The effect of plant growth regulators, basal medium and light on micropropagation of Allium jesdianum Boiss. &amp; Buhse

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The effect of plant growth regulators, basal medium and light on micropropagation of Allium jesdianum Boiss. & Buhse

https://doi.org/10.21203/rs.3.rs-2863395/v1

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Allium jesdianum Boiss. & Buhse. is a pharmaceutically valuable plant that is threatened with extinction due to over-exploitation of wild plants. In vitro culture of plants is a favorable alternative for the conservation of endangered plant species. To establish an efficient protocol for micropropagation of Allium jesdianum, the effects of plant growth regulators, basal medium, and light were studied in this experiment. Basal parts of collected bulbs from Zagros Mountains in Iran were surface sterilized and placed on five different basal media with different concentrations of IBA, NAA, BAP and kinetin and kept under dark and light conditions. The results showed that among the exogenously applied PGRs, IBA at concentrations of 1 and 2 mg/l was the most convincing PGR for shoot and bulblet regeneration. The best results were obtained with CLC and MS media under light. Higher NH4+ also increased regeneration. Our experiment may have implications for plant growth and conservation, as well as for in vitrobreeding and drug development.

Auxein

Cytokinin

Basal medium

NH4+/ NO3- ratio

IBA enhances bulblet regeneration in Allium species, especially in a medium which has a higher NH4+/NO3 ratio. In vitro bulblet regeneration also depends on white light irradiation.

Allium jesdianum Boiss. & Buhse. belongs to the family Amaryllidaceae and is a native Iranian plant that has important pharmaceutical properties (USDA, 2023). The leaves and bulbs of Allium jesdianum are commonly used by locals as a natural medicine for the treatment of kidney, abdominal and rheumatic diseases (Pirbalouti 2019). Various therapeutic uses of Allium jesdianum have been reported, including pain relief (Mimaki et al. 1999), antibacterial (Ekrami et al. 2022), antifungal (Naeini et al. 2019), and cytotoxic (Grases et al. 2009) effects. Mimaki et al. (1999) isolated a new spirostanol saponin and a new plant cholestane glycoside from the extract of Allium jesdianum. Many studies have promoted the use of Allium species in daily diet due to their therapeutic effects without side effects and possible health-promoting properties (Praticò et al. 2018; Zhao et al. 2021; Yan et al. 2022).

Allium jesdianum (locally known as "Bonsorkh") grows in the high altitudes of the Zagros Mountains in western and southwestern Iran and is at high risk of extinction due to continuous grazing, high harvest levels, inadequate conservation monitoring, and lack of compensatory planting. Therefore, conservation and propagation of this plant are in demand. Until now, there is no report of field or in vitro culture of this plant. In vitro propagation of plants facilitates tissue growth and differentiation and is a basic alternative to large-scale plant propagation. Both chemical and physical factors such as nutrients in the medium, growth regulators, light, temperature and pH are the key factors that influence the success of micropropagation. Optimization of in vitro conditions plays an important role in obtaining high numbers of propagules in each culture. In vitro micropropagation of other Allium species has been reported, such as in Allium hirtifolium (Farhadi et al. 2017), (Azeri and Öztürk 2021), (Yang et al. 2021). The only in vitro study of Allium jesdianum focused on callus induction and elicitation of some secondary metabolites (Yazdanian et al. 2022).

In this study, we aimed to investigate in vitro micropropagation of Allium jesdianum for the first time. The success in micropropagation of this plant serves as a reliable way for its conservation and propagation. In vitro culture also makes the plant more amenable to manipulation for inclusion in breeding programs and secondary metabolite utilization. For this purpose, we have studied the effect of growth regulators, basal medium, and light on in vitro growth and multiplication of Allium jesdianum.

2.1. Plant material

The bulbs of Allium jesdianum were collected in their natural habitat in the mountains of Sabzekoh in southwestern Iran. This region is a rich source of important medicinal plants such as Allium species. The collected bulbs were kept in the soil under dark and cold conditions for about 3 months until their dormancy period was over. Then, the intact bulbs were washed under running tap water for 20 minutes before disinfection. A two-step disinfection method was optimized to achieve a low contamination rate and high viability of the sterilized explants. First, the outer surface of the bulbs was treated with 70% ethanol for 1 minute and 1% sodium hypochlorite for 5 minutes. In the second step, the bulbs were cut into two or three pieces and were again surface sterilized with sodium hypochlorite at the same concentration and time. The disinfected explants were rinsed twice with sterile water (for 5 minutes each time).

2.2. Treatment with plant growth regulators (PGR)

In the first experiment, the sterilized explants were cultured on MS basal medium enriched with 1-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) in combination with 6-benzylaminopurine (BAP) and kinetin. All of these plant growth regulators (PGRs) were used at concentrations of 0, 1, 2, and 5 mg/l and added to the media before autoclaving, except for IBA, which was filtered through a 0.22 µm sterile filter. A factorial experiment was performed in the form of completely randomized experimental design with three replicates (each replicate contained 10 explants), considering auxins (NAA and IBA) and cytokinin (BAB and kinetin) PGRs as two independent factors. The post hoc LSD test was used at the 0.05 probability level to detect significant differences between means.

2.3. Treatment of basal nutrient culture medium and light

Nutrient composition plays an important role in growth and differentiation under in vitro conditions. Therefore, different basal media are developed for specific purposes. In the second experiment, we investigated the effect of different basal media such as MS (Murashige and Skoog 1962), CLC/Ipomoea (Chée et al. 1992), B5 (Gamborg et al. 1976), WPM (Lloyd and McCown 1980), and N6 (Chu et al., 1975) on micropropagation of Allium jesdianum under dark and light conditions. Under light conditions, cultures were incubated at 25^oC, a photoperiod of 16/8 (light/dark), and a light intensity of 14 µmol m^− 2 s^− 1 (White cool fluorescent light, Philips). Under dark conditions, cultures were maintained at the same temperature and without light exposure for two weeks. The composition of each media is shown in Table 1. All media were enriched with the best PGR combination derived from the previous experiment. The obtained data were analyzed based on a factorial experiment in the form of a completely randomized experimental design with three replicates. The type of basal medium and light conditions was assumed to be two independent factors. Comparison of mean values was performed based on the LSD test at the 0.05 probability level.

Table 1

Concentration of macro- and microelements (mg/l) in five different media used in this experiment
	Medium
Nutrient	MS	N6	CLC	B5	WPM
Macro nutrient
CaCl2	332.02	125.33	332.02	113.23	72.5
KH2PO4	170	400	170	-	170
KNO3	1900	2830	1900	2500
MgSO4	180.54	90.27	180.54	121.56	180.54
NH4NO3	1650		800.4		400
(NH4)2SO4	-	463	-		-
(NH4)2SO4	-	-	-	134	-
NaH2PO4	-	-	-	130.44
Ca(NO3)2 .4H2O	-	-	-	-	471.26
K2SO4					990
Micro nutrient
CoCl2.6H2O	0.025	-	0.025	0.025	-
CuSO4.5H2O	0.025	-	0.025	0.025	0.25
FeNaEDTA	36.7	36.7	36.7	36.7	36.7
H3BO3	6.2	1.6	6.2	3	6.2
KI	0.83	0.8	0.83	0.75
MnSO4.H2O	16.9	3.33	16.9	10	22.3
Na2MoO4.2H2O	0.25		0.25	0.25	0.25
ZnSO4.7H2O	8.6	1.5	8.6	2	8.6

2.4. Growth conditions

All cultures were incubated under light conditions at a 16/8 photoperiod with a light intensity of 1000 lux at 25^oC and subcultured every 3 weeks. After 2 months of culture, the percentage of root, shoot and bulblet regeneration per explant were measured. For adaptation, regenerants were moved to covered pots and maintained at 20 ^oC with regular irrigation.

3.1. Effect of plant growth regulators

Dormancy in bulbous plants is a common phenomenon (Okubo 1992; Liu et al. 2015; Cai et al. 2019); therefore, it is important to collect explants from bulbs whose short-term dormancy has been overcome. For this reason, we kept the collected bulbs in soil under dark and cold conditions for two months before in vitro culture; on the other hand, no regeneration was observed when the collected bulbs were used directly for explant collection. The results showed that a cross-section at the basal plate is a prerequisite for bulblet induction, as almost no regenerated bulblets could be obtained from uncut bulbs (Ren et al. 2018).

Several parts of disinfected bulbs were used in this experiment, but only explants having basal part exhibited various level of regeneration. Interaction effect of auxins and cytokinins on different organ regeneration is presented in Table 1. No root and bulblet was regenerated in auxin-free media. There was no significant difference among treatments in the sense of regenerated roots. Highest number of shoots was produced in the media supplemented with IBA in combination with different concentrations of kinetin and BAP. Moreover, direct bulblets were exclusively produced in media enriched with IBA in combination of BAP. In general, amongst the auxin hormones that were exogenously supplied to MS media in this experiment, IBA in concentration of 1 and 2 mg/l were the most potent PGRs for shoot and bulblet regeneration.

Table 2

Percent of roots, shoots, and bulblet regeneration in media supplemented with different concentrations of auxins and cytokinins in in vitro culture of Allium jesdianum
Plant growth regulators		Regenerated organs
Auxin (mg/l)	Cytokinin (mg/l)	Root (%)		Shoot (%)		Bullblet (%)
0	0	0.00	b	0.00	e	0.00	c
0	KIN (1)	0.00	b	13.33	de	0.00	c
0	KIN (5)	0.00	b	13.33	de	0.00	c
0	BAP (1)	0.00	b	26.67	bcde	0.00	c
0	BAP (5)	0.00	b	6.67	e	0.00	c
NAA (1)	0	6.67	ab	13.33	de	0.00	c
NAA (1)	KIN (1)	6.67	ab	26.67	bcde	6.67	bc
NAA (1)	KIN (5)	6.67	ab	26.67	bcde	6.67	bc
NAA (1)	BAP (1)	6.67	ab	26.67	bcde	6.67	bc
NAA (1)	BAP (5)	6.67	ab	20.00	cde	0.00	c
NAA (2)	0	0.00	b	6.67	e	0.00	c
NAA (2)	KIN (1)	6.67	ab	26.67	bcde	6.67	bc
NAA (2)	KIN (5)	6.67	ab	26.67	bcde	6.67	bc
NAA (2)	BAP (1)	13.33	ab	33.33	bcd	13.33	bc
NAA (2)	BAP (5)	6.67	ab	13.33	de	0.00	c
NAA (5)	0	6.67	ab	13.33	de	0.00	c
NAA (5)	KIN (1)	13.33	ab	20.00	cde	0.00	c
NAA (5)	KIN (5)	6.67	ab	20.00	cde	0.00	c
NAA (5)	BAP (1)	6.67	ab	20.00	cde	6.67	bc
NAA (5)	BAP (5)	6.67	ab	13.33	de	0.00	c
IBA (1)	0	6.67	ab	33.33	bcd	6.67	bc
IBA (1)	KIN (1)	20.00	ab	46.67	ab	13.33	bc
IBA (1)	KIN (5)	13.33	ab	40.00	abc	13.33	bc
IBA (1)	BAP (1)	26.67	a	46.67	ab	20.00	ab
IBA (1)	BAP (5)	13.33	ab	33.33	bcd	20.00	ab
IBA (2)	0	6.67	ab	40.00	abc	6.67	bc
IBA (2)	KIN (1)	13.33	ab	46.67	ab	13.33	bc
IBA (2)	KIN (5)	13.33	ab	40.00	abc	6.67	bc
IBA (2)	BAP (1)	20.00	ab	60.00	a	26.67	a
IBA (2)	BAP (5)	13.33	ab	40.00	abc	20.00	ab
IBA (5)	0	6.67	ab	13.33	de	6.67	bc
IBA (5)	KIN (1)	13.33	ab	26.67	bcde	13.33	bc
IBA (5)	KIN (5)	13.33	ab	20.00	cde	6.67	bc
IBA (5)	BAP (1)	6.67	a	26.67	bcde	13.33	bc
IBA (5)	BAP (5)	20.00	ab	20.00	cde	13.33	bc
^{Means in each column indicated by the same letter are not significantly different from each other based on the LSD test at the 0.05 probability level}

3.2. The effect of light and medium composition

In the second experiment, the basal parts of the disinfected bulbs were excised and placed on five different media: MS, CLC, N6, WPM and B5. All media were enriched at 1 mg/l of both IBA and BAP, which had been shown to be the best PGR treatment in the previous experiment. To evaluate the effect of light and darkness, the experiment was repeated and the explants were then incubated under dark conditions. The results in Table 2 show that the highest number of regenerates was obtained from explants cultured on CLC and MS media, particularly when the cultures were maintained under light conditions. Although no bulb was observed under dark conditions, a different number of roots and shoots were regenerated. In general, the highest regeneration rate was obtained when explants were placed on CLC medium and then maintained under light conditions. All regenerated shoots and bulblets were transferred to PGR-free medium with half sucrose concentration to accelerate the adaptation. Then they were potted in sterilized soil under humid conditions with regular irrigation. The results showed that 90% of the shoots and all the bulblets produced full-sized plantlets. Figure 1 shows the different stages of the experiment, from explant source selection to regenerated plantlets.

Table 3

Effect of different basal media and light conditions on percent of roots, shoots and bullblet regeneration in in vitro culture of Allium jesdianum
Light condition	Medium	Percent of root regeneration		Percent of shoot regeneration		Percent of bulbus regeneration
Light	MS	26.6	b	46.6	ab	26.6	b
Light	N6	0	d	13.3	cd	0	c
Light	WPM	0	d	20	cd	0	c
Light	CLC	46.6	a	60	a	60	a
Light	B5	0	d	6.6	d	0	c
Dark	MS	6.6	cd	33.3	bc	0	c
Dark	N6	0	d	13.3	cd	0	c
Dark	WPM	0	d	6.6	d	0	c
Dark	CLC	13.3	c	53.3	ab	0	c
Dark	B5	0	d	6.6	d	0	c
^{Means in each column indicated by the same letter are not significantly different from each other based on the LSD test at the 0.05 probability level}.

Auxins and cytokinins are the most commonly used PGRs in plant tissue culture (George et al. 2008); therefore, regeneration is usually limited when PGRs are removed from the medium. In this experiment, only some shoots were regenerated in PGR-free media, but most of them showed albino symptoms, which is consistent with the results of Farhadi et al. (2017) and Hailekidan et al. (2013).

Our results showed that the combination of auxins with cytokinins increased the number of regenerates. The type and number of organs regenerated from explants directly depended on the type and concentration of PGRs and also on the plant species. In contrast to other studies, we found that IBA has a better effect on the proliferation rate, especially through bulblet production. Ren et al. (2018) emphasized that bulblet production in Lycoris sprengeri was enhanced when 1 mg/l of NAA was used. In addition, it is reported that the simultaneous use of NAA and BAP was the best hormone combination for bulblet proliferation in Lilium Hybrid Cv. 'Ravenna' (Rafiq et al. 2021) and Narcissus tazzeta L. (Rahimi Khonakdari et al. 2020). Although IBA is basically used for root induction in propagated regenerates (George et al. 2008), in our experiment, the number of roots, shoots and bulblets increased significantly with the addition of IBA.

No callus was obtained in either experiment, indicating that regenerates were produced by direct organogenesis. The absence of callus in the basal parts of the regenerants is very fruitful in adaptation because the vascular system of the root and the vascular system of the plantlet are better connected. Although the effect of different basal media on the regenerative ability of in vitro cultured plants has been investigated in numerous studies (Kairuz et al. 2021; Tahiri et al. 2022; Pullaiah et al. 2022), there have been no reports on the effectiveness of basal media in bulbous plants under in vitro conditions. The use of five different media in this study showed that some media, including N6, B5, and WPM, limited the regenerative ability of explants. On the other hand, the CLC and MS media showed the highest regeneration potential for all types of regenerations under light conditions. The CLC medium was specifically developed by Chée et al. (1992) to enhance somatic embryogenesis of Ipomoea batatas. Considering composition of the different incorporated media, we find that CLC and MS media have similar compositions, except for NH4NO3 and MgSo4.7H2O, which are present in about half the concentration in the CLC medium than in the MS medium. In order to clearly compare the studied media, the concentration of macroelements in each medium was calculated (Table 4). A principal component analysis was then performed to reduce the dimensionality of the obtained data and to increase interpretability. A biplot was then generated based on the first two components (Fig. 1), which explained more than 98 percent of the variability. The distribution of media and elements on the biplot diagram shows that there is a close relationship between the media CLC and MS; moreover, the element N is located near these two media, suggesting a high effectiveness of the element N on the mentioned media composition. The total concentration of N and the ratio of nitrate (NH4⁺) to ammonium (NO3⁻) ions are two important factors to consider when formulating a media for different plant species is in demand (George et al., 2008). The amount of NH4⁺ and also total nitrogen is high in MS and CLC medium compared to other media. Reduced nitrogen (NH4⁺) is generally available in lower amounts than nitrate in various media compositions, but organogenesis is highly dependent on its presence (Drew 2015). Luciani et al. (2001) demonstrated that proliferation rates of garlic clones can be improved by using higher amounts of nitrogen. (Kumar et al. 2007) concluded that nitrogen-containing compounds have a crucial effect on bulblet initiation and enlargement in in vitro culture of Oriental lilies.

Table 4

The concentration of the different elements (mM) in five basal media used in this experiment
Element	Media
Element	MS	N6	CLC	B5	WPM
Ca	2.99	1.13	2.99	1.02	3
Cl	5.98	2.26	5.98	2.04	1.3
K	20.04	30.93	20.04	24.73	12.61
P	1.25	2.94	1.25	1.09	1.25
N	60.01	34.99	38.79	26.75	14.7
MG	1.5	0.75	1.5	1.01	1.5

Plant species respond specifically to light regime and intensity, so it is important to evaluate light efficacy in each plant in vitro. Our results showed that in darkness, root production was reduced, shoot multiplication was moderately affected, but bulblet production was completely reduced. According to George et al. (2008), adventitious shoots can regenerate in the dark, but they are basically stronger in the light.

Root production can be stimulated or inhibited in light (Li et al. 2021). Regeneration of adventitious roots on explants appears to be affected by light energy. However, in some plants, root induction is enhanced in darkness, possibly due to natural auxin enhancement under these conditions.

The effect of light on bulblet regeneration has been emphasized in numerous reports. Rahimi Khonakdari et al. (2020) reported that the average bulblet production ratio under light conditions was twice that under dark conditions. Cheesman et al. (2010) showed that bulblet formation was completely suppressed in Eucomis zambesiaca in total darkness, which is consistent with our results. In contrast, bulblet regeneration was increased in Lilium longiflorum (Kumar et al. 2006) in continuous darkness.

According to the results of this experiment it could be concluded that using cross-cutting of basal plates that have passed their dormant stage is absolutely necessary. Contrary to other reports, the high effectiveness of IBA on regeneration, especially through the production of bulblets, was found in our experiment. In addition, the concentration of total nitrogen and also the NO3⁻/NH4⁺ ratio play an important role in the type and number of regenerations. Although some types of regenerations occurred in the dark, irradiation with white light proved to be superior, especially in terms of bulblet production. Since Allium jesdianum is threatened with extinction in its narrow habitat, the results of our experiment could be fruitfully used for the propagation and survival of the plant and also facilitate in vitro breeding programs and pharmaceutical exploitation. The results of this experiment will facilitate the propagation, conservation and improvement of Allium jesdianum.

IBA

indole-3-butyric acid

NAA

1-Naphthaleneacetic acid

BAP

6-benzylaminopurine

Competing interests: The authors have no competing interests to declare that are relevant to the content of this article.

Funding: Partial financial support was received from Shahrekord University.

Author Contributions: All authors contributed to the study conception and design. Material preparation and data collection were performed by Mahdi Zare. Data analysis was performed by Mohammad Rabiei. The first draft of the manuscript was written by Mohammad Rabiei. All authors read and approved the final manuscript

Data Availability Statements

All data generated or analysed during this study are included in this published article and its supplementary information file.

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The effect of plant growth regulators, basal medium and light on micropropagation of Allium jesdianum Boiss. & Buhse

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Abstract

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Key Message

1. Introduction

2. Materials and methods

2.1. Plant material

2.2. Treatment with plant growth regulators (PGR)

2.3. Treatment of basal nutrient culture medium and light

2.4. Growth conditions

3. Results

3.1. Effect of plant growth regulators

3.2. The effect of light and medium composition

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

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