In Vitro Cultivation of Purple-Fleshed Potato Varieties: Insights into Their Growth and Development

Abstract

Purple-fleshed potatoes (PFP) are varieties of Solanum tuberosum L., which recently have been recorded to be more and more cultivated and consumed in all European countries, including Romania, as they are promoted for their content in bioactive compounds and benefits to human health. This paper presents a micropropagation protocol study for PFP varieties already traded into the Romanian market, namely Blue Danube (BD), Salad Blue (SB), Violet Negretin (VN), and Violet Queen (VQ). These varieties were tested for in vitro micropropagation also considering asepsis, initiation, callus formation, and microtuberization. To establish the optimum asepsis treatment, a preliminary experiment was performed and, the best results were obtained by using 70% EtOH (1 min) followed by 20% (v/v) Domestos^® (20 min). The MS formula (Murashige and Skoog 1962) was tested as the basic culture medium without growth regulators for all tested stages except for callus initiation and its further multiplication stages. The effect of glycine on direct organogenesis and shoot multiplication was evaluated for propagated micro-cuttings. We emphasize that the addition of glycine at a concentration of 15 mg/L to the culture medium induced a better plantlet vigor for all four varieties. Regarding the indirect organogenesis, culture medium supplemented with NAA (5.00 mg/L), GA₃ (1.00 mg/L), TDZ (1.00 mg/L) and glycine (15.00 mg/L) induced the best results for shoot cluster regeneration as well as turning of white callus from control to purple callus. Further, the microtuberization was successfully produced when sucrose was supplemented at 8% (w/v) into the culture medium. Among all four tested PFP varieties, SB has proven to give the best results regarding the adaptability for in vitro cultivation.

1. Introduction

As is it well established, Solanum tuberosum L. is a vegetable native to South America and imported to the European continent after the 16th century [1]. Potato has achieved significant world importance as a food and feed resources due to its extraordinary yield per unit area compared to other crops [2]. Potato is also among the four most important crops worldwide [3]. Another strength that attracts the large consumption of potatoes globally is the diversity through which they contribute to the preparation of different culinary recipes; potatoes can be cooked boiled, flaked, air-fried, baked, mashed, and new data are suggesting that potato sprouts can be a valuable food resource [4,5]. On the other hand, climate change has generated serious problems worldwide related to water shortages, flooding, and extreme weather having a major impact on the environment, especially on agriculture and food security [6] As a consequence, lot of varieties have been developed from this species considering cultivation techniques and plant physiology, but the most relevant are the ones related to the characteristics of tubers (i.e., size, shape, color, texture, taste). Tubers can have different flesh colors from white, yellow, and red, to purple and dark blue [7]. Moreover, recent studies have shown that a new purple-fleshed variety named Hongmei, which can be eaten as a raw vegetable, has been developed in China [8]; in Korea there has been created a clonal selection with yellow-fleshed named Gogu Valley, a medium-to-late potato cultivar that is also ready to be consumed as raw or fried food [9]. Another use of potato production obtained from the autumn crops is in the starch and alcohol industry [10], as well as animal feed [11]. More recently, in Bangladesh, potato peeling wastes have been used in the green energy field to produce bio-oil [12].

Based on the current trends, it is possible to switch the culture of traditional potato varieties to PFP varieties for their nutritional and health benefits [13]. A major propriety of these PFP varieties is related to the presence of antioxidants and phenolic compounds.

Anthocyanins are organic compounds that are produced into the shikimate pathways and are considered very important plant pigments in connection to plant response to environmental factors [14]. Due to their proven antioxidant effect [15], purified anthocyanins are increasingly used in the pharmaceutical [16], cosmetic [17], and food industries [18].

In the past decade, scientific research on PFPs has emphasized the importance of bio-compounds and their effects on human health, especially phenols, carotenoids, anthocyanins, specific proteins, minerals, and vitamins [19,20]. According to different authors, PFPs are among the richest sources of anthocyanins, which can be an important antioxidant and anti-inflammatory food supplement [21,22].

Due to prolonged drought, European countries including Romania have started to pay attention to excessive consumption of water, rationalized its consumption in some areas, and prohibited the irrigation of agricultural crops. To ensure food security and, at the same time to rationally exploit the agricultural land, farmers began to introduce and cultivate different species from exotic areas that can adapt to new climate conditions [23]. For example, in the past decade the cultivation of PFPs was adopted by some Romanian farmers. The clonal selection system for obtaining potato seeds has long been replaced by micropropagation and mini-propagation [24], and at the present time in the potato industry a special emphasis is placed on in vitro multiplication and microtuberization.

The purpose of this article was to study micropropagation and microtuberization for four varieties of PFPs: Blue Danube (BD), Salad Blue (SB), Violet Negretin (VN), and Violet Queen (VQ).

As models, micropropagation protocols of classical (white-fleshed) potato were used as starting point [24]. In the first stage, this study aimed to test and improve the old micropropagation protocol that was applied in our laboratory for more than 30 years [25]. All micropropagation stages will be discussed, starting with the asepsis of plant material. For micropropagation, glycine and different growth regulator concentrations and ratios were tested using the classical MS culture medium (Murashige and Skoog 1962). In case of microtuberization, different concentrations of sucrose were tested using the same culture medium.

2. Materials and Methods

2.1. Plant Materials and Explants

The biological material consisted in four varieties of PFPs (Solanum tuberosum L.) as certified seed tubers (

Table 1. Summary characterization of Solanum tuberosum L. varieties studied according to the Potato Variety Database [26].

Variety	Country of Origin	Provider	Type of Variety	Shape of Tuber	Epidermis Color	Flesh Color
Blue Danube	Germany	Romanian Farmer 46°12′07.9″ N; 23°41′16.1″ E 1	Early	Oval	Blue	White
BD
Salad Blue SB	U.K. (Scotland)	The National Research and Development Institute for Potato and Sugar Beet from Brasov, Romania	Early	Oval	Dark blue shade	Purple to blue with white insertions
Violet Negretin VN	France	A nationally recognized chain of stores from Romania	Late	Elongated	Dark blue, almost black	Dark blue or dark purple
Violet Queen VQ	The Netherlands	A nationally recognized chain of stores from Romania	Early	Oval-long	Blue particolored	Blue particolored

Note: ¹ GPS coordinates of the Romanian farmer. BD, SB, VN, VQ—the short names of PFP varieties.

). These four varieties were selected to be studied because they were already introduced for food consumption into the Romanian market.

The country of origin of all four varieties was established based on the Potato Variety Database [26].

All collected tubers were first investigated for their healthy status and only healthy tubers were used in our experiments. Thus, they were selected based on the absence of bacteria and fungus attacks and worms. The biological material used to initiate the in vitro culture was represented by the apexes collected from sprouts of PFP tubers.

2.2. Explant Asepsis

The healthy collected potato sprouts were obtained after 3 weeks of being kept under dark conditions. Potato tubers were kept in the dark at room temperature in Petri dishes with filter paper soaked into distilled water to stimulate the sprouting growth [27].

The collected sprouts were sterilized using six different treatments (

Table 2. Asepsis treatments experiment for testing the sterilization efficiency of SB sprouts.

Treatment	Pretreatment	Treatment
		Chemical Agent	Conc.2	Duration (min)
T1	EtOH 70% (1 min)	Domestos® 1	10% (v/v)	10
T2				20
T3			15% (v/v)	10
T4				20
T5			20% (v/v)	10
T6				20

Note: ¹ Domestos^®, Unilever U.K. with NaOCl in 5% concentration; ² Concentration (Conc.). T₁, T₂, T₃, T₄, T₅, T₆ asepsis treatment variant used for PFP.

). To identify the optimal disinfection treatment, the concentration of disinfectant solution and the time of immersion were tested. In this experiment, 270 tubes were inoculated with sterile apexes for Salad Blue variety on MS medium (i.e., 15 tubes for 6 treatments in 3 repetitions).

The asepsis started with potato sprouts being washed under tap water for 5 min. The process continued with explants’ immersion in EtOH 70% for 1 min, after which they were rinsed with sterile distilled water three times under continuous agitation for 1 min. each. The next step was the treatment with Domestos^® solution in different concentrations (10, 15, and 20%) for two different exposure times (10 and 20 min), followed by three rinses in sterile distilled water for 1 min each under the laminar flow hood, in sterile conditions.

2.3. Sprouts Asepsis, Explant Initiation, and Micro-Cuttings Subcultivation

Explants consisting in small apexes taken from the sprouts of PFPs [28] represented the starting material for culture initiation. For in vitro culture initiation, T₆ treatment for explants’ asepsis and 160 apexes for all four PFP varieties were used. After the asepsis process, the apexes were inoculated on MS culture medium. At the initiation stage, 40 explants for each variety were inoculated on two variants of culture media.

The experimental model of the micro-cuttings’ subculture aimed to test whether the culture media used could help to obtain vigorous plantlets from all four varieties of PFP. For PFP subculture, micro-cuttings provided from in vitro regenerated shoots were multiplied on fresh culture medium. Therefore, the culture media MS supplemented with glycine were used for micro-cuttings subculture in parallel with MS as a control. The first subculture was started four weeks from the initiation moment and the successive subcultures were performed every three weeks. A total of 960 micro-cuttings for all three subcultures were used (40 per variety in one subculture).

2.4. In Vitro Culture Media and Growth Conditions

To initiate in vitro culture, explants from PFPs were cultured in MS medium (Murashige and Skoog 1962) [29] including vitamins (MS), 20.00 g/L sucrose and 3.00 g/L Gelrite (Duchefa), in plant growth regulators’ (PGRs) absence.

The subculture was performed using two variants of culture medium for control and MS supplemented with 15.00 mg/L glycine (Sigma-Aldrich, St. Louis, MO, USA) (

Table 3. Culture media and growth conditions for subculture of PFP.

	MS62 Including Vitamins	Glycine	Sucrose	Gelrite	pH Value	Growth Room Condition
MS (control) MS	4.40 g/L	-	20.00 g/L	3.00 g/L	5.7	Irradiance capacity 100–112 μmol/m2/s Temperature 23 °C ± 2 °C Photoperiod 16/8 h
MS with glycine MS + Gly	4.40 g/L	15.00 mg/L	20.00 g/L	3.00 g/L	5.7

Note: MS; MS + Gly type of culture media used for subculture process.

). Glycine was added to the culture medium from stock solution prior to autoclaving.

In parallel to these experiments, the callus initiation and multiplication for all four varieties were performed in five types of MS culture medium (

Table 4. MS culture media composition for PFP callus initiation and subcultivation. Sucrose was added at 20 g/L, Gelrite at 3 g/L, and the pH value was 5.7 before autoclaving for all tested culture media.

Type of Callus Culture Media	Plant Growth Regulators (PGRs)					Amino Acid
	2,4-D (mg/L)	NAA (mg/L)	GA3 (mg/L)	BAP (mg/L)	TDZ (mg/L)	Glycine (mg/L)
C1	2.50	-	-	-	-	-
C2	-	5.00	1.00	1.00	-	-
C3	-	5.00	1.00	-	1.00	-
C4	-	5.00	1.00	1.00	-	15.00
C5	-	5.00	1.00	-	1.00	15.00

Note: 2, 4-dichlorophenoxyacetic acid (2,4-D); 1-naphthaleneacetic acid (NAA); gibberellic acid (GA₃); 6-benzylaminopurine (BAP); thidiazuron (TDZ); C₁, C₂, C₃, C₄, C₅ culture media used for callus initiation and subculture.

), with PGRs (Duchefa) in different combinations and concentrations for indirect organogenesis (shooting).

The next experiment consisted of in vitro microtuberization of PFPs using micro-cuttings collected from in vitro plantlets cultivated on MS + Gly medium for 4 weeks. For this process, four types of culture medium with addition of different concentration of sucrose were used (

Table 5. MS culture media composition for PFP in vitro microtuberization process.

	Sucrose (w/v)	Growth Room Condition
MT1	6% 8% 10%	Irradiance capacity 100–112 μmol/m2/s Temperature 23 °C± 2 °C Photoperiod 16/8 h
MT2
MT3
MT4	12%

Note: MT₁, MT₂, MT₃, MT₄ culture media used for in vitro microtuberization process.

).

For all type of culture media used, the pH was adjusted to 5.7 using 1 N NaOH and/or 1 N HCl solutions before adding the Gelrite. The obtained solution was distributed to test tubes (10 mL/tube) with total volume of 50 mL, height 14.5 cm, Ø of 2.5 cm. The culture medium sterilization was performed by autoclaving at 121 °C under a pressure of 101,325–121,590 Pa for 20 min.

Following inoculation, all test tubes containing the explants were maintained in the growth room at 23 ± 2 °C during day/night, with a photoperiod of 16 h light and 8 h darkness. Gro-Lux F36W/GRO fluorescent tubes (Sylvania, Germany) with an irradiance capacity of 100–112 μmol/m²/s were used.

The same parameters of the growth room (23 ± 2 °C during day/night) were used following callus initiation and subculture. Out of initiated and multiplicated callus material, the light effect was tested in two variants, one variant with a photoperiod of 16 h light and 8 h darkness, and a second one for 24 h under full darkness conditions.

2.5. Tuber Asepsis, Callus Initiation, and Subcultivation

Potato tubers from all four varieties were used for callus initiation. T₆ treatment was applied for explants’ asepsis. After asepsis, slices of ~2 cm length and 0.1–0.3 mm in height were excised and cultivated separately on all five culture media variants [30]. All these operations were performed under the laminar flow hood in sterile conditions. For callus initiation, 15 explants of each variety on each variant of culture medium were inoculated.

The callus subculture had the objective to test the in vitro capacity to direct organogenesis regeneration of PFPs. After four weeks of initiation, subcultures of calli were obtained from the same variant of culture medium used. Three callus subcultures were made at intervals of six weeks.

2.6. In Vitro Microtuberization

An essential source of tissue in regard to the processes of conservation and exchange of germplasm is potato microtubers [31]. The effect of sucrose (6, 8, 10, and 12%) on microtuber induction was evaluated using the MS basal growth medium supplemented with vitamins and without PGRs on the four PFP varieties. Micro-cuttings (~2 cm length with one or two leaves) after 8 weeks’ growth on MS + Gly were transferred to the fresh media for microtuberization (Table 4).

For each variety, 10 test tubes were inoculated, in 3 repetitions, which created a total of 480 plantlets. They were placed in the growth room conditions mentioned on Table 4.

2.7. Experimental Design and Data Analysis

To determine the most efficient disinfection treatment, a completely randomized experimental design was chosen. Each treatment was applied in 3 repetitions and each repetition consisted of 15 samples per replicate. To test the degree of contamination, simple tests were performed.

Biometric parameters were determined for each subculture on MS and MS + Gly culture media. Statistical analyses were performed for subcultures 2 to 4. For this experiment, 40 samples for each variety were used, which created a total of 960 micro-cuttings. Bifactorial experiments were used, where the first experimental factor was the potato variety, and the second experimental factor was the culture medium. Every 4 weeks the following biometric parameters were determined: length of shoots (cm), node number, and distance between nodes (cm).

To establish the regeneration from callus, the data provided from the third subculture were analyzed. In case of studying calli, trifactorial experiments were used, where the first experimental factor was the potato variety, the second experimental factor was the culture medium, and the third experimental factor was the growth conditions. Four weeks after callus initiation, the rate of callus formation was determined. Six weeks after the inoculation of the third subculture, the following biometric parameters were determined: shoot regeneration rate (%), fresh weight (g), and the calli’s main characteristics such as color and texture.

In case of microtuberization process, a bifactorial experiment was used, where the first experimental factor was the potato variety, and the second experimental factor was the culture medium used for the microtuberization process. Eight weeks after the micro-cuttings’ inoculation, the following biometric parameters were analyzed: tuberization rate (%); number of microtubers; weight of microtubers (g); diameter of microtubers (mm).

All collected data were analyzed by using the POLIFACT Statistical Software (USAMV Cluj-Napoca). To evaluate the differences between the data, two-way ANOVA was considered. If statistically significant values between averages were registered, the Duncan’s significance test (Duncan’s MRT, p < 0.05) was applied.

3. Results

3.1. Explant Asepsis

Analyzing the asepsis treatments (

Table 6. Influence of disinfection treatments of the PFP sprout explants.

Treatment	Survival (%)	Contamination (%)	Incubation Period (Days)	Contaminant Abundance
T1	24.44 c	75.56	2–3	+ + + +
T2	42.22 b,c	57.78	2–3	+ + +
T3	35.55 b,c	64.45	2–6	+ + + +
T4	68.89 a	31.11	2–4	+ + +
T5	51.11 b	48.89	3–5	+ +
T6	79.99 a	20.34	3–6	+
DS 5%	17.38–19.15

Note: + + + +, very high contamination; + + +, high contamination; + +, low contamination; +, very low contamination. The means in the column inside the table followed by different letters are significant according to Duncan’s MR test (p < 0.05).

), the inocula survival percentage was between 24.44% at T₁ and 79.99% at T₆. The expression of contamination period varied between 2 and 6 days and depended on the disinfectant concentration. At the lower concentration treatment, the contamination period was 2–3 days and very high and at the higher concentration treatment, the contamination period was increased to 3–6 days and the contaminant abundance was reduced to a very low infection rate.

The treatment for explant asepsis with the best results was recorded for T₆ disinfection method at a 79.99% rate of survival and a very low contamination in the test tubes. In case of other tested methods, lower values of the rate of survival were obtained. Thus, T₁ was the lowest efficient treatment with 75.56% rate of contamination, succeeded by T₃ with 64.45% plant material with contamination, then T₂ (57.78%), T₅ (48.89%), and T₄ (31.11%). They presented contaminations in the culture medium ranged from a very high abundance (T₁ and T₃), to high (T₂ and T₄), low (T₅), and very low (T₁) abundance.

Following the asepsis treatments applied on the potato sprouts from the Salad Blue variety (see on Table 3), the best results were from T₆, which means a pretreatment with 70% EtOH for 1 min, followed by a treatment with 20% Domestos^® for 20 min, and three rinses with sterile distilled water under the laminar flow hood, in sterile conditions.

3.2. Sprout Asepsis, Explant Initiation, and Micro-cutting Subcultivation

The explants from potato sprouts (Figure 1a) were successfully disinfected using T₆ treatment and inoculated on MS medium (Figure 1b). Three days after the initiation, hypertrophic processes were observed in all explants (Figure 1c), then small callus formation was followed by emergence of shoots, and three weeks later potato plantlets were developed enough to be subcultivated (Figure 1d) on fresh culture media as micro-cuttings.

Regarding the explants’ initiation, the highest success was recorded for SB variety (87.50%), followed by VQ (82.50%), BD (77.50%), and VN (72.50%) varieties. It can be considered that we obtained an 80.93% success rate for the whole experiment.

Three weeks after initiation, the highest mean shoot height was for the SB variety at 8.63 cm, followed by BD (6.97 cm), VN (6.63 cm), and VQ (4.80 cm) varieties (

Table 7. Rate of survival after three weeks from initiation moment.

Variety	Survival (%)	Contamination (%)	Length Shoots (cm)
BD	77.50 a,b	22.50	6.97 b
SB	87.50 a	12.50	8.63 a
VN	72.50 b	27.50	6.63 b
VQ	82.50 a,b	17.50	4.80 c
DS 5%	11.25–11.30	-	1.60–1.68

Note: BD, SB, VN, VQ—the name of PFP varieties studied; the means in the column inside the table followed by different letters are significant according to Duncan’s MR test (p < 0.05).

). These results are significant from a statistical point of view according to Duncan’s MR test (p < 0.05).

The next step was to transfer the micro-cuttings to the fresh media, to observe which was the optimum treatment for mass micropropagation. The plantlets were transferred from the initiation media by micro-cuttings to MS (Ct) and MS + Gly media (Figure 2).

Regarding the length of shoots resulted from the micro-cuttings’ subculture (

Table 8. Morphometry of plantlets after the third subcultivation on MS and MS + Gly culture media.

Variety	Shoot Length (cm)		Node Number		Internode Distance (cm)
	MS (Ct)	MS + Gly	MS (Ct)	MS + Gly	MS (Ct)	MS + Gly
BD	3.30 e	8.40 b	5.00 b	7.33 a	0.69 d	1.15 b,c
SB	3.93 d,e	9.00 a,b	2.67 c	6.00 a,b	1.51 a,b	1.53 a,b
VN	3.60 d,e	9.50 a	3.33 c	5.67 b	1.10 c	1.73 a
VQ	7.33 c	4.27 d	6.00 a,b	2.67 c	1.24 b,c	1.64 a
DS 5%	0.87–0.97		1.27–1.43		0.35–0.40

Note: BD, SB, VN, VQ—the name of PFP varieties studied. The means in the column inside the table followed by different letters are significant according to Duncan’s MR test (p < 0.05). The values are means ± SE (n = 3).

), significant statistical differences were obtained by applying the Duncan test. The glycine added to the culture medium contributed to obtaining longer shoots for BD, SB, and VN varieties, with significant statistical differences. In MS (Ct), the best results were obtained for the VQ variety (7.33 cm), followed by SB (3.93 cm), VN (3.60 cm), and BD (3.30 cm) varieties. Regarding the MS + Gly medium, VN had a significant growth of length shoot (9.50 cm) approximately three times more than plantlets grown on MS (Ct), succeeded by SB (9.00 cm) and BD (8.40 cm) varieties. For the VQ variety, MS + Gly had an opposite effect compared to the other three varieties and the shoot length was 4.27 cm, approximately two times lower than on the MS (Ct) culture medium. On the other hand, plantlets were more vigorous and showed formed leaves on MS + Gly in all four varieties of PFP.

Regarding the analysis of node numbers and distance between nodes, our best results were obtained on MS + Gly. The number of nodes was similar with MS in case of BD, SB, and VN varieties; the exception was the VQ variety, with significant statistical differences registered for another three PFP varieties when applying Duncan’s test (p < 0.05). For the SB variety, we recorded the highest difference between number of nodes per plantlet (6.00 on MS + Gly and 2.67 on control), followed by VN (5.67 on MS + Gly and 3.33 on control) and BD (7.33 on MS + Gly and 5.00 on control) varieties. For the VQ variety, we recorded the highest number of nodes on MS (6.00) compared to MS + Gly (2.67).

In the case of the length of the internodes, it was higher on MS + Gly medium than on MS medium for all four varieties studied and differences were significantly positive, except for the SB variety.

3.3. Tuber Asepsis, Callus Initiation, and Subcultivation

Callus culture of PFPs was tested on five variants of culture media with different concentrations of PGRs and/or glycine amino acid (Table 3). For initiation culture, a trifactorial analysis was performed regarding the percentage of explants producing calli and percentage of shoots produced from calli, reporting the following parameters: type of culture medium, variety of PFP, growth conditions (

Table 9. Rate of callus formation and plant regeneration after callus initiation.

Culture Media	Variety	Callus Initiation
		% of Explants Producing Callus		% of Callus Producing Shoots
		Light	Dark	Light	Dark
C1	BD	63.33	23.33	0.00	0.00
	SB	76.66	30.00	0.00	0.00
	VN	83.33	26.66	4.00	0.00
	VQ	53.33	36.66	0.00	9.09
C2	BD	56.66	16.66	47.05	0.00
	SB	83.33	36.66	44.00	18.18
	VN	80.00	23.33	37.50	0.00
	VQ	60.00	43.33	46.66	7.69
C3	BD	86.66	36.66	42.30	9.09
	SB	76.66	46.66	43.47	14.28
	VN	73.33	13.33	36.36	0.00
	VQ	56.66	20.00	35.29	0.00
C4	BD	83.33	26.66	72.00	0.00
	SB	90.00	30.00	59.25	0.00
	VN	86.66	36.66	57.69	9.09
	VQ	63.33	16.66	57.89	0.00
C5	BD	90.00	30.00	74.07	0.00
	SB	86.66	36.66	73.07	9.09
	VN	86.66	43.33	65.38	7.69
	VQ	66.66	33.33	60.00	0.00

Note: BD, SB, VN, VQ—the name of PFP varieties studied. C₁, C₂, C₃, C₄, C₅—type of culture media for callus.

).

In the case of all five varieties of culture media, good results were obtained. Regarding the culture medium used, a high percentage of callus production on MC₅ was registered and giving the best results. For example, BD produced 90% callus compared with C₁ (63.33%) on light conditions. In dark conditions, the results for callus initiation were diminished around two times compared with those of the same culture medium and variety maintained in the light during the growth process.

Regarding the shoots produced from calli, it was obvious that dark conditions were not an auspicious factor because no shoots were obtained and explants were only producing callus, except for the VQ variety on C₁ (9.09%), the SB variety (18.18%) and VQ variety (7.69%) on C₂, the BD variety (9.09%) and SB variety (14.28%) on C₃, the VN variety (9.09%) on C₄, and the SB variety (9.09%) and VN variety (7.69%) on C₅, respectively. Under light conditions, shoots from calli were commonly obtained, except on C₁, where for the VN variety just 4% of calli generated new shoots. On C₅, the best results were obtained as follows: 74.07% for the BD variety, 73.07% for the SB variety, 65.38% for the VN variety, and 60.00% for the VQ variety.

Three weeks after the moment of callus initiation, they were transferred to fresh culture medium of the same variant used for initiation but were exposed just to light conditions (16 h light and 8 h dark) in the growth room, at 23 ± 2 °C. The calli obtained on the third subculture were analyzed regarding the shoots’ regeneration, fresh weight, and morphology aspects (

Table 10. Morphological aspects of calli obtained from the third subculture.

Culture Media	Variety	Callus from the Third Subculture
		Shoots Regeneration (%)	Callus Fresh Weight (g)	Callus Morphology (Color and Texture)
C1	BD	18.89	1.98 a	green, friable
	SB	14.44	1.96 a	green/brown, friable, with roots
	VN	16.67	1.66 a–c	green, friable
	VQ	12.22	1.80 a,b	green/brown, with roots
C2	BD	41.11	0.61 i,j	white/green, soft
	SB	45.56	1.10 e–h	white/purple, soft
	VN	37.78	0.44 j	yellow/green, friable
	VQ	42.22	0.94 g–i	white/purple, rough
C3	BD	47.78	0.94 g–i	green/white, rough
	SB	51.11	1.35 c–g	green/yellow, rough
	VN	53.33	0.93 g–i	green, rough
	VQ	44.44	1.07 f–h	green/white, rough
C4	BD	74.44	1.55 b–d	white, soft
	SB	71.11	1.15 d–h	green/purple, soft
	VN	62.22	0.84 h, i	green/white, soft
	VQ	56.67	1.46 b–f	purple/white, soft
C5	BD	76.67	1.10 e–h	white/green, soft
	SB	73.33	1.53 b–e	white/purple, soft
	VN	64.44	1.27 c–h	yellow/purple, soft
	VQ	58.89	1.20 d–h	purple, soft
DS 5%			0.38–0.45

Note: BD, SB, VN, VQ—the name of PFP varieties studied. C₁, C₂, C₃, C₄, C₅—type of culture media for callus. The means in the column inside the table followed by different letters are significant according to Duncan’s MR test (p < 0.05). The values are means ± SE (n = 3).

).

On the third subculture of calli, shoots regenerated in all cases. On C₁ the VQ variety showed the lowest process of shoot regeneration (12.22%) and the highest shoot regeneration was registered on MC₅ for the BD variety (76.67%, respectively).

Regarding callus fresh weight, statistically significant differences were obtained according to Duncan’s MR test (p < 0.05). The highest value was obtained on C₁ for the BD variety (1.98 g) and the lowest value was registered on C₂ for the VN variety (0.44 g). The predominant color of the calli was green, except in the case of C₅, where a purple color was evident in calli of the VQ variety. The texture of calli was different, ranging from soft, to friable, and rough. In Figure 3 are presented the aspects of SB variety callus formation in all five varieties of culture media used.

The optimum culture medium for callus production and callus fresh weight was C₁; C₅ was the optimum culture medium to obtain shoots via callus regeneration.

3.4. In Vitro Microtuberization

In vitro microtuberization was tested in four variants of culture media (MS) supplemented by different sucrose concentrations (6, 8, 10, and 12%). Eight weeks from inoculation, biometry was performed to establish the optimum medium concentration for microtuberization. All four variants used for microtuberization gave results (

Table 11. Results after microtuberization process of PFP.

Culture Media	Variety	Tuberization (%)	Number of Microtubers Per Plantlet	Weight of Microtuber (g)	Diameter of Microtuber (mm)
MT1	BD	31.11	1.00 d	0.46 c	4.50 e–h
	SB	37.78	1.00 d	0.53 c	4.60 e–h
	VN	36.67	1.00 d	0.53 c	4.83 e,f
	VQ	33.33	1.00 d	0.49 c	4.70 e–g
MT2	BD	54.44	1.67 c,d	1.01 a,b	8.03 b
	SB	67.78	3.67 a	1.11 a	8.70 a
	VN	55.56	3.00 a,b	0.98 a,b	7.23 c
	VQ	57.78	2.33 b,c	0.87 b	6.23 d
MT3	BD	25.56	1.33 c,d	0.89 b	7.13 c
	SB	32.22	2.00 b–d	1.07 a,b	7.17 c
	VN	24.44	1.67 c,d	1.16 a	6.87 c
	VQ	27.78	2.33 b,c	0.98 a,b	6.93 c
MT4	BD	2.22	1.00 d	0.40 c	4.27 f–h
	SB	6.67	1.00 d	0.36 c	4.97 e
	VN	5.56	1.00 d	0.47 c	4.14 g,h
	VQ	4.44	1.00 d	0.47 c	4.07 h
DS 5%			1.12–1.33	0.19–0.23	0.54–0.64

Note: BD, SB, VN, VQ—the name of PFP varieties studied. MT₁, MT₂, MT₃, MT₄—type of culture media for microtuberization. The means in the column inside the table followed by different letters are significant according to Duncan’s MR test (p < 0.05). The values are means ± SE (n = 3).

).

Regarding the microtuberization process, MT₂ showed the best results for all four varieties (67.78% on the SB variety, 57.78% on the VQ variety, 55.56% on the VN variety, and 54.44% on the BD variety) and the lowest results were registered on MT₄ with a microtuberization percentage under 7% for all four varieties.

In the case of number of microtubers per plantlet, MT₂ with MT₃ was superior to MT₁ and MT₄. The best results for microtubers per plantlet were registered on MT₂ for the SB variety (3.67), succeeded by VN (3.00), VQ (2.33), and BD (1.67) varieties. In the case of MT₁ and MT₄, all four varieties had just one microtuber per plantlet. All the results were significant according to Duncan’s MR test (p < 0.05).

On MT₂ and MT_3, the weight of microtubers was at a similar value for all four varieties of PFP and superior to the weight obtained on MT₁ and MT₄. The greatest value of weight microtuber was on MT₂ for the SB variety (1.11 g) and the smallest on MT₄ for the same variety (0.36 g). All the results were significant according to Duncan’s MR test (p < 0.05).

Regarding the diameter of microtubers, similar results were obtained from MT₂ with MT₃, and from MT₁ with MT₄, but with significant differences from a statistical point of view. The largest diameter was registered on MT₂ for the SB variety with 8.70 mm, followed by BD (8.03 mm) and VN (7.23 mm) on the same culture medium. The smallest diameter was obtained on MT₄ for the VQ variety (4.07 mm) followed by VN (4.14 mm) and BD (4.27 mm) varieties on the same culture medium.

The optimal culture medium for obtaining microtubers of PFP was MT₂ and the more productive variety was SB (Figure 4).

4. Discussion

The aim of this study was to develop a micropropagation protocol for different varieties of PFP by adapting micropropagation protocols already developed on the classical MS potato (white-fleshed) varieties in our laboratory. Among the previous investigations of other authors, we did not find any report regarding complete in vitro micropropagation and tuberization protocols on the selected PFP varieties.

An analysis was performed on the articles generated by searching scientific databases including the Web of Science Core Collection (WOS) [32] using as keywords ‘purple-fleshed potato’. This scientific documentation was relevant for emphasizing the importance of studying these potato varieties. Of 408 scientific found papers, 231 had as their main subject PFP (Solanum tuberosum L.) (i.e., 56.61%). However, the remaining 173 scientific papers were focused on purple sweet flesh potatoes (Ipomea batatas L.), 3 on purple varieties of maize (Zea mays L.), and one on the topic of carrots (Daucus carota L.).

All 231 scientific papers on PFPs, consisting in articles, reviews or short communications collected from electronic libraries, were published between 1997 and 2022 (Figure 5). Thus, 23 were published between 1997 and 2007, 67 between 2008 and 2014, and 141 between 2015 and 2022. It can be assumed that PFP varieties are becoming more and more relevant for research, which is why the number of publications is constantly increasing.

In the second stage, all articles were analyzed based on their keywords and the most used keyword was ‘anthocyanins’ (Figure 6). There were six clusters of identified keywords following the interest of different research groups. Therefore, other keywords used and relevant for our study were the following: cultivars, polyphenols, purple-fleshed potatoes, gene expression, storage, stability.

According to all surveyed scientific articles, PFPs are important as they are rich in anthocyanins [34]. The scientific papers deal with aspects related to the importance of the content of anthocyanins in PFPs, as well as extracting methods [35] or the maintenance of their active biological properties after cooking [36]. We also found studies related to the analysis of the anthocyanins in different PFP varieties [37]. Thus, it was discovered that potatoes, which were thought to be a major contributor to the emergence of many chronic diseases, really have health-promoting qualities due to different biological active substances including phenolics and anthocyanins [38,39]. In this regard, a recent study examined the antioxidant properties of the Blue Congo PFP variety in rats with diabetes induced by streptozotocin. The study proved that the administration into transfusion of the extract of PFPs decreased in the end the amount of glycated hemoglobin and improved glucose tolerance for diabetic rats [40]. Regarding their positive effects on human health, it was proved that PFPs have a positive impact on visual quality and cognition as well as in the management of diabetes, obesity, and cardiovascular diseases, and they are also recommended as an integral part of chemoprevention against human cancers [40]. Other scientific topic discussed in the investigated papers were crop production for PFPs [41].

None of the selected scientific papers seems to cover aspects related to in vitro culture or in vitro stress response of PFPs.

No report has been published at the European level regarding PFP cultivation and production. In Romania, all production statistics for potato include all possible varieties of the species Solanum tuberosum L. in accordance with the monitoring system of crop production for all the European Union countries.

4.1. Explant Asepsis

The success of in vitro plant culture is first due to the complete asepsis of desired explants. In this regard, in the first stage scientists tried to remove all potential microorganisms inside or outside the explants. Another source of contamination may be the working environment that is due to deficiencies in working sterile conditions [42]. In the case of potato, the removing of pathogenic viruses was also realized to improve the quality of the seed potatoes [43]. Different asepsis reagents and protocols have been published as tested as the best formula for specific conditions [44,45,46,47,48,49].

For more than 100 years, scientists tried to use different sterilizing reagents for plant species such as: ethanol, sodium hypochlorite, and mercuric chloride [44]. For example, wheat caryopses in the case of Triticum aestivum were successfully sterilized with 5% sodium hypochlorite solution of commercial Domestos^® [45]. To develop a micropropagation protocol for Pinus tecunumanii, the effect of a range of dilutions between 0.05 and 1.00% mercuric chloride was studied [46] and for the Oriental Lillum hybrid, a bulb’s asepsis treatment with 0.1% mercuric chloride followed by 70% ethyl alcohol gave the best results [47].

The potato sprouts from the four potato genotypes Lady Rosetta, Jaerla, Cara, and Hermis, from Egypt, were sterilized using the disinfectant sodium hypochlorite (Clorox^®) at different concentrations and time exposures. A 20% Clorox^® concentration for 20 min gave the best survival rate and disinfection success for these potato varieties [48]. In the case of Christian and Desiree potato varieties, during the asepsis process shoots were immersed for 3 min in ethylene (96%) followed by immersion for 15 min in Domestos^® solution (20%) supplemented with two drops of Tween-20 and further followed by three washes with sterile water for 3 min. [49].

The asepsis experiment we followed used the recommended sodium hypochlorite (the commercial Domestos^® brand) with excellent results for the following protocol: 1 min immersed in ethanol 70%, followed by immersion for 20 min in Domestos^® sterile solution of 20% (v/v) and sterile water washing three times for 1 min under continuous agitation. This protocol was constantly and professionally applied in our laboratory for classic potato. Based on these results, it is proved that the asepsis treatment was successful for potato varieties including Salad Blue. Therefore, Salad Blue was successfully sterilized, and the same treatment was applied for the other three varieties: Blue Danube, Violet Negretin, and Violet Queen, with similar results.

4.2. Sprout Asepsis, Explant Initiation, and Micro-cutting Subcultivation

For the sprouts’ asepsis, the following protocol was applied: 1 min immersed in ethanol 70%, followed by immersion for 20 min in Domestos^® sterile solution of 20% (v/v), and washing three times in sterile water for 1 min each time under continuous agitation. The obtained results regarding the rate of survival confirm that the asepsis method was the right choice.

In 1955, Chapman started potato tissue culture from nodes excised from potato sprouts on White’s culture medium and recommended it as an excellent growth culture medium for potato tissue [50]. At the starting point, he established cultures from several types of plant parts: root tips, tuber parenchyma, excised embryos, tuber eyes, and sprout nodes. After twenty years, Roca et al. described a method for the rapid propagation of potatoes by in vitro culture of nodal section on MS basal medium supplemented with 6-benzylaminopurine, gibberellic acid, and naphthalene acetic acid [51]. Bostan and Demirel (2004) indicated that the best medium for in vitro potato culture is MS medium without any growth regulators [43]. In our research, the explant initiation consisted in inoculation of sprouts’ apexes on classically MS culture medium without any PGRs and gave satisfactory results. Other researchers obtained similar results regarding explant initiation [43,52]; therefore, it can be considered that the protocol used on classical potato varieties can be also performed with success on PFP varieties.

For more than 70 years, scientists developed experimental models for investigating nitrogen’s effects on different plant species during in vitro cultivation [53]. Among the substances providing nitrogen, they used different inorganic salts and amino acids [54]. Although most plants can often synthesize the essential amino acids for the growing process, explants demand the inclusion of specific amino acid combinations [55]. The researchers also described toxic effects of high nitrogen concentration on plants (i.e., developmental changes such as elongation of the vegetative phase, delayed maturity, elongation of plant life cycle, and increased succulence) [56]. According to Gamborg [57] and Murashige and Skoog [29], almost all micropropagation protocols used glycine, but at the low concentration level proposed by the abovementioned authors. Glycine is an abundant free amino acid that can be assimilated by plants faster than other amino acids [58] and increases the growth and development of plants by exogenous applications [59].

The subcultivation of PFP plantlets was performed by transferring the micro-cuttings into two fresh variants of culture medium as follows: MS (control) and MS with 15 mg/L glycine addition (MS + Gly). Usually, in subcultivation of regular varieties of potato, reduced concentration of glycine was used [60]. In the case of PFPs cultivated on MS, negative effects were registered (i.e., frail plantlets, insufficiently developed and etiolated leaves), compared to those cultivated on MS + Gly, which had favorable effects regarding the plantlets’ development (i.e., vigorous plantlets, well developed leaves, greed leaves).

The high concentration of glycine tested (15 mg/L) did not generate a toxic effect on the PFP plantlets as was reported in other cases [56]; on the contrary, we observed an improvement of plantlets’ vigor and the expression of purple color in callus compared to control. It is possible that for in vitro cultivation, the synthesis of anthocyanins requires a larger amount of nitrogen, which can be supported by these results.

4.3. Tuber Asepsis, Callus Initiation and Subcultivation

For the tubers’ asepsis, the same protocol used for the sprouts was successfully applied.

For callus initiation and subcultivation, five types of culture media were used to establish the best formula for indirect organogenesis. Glycine and plant growth regulators from auxins (2,4-D and NAA) to cytokinins (BAP and TDZ) to gibberellin (GA₃) were added in different concentrations and combinations to MS culture medium. Generally, potato callus initiation is realized with a low concentration of glycine. Taking into consideration our best results obtained on PFP plantlets subcultured by adding 15 mg/L glycine into the culture medium, we tested it on callus initiation and subculturing.

The use of the synthetic plant growth regulator 2,4-D for in vitro plant experiments was successfully applied after 1945 and it is responsible for the production of callus tissue [61]. In the case of Oxalis reclinata, the addition of 1.00 mg/L of 2,4-D inhibited the anthocyanin pigments’ production on white callus and decreased anthocyanin accumulation in the red callus [62]. Sherkar and Chavan (2014) showed that MS media supplemented with 2.0–4.0 mg/L 2,4-D had a major influence regarding callus formation [63]. For a similar study regarding regular potatoes, good results were obtained on callus formation and callus proliferation with MS supplemented by 2.00 mg/L 2,4-D [30]. Similar results were obtained in our study for all four varieties of PFP by using MS supplemented with 2.50 mg/L 2,4-D.

Skoog and Tsui (1948) tested the use and importance of chemical substances in tobacco callus culture, and among these substances NAA was included [64]. After this research, more researchers started to use different concentrations of NAA in the culture medium for root formation and callus induction, including regular potato cultivars [43,65]. In our study, four variants of culture media used for callus initiation and subcultivation were supplemented with 5.00 mg/L NAA. All four variants of culture media produced good results in the studied PFP varieties.

BAP and TDZ are cytokines that play an important role in plant micropropagation [43] and their effects regarding in vitro potato culture have been studied since 1969 by Palmer and Smith [66]. Generally, they are used in combination with auxins (i.e., NAA) in order to enable plantlets’ regeneration by callus. The regeneration of regular potato shoots was observed when calli were subcultured on MS supplemented with 1.4 mg/L TDZ and 1.4 mg/L BAP [63]. We used 1.00 mg/L of BAP or TDZ during the initiation and subcultivation of the callus process and both gave similar results for PFP varieties compared to the regular potato varieties. GA₃ is a gibberellin used frequently in MS culture media to promote shoot development and elongation [30]. In our study, it was used by supplementing the MS medium with 1.00 mg/L in combination with others PGRs (NAA and BAP or TDZ).

All five variants of callus culture media showed good results for all four PFP varieties. The culture medium with 2,4-D showed the lowest shoot regeneration but gave a good callus proliferation. The callus texture was friable, and the color was from green to brown. Similar results were obtained for regular potatoes using 2,4-D concentrations on MS culture medium [30]. The highest shoot regeneration was obtained on MS supplemented with NAA (5.00 mg/L), GA₃ (1.00 mg/L), TDZ (1.00 mg/L), and glycine (15.00 mg/L) and the calli presented a purple color and a soft texture. It is possible that glycine had a role in stimulating the production of anthocyanins. These issues can be further subjects in study of the importance of glycine in anthocyanins production, where the callus can be tested for anthocyanins’ quantification.

4.4. In Vitro Microtuberization

In vitro microtuberization represents a method developed to obtain healthy material [67]. Microtubers are an innovative approach for potato seed production, being produced under in vitro conditions using different protocols [68]. They are considered the first generation of potato seeds from tissue culture and present advantages due to their little size and reduced weight (i.e., in storage, transport, and mechanization) [69]. For the formation and development of in vitro microtubers, sucrose is a requirement for an external carbon substrate [70]. The present study aimed to evaluate the potential role of sucrose and its relationship in microtuber formation in PFP in vitro culture.

Magrou in 1938 started to work on and discuss the tuberization process in potatoes using the Knop’s solution as culture medium [71]. After twenty years, it was established that the varying N level of the medium can have no effect on tuberization rate [50]. In 1954, Mes and Menge showed that a high sucrose concentration (5%) favored the microtuberization process [72]. Even though the potato microtuberization process began to be studied about 100 years ago, it can be considered an important topic nowadays. In our country, the researchers from The National Institute of Research and Development for Potato and Sugar Beet in Brașov tested a series of culture media to establish a variant that favors obtaining healthy, vigorous potato microtubers in as large a quantity as possible [49,67].

In our study, it was important to follow the micropropagation protocol that is used for regular potato and in this way, four types of culture media were tested. The MS was supplemented with different concentration of sucrose (6, 8, 10, and 12%). At 8% concentration of sucrose, the best results were obtained regarding the number of microtubers per plantlet, weight, and diameter. Similar results were obtained by Khuri and Moorby for regular potato [73].

The results revealed that this species, regardless of the variety, can provide microtubers on the culture medium supplemented with 6% sucrose.

5. Conclusions

In the present research work, the full micropropagation and microtuberization protocols of in vitro culture for some PFP varieties (Solanum tuberosum L.), starting from the apex cultivation initiation, are presented. Based on the analysis of our results, the best results for micropropagation were obtained for Salad Blue followed by Violet Queen, Violet Negretin, and Blue Danube.

Old protocols developed for classical Solanum tuberosum L. varieties need to be improved regarding the culture medium composition, especially related to the increase of nitrogen concentration in case of PFP varieties.

By adding glycine at 15 mg/L concentration into the culture medium, we induced in in vitro plantlets a better vigor, with very well-developed leaves. In case of calli treated with 15 mg/L glycine, the texture became rougher with a more intense purple color. On one hand, it can be considered that glycine in higher concentrations (15 compared to 2 mg/L) can stimulate PFP plantlets’ development, and on the other hand it can stimulate anthocyanin production in the callus.

In the microtuberization process, the addition of 8% sucrose gave similar results to those that were obtained from regular potato varieties.

Further work is in progress regarding optimization of mass production and more complex analyses to obtain virus-free plant material starting from this micropropagation protocol. To fully understand the anthocyanin production process in PFPs correlated with the addition of glycine to culture medium, and the potential health benefits of consuming PFPs, it will be necessary to analyze anthocyanins in tubers, plantlets, and microtubers.