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Article

The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures

by
Doina Clapa
1,
Monica Hârța
1,*,
Katalin Szabo
2,*,
Bernadette-Emőke Teleky
3 and
Doru Pamfil
1,4
1
Faculty of Horticulture and Business in Rural Development, BIOCERA Research Centre, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania
2
Technological Transfer Center COMPAC, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania
3
Life Science Institute, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania
4
Romanian Academy, Cluj-Napoca Branch, Republicii St. 9, 400015 Cluj-Napoca, Romania
*
Authors to whom correspondence should be addressed.
Horticulturae 2023, 9(8), 902; https://doi.org/10.3390/horticulturae9080902
Submission received: 4 July 2023 / Revised: 28 July 2023 / Accepted: 4 August 2023 / Published: 8 August 2023
(This article belongs to the Special Issue In Vitro Technology and Micropropagated Plants)
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Abstract

:
Micropropagation has an important role in the large-scale production of blackberry plant material, given the high proliferation rates of this species. The aim of the present study was to evaluate the proliferative capacity of blackberry grown in vitro on wheat starch-gelled culture medium compared to classical agar-gelled medium and to assess the genetic fidelity between the proliferated shoots in starch-gelled culture medium and their mother plants. Six blackberry varieties (‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’) were tested. For the in vitro shoots proliferation, Murashige and Skoog (MS) medium supplemented with 0.5 mg dm−3 6-benzyladenine (BA) was used. The conventional medium was gelled with 0.5% plant agar, and wheat starch was used as an alternative gelling agent in a concentration of 5%. The results showed that for all blackberry cultivars, the highest number of shoots/inoculum was obtained in wheat starch-gelled culture medium, with a maximum value of 54.42 ± 4.18 presented by ‘Karaka Black’. Considering the length of the proliferated shoots, all tested cultivars presented outstanding results on the culture medium gelled with 5% wheat starch. The highest values regarding shoots length were observed on the ‘Chester Thornless’ followed by ‘Čačanska Bestrna’, and ‘Loch Ness’ with values of 5.55 ± 0.04 cm, 5.46 ± 0.06 cm, and 5.37 ± 0.09 cm, respectively. The genetic uniformity of the micropropagated shoots in relation to their mother plants was confirmed by sequence-related amplified polymorphism (SRAP) and start codon targeted (SCoT) molecular markers.

1. Introduction

Blackberry (Rubus fruticosus L.) is a well-known shrub from the Rubus genus with edible fruits having a delicious taste and a pleasant aroma due to its specific biochemical composition. Fruits are widely consumed as they have a high content in vitamins, minerals, antioxidants, and dietary fibers that are beneficial for human health and well-being [1,2,3,4].
Similar to other Rubus species with edible fruits, blackberry varieties have been commercially propagated by classical methods of vegetative propagation, e.g., by hardwood and softwood cuttings, by layering, and/or by bush division [5,6,7,8]. Another successful strategy for large-scale commercial micropropagation of blackberry plant material includes in vitro tissue culture-based techniques [9,10,11,12,13,14].
The micropropagation process involves several distinct stages: initiation or the establishment of culture, proliferation of the shoots, elongation of the shoots, and rooting, followed by the acclimatization of in vitro-grown plants [15,16,17,18]. Among the aforementioned stages, shoot proliferation is considered crucial for successful mass propagation linked to the quantity and quality of the obtained in vitro plant material [19,20].
Prior research on the multiplication of Rubus species has demonstrated that blackberry varieties can be successfully propagated using a wide range of culture media. Among these, the Murashige and Skoog (MS) medium have emerged as the most commonly employed options [21,22,23,24,25], followed by McCown’s Woody Plants medium [4,13,25,26,27], Gamborg’s B5 medium [4,28], Juglans Medium [29,30,31,32], Quoirin–Lepoivre [25,33,34], Anderson medium [25,35,36] and Murashige and Tucker medium [17,37].
The multiplication of blackberry shoots is usually made on media supplemented with cytokinins, in concentrations between 0.3 and 2.0 mg dm−3 as the major plant growth regulator, and with smaller concentrations of auxins, and occasionally with gibberellins [13,31,32]. Thus, the highest values of shoot proliferation rates were reported in the presence of 6-benzylaminopurine (BAP) alone or in combination with indole-3-butyric acid (IBA) and/or gibberellic acid (GA3) [12,14,38,39,40,41,42].
The large-scale gelling agent used for in vitro propagation of blackberry is agar [13,14,43,44]. Given the relatively high cost of agar, several alternatives were tested in the blackberry multiplication phase previously. For example, Gelcarin GP-812, Isubgol, and Guar gum were used for the in vitro multiplication phase of ‘Thornless evergreen’ [31]. The culture media gelled with agents such as wheat starch and corn starch were also successfully tested on the blackberry varieties ‘Navaho’, and ‘Čačanska Bestrna’ [12].
During tissue culture for the large-scale propagation of commercially important plants, somaclonal variations might occur at any stage of the plantlet’s development, especially in the multiplication stage. These variations can be caused by environmental conditions, explant type, the number of successive subcultures, and culture media type [45,46]. First of all, the use of plant growth regulators in high doses, combined with the number of subcultures, causes stress that leads to cellular instability, triggering genetic or epigenetic variations in plants in vitro [47]. Another aspect to consider is the morphogenetic pathway utilized for clone production. Obtaining plants through axillary branching typically does not lead to the generation of variants, whereas cultures that undergo a callus phase are the ones that theoretically promote a higher mutation rate [46,48]. Therefore, it is important to check the genetic uniformity of the multiplied plants in relation to the mother plants to confirm their quality for commercial use [49].
The assessment of genetic fidelity between the micropropagated plantlets and their mother plants using molecular markers such as Sequence-related amplified polymorphism (SRAP) and Start codon targeted polymorphism (SCoT) serve as valuable tools to test the uniformity of plant material [50,51,52,53].
In this context, the first objective of the present study was to investigate the in vitro proliferative capacity of six Rubus fruticosus L. cultivars grown on wheat starch-gelled culture medium in comparison with the same cultivars grown on the classical agar-gelled medium. The second objective of the study was to evaluate the genetic fidelity between shoots propagated in starch-gelled culture medium after twelve successive subcultures and their mother plants using SRAP and SCoT molecular markers.

2. Materials and Methods

2.1. Plant Material and In Vitro Shoot Cultures

The studied blackberry cultivars were ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’ (thornless blackberry), and ‘Karaka Black’ (thorny blackberry), having a tetraploid genetic structure (2n = 4x = 28).
In vitro culture initiation was carried out on annual shoots at the end of October on MS medium supplemented with 3% sucrose and 0.5 mg dm−3 6-benzyladenine (BA), gelled with 0.5% plant agar, and pH = 5.8. The small cuttings were washed with tap water and rinsed with sterile deionized water. Disinfection was made with 20% bleach (ACE Procter and Gamble, București, Romania; <5% active ingredient) for 20 min and rinsed three times with sterile water. The axillary and apical buds were excised and inoculated into the culture medium, one bud/test tube. After six weeks, the regenerated shoots were transferred to a culture medium with the same composition for in vitro culture stabilization. Further, eleven successive subcultures were carried out (each subculture lasting for 10 weeks) on the same agar-gelled culture medium, and in parallel, on culture media with 5% wheat starch.
In this experiment, specifically during the twelfth subculture, the in vitro culture of blackberry was conducted under identical conditions. Two different variants of the culture media were prepared: one was solidified with plant agar at a concentration of 0.5%, while the other utilized wheat starch at a concentration of 5%. The plants were cultured in 720 mL jars with a diameter of 9 cm and a height of 13.5 cm, with screw caps equipped with leukopore tape filters, using 100 mL culture medium/jar.
The culture medium gelled with agar was sterilized for 20 min in an autoclave at 121 °C and 0.11 MPa, while the other one gelled with wheat starch was sterilized for 30 min under the same conditions. The medium gelled with starch needs a longer sterilization period to avoid infections. All the components of the culture media were added prior to sterilization, as well the pH was adjusted to 5.8.
In each jar, four shoot fragments of approx. 2 cm were inoculated.
The in vitro cultures were incubated in the growth room at 16 h photoperiod, 32.4 μmol m−2s−1 light intensity (Philips CorePro LEDtube 1200 mm 16W865 CG, 1600 lm Cool Daylight) at 22 ± 3 °C and 50 ± 5% humidity.
All the necessary components of the culture media were purchased from Duchefa (Biochemie B.V, Haarlem, Netherlands), and the wheat starch was purchased from SanoVita (https://sanovita.ro/, accessed on 15 February 2023).

2.2. Estimation of the Costs per Liter of Culture Medium

The cost per liter of medium was calculated based on the current prices of various types of agar and wheat starch (Table 1).

2.3. Rheological Analyses

Dynamic rheological analyses were carried out on both wheat starch-gelled culture medium and agar-gelled medium using an Anton Paar MCR 72 modular compact rheometer (Anton Paar, Graz, Austria). The rheometer was equipped with a Peltier plate-plate system (P-PTD 200/Air) that allowed temperature control. However, for this study, the samples’ viscosity was measured at room temperature of 22 ± 3 °C. Each sample, approximately 3 mL in volume, was placed between two plates. The upper plate had a smooth parallel plate geometry with a diameter of 50 mm (PP-50-67300), while the lower plate was at a gap of 1 mm [54]. Prior to measurement, any excess sample was removed, and the samples were left undisturbed for 5 min to ensure thermal equilibrium before testing. Duplicate measurements were conducted using a linearly increasing shear rate, ranging from 5 to 300 1/s [55].

2.4. Genetic Fidelity Analysis Using SRAP and SCoT Markers

Each analyzed cultivar was represented by 12 samples obtained from three jars, each containing four initial inoculums. From each inoculum, a shoot was randomly selected, and the leaves were used for DNA isolation. The harvested leaves were dried, ground into a fine powder (TissueLyser II, Qiagen, Hilden, Germany), and stored at 4 °C until DNA isolation.

2.4.1. DNA Isolation

Total genomic DNA was isolated from 0.1 g of dried powder using a protocol based on the CTAB (cetyltrime-thylammonium bromide) method according to Lohdi et al., 1994 [56] and slightly modified by Pop et al., 2003 [57] and Bodea et al., 2016 [58]. The DNA purity and concentration were evaluated using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Prior to performing the polymerase chain reaction (PCR) amplifications, all the DNA samples were diluted to 50 ng/µL using sterile double distilled water.

2.4.2. SCoT and SRAP Analysis

For the SRAP analysis, the PCR amplification reactions were carried out according to Li and Quiros’ (2001) [59] protocol, and the reaction volumes were adjusted to 15 μL. The reaction mixture consisted of 50 ng/μL of gDNA, 5X Green Go Taq flexi buffer, 1.5 mM MgCl2, 0.2 mM of dNTPs, 0.3 µM of both forward and reverse primer, nuclease-free water and 1 U of Taq DNA polymerase (Promega, Madison, WI, USA).
The DNA amplification was carried out in a Gradient thermal cycler, SuperCycler Trinity (Kyratec, Mansfield, Australia), programmed for 1 cycle of 5 min at 94 °C for initial denaturation, followed by five cycles of 1 min of denaturation at 94 °C, 1 min of annealing at 35 °C and 1 min of elongation at 72 °C and then 35 cycles (94 °C for 1 min; 50 °C for 1 min and 72 °C for 1 min) with a final elongation step of 10 min at 72 °C.
For the SCoT analysis, the PCR amplification reactions were performed using the protocol described by Collard and Mackill (2009) [60]. The reaction mixture (a total volume of 15 μL) consisted of 50 ng/μL of gDNA, distilled H2O for the PCR reactions, 5X GoTaq Flexi Green buffer (Promega, Madison, WA, USA), 1.5 mM MgCl2 (Promega, Madison, WA, USA), 0.2 mM of dNTP mix (Promega, Madison, WA, USA), 1 µM SCoT primer (GeneriBiotech, Hradec Králové, Czechia), and 1U of GoTaq polymerase (Promega, Madison, WA, USA). The PCR temperature cycling conditions were: (a) 1 cycle of 5 min at 94 °C for initial denaturation, (b) 35 cycles of denaturation at 94 °C for 1 min, annealing at 50 °C for 1 min and elongation at 72 °C for 2 min, and (c) the final elongation step of 7 min at 72 °C.
The list of SRAP primer combinations and SCoT primers used in this study is shown in Table S1. The PCR amplifications were repeated twice for each SCoT primer and each SRAP primer combination to ensure the reproducibility of the results. Separation of the PCR amplicons for both techniques was performed by electrophoresis on 1.4% agarose gels (Promega, Madison, WA, USA) stained with RedSafeTM Nucleic Acid staining solution (iNtRON Biotech, Seoul, Republic of Korea) in 1X TBE (Tris Borate-EDTA buffer), at 110 V and 136 mA for 2.5–3 h. The electrophoretic profiles were visualized under UV (ultraviolet in UVP Biospectrum AC Imaging System (UVP BioImaging Systems, Upland, CA, USA).

2.5. Data Collection and Statistical Analysis

Data regarding shoot number (SN) and shoot length (SL) were collected after twelve subcultures of the blackberry cultivars, and each subculture’s duration was 10 weeks.
The in vitro experiments were carried out in a completely randomized design (CRD) in factorial with two factors (two gelling agents and six cultivars), and two-way ANOVA was performed to check the differences between the experimental variants. When the null hypothesis was rejected, ANOVA was completed with Duncan’s test (α < 0.05) to separate and highlight the differences between means [61]. The presented values are means ± S.E.
For the SCoT and SRAP analysis, the gel images were analyzed using TotalLab120 software (Nonlinear Dynamics, Newcastle upon Tyne, UK) to determine the number and molecular weight range of amplified bands. The number and size range in base pairs (bp) of the PCR-amplified bands were recorded with the statement that the low intensity of some amplified bands in gels was not considered an eliminative factor while scoring. The genetic distances between analyzed blackberry genotypes were calculated using the Euclidean coefficient of similarity. Cluster analysis was performed with the UPGMA algorithm using PAST software (PAle-ontological STatistics Version 4.11, Natural History Museum, Oslo, Norway) [62]. Its consistency was assessed using the bootstrap method with 10,000 repetitions.

3. Results

3.1. In Vitro Shoot Cultures in Starch-Gelled Culture Media and Agar-Gelled Culture Media

The wheat starch-gelled culture media compared to the classical agar-gelled medium (Figure 1) had a distinct influence on the number of shoots/inoculum and the shoot length for all the studied cultivars, as presented in Figure 2 and Figure 3.
On the agar-gelled culture medium, the highest SN was shown by ‘Čačanska Bestrna’ with 33.92 ± 4.44, followed by ‘Chester Thornless’ (32.42 ± 4.62) and ‘Loch Ness’ (32.08 ± 4.70). A decreased SN was observed in cultivars ‘Driscoll’s Victoria’ (21.42 ± 2.24) and ‘Polar’ (24.25 ± 6.08), as shown in Figure 2
On wheat starch-gelled culture medium, the highest SN was attained by ‘Karaka Black’ (54.42 ± 4.18), followed by ‘Chester Thornless’ (42.58 ± 4.92), as shown in Figure 2. The ‘Polar’ variety had the lowest SN on the culture medium gelled with starch, respectively 26.50 ± 3.71.
Considering the length of the obtained shoots, it can be observed that all of the tested cultivars presented outstanding results on culture medium gelled with 5% wheat starch. The highest values regarding shoots length were observed on the ‘Chester’ cultivar, followed by ‘Čačanska Bestrna’, and ‘Loch Ness’ with values of 5.55 ± 0.08 cm, 5.46 ± 0.07 cm and 5.37 ± 0.09, respectively, as illustrated in Figure 3. On the culture media gelled with agar, the longest shoots were recorded by the ‘Loch Ness’ cultivar (5.11 ± 0.08 cm), statistically significant compared to the other studied varieties (Figure 3).

3.2. Rheological Analyses

The rheological properties of the two samples (wheat starch-gelled culture medium and agar-gelled medium) can be seen in Figure 4, assessed across shear rates ranging from 5 to 300 s−1. This evaluation aimed to determine the relationship between viscosity and shear rate for the samples.
The results showed that the agar-gelled medium samples had a higher viscosity than the wheat starch-gelled medium samples. In the case of the agar-gelled medium samples, the viscosity started from 18,160 ± 29 and decreased to 59 ± 21, while in the case of the wheat starch-gelled medium samples, it started from 3739 ± 28 and decreased to 120 ± 30. Both samples presented a shear-thinning (pseudo-plastic) behavior.

3.3. Genetic Fidelity Evaluation Using SRAP and SCoT Molecular Markers

In the present study, SRAP and SCoT markers were used to assess the genetic uniformity of blackberry shoots proliferated on wheat starch-gelled medium and their mother plants.
SRAP analysis. Ten SRAP primer combinations were used for the genetic fidelity analysis. However, only eight primer combinations produced clear and scorable PCR bands (Table 2).
Each SRAP primer combination generated monomorphic bands that ranged in size from 150 bp (Em7-Me1; Em5-Me4; Em8-Me5) to 2000 bp (Em3-Me8; Em2-Me8; Em8-Me5). The lowest number of monomorphic bands was recorded by ‘Loch Ness’ and ‘Karaka Black’ generated by the Em5-Me4 primer combination, and the highest number of bands was detected in ‘Driscoll’s Victoria’ cultivar with the primer combinations Em4-Me6 and Em8-Me5, as presented in Table 2.
Regarding the total number of monomorphic bands/cultivar, the highest score was detected in ‘Polar’ and ‘Driscoll’s Victoria’ cultivars with 98 PCR bands, and the lowest number of bands (86) was detected in ‘Loch Ness’ cultivar (Table 2).
Despite the fact that no differences were revealed between the mother plants and their in vitro proliferated shoots of each cultivar, the six analyzed cultivars had different SRAP profiles, as shown in Figure 5.
SCoT analysis. From nine SCoT primers used for the initial screening of the genetic fidelity between the mother plants and the proliferated shoots, only seven SCoT primers generated clear and reproducible bands. The number and size range of SCoT amplified bands in the analyzed R. fruticosus L. cultivars are presented in Table 3.
The generated PCR bands ranged in size from 210 bp (SCoT-3) to 2350 bp (SCoT-6). It is noteworthy that no polymorphism was observed between the in vitro proliferated shoots and their mother plants (Figure 6). The number of scorable monomorphic amplified bands varied between 7 and 16. A total of 7 bands were observed for SCoT-2 in samples ‘Čačanska Bestrna’ and 16 bands for SCoT-9 in samples ‘Karaka Black’ and ‘Polar’ (Table 3). Regarding the total number of monomorphic bands generated after the PCR amplifications, the highest number of amplified bands were detected at ‘Karaka Black’ (79), and the lowest number of bands was recorded at ‘Driscoll’s Victoria’ (69) (Table 3).
The results of our study confirm that the SRAP and SCoT markers can be successfully used to rapidly assess the genetic stability of blackberry in vitro grown plants at the proliferation stage. These findings are confirmed by UPGMA cluster analysis. Thus, the constructed dendrograms based on SRAP and SCoT analysis revealed the genetic uniformity between the proliferated shoots and their mother plants. Moreover, the UPGMA cluster analysis showed that there were differences between the genetic profiles of the analyzed blackberry cultivars (Figure 7 and Figure 8).

4. Discussion

Generally, the mass propagation of berry fruit plants can be achieved by using plant tissue culture techniques to produce high-quality plant material characterized by genetic uniformity and free of contamination [63]. Furthermore, micropropagation strategies aim to achieve two goals simultaneously: to increase the proliferation rate of the targeted plant material and to reduce production costs at the same time. Replacing agar, the most expensive component of the culture medium, with alternative low-priced gelling agents can lead to reduced production costs of the in vitro cultures [64].
In this context, the outcomes of our study demonstrated two positive aspects when replacing agar with wheat starch in the in vitro multiplication stage of six blackberry cultivars: (1) reduction in cost per liter of culture medium and (2) increased proliferation rate and shoot length. According to the data presented in Table 3, substituting agar with wheat starch can lead to a cost reduction of up to 2.45 euros per liter of culture medium. In our case, replacing plant agar (0.5%) with 5% wheat starch reduced the cost of one liter of the medium by 0.34 euro.
Aside from cost reduction, achieving the appropriate hardness of the medium is crucial for successful plant tissue cultures. If the medium is excessively soft, it can lead to hyperhydricity in tissue cultures, while if it is excessively hard, it can impede shoot development. [64].
In our study, the viscosity of the culture medium gelled with 5% wheat starch was almost five times lower than that of the medium gelled with 0.5% plant agar.
The distinctive structural properties of agar molecules and their interactions within the culture medium account for the variation in viscosity and shear-thinning behavior. Conversely, in the case of starch, these characteristics can be attributed to the molecular structure and interactions within the substrate [65].
Agar is a polysaccharide derived from seaweed, consisting of long chains of repeating sugar units. In its natural state, agar forms a gel-like structure due to the formation of physical cross-links between the agar molecules. These cross-links contribute to the viscosity and elasticity of the agar gel [66]. When shear is applied to the agar-based substrate, such as during stirring or flow, the shearing forces disrupt the physical cross-links between the agar molecules. This results in the temporary reduction of viscosity, allowing the substrate to flow more easily. As the shearing forces increase, the structural network of agar molecules continues to break down, leading to a further reduction in viscosity [67].
Starch is a complex carbohydrate composed of glucose units organized into two main types of polymers: amylose and amylopectin. The amylose component consists of linear chains, while amylopectin has a branched structure [68]. These molecular arrangements contribute to the unique rheological properties of starch-based substrates. When subjected to shear stress, the starch molecules undergo various changes that result in shear thinning behavior, like molecular alignment, disruption of granule structure, and solvent interactions [69].
Our results show that the lower viscosity of the wheat starch-gelled medium caused the inocula to sink into the culture medium as they developed and became heavier. Consequently, the proliferated shoots had a larger surface in contact with the culture medium, which could explain their better development in starch-gelled media compared to agar-gelled media. This phenomenon was observed in a study conducted by Jain and Babbar (2002) [70] when katira gum was used as a gelling agent in culture media for in vitro shoot formation and rooting at Syzygium cuminii. In this case, the viscosity of the culture medium gelled with katira gum was less than one-sixth of the viscosity of the medium gelled with agar, and frequent shaking of the culture vessel often caused the explants to sink, with submerged explants showing a positive response.
Similar results were obtained in a previous study conducted by Amlesom et al. in 2021 [71], who evaluated the efficacy of three types of starch (corn, potato, and barley) in both laboratory and commercial grades as alternatives to agar for potato micropropagation. In terms of physical parameters, including plant height, root length, fresh weight, and dry weight, the media containing laboratory-grade potato starch, commercial corn starch, and laboratory-grade corn starch yielded superior results compared to the control medium gelled with agar. Both laboratory and commercial-grade starch-based media led to cost reductions of 15–22% and 61–66%, respectively. These findings indicate that both corn and potato starches can be considered reliable and cost-effective substituents for agar in potato micropropagation. In a recent study, plantain and banana explants were propagated on 16 starch-based substrates (mung bean, sago, xanthan, Isabgol, guar gum, pear sago, cassava starch, tapioca starch and their combinations with agar) to evaluate their suitability as tissue culture gelling agents [72]. Two of the substrates, mung bean and Isabgol, had suitable gelling properties and cost less than one euro, and were more economical than agar.
In the present study, the type of gelling agent had a significant effect on the proliferation rate and shoot length in all six blackberry cultivars, as shown in Figure 2 and Figure 3. The highest proliferation rate was obtained in the culture medium gelled with wheat starch in all blackberry cultivars. In this regard, the ‘Karaka Black’ cultivar exhibited the highest proliferation rate, nearly twice as high as that on the agar-gelled medium. The smallest differences in proliferation rate were observed in the ‘Polar’ variety, with only a 1.09-fold increase in the starch-gelled medium compared to the agar-gelled medium. Also, even if the consistency of the culture media gelled with wheat starch was softer, no hyperhydrated cultures or other physiological disorders were observed.
Since the culture medium gelled with 5% wheat starch is opaque, possible endogenous bacterial contamination cannot be observed. For this reason, this medium is indicated to be used only in the in vitro multiplication stage of blackberry. Our previous investigations regarding in vitro multiplication of Rubus sp. showed that the culture medium, solidified with different alternative gelling agents, including wheat starch and potato starch, generated high proliferation rates and vigorous shoots suitable for concomitant acclimation and ex vitro rooting to different blackberry varieties: ‘Thornless Evergreen’ [33], ‘Loch Ness’, ‘Čačanska bestrna’, ‘Chester Thornless’, and ‘Navaho’ [12].
Previous research results show that the assessment of genetic fidelity is one of the most important requirements in tissue culture-based propagation of any fruit plant species [50] due to the possible presence of somaclonal variations. Chromosomal rearrangements are an important source of somaclonal variations that can occur between the in vitro plants regenerated from one parental line as a result of the successive subculture of plant organs, tissues, and cells [46]. In this particular context, molecular marker techniques have emerged as valuable tools for two purposes: verifying the genetic fidelity of in vitro propagated plants in comparison to their mother plants and confirming their uniformity for commercial applications [73,74,75,76].
During the last decades, several research articles have been published regarding the evaluation of the genetic uniformity of in vitro propagated plants by SRAP markers on different fruit species like Vitis vinifera L. [77], Musa sp. [78], Rubus fruticosus L. [50], Ribes grossularia L. [79], Aronia melanocarpa (Michx.) [80]. The results of these studies are consistent with the findings of the present research, showing that SRAP markers were adequate to assess the genetic fidelity of the proliferated shoots after 12 consecutive subcultures. The experimental design included the genetic fidelity analysis for the in vitro proliferated shoots in the starch-gelled medium and confirmed the uniformity of the proliferated shoots with their mother plant, as illustrated by the DNA fingerprinting profiles (Figure 6).
It is worth mentioning that the SRAP technique is an easily accessible tool for different laboratories to assess the genetic fidelity of in vitro grown plant material, and the reproducibility of the results is satisfactory [79].
In the present study, SCoT markers were used to confirm the SRAP results, given that this type of DNA-based molecular marker is highly reproducible. SCoT technique amplifies the short, conserved regions around the ATG translation start codon located in plant genes, and previous studies confirm the complementary validation of the two techniques regarding genetic fidelity on other micropropagated plant species [51]. Unlike RAPD or ISSR markers that amplify DNA fragments from non-coding regions of the genome, the advantage of using SCoT markers is associated with functional genes and their corresponding traits [81,82,83]. The results presented in Figure 7 confirm the genetic fidelity of the in vitro grown shoots after 12 concomitant subcultures and their mother plant and represents a complementary validation of the SRAP results at the same time. To the best of our knowledge, this study is the first report on blackberry that applies SCoT markers to assess the genetic uniformity of the in vitro propagated shoots.
Furthermore, the results of this study show that both the SRAP and SCoT marker systems are useful for cultivar differentiation at the DNA molecular level.

5. Conclusions

In conclusion, our study on in vitro shoot proliferation of six blackberry cultivars (‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’) indicates that wheat starch is an efficient gelling agent. The use of wheat starch for agar replacement in blackberry culture media resulted in a cost reduction of 0.34 euro per liter (compared to plant agar). Additionally, it promoted a greater number of shoots, longer and physiologically suitable for acclimation. The genetic uniformity of the micropropagated shoots with their mother plants was confirmed by SRAP and SCoT molecular markers, thus, revealing the sustainability of the wheat starch as an effective gelling agent for the production of clonal blackberry planting material.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9080902/s1, Table S1: Sequences of SCoT primers and SRAP primer combinations used in this study.

Author Contributions

Conceptualization, D.C., K.S. and M.H.; methodology, D.C. and M.H.; software, D.C. and M.H.; validation, D.P.; investigation, D.C., M.H., B.-E.T. and K.S.; resources, D.C.; data curation, D.C.; writing—original draft preparation, D.C., M.H. and B.-E.T.; writing—review and editing, K.S. and D.P.; supervision, D.P.; project administration, D.C.; funding acquisition, D.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Research Development Projects to finance Excellence (PFE)—14/2022-2024, granted by the Romanian Ministry of Research and Innovation.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. In vitro cultures of blackberry, cultivated on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 5% wheat starch (left-sided jar) and 0.5% plant agar (right-sided jar): (a) ‘Chester Thornless’; (b) ‘Čačanska Bestrna’; (c) ‘Loch Ness’; (d) ‘Polar’; (e) ‘Driscoll’s Victoria’; and (f) ‘Karaka Black’.
Figure 1. In vitro cultures of blackberry, cultivated on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 5% wheat starch (left-sided jar) and 0.5% plant agar (right-sided jar): (a) ‘Chester Thornless’; (b) ‘Čačanska Bestrna’; (c) ‘Loch Ness’; (d) ‘Polar’; (e) ‘Driscoll’s Victoria’; and (f) ‘Karaka Black’.
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Figure 2. The number of shoots/inoculum of the in vitro blackberry cultivars ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’ grown on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 0.5% plant agar (agar), and 5% wheat starch (starch). Different lowercase letters above the bars indicate significant differences between the means of the proliferation rate among cultivars, according to Duncan’s test (α < 0.05).
Figure 2. The number of shoots/inoculum of the in vitro blackberry cultivars ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’ grown on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 0.5% plant agar (agar), and 5% wheat starch (starch). Different lowercase letters above the bars indicate significant differences between the means of the proliferation rate among cultivars, according to Duncan’s test (α < 0.05).
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Figure 3. Shoot length of the in vitro blackberry cultivars ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’ grown on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 0.5% plant agar (agar), and 5% wheat starch (starch). Different lowercase letters above the bars indicate significant differences between the means of the proliferation rate among cultivars, according to Duncan’s test (α < 0.05).
Figure 3. Shoot length of the in vitro blackberry cultivars ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’ grown on MS medium supplemented with 0.5 mg dm−3 BA, gelled with 0.5% plant agar (agar), and 5% wheat starch (starch). Different lowercase letters above the bars indicate significant differences between the means of the proliferation rate among cultivars, according to Duncan’s test (α < 0.05).
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Figure 4. The viscosity of wheat starch-gelled medium (yellow) and agar-gelled medium (blue) samples (in duplicates).
Figure 4. The viscosity of wheat starch-gelled medium (yellow) and agar-gelled medium (blue) samples (in duplicates).
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Figure 5. The monomorphic SRAP profiles of mother plants (M1–M6) from each of the six Rubus fruticosus L. cultivars and their in vitro proliferated shoots (1–12) generated by the primer combination Me4-Em5; (a) ‘Čačanska Bestrna’, ‘Chester Thornless’; (b) ‘Loch Ness’, ‘Polar’; (c) ‘Driscoll’s Victoria’, ‘Karaka Black’. Lane L—indicates the molecular marker (100 bp Ladder, Promega, USA); NC—sample controls without DNA.
Figure 5. The monomorphic SRAP profiles of mother plants (M1–M6) from each of the six Rubus fruticosus L. cultivars and their in vitro proliferated shoots (1–12) generated by the primer combination Me4-Em5; (a) ‘Čačanska Bestrna’, ‘Chester Thornless’; (b) ‘Loch Ness’, ‘Polar’; (c) ‘Driscoll’s Victoria’, ‘Karaka Black’. Lane L—indicates the molecular marker (100 bp Ladder, Promega, USA); NC—sample controls without DNA.
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Figure 6. The monomorphic SCoT profiles of mother plants (M1–M6) from each of the six Rubus fruticosus L. cultivars and their in vitro proliferated shoots (1–12) generated by the primer SCoT 9, (a) ‘Čačanska Bestrna’, ‘Chester Thornless’, (b) ‘Loch Ness’, ‘Polar’ (c) ‘Driscoll’s Victoria’, ‘Karaka Black’. Lane L—indicates the molecular marker (100 bp Ladder, Promega, USA); NC—sample controls without DNA.
Figure 6. The monomorphic SCoT profiles of mother plants (M1–M6) from each of the six Rubus fruticosus L. cultivars and their in vitro proliferated shoots (1–12) generated by the primer SCoT 9, (a) ‘Čačanska Bestrna’, ‘Chester Thornless’, (b) ‘Loch Ness’, ‘Polar’ (c) ‘Driscoll’s Victoria’, ‘Karaka Black’. Lane L—indicates the molecular marker (100 bp Ladder, Promega, USA); NC—sample controls without DNA.
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Figure 7. UPGMA dendrogram based on SRAP analysis, showing the genetic fidelity between in vitro proliferated shoots from the analyzed cultivars (M1.1–M1.12; M2.1–M2.12; M3.1–M3.12; M4.1–M4.12; M5.1–M5.12; M6.1–M6.12) and their mother plants (M1–M6). Numbers on the branches show bootstrap values computed from 10,000 replications. M1—‘Čačanska Bestrna’; M2—Chester Thornless’; M3—‘Loch Ness’; M4—‘Polar’; M5—‘Driscoll’s Victoria’; M6—‘Karaka Black’.
Figure 7. UPGMA dendrogram based on SRAP analysis, showing the genetic fidelity between in vitro proliferated shoots from the analyzed cultivars (M1.1–M1.12; M2.1–M2.12; M3.1–M3.12; M4.1–M4.12; M5.1–M5.12; M6.1–M6.12) and their mother plants (M1–M6). Numbers on the branches show bootstrap values computed from 10,000 replications. M1—‘Čačanska Bestrna’; M2—Chester Thornless’; M3—‘Loch Ness’; M4—‘Polar’; M5—‘Driscoll’s Victoria’; M6—‘Karaka Black’.
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Figure 8. UPGMA dendrogram based on SCoT analysis, showing the genetic fidelity between in vitro proliferated shoots from the analyzed cultivars (M1.1–M1.12; M2.1–M2.12; M3.1–M3.12; M4.1–M4.12; M5.1–M5.12; M6.1–M6.12) and their mother plants (M1–M6). Numbers on the branches show bootstrap values computed from 10,000 replications. M1—‘Čačanska Bestrna’; M2—‘Chester Thornless’; M3—‘Loch Ness’; M4—‘Polar’; M5—‘Driscoll’s Victoria’; M6—‘Karaka Black’.
Figure 8. UPGMA dendrogram based on SCoT analysis, showing the genetic fidelity between in vitro proliferated shoots from the analyzed cultivars (M1.1–M1.12; M2.1–M2.12; M3.1–M3.12; M4.1–M4.12; M5.1–M5.12; M6.1–M6.12) and their mother plants (M1–M6). Numbers on the branches show bootstrap values computed from 10,000 replications. M1—‘Čačanska Bestrna’; M2—‘Chester Thornless’; M3—‘Loch Ness’; M4—‘Polar’; M5—‘Driscoll’s Victoria’; M6—‘Karaka Black’.
Horticulturae 09 00902 g008
Table 1. Comparing the costs of various gelling agents per liter of culture medium.
Table 1. Comparing the costs of various gelling agents per liter of culture medium.
Gelling AgentPrice/kg
(Euro)		g/L Culture MediumPrice/L Culture Media (Euro)Price Difference (Euro)
Wheat starch SANOVITA (used in the present study)2500.10-
Plant agar * (used in the present studies) 87.450.440.34
Daishin agar *206.571.451.35
Micro agar *111.760.670.55
Phyto agar *10160.610.51
Gelrite *174.430.520.42
Agar Sigma A1296 **4385.62.452.35
Catalog prices * Duchefa and ** Sigma.
Table 2. Number and size range of SRAP amplified bands in the analyzed R. fruticosus L. cultivars.
Table 2. Number and size range of SRAP amplified bands in the analyzed R. fruticosus L. cultivars.
Primer
Combinations		Size Range of
Bands (bp)		No. of Scorable Monomorphic Bands
Čačanska BestrnaChester ThornlessLoch NessPolarDriscoll’s VictoriaKaraka Black
Em4-Me6180–1450121012151613
Em7-Me1150–1350141511101011
Em3-Me8200–2000111211131210
Em5-Me4150–18001011810128
Em2-Me8300–200012910121011
Em8-Me5150–2000151513151614
Em1-Me7200–1900101012111013
Em6-Me3200–170011119121210
Total no. of bands/cultivar959386989890
Table 3. Number and size range of SCoT amplified bands in the analyzed R. fruticosus L. cultivars.
Table 3. Number and size range of SCoT amplified bands in the analyzed R. fruticosus L. cultivars.
Primer NameSize Range of
Bands (bp)		No. of Scorable Monomorphic Bands
Čačanska BestrnaChester ThornlessLoch NessPolarDriscoll’s VictoriaKaraka Black
SCoT-1250–1900810811119
SCoT-2320–2100799889
SCoT-3210–2200111197911
SCoT-4300–230089119812
SCoT-5250–1800131213111211
SCoT-6280–2350121311131211
SCoT-9220–190015141016916
Total no. of bands/cultivar747871756979
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Clapa, D.; Hârța, M.; Szabo, K.; Teleky, B.-E.; Pamfil, D. The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures. Horticulturae 2023, 9, 902. https://doi.org/10.3390/horticulturae9080902

AMA Style

Clapa D, Hârța M, Szabo K, Teleky B-E, Pamfil D. The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures. Horticulturae. 2023; 9(8):902. https://doi.org/10.3390/horticulturae9080902

Chicago/Turabian Style

Clapa, Doina, Monica Hârța, Katalin Szabo, Bernadette-Emőke Teleky, and Doru Pamfil. 2023. "The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures" Horticulturae 9, no. 8: 902. https://doi.org/10.3390/horticulturae9080902

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