr;8(3):203-208.
9. Hasimi NR, Poerwanto R, Suketi K. Siamese citrus fruit degree (Citrus Nobilis) on several concentrations and duration of ethylene exposure. J Hortikultura Indonesia. 2016 Aug;7(2):111-120.
10. Iqbal MA, Cheema ZA, Afzal IM. Evaluation of forage soybean (Glycine max L.) germination and seedling growth enhancement by seed priming techniques. American-Eurasian J. Agric. & Environ. Sci. 2015 Oct;15(6):1198-1203.
11. Kosmiatin M, Husni A. Breeding seedless citrus varieties through conventional and non-conventional breeding. J. litbang Pertanian. 2018 Dec;37(2):91-100.
12. Langhans M, Niemes S, Pimpl P, Robinson DG. Oryzain bodies: in addition to its ant microtubule properties, the dinitroaniline herbicide oryzalin causes nodulation on the endoplasmic reticulum. J. Protoplasm A. 2009 Jun;236:73-84.
13. Lelanga MA, Seran MK. Effect of colchicine concentration on phenotype performance of local bird's eye chili (Capsicum frutescens L.) from Timor Island. J. Savana Cendana. 2020 Jan;4(1):15-17.
14. Liu S, Chen S, Chen Y, Guan Z, Yin D, Chen F. In vitro induced tetraploid of dendrathema nakingense (nakai) tzvel, shows an Improved level of abiotic stress tolerance. J. Scientia Horticulturae. 2011 Jan;127(3):411-419.
15. Mahyuni R, Girsang ESB, Hanafiah DS. Effect of colchicine administration on morphology and chromosome number of binahong plants (Anredera cordifolia (Ten) Steenis). J. Agroekoteknologi. 2015 Dec;4(1):23-33.
16. Maulana E, Efendi D, Sari L. Growth evaluation of polyploid chlorophyll and carotenoid content of torbangun (Coleus amboinicus L.) through in vitro culture. J. Bioteknol dan Biosains Indonesia. 2021 Dec;8(2):230-243.
17. Miguel TP, Leonhardt KW. In vitro poliploid induction of orchids using oryzalin. J. Scientia Horticulturae. 2011 Aug;130:314-319.
18. Antil R. Global and national climate change concerns in India. Int. J Geogr Geol. Environ. 2022;4(2):187-190. DOI: 10.22271/27067483.2022.v4.i2b.161
19. Nofitahesti, I., Daryono, B.S. Phenotypic characters of soybean (glycine max (L.) Merr.) polyploidized with colchicine. J. Sains Dan Pendidikan Sains. 2016;5(2):90-98.
20. Omidbaigi R, Mirzaee M, Hassani ME, Moghadam MS. Induction and identification of polyploidy in basil (ocimum basilicum L.) medicinal plant by colchicine treatment. International Journal of Plant Production. 2010 May;4(2):87-98.
21. Pardede E. Review of the chemical composition of fruits and vegetables: Role as a nutrient and its relation to preservation and processing technology. Jurnal Visi Universitas HKBP Nommensen. 2013 Oct;21(3):14521463.
22. Ramadhani R, Damanhuri, Purnamaningsih SL. Appearance of ten genotypes of red chili (Capsicum annuum L.). J. Plant production. 2013 Mei;1:2, ISSN: 2338-3976.
23. Riandoni G, Sugiharto AN, Saptadi D. Increasing the number of tetraploid watermelon seeds (Citrullus Vulgaris) by manipulating pollination time and frequency. J Plant production. 2020 Sep;8(7):669-673.
24. Rochmat SM. Rahayu T, Laili S. The effect of giving various concentrations of colchicine with soaking time on the phenotypic response of olives (Olea Europaea). J. Biosaintropis. 2017 Dec;2(2):36-41.
25. Sartika D, Agustrina R, Ernawiati E, Irawan B. The Role of colchicine in the multiplication of polyploidy banana kapok plantlets in vitro. J. Natural Indonesia. 2020 Oct;18(2):78-81.
26. Setyanto P. Standard operating procedures for cayenne pepper cultivation. Agriculture department. Directorate of Vegetables and Medicinal Plants Press. 2020 Mei. 3.
27. Setyawan KF, Adiredjo AL, Ashari S. Pollination of watermelon flowers (citrullus vulgaris) as an effort to form superior seeds. J. Produksi Tanaman. 2018 Jan;6(7):1427-1432.
28. Silvia M, Susanti H, Samharinto S, Noor GMS. Production of cayenne pepper (Capsicum frutescens l.) on ultisol soil using household organic waste bokashi and NPK. J. Enviro Scienteae. 2016 Apr;12(1):19-28.
29. Simonsvsks J, Skerget M, Knez Z, Srbinoska M, Kavrakovski Z, Grozdanov A, et al. Physicochemical characterization and bioactive compounds of stalk from hot fruits of capsicum annum L. Macedonian Journal of Chemistry and Chemical Engineering. 2016 Dec;35(2):199-208.
30. Sukamto LA. Endosperm In vitro culture, an efficient protocol for obtaining triploid plants directly. J. Agro Biogen. 2010 Okt;6(2):107-112.
31. Suminah, Sutarno, Setyawan AD. Induction of polyploidy in shallots (Allium ascalonicum L.) by administering colchicine. J. Biodiversitas. 2002 Jan;3(1):174-180.
32. Syaifudin A, Ratnasari E, Isnawati. The effect of giving various concentrations of colchicine on the growth and production of chili (Capsicum annum L.) Lado F1 variety. Lentera Bio. 2013 Mei;2(2):167-171.
33. Zaitun, Zulfahrizal, Susanti E. Counseling on drip irrigation technology to increase chili productivity in Ie Suum Village, Mesjid Raya Aceh Besar District. J. Aplikasi Ipteks untuk Masyarakat. 2021 Dec;10(4):363366.
34. Zuyasna, Marliah A, Rahayu A, Hayati E, Husna R. The Growth of MV1 Patchouli Plants in Lhokseumawe Variety Due to Concentration and Immersion Time of Colchicine. J. Agro Bali Agricultural. 2021 Marc;4(1):23-33.
35. Murni, D. Effect of colchicine treatment on the number of chromosomes and phenotypes of curly chili plants (capsicum annuum L.). J Agroekotek. 2010 Jul;2(1):4348.