Next Article in Journal
The Variability of Proximate Composition, Sugars, and Vitamin C in Natural, Organic, and Biodynamic, and Fermented Leaves of Fireweed (Chamerion angustifolium (L.) Holub (Onagraceae))
Previous Article in Journal
Physiological and Molecular Responses of Camellia oleifera Seedlings to Varied Nitrogen Sources
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Horticulturae
Volume 9
Issue 11
10.3390/horticulturae9111244
horticulturae-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effect of Organic Additives on the Micropropagation of Asparagus officinalis

by
Preekamol Klanrit
1,2,
Khanittha Lila
3,
Paramaporn Netsawang
3,
Phakamas Siangsanor
3,
Pornthap Thanonkeo
1,2 and
Sudarat Thanonkeo
4,*
1
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
2
Fermentation Research Center for Value Added Agricultural Products (FerVAAPs), Khon Kaen University, Khon Kaen 40002, Thailand
3
Agricultural Promotion and Development Centre Suphan Buri, Suphan Buri 72160, Thailand
4
Walai Rukhavej Botanical Research Institute, Mahasarakham University, Maha Sarakham 44150, Thailand
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(11), 1244; https://doi.org/10.3390/horticulturae9111244
Submission received: 28 October 2023 / Revised: 17 November 2023 / Accepted: 17 November 2023 / Published: 18 November 2023
(This article belongs to the Section Propagation and Seeds)
Browse Figure
Versions Notes

Abstract

:
In vitro propagation is one of the most promising techniques for the large-scale clonal propagation of Asparagus officinalis. The aim of this study was to investigate the effect of organic additives, including coconut water (CW), banana homogenate (BH), and potato homogenate (PH), on A. officinalis shoot and root proliferation. The results revealed that CW, specifically at 20% (v/v), was the most effective organic additive for promoting shoot and root formation in the in vitro plantlets. Furthermore, the longest shoot and root lengths were also observed in the MS medium supplemented with 20% (v/v) CW. In the supporting medium of 1:1 peat moss and vermiculite, the in vitro plantlets exhibited a high survival rate with a morphology comparable to that of the mother plant. The results of this study demonstrate that CW can be applied as a supplemental material for large-scale A. officinalis micropropagation.

1. Introduction

Asparagus is one of the most important genera of the family Asparagaceae, comprising approximately 200 species of herbaceous perennial and tender woody shrubs that are native to Asia, Europe, and Africa [1,2]. Among the different species of asparagus, garden asparagus (Asparagus officinalis L.) is the most important in terms of economic, human nutritional, and medicinal value [3,4]. The aerial parts, roots, and seeds of A. officinalis contain several bioactive compounds, such as saponins, vitamins, flavonoids, steroids, glutathione, and spirostanol glycoside, which possess a variety of biological properties, including antioxidant, anti-inflammatory, antihepatotoxic, antimicrobial, antidiarrheal, antiulcerogenic, and anticancer properties [5,6,7,8,9,10,11].
A. officinalis is cultivated worldwide, and global production is estimated to reach 10.7 million metric tons by 2026. China is the world’s largest producer of asparagus at approximately 7.3 million metric tons in 2021 [12]. Conventional propagation of A. officinalis is performed using seeds or sometimes vegetatively via separation of the plant rhizome. However, despite the low viability of plant seeds, the slow growth rate of plantlets obtained from seed propagation, and the risk of spreading diseases, such as anthracnose, soft rot, and Fusarium rot, vegetative propagation [4,13] is not often used in commercial production. In vitro propagation or micropropagation techniques, such as direct or indirect organogenesis and somatic embryogenesis, can be used to overcome these obstacles. These propagation techniques provide large uniform plantlets with genetic stability; they are considered cost-effective methods for commercial propagation and can eliminate systemic diseases caused by plant pathogens.
In vitro propagation of A. officinalis has previously been reported, and most studies have focused on the effects of plant growth regulators (PGRs) on plant regeneration and development. For instance, Esmaeili et al. [3] investigated the effect of PGRs on the in vitro regeneration of A. officinalis. The authors reported that the highest number of shoots (4.25 shoots/explant) and roots (2.0 roots/explant) were obtained with Murashige and Skoog’s (MS) medium supplemented with 1.5 mg/L 6-benzylaminopurine (BAP) and 0.05 mg/L 1-naphthaleneacetic acid (NAA) and with 0.4 mg/L indole-3-butyric acid (IBA), respectively. Rasad et al. [14] reported the in vitro propagation of A. officinalis using the stem and root explants of sterile plantlets. The authors reported that MS medium supplemented with 2.0 mg/L BAP and 1.0 mg/L NAA resulted in the highest number of shoots (6.233 shoots/explant and 4.967 shoots/explant for stem and root explants, respectively). The highest number of roots was observed in MS medium supplemented with 0.5 mg/L BAP and 1.5 mg/L NAA for microshoots regenerated from stem explants and in MS medium supplemented with 2.0 mg/L BAP and 1.0 mg/L NAA for microshoots regenerated from root explants. Another study by Maung et al. [15] reported that MS medium supplemented with 2.0 mg/L BAP produced the highest values for the number of shoots (9 shoots/explant), shoot length (2 cm), and number of nodes (2 nodes/shoot), while the highest rooting rate (40%) was observed in medium supplemented with 2.5 mg/L IBA. Most recently, Minh et al. [16] demonstrated that MS medium supplemented with 2.0 mg/L BAP and 1.0 mg/L NAA resulted in the highest shoot formation rate (87.2%), while MS medium supplemented with 0.5 mg/L NAA yielded the highest rooting rate (74.59%) of in vitro A. officinalis plantlets.
The cultivation media for in vitro plant propagation comprise macro- and micronutrients, amino acids, vitamins, carbon sources, PGRs, and gelling agents, which are generally expensive [17]. Therefore, modifying the cultivation media composition by adding low-cost organic substances to replace expensive materials is considered an efficient approach for large-scale clonal plant propagation [18]. A variety of organic additives, such as coconut water (CW), banana homogenate (BH), potato homogenate (PH), yeast extract (YE), tomato juice (TJ), pineapple juice (PJ), and pineapple pulp (PP), have been widely used as supplements in plant tissue culture medium [18]. These organic additives provide not only natural sources of carbon, such as sucrose and fructose, but also natural vitamins, amino acids, lipids, minerals, and PGRs, specifically plant hormones, which are essential for plant growth and development [19,20]. However, based on a literature review, the effect of organic additives on the in vitro propagation of A. officinalis has never been documented; in most studies, organic additives were added to culture media for the in vitro propagation of orchids [20,21,22]. Therefore, to overcome the limitations of the conventional propagation of A. officinalis, which is generally based on plant seeds and rhizomes, and to minimize the in vitro propagation cost of plant tissue culture medium, this research aimed to employ an in vitro culture technique for the micropropagation of A. officinalis. Furthermore, the effect of low-cost and readily available organic additives, including CW, BH, and PH, on in vitro shoot and root proliferation in A. officinalis was also evaluated. The present study could provide useful information for the sustainable large-scale production of A. officinalis plantlets.

2. Materials and Methods

2.1. Chemical and Explant Preparation

Chemicals, including the plant tissue culture medium (Murashige & Skoog (MS) basal medium), phyto agar, and plant hormones (NAA and Kinetin (N6-furfuryladenine) were acquired from PhytoTech Labs, Lenexa, KS, USA, and Sigma Aldrich Corporation, Burlington, MA, USA, respectively. CW, banana (cv. Hong Thong), and potato were purchased from the local market in Khon Kaen, while peat moss and vermiculite were procured from Bee Garden and Farm Co., Ltd., Khon Kaen Province, Thailand.
Two-year-old plants of A. officinalis cultivar Brock’s Improved were used in this study. Plants were collected and washed thoroughly with running tap water, and then the stems of the plants were cut to approximately 5 cm long. For surface sterilization, the explants were washed with 70% (v/v) ethanol for 1 min and then washed three times with sterilized distilled water to remove the excess ethanol. The resulting explants were soaked in 15% (v/v) sodium hypochlorite for 15 min, followed by 10% (v/v) sodium hypochlorite for 10 min. After washing with sterilized distilled water, the axillary buds were excised and placed on MS basal medium supplemented with 1.0 mg/L kinetin and 0.05 mg/L NAA and cultured in a standard culture room at 25 ± 2 °C with a light intensity of 3000 lux and a 16-h photoperiod. The explants were subcultured on the same fresh solid MS basal medium before being cultivated for 28 days under the specified conditions.

2.2. Preparation of Organic Additives and Culture Medium

CW was taken directly from a young tender coconut. After filtration through four layers of cheesecloth to remove impurities, such as coconut bark or palm kernel, it was stored at −20 °C before use. The ripe banana was used in this study. The peeled fruit (100 g) was cut into small pieces and ground using a fruit blender (HR2115, Philips, Bangkok, Thailand). The resulting BH was collected and kept at −20 °C for future use. To prepare PH, 100 g of peeled potato was cut into small pieces and ground using a fruit blender. The resulting homogenate was collected and stored at −20 °C before use.
The culture medium was prepared by dissolving 4.43 g MS basal medium and 30 g of sucrose in distilled water. CW, BH, or PH at various concentrations was added, and the pH of the medium was adjusted to 5.8 using 0.1 N NaOH/HCl. Seven grams of phyto agar powder was added, and the volume of the culture medium was adjusted to 1 L using distilled water. After boiling using a microwave, 30 mL of the medium was aliquoted into a culture vessel and later sterilized by autoclaving at 121 °C, 15 kPa for 15 min.

2.3. Effect of Organic Additives on Shoot Proliferation

The sterile explants of A. officinalis were placed on MS basal agar medium containing 0.05 mg/L NAA and supplemented with different concentrations of CW (5, 10, 15, and 20%, v/v), BH (5, 10, 15, and 20%, w/v), and PH (5, 10, 15, and 20%, w/v). The MS basal medium was used as a control without PGRs and organic additives. After 4 weeks of cultivation, the number of shoots and the average shoot length were measured. One explant was placed in each culture vessel. All experiments were carried out twice, with ten replicates for each treatment.

2.4. Effect of Coconut Water on Root Proliferation

The sterile explants (stems) of A. officinalis were placed on MS basal agar medium supplemented with different concentrations of CW (5, 10, 15, and 20%, v/v). MS basal medium without PGR supplementation and medium supplemented with 0.05 mg/L kinetin and 0.35 mg/L NAA were used as controls. The number and length of the induced roots were determined after 4 weeks of cultivation. One explant was placed in each culture vessel. The experiment was repeated twice, each with ten replicates.

2.5. Ex Vitro Acclimatization of the A. officinalis Plantlets

Four-week-old plantlets of A. officinalis cultivated in MS basal medium supplemented with CW were selected and subjected to ex vitro acclimatization. Roots were washed with tap water after being removed from the culture vessel to remove the adhering culture medium. The resulting in vitro plantlets were transplanted in 10.0 cm diameter plastic pots filled with peat moss and vermiculite in a 1:1 ratio. All of the potted plants were transferred to a growth chamber at 25 ± 2 °C with a photoperiod of 16 h, a light intensity of approximately 3000 lux, and a relative humidity of approximately 45–55%. The survival rate and plant growth (plant height) were monitored after 30 days of cultivation. The experiments were performed twice, each with ten replicates.

2.6. Experimental Design and Statistical Analysis

The statistical experimental design, namely, a completely randomized design (CRD), was used in this study with 10 replicates for each treatment. The experimental data were manipulated using analysis of variance (ANOVA) with the SPSS application for Windows. The mean difference between each treatment was determined using Duncan’s multiple range test (DMRT) at the 95% confidence level or a probability of p ≤ 0.05.

3. Results and Discussion

3.1. Effect of Organic Additives on Shoot Proliferation

Several organic additives, such as CW, BH, PH, YE, TJ, PJ, and PP, have been used for plant micropropagation; however, their effects on plant growth and development vary depending on many factors, including plant species and cultivar, plant age, explant type, and cultivation conditions [19,23,24]. In this study, three organic additives, CW, BH, and PH, which are widely used and readily available at low cost, especially in Thailand, were chosen, and their effect on shoot proliferation in A. officinalis was evaluated. The effect of CW on shoot proliferation in A. officinalis is summarized in Table 1. MS medium without PGR and CW supplementation yielded the lowest number of shoots (1.55 shoots/explant) and average shoot length (2.19 cm). Supplementation of MS medium with 0.05 mg/L NAA and 1.0 mg/L kinetin improved shoot proliferation in A. officinalis, leading to an increase in the number of shoots (2.55 shoots/explant) and the average shoot length (3.70 cm). This finding suggests that NAA and kinetin are essential for shoot formation and development, since these PGRs are involved in cell division, differentiation, and elongation [25]. Notably, in the current study, the replacement of kinetin with CW significantly increased the number of shoots and the shoot lengths of A. officinalis. Supplementation with CW at concentrations of 15 and 20% (v/v) yielded the highest values for the number of shoots (4.00–4.10 shoots/explant) and shoot length (7.25–7.26 cm), implying that CW is more effective for promoting shoot formation and development in A. officinalis than kinetin at 1.0 mg/L. Apart from natural sources of carbon, mainly sugars, CW also contains some vitamins (thiamin, pyridoxine, ascorbic acid), amino acids, minerals (phosphorus, magnesium, potassium, calcium, iron, and manganese), and PGRs, such as cytokinins, zeatin, auxins (indoleacetic acid, IAA), gibberellic acid (GA), and abscisic acid (ABA), which are essential for plant cell division, differentiation, and development [18,20,24,26,27]. CW has been successfully used to promote shoot proliferation in several plants, such as Musa acuminata [24], Dendrobium chryseum [28], Dianthus caryophyllus [29], Hylocereus polyrhizus [30], Coelogyne pandurata [21], Echinacea purpurea [31], Cymbidium aloifolium [32], and D. cruentum [22].
The effect of BH on A. officinalis shoot proliferation was investigated, and the results are summarized in Table 2. MS medium without PGR and BH supplementation yielded 1.60 shoots/explant and a shoot length of 2.16 cm. Supplementation of MS medium with NAA and kinetin at 0.05 and 1.0 mg/L promoted shoot proliferation in A. officinalis. Similarly, supplementation of the MS medium with BH at 5% (w/v) also promoted shoot proliferation compared with MS medium without PGR and BH supplementation, suggesting that BH can compensate for the lack of kinetin. Notably, the number of shoots and the shoot lengths of A. officinalis in MS medium supplemented with 5% (w/v) BH were still lower than those of MS medium supplemented with NAA and kinetin. Furthermore, increasing the concentration of BH in the MS medium over 5% (w/v) yielded a lower number of shoots and lower shoot lengths, indicating that BH had a negative effect on A. officinalis shoot proliferation. Like other organic additives, such as CW or PH, BH contains high amounts of sugars and trace elements, such as potassium, calcium, sodium, iron, manganese, and bromine, as well as vitamins (thiamin, riboflavin, niacin, pyridoxine, pantothenic acid, and ascorbic acid), phenolic acid, carotenoids, and unidentified substances [19,33,34,35]. These substances are necessary for plant growth and development, and the optimal concentration of each substance depends on plant species, plant age, explant type, and cultivation conditions. For instance, BH at a concentration of 5% (v/v) was the best for shoot regeneration in Celosia sp. [36], while a concentration of 10% (w/v) was suitable for in vitro regeneration of Dendrobium sp. [37]. Another study by Gansau et al. [38] reported that 2.5% (w/v) BH was effective for shoot and root development during in vitro propagation of D. lowii. According to De Stefano et al. [39], BH at a concentration of 1% (w/v) promoted shoot and root proliferation in Epidendrum nocturnum. A recent study by Samala and Thipwong [22] also demonstrated that BH yielded the highest number of in vitro D. cruentum plantlets. Notably, the excess of some substances in BH, specifically sugars or some trace elements, such as calcium or sodium, may inhibit cell growth and development. The adverse effects of high concentrations of BH on the growth and development of some plant species have also been reported, e.g., protocorm-like bodies (PLBs) of Dendrobium sp. [40] and Raphanus sativus [27].
Considering the effect of PH on A. officinalis shoot proliferation, supplementation with this organic additive promoted shoot formation and shoot length compared to MS medium with and without PGR supplementation (Table 3). The maximum number of shoots (4.00 shoots/explant) and shoot length (7.22 cm) of A. officinalis were determined in MS medium supplemented with 0.05 mg/L NAA and 10% (w/v) PH. Higher concentrations of PH at 15 and 20% (w/v) reduced the number of shoots and the shoot lengths, indicating that this organic additive had a negative effect on plant cell growth and development, possibly due to an oversupply of the trace elements, such as potassium, iron, or magnesium, contained in the PH, as reported in other studies. As previously reported, PH contains several organic and inorganic substances, such as carbohydrates, amino acids, vitamins (thiamin, pyridoxine, and ascorbic acid), minerals (potassium, iron, and magnesium), PGRs, and unidentified substances that are involved in cell growth, differentiation, and development [41,42]. This organic additive has been widely used for the in vitro propagation of several plants, and the response of plant cells to PH depends on plant species and explant types. For example, Ouyang et al. [43] reported that PH at 5% (w/v) promoted callus formation and green shoot regeneration in Triticum aestivum. However, a higher concentration of this organic additive resulted in reduced green shoot regeneration. According to a study by Islam et al. [44], 100 mL/L PH promoted seedling growth for in vitro cultures of Vanda roxburgii. Increasing the concentration of PH led to a reduction in seedling growth in this orchid. A recent study by Rohmah et al. [32] demonstrated that supplementing MS medium with 10 g/L PH and 100 mL/L CW or 5 g/L PH and 150 mL/L CW yielded the highest number of shoots in C. aloifolium, while increasing the concentration of PH yielded a lower number of shoots.
Among the different types of organic additives tested in this study, CW exhibited superior performance in promoting A. officinalis shoot proliferation, followed by PH and BH. These organic additives contain several chemical constituents, including carbohydrates, vitamins, amino acids, trace elements, and PGRs [18,19,20,24,26,27,33,34,35,41,42]. The outperforming of CW for promoting shoot proliferation in A. officinalis could be attributed to the types of carbohydrates found in this material. The prime carbohydrates found in CW are soluble carbohydrate compounds, specifically sucrose, glucose, and fructose, which are efficiently taken up by plant cells, while those contained in BH and PH are starches, which have to be broken down into simple sugars before being taken up by plant cells. Another possibility is that CW is rich in PGRs, especially auxin and cytokinin, as well as vitamins, compared to BH and PH. Adding CW to the culture medium may provide sufficient phytohormones and vitamins to accelerate plant cell growth, differentiation, and development [25,45].
It should be noted in the current study that the number of shoots and the shoot lengths of A. officinalis cultivated in MS medium supplemented with CW and PH were nearly identical. However, adding PH to the MS medium caused the young plantlets to turn yellow, while CW produced greener and healthier plants. Therefore, CW was chosen to further investigate its effect on root proliferation in A. officinalis.

3.2. Effect of Coconut Water on Root Proliferation

In addition to sugars, vitamins, amino acids, organic acids, and minerals, CW is also rich in phytohormones, specifically natural auxins, which play an essential role in all plant physiological processes, including the induction of adventitious and lateral roots [25]. The major auxin predominantly found in CW is indole acetic acid (IAA) [45]. Based on the literature review, CW has been successfully utilized as a growth supplement for in vitro and ex vitro root proliferation in several plants, such as kiwifruit (Actinidia deliciosa) [46], mangrove (Rhizopora stylosa) [47], Tribulus (Tribulus terrestris) [48], and banana (Musa cv. Rajabulu) [49]. Notably, the application of CW for root induction in asparagus has never been documented. Therefore, the effect of CW as a growth supplement for in vitro root proliferation in A. officinalis was investigated in the current study. As shown in Table 4, the formation of A. officinalis roots was also observed in the control treatment, yielding 2.50 roots/explant with a root length of 1.55 cm. This finding suggested that this plant rooted easily in MS medium without PGR supplementation, similar to other plants, such as Philodendron sp. [50]. MS medium supplemented with 0.35 mg/L NAA and 0.05 mg/L kinetin yielded a higher number of roots (3.10 roots/explant) than the control MS medium, while root length was not significantly different from the control, indicating that a combination of auxins and cytokinins could enhance A. officinalis root formation, consistent with the findings of Desjardins et al. [51], Shigeta et al. [52], and Rasad et al. [14]. It has also been reported that auxins alone can promote root formation in A. officinalis. For instance, Maung et al. [15] showed that 2.5 mg/L IBA resulted in the highest rooting rate of 44%, while Minh et al. [16] discovered that NAA at a concentration of 0.5 mg/L substantially improved the rooting rate by up to 74.59%.
Considering the effect of CW on the in vitro rooting of A. officinalis, the concentrations of 5% and 10% (v/v) CW did not significantly induce root formation in the plant, but instead promoted root elongation, yielding approximately root lengths of 4.54 and 4.88 cm, respectively. Increasing the concentrations of CW to 15 and 20% (v/v) significantly improved A. officinalis root proliferation, suggesting that CW at these concentrations is suitable for root formation in this plant, possibly due to the hormonal balance between auxins and cytokinins, which could promote root formation in the explants [25]. The maximum number of roots (3.20 roots/explant) and root lengths (5.50 cm) were observed in the MS medium supplemented with 20% (v/v) CW. These results are consistent with the findings reported for other plants, such as Daphne sp. [53], R. stylosa [47], and Epidendrum nocturnum [39]. Based on the results obtained in this study, CW can be utilized as an alternative root-setting medium instead of using commercial rooting hormones for the in vitro propagation of A. officinalis.

3.3. Ex Vitro Acclimatization of In Vitro Regenerated A. officinalis

Like other in vitro propagated plants, an ex vitro acclimatization process is required to ensure plant growth and survival of well-developed in vitro A. officinalis plantlets when transplanted to soil or field environments. A variety of supporting materials or rooting substrates have been utilized as planting media for ex vitro acclimatization, such as sand, coco peat, rockwool, rice husk, peat moss, vermiculite, and perlite [54,55,56,57]. Among these materials, peat moss and vermiculite are the most commonly used, since they have a higher water-holding capacity than the others. Furthermore, peat moss is also rich in nutrients, which could promote plant growth and development [57,58,59]. The ex vitro acclimatization of micropropagated A. officinalis was investigated in this study by using peat moss and vermiculite at a ratio of 1:1 as supporting materials. As shown in Table 5 and Figure 1, all of the in vitro plantlets were successfully acclimatized with 100% survival rates. Furthermore, the acclimatized plants also exhibited morphology comparable to that of the mother plants. The in vitro plantlets from the control treatment or the MS-free hormone produced the lowest number of shoots and roots compared to those from other treatments. However, the plantlets from this control medium exhibited relatively longer shoot and root lengths than those from MS medium supplemented with 0.35 mg/L NAA and 0.05 mg/L kinetin. Notably, the plantlets from MS medium supplemented with CW exhibited significantly higher numbers of shoots and roots as well as longer shoot and root lengths than those from the MS-free hormone, particularly in the medium with high concentrations of CW. Among different concentrations of CW, the plantlets from the medium supplemented with 20% (v/v) CW yielded the highest values for the number of shoots (6.60 shoots/explants), shoot length (12.79 cm), number of roots (4.80 roots/explant), and root length (6.03 cm). These results demonstrated that CW not only promotes in vitro plant propagation but also accelerates ex vitro plant growth and development, in agreement with findings reported for Dracaena purplecompacta [60] and R. stylosa [47].
The present results clearly demonstrated that CW is a promising material that can be used as a growth supplement for the micropropagation of A. officinalis. In addition to being a natural carbon source, it is also rich in natural vitamins, amino acids, minerals, and phytohormones, which play an essential role in plant growth and development. Furthermore, CW is cheap, readily available, and easy to prepare and apply to the plant cultivation medium, promoting the sustainability of the commercial propagation of A. officinalis.
Although the regenerated plants revealed morphological traits similar to those of the mother plant (Figure 1), the in vitro culture environment can trigger mutations in the new plantlets, known as somaclonal variations [18]. Identifying somaclonal variations is crucial in in vitro propagating plants. Several techniques have been employed; among these, PCR-based molecular markers, such as random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), simple sequence repeats (SSR), and start codon targeted (SCoT) polymorphism markers, are widely used [18]. Therefore, further investigation of their genetic stability is needed to verify the new plantlets of A. officinalis.

4. Conclusions

As demonstrated in this study, organic additives, including CW, BH, and PH, could be used as growth-promoting materials for the in vitro propagation of A. officinalis. Among these organic additives, CW exhibited great potential for promoting shoot and root proliferation in A. officinalis compared to BH and PH. Supplementation of the MS basal medium with 20% (v/v) CW yielded the highest number of shoots (4.10 shoots/explant) and roots (3.20 roots/explant) and the longest shoots (7.26 cm) and roots (5.50 cm); these values were higher than those obtained from MS-free hormone and MS supplemented with NAA and kinetin. Furthermore, the in vitro plantlets regenerated from the MS medium supplemented with CW, especially at 20% (v/v), also exhibited higher growth performance than those regenerated using other treatments, with morphological characteristics similar to those of the mother plant. This report is the first to demonstrate that organic additives, specifically CW, are the best growth supplement material for the in vitro propagation of A. officinalis and can be applied instead of commercial phytohormones to minimize the costs of large-scale plant production.

Author Contributions

Conceptualization, P.K., P.T. and S.T.; methodology, P.K., K.L., P.N., P.S. and S.T.; software, P.K. and P.T.; validation, P.K., P.T. and S.T.; formal analysis, P.K., K.L., P.N., P.S. and S.T.; investigation, P.K., K.L., P.N., P.S.,and S.T.; resources, P.K., K.L., P.N., P.S. and S.T.; data curation, P.K., P.T. and S.T.; writing—original draft preparation, P.K., P.T. and S.T.; writing—review and editing, P.K., P.T. and S.T.; visualization, P.K., P.T. and S.T.; supervision, P.K., K.L., P.N., P.S., P.T. and S.T.; project administration, P.K., K.L., P.T. and S.T.; funding acquisition, P.K., K.L., P.T. and S.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Research and Graduate Studies and Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University, under the Research Program.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors thank Saowaluk Chuakuna for her technical support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kubota, S.; Konno, I.; Kanno, A. Molecular phylogeny of the genus Asparagus (Asparagaceae) explains interspecific crossability between the garden asparagus (A. officinalis) and other Asparagus species. Theor. Appl. Genet. 2012, 124, 345–354. [Google Scholar] [CrossRef]
  2. Kubituki, K.; Rudall, P.J. Asparagaceae. In The Families and Genera of Vascular Plants; Kubituki, K., Ed.; Springer: Berlin/Heidenberg, Germany, 1998; pp. 125–128. [Google Scholar]
  3. Esmaeili, A.K.; Taha, R.M.; Mohajer, S.; Banisalam, B. In vitro regeneration and comparison of phenolic content, antioxidant and antityrosinase activity of in vivo and in vitro grown Asparagus offinalis. Sains Malays. 2016, 45, 373–381. [Google Scholar]
  4. Encina, C.L.; Regalado, J.J. Aspects of in vitro plant tissue culture and breeding of asparagus: A review. Horticulturae 2022, 8, 439. [Google Scholar] [CrossRef]
  5. Shimoyamada, M.; Suzuki, M.; Sonta, H.; Maruyama, M.; Okubo, K. Antifungal activity of the saponin fraction obtained from Asparagus officinalis L. and its active principle. Agric. Biol. Chem. 1990, 54, 2553–2557. [Google Scholar] [CrossRef]
  6. Terada, K.; Honda, C.; Suwa, K.; Takeyama, S.; Oku, H.; Kamisako, W. Acetylenic compounds isolated from cultured cells of Asparagus officinalis. Chem. Pharm. Bull. 1995, 43, 564–566. [Google Scholar] [CrossRef]
  7. Makoto, S.; Masayuki, S.; Makiko, M.; Watanabe, K. An antifungal saponin from white asparagus (Asparagus officinalis L.) bottoms. J. Sci. Food. Agric. 1996, 72, 430–434. [Google Scholar]
  8. Shao, Y.; Poobrasert, O.; Kennelly, E.J.; Chin, C.K.; Ho, C.T.; Huang, M.T.; Garrison, S.A.; Cordell, G.A. Steroidal saponins from Asparagus officinalis and their cytotoxic activity. Planta. Med. 1997, 63, 258–262. [Google Scholar] [CrossRef]
  9. Jang, D.S.; Cuendet, M.; Fong, H.H.; Pezzuto, J.M.; Kinghorn, A.D. Constituents of Asparagus officinalis evaluated for inhibitory activity against cyclooxygenase-2. J. Agric. Food Chem. 2004, 52, 2218–2222. [Google Scholar] [CrossRef]
  10. Negi, J.S.; Singh, P.; Joshi, G.P.; Rawat, M.S.; Bisht, V.K. Chemical constituents of Asparagus. Pharmacogn. Rev. 2010, 4, 215–220. [Google Scholar]
  11. Bousserouel, S.; Grandois, J.; Gosse, F.; Werner, D.; Barth, S.W.; Marchioni, E.; Marescaux, J.; Raul, F.A. Methanolic extract of white asparagus shoots activates trail apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model. Int. J. Oncol. 2013, 43, 394–404. [Google Scholar] [CrossRef]
  12. Statista. Global Leading Asparagus Producing Countries in 2021. Available online: https://www.statista.com/statistics/279556/global-top-asparagus-producing-countries/ (accessed on 6 January 2023).
  13. Corpas-Hervias, C.; Melero-Vara, J.M.; Molinero-Ruiz, M.L.; Zurera-Munoz, C.; Basallote-Ureba, M.J. Characterization of isolates of Fusarium spp. obtained from asparagus in Spain. Plant Dis. 2006, 90, 1441–1451. [Google Scholar] [CrossRef]
  14. Rasad, F.M.; Hasbullah, N.A.; Azis, N.A.; Daud, N.F.; Lassim, M.M. Micropropagation of Asparagus officinalis L. (garden asparagus) in vitro. Int. J. Life Sci. Res. 2019, 7, 123–129. [Google Scholar]
  15. Maung, M.; Myint, K.T.; Thu, M.K. Effects of different explant types, plant growth regulators and shoot density on in vitro regeneration of asparagus (Asparagus officinalis L.). J. Agric. Res. 2019, 6, 109–115. [Google Scholar]
  16. Minh, L.N.; Nhat, T.N.T.; Diem, S.L.H.; Mironov, A.; Vorobyev, M.; Orlova, E. Micropropagation of Asparagus officinalis L. through callus development. Bio. Web Conf. 2022, 47, 04001. [Google Scholar]
  17. Häkkinen, S.T.; Nygren, H.; Nohynek, L.; Pimiä, R.P.; Heiniö, R.L.; Maiorova, N.; Rischer, H.; Ritala, A. Plant cell cultures as food-Aspects of sustainability and safety. Plant Cell Rep. 2020, 39, 1655–1668. [Google Scholar] [CrossRef]
  18. Hamdeni, I.; Louhaichi, M.; Slim, S.; Boulila, A.; Bettaieb, T. Incorporation of organic growth additives to enhance in vitro tissue culture for producing genetically stable plants. Plants 2022, 11, 3087. [Google Scholar] [CrossRef]
  19. Khorsha, S.; Alizadeh, M.; Mashayekhi, K. The usefulness of apricot gum as an organic additive in grapevine tissue culture media. Adv. Hortic. Sci. 2016, 30, 111–118. [Google Scholar]
  20. Thejaswini, R.; Narasimhan, S. Undefined organic additives stimulate in vitro seed germination of Dendrobium ovatum (willd.) Kraenzl, a medicinal orchid. Int. J. Pharma. Med. Biol. Sci. 2017, 6, 29–31. [Google Scholar] [CrossRef]
  21. Hartati, S.; Arniputri, R.B.; Soliah, L.A.; Cahyono, O. Effects of organic additives and naphthalene acetic acid (NAA) application on the in vitro growth of black orchid hybrid (Coelogyne pandurata Lindley). Bulg. J. Agric. Sci. 2017, 23, 951–957. [Google Scholar]
  22. Samala, S.; Thipwong, J. Influences of organic additives on asymbiotic seed germination of Dendrobium cruentum Rchb. F. for in vitro micropropagation. Trends Sci. 2022, 19, 4181. [Google Scholar] [CrossRef]
  23. Wu, K.; Zeng, S.; Lin, D.; Teixeira da Silva, J.A.; Bu, Z.; Zhang, J.; Duan, J. In vitro propagation and reintroduction of the endangered Renanthera imschootiama Rolfe. PLoS ONE 2014, 9, e110033. [Google Scholar]
  24. Selakorn, O.; Phasinam, K.; Kassanuk, T.; Sutaphan, S. Influence of organic additives on multiple shoot formation of Musa (AA group) ‘Kluai Nam Thai’ in vitro. Rajabhat J. Sci. Humanit. Soc. Sci. 2020, 21, 347–353. [Google Scholar]
  25. Sosnowski, J.; Truba, M.; Vasileva, V. The impact of auxin and cytokinin on the growth and development of selected crops. Agriculture 2023, 13, 724. [Google Scholar] [CrossRef]
  26. Yong, J.W.H.; Ge, L.; Ng, Y.F.; Tan, S.N. Chemical composition and biological properties of coconut (Cocos nucifera L.) water. Molecules 2009, 14, 5144–5164. [Google Scholar] [CrossRef] [PubMed]
  27. Manawadu, I.; Dahanayake, N.; Senanayake, S.G.N. Effects of different natural organic additives on in vitro shoot regeneration of Raphanus sativa L. var. beeralu. J. Agric. Sci. Technol. 2014, 4, 219–223. [Google Scholar]
  28. Maharjan, S.; Thakuri, L.S.; Thapa, B.B.; Pradhan, S.; Pant, K.K.; Joshi, G.P.; Pant, B. In vitro propagation of the endangered orchid Dendrobium chryseum Rolfe from protocorms culture. Nepal. J. Sci. Technol. 2020, 19, 39–47. [Google Scholar] [CrossRef]
  29. Khatun, M.; Roy, P.K.; Razzak, M.A. Additive effect of coconut water with various hormones on in vitro regeneration of carnation (Dianthus caryophyllus L.). J. Anim. Plant Sci. 2018, 28, 589–596. [Google Scholar]
  30. Ng, Z.C.; Tan, S.H.; Shiekh Mahmud, S.H.R.; Ma, N.L. Preliminary study on micropropagation of Hylocereus polyrhizus with waste coconut water and sucrose. Mater. Sci. Forum. 2020, 981, 316–321. [Google Scholar] [CrossRef]
  31. Nilanthi, D.; Yang, Y.S. Effects of sucrose and other additives on in vitro growth and development of purple coneflower (Echinacea purpurea L.). Adv. Biol. 2014, 2014, 402309. [Google Scholar] [CrossRef]
  32. Rohmah, K.N.; Taratima, W. Effect of chitosan, coconut water and potato extract on protocorm growth and plantlet regeneration of Cymbidium aloifolium (L.) Sw. Curr. Appl. Sci. Technol. 2022, 22, 1–10. [Google Scholar]
  33. Anhwange, B.A.; Ugye, T.J.; Nyiaatagher, T.D. Chemicals composition of Musa sapientum (banana) peels. Electron. J. Environ. Agric. Food Chem. 2009, 8, 437–442. [Google Scholar]
  34. Fernando, H.R.P.; Srilaong, V.; Pongprasert, N.; Boonyaritthongchai, P.; Jitareerat, P. Changes in antioxidant properties and chemical composition during ripening in banana variety ‘Hom Thong’ (AAA group) and ‘Khai’ (AA group). Int. Food Res. J. 2014, 21, 749–754. [Google Scholar]
  35. Ssamula, A.; Arinaitwe, G.; Mukasa, S.B. Banana juice as an alternative energy source for banana in vitro growth medium. Afr. Crop Sci. J. 2015, 23, 59–66. [Google Scholar]
  36. Daud, N.; Taha, R.M.; Noor, N.N.M.; Alimon, H. Effects of different organic additives on in vitro shoot regeneration of Celosia sp. Pak. J. Biol. Sci. 2011, 14, 546–551. [Google Scholar] [CrossRef]
  37. Aktar, S.; Nasiruddin, K.M.; Hossain, K. Effects of different media and organic additives interaction on in vitro regeneration of Dendrobium orchid. J. Agric. Rural Dev. 2008, 6, 69–74. [Google Scholar] [CrossRef]
  38. Gansau, J.A.; Indan, H.; Abdullah, S.N.; David, D.; Marbawi, H.; Jawan, R. Effects of organic additives and plant growth regulators on protocorm development of Dendrobium lowii. Trans. Sci. Technol. 2016, 3, 462–468. [Google Scholar]
  39. De Stefano, D.; Costa, B.N.S.; Downing, J.; Fallahi, E.; Khoddamzadeh, A.A. In vitro micropropagation and acclimatization of an endangered native orchid using organic supplements. Am. J. Plant Sci. 2022, 13, 380–395. [Google Scholar] [CrossRef]
  40. Islam, M.O.; Islam, M.S.; Saleh, M.A. Effect of banana extract on growth and development of protocorm like bodies in Dendrobium sp. orchid. Agriculturists 2015, 13, 101–108. [Google Scholar] [CrossRef]
  41. Bartova, V.; Barta, J. Chemical composition and nutritional value of protein concentrates isolated from potato (Solanum tiberosum L.) fruit juice by precipitation with ethanol or ferric chloride. J. Agri. Food Chem. 2009, 57, 9028–9034. [Google Scholar] [CrossRef]
  42. Molnar, Z.; Virag, E.; Ordog, V. Natural substances in tissue culture media of higher plants. Acta. Biol. Szeged. 2011, 55, 123–127. [Google Scholar]
  43. Ouyang, J.W.; Liang, H.; Zhang, C.; Zhao, T.H.; Jia, S.E. The effect of potato extract used as additive in anther culture medium on culture responses in Triticum aestivum. Cereal Res. Commun. 2004, 32, 501–508. [Google Scholar] [CrossRef]
  44. Islam, M.O.; Akter, M.; Prodhan, A.K.M.A. Effect of potato extract on in vitro seed germination and seedling growth of local Vanda roxburgii orchid. J. Bangladesh Agric. Univ. 2011, 9, 211–215. [Google Scholar] [CrossRef]
  45. Aishwarya, P.P.; Seenivasan, N.; Naik, D.S. Coconut water as a root hormone: Biological and chemical composition and applications. Pharma Innov. J. 2022, 11, 1678–1681. [Google Scholar]
  46. Asma, N.; Kashif, A.; Saifullah, K. Optimized and improved method for the in vitro propagation of kiwifruit using coconut water. Pak. J. Bot. 2007, 40, 2355–2360. [Google Scholar]
  47. Ogatis, R.A. A comparative evaluation of coconut water as root setting medium for Rhizopora stylosa hypocotyl propagation. Int. J. Sci. Res. 2016, 5, 2061–2063. [Google Scholar]
  48. Akhiriana, E.; Samanhudi; Yunus, A. Coconut water and IAA effect on the in vitro growth of Tribulus terrestris L. Acta Univ. Agric. Silvic. Mendelianae Brun. 2019, 67, 9–18. [Google Scholar] [CrossRef]
  49. Mardhikasari, S.; Yunus, A.; Samanhudi, S. Modification of media for banana in vitro propagation with foliar fertilizer and coconut water in cv. Rajabulu. Caraka Tani J. Sustain. Agric. 2020, 35, 23–32. [Google Scholar] [CrossRef]
  50. Sreekumar, S.; Mukunthakumar, S.; Seeni, S. Morphogenetic response of six Philodendron cultivars in vitro. Indian J. Exp. Biol. 2001, 39, 1280–1287. [Google Scholar] [PubMed]
  51. Desjardins, Y.; Tiessen, H.; Harney, P.M. The effect of sucrose and ancymidol on the in vitro rooting of nodal sections of asparagus. HortScience 1987, 22, 131–133. [Google Scholar] [CrossRef]
  52. Shigeta, J.; Sato, K.; Tanaka, S.; Nakayama, M.; Mii, M. Efficient plant regeneration of asparagus by inducing normal roots from in vitro multiplied shoot explants using gellan gum and glucose. Plant Sci. 1996, 113, 99–104. [Google Scholar] [CrossRef]
  53. Wiszniewska, A.; Fajerska, E.H.; Grabski, K.; Tukaj, Z. Promoting effects of organic medium supplements on the micropropagation of promising ornamental Daphne species (Thymelaeaceae). In Vitro Cell. Dev. Biol. Plant. 2013, 49, 51–59. [Google Scholar] [CrossRef]
  54. Chen, F.C.; Wang, C.Y.; Fang, J.Y. Micropropagation of self-heading Philodendron via direct shoot regeneration. Sci. Hortic. 2012, 141, 23–29. [Google Scholar] [CrossRef]
  55. Hassan, H.M.S.; Ali, M.A.M.; Soliman, D.A. Effect of low cost gelling agents and some growth regulators on micropropagation of Philodendron selloum. J. Plant Prod. 2016, 7, 169–176. [Google Scholar] [CrossRef]
  56. Alawaadh, A.A.; Dewir, Y.H.; Alwihibi, M.S.; Aldubai, A.A.; El-Hendawy, S.; Naidoo, Y. Micropropagation of lacy tree Philodendron (Philodendron bipinnatifidum Schott ex Endl.). HortScience 2020, 55, 294–299. [Google Scholar] [CrossRef]
  57. Hoang, N.N.; Kitaya, Y.; Shibuya, T.; Endo, R. Effects of supporting materials in in vitro acclimatization stage on ex vitro growth of wasabi plants. Sci. Hortic. 2020, 261, 109042. [Google Scholar] [CrossRef]
  58. Choi, J.M.; Chung, H.J.; Choi, J.S. Physico-chemical properties of organic and inorganic materials used as container media. Hortic. Sci. Technol. 2000, 18, 529–535. [Google Scholar]
  59. Oh, M.M.; Seo, J.H.; Park, J.S.; Son, J.E. Physicochemical properties of mixtures of inorganic supporting materials affect growth of potato (Solanum tuberosum L.) plantlets cultured photoautotrophically in a nutrient-circulated micropropagation system. Hortic. Environ. Biotechnol. 2012, 53, 497–504. [Google Scholar] [CrossRef]
  60. Agampodi, V.A.; Jayawardena, B. Effect of coconut (Cocos nucifera L.) water extracts on adventitious root development in vegetative propagation of Dracaena purplecompacta L. Acta Physiol. Plant. 2009, 31, 279–284. [Google Scholar] [CrossRef]
Horticulturae 09 01244 g001
Figure 1. The plantlets of A. officinalis after ex vitro acclimatization for 4 weeks using peat moss and vermiculite at a ratio of 1:1 as supporting materials. The in vitro plantlets grown from MS-free hormone (A), MS medium supplemented with 0.35 mg/L NAA and 0.05 mg/L kinetin (B), MS medium supplemented with 5% (v/v) CW (C), MS medium supplemented with 10% (v/v) CW (D), MS medium supplemented with 15% (v/v) CW (E), and MS medium supplemented with 20% (v/v) CW (F).
Figure 1. The plantlets of A. officinalis after ex vitro acclimatization for 4 weeks using peat moss and vermiculite at a ratio of 1:1 as supporting materials. The in vitro plantlets grown from MS-free hormone (A), MS medium supplemented with 0.35 mg/L NAA and 0.05 mg/L kinetin (B), MS medium supplemented with 5% (v/v) CW (C), MS medium supplemented with 10% (v/v) CW (D), MS medium supplemented with 15% (v/v) CW (E), and MS medium supplemented with 20% (v/v) CW (F).
Horticulturae 09 01244 g001
Table 1. Effects of coconut water (CW) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
Table 1. Effects of coconut water (CW) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
MediumNumber of Shoots
(Shoots/Explant)		Shoot Length
(cm)
MS (control)1.50 c2.19 e
MS + 0.05 mg/L NAA + 1.0 mg/L kinetin2.60 b3.70 d
MS + 0.05 mg/L NAA + 5% (v/v) CW2.60 b5.76 c
MS + 0.05 mg/L NAA + 10% (v/v) CW3.40 a6.53 b
MS + 0.05 mg/L NAA + 15% (v/v) CW4.00 a7.25 a
MS + 0.05 mg/L NAA + 20% (v/v) CW4.10 a7.26 a
According to DMRT analysis, means followed by distinct letters within a column are significantly different at p ≤ 0.05.
Table 2. Effects of banana homogenate (BH) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
Table 2. Effects of banana homogenate (BH) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
MediumNumber of Shoots
(Shoots/Explant)		Shoot Length
(cm)
MS (control)1.60 bc2.16 c
MS + 0.05 mg/L NAA + 1.0 mg/L kinetin2.70 a3.69 a
MS + 0.05 mg/L NAA + 5% (w/v) BH2.00 b3.08 a
MS + 0.05 mg/L NAA + 10% (w/v) BH1.30 c1.55 d
MS + 0.05 mg/L NAA + 15% (w/v) BH0.60 d0.31 e
MS + 0.05 mg/L NAA + 20% (w/v) BH0.20 d0.06 e
Means followed by different letters within a column are significantly different at p ≤ 0.05 based on DMRT analysis.
Table 3. Effects of potato homogenate (PH) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
Table 3. Effects of potato homogenate (PH) on shoot proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
MediumNumber of Shoots
(Shoots/Explant)		Shoot Length
(cm)
MS (control)1.60 c2.17 e
MS + 0.05 mg/L NAA + 1.0 mg/L kinetin2.50 b3.67 d
MS + 0.05 mg/L NAA + 5% (w/v) PH2.70 b5.27 c
MS + 0.05 mg/L NAA + 10% (w/v) PH4.00 a7.22 a
MS + 0.05 mg/L NAA + 15% (w/v) PH3.80 a6.23 b
MS + 0.05 mg/L NAA + 20% (w/v) PH3.70 a6.29 b
Means followed by different letters within a column are significantly different at p ≤ 0.05 based on DMRT analysis.
Table 4. Effect of coconut water (CW) on root proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
Table 4. Effect of coconut water (CW) on root proliferation in A. officinalis after 4 weeks of cultivation in different MS media.
MediumNumber of Roots
(Roots/Explant)		Root Length
(cm)
MS (control)2.50 ab1.55 c
MS + 0.35 mg/L NAA + 0.05 mg/L kinetin3.10 a1.33 c
MS + 5% (v/v) CW1.80 c4.54 b
MS + 10% (v/v) CW2.30 bc4.88 b
MS + 15% (v/v) CW2.60 ab5.35 a
MS + 20% (v/v) CW3.20 a5.50 a
Means followed by different letters within a column are significantly different at p ≤ 0.05 based on DMRT analysis.
Table 5. The growth of A. officinalis after 4 weeks of acclimatization using peat moss and vermiculite at a ratio of 1:1 as supporting material.
Table 5. The growth of A. officinalis after 4 weeks of acclimatization using peat moss and vermiculite at a ratio of 1:1 as supporting material.
In Vitro Plantlet from the MediumNumber of Shoots
(Shoots/Plantlet)		Shoot Length
(cm)		Number of Roots
(Roots/Explant)		Root Length
(cm)
MS (control)2.30 c11.15 c2.90 d2.74 b
MS + 0.35 mg/L NAA + 0.05 mg/L kinetin6.30 a9.23 e4.10 bc2.27 c
MS + 5% (v/v) CW4.80 b10.62 d3.70 c5.73 a
MS + 10% (v/v) CW5.80 a11.66 b4.50 ab5.75 a
MS + 15% (v/v) CW6.50 a12.56 a4.70 ab5.92 a
MS + 20% (v/v) CW6.60 a12.79 a4.80 a6.03 a
Means followed by different letters within a column are significantly different at p ≤ 0.05 based on DMRT analysis.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Klanrit, P.; Lila, K.; Netsawang, P.; Siangsanor, P.; Thanonkeo, P.; Thanonkeo, S. Effect of Organic Additives on the Micropropagation of Asparagus officinalis. Horticulturae 2023, 9, 1244. https://doi.org/10.3390/horticulturae9111244

AMA Style

Klanrit P, Lila K, Netsawang P, Siangsanor P, Thanonkeo P, Thanonkeo S. Effect of Organic Additives on the Micropropagation of Asparagus officinalis. Horticulturae. 2023; 9(11):1244. https://doi.org/10.3390/horticulturae9111244

Chicago/Turabian Style

Klanrit, Preekamol, Khanittha Lila, Paramaporn Netsawang, Phakamas Siangsanor, Pornthap Thanonkeo, and Sudarat Thanonkeo. 2023. "Effect of Organic Additives on the Micropropagation of Asparagus officinalis" Horticulturae 9, no. 11: 1244. https://doi.org/10.3390/horticulturae9111244

APA Style

Klanrit, P., Lila, K., Netsawang, P., Siangsanor, P., Thanonkeo, P., & Thanonkeo, S. (2023). Effect of Organic Additives on the Micropropagation of Asparagus officinalis. Horticulturae, 9(11), 1244. https://doi.org/10.3390/horticulturae9111244

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop