Over Half a Century of Research on Blackberry Micropropagation: A Comprehensive Review

Abstract

Micropropagation of blackberry (Rubus spp.) has emerged as a key technique for large-scale production of genetically uniform, disease-free plants. This review summarizes more than half a century of in vitro blackberry culture research, covering fundamental aspects such as establishment, proliferation, rooting, acclimation, genetic stability and conservation. Optimization of culture media, plant growth regulators and environmental conditions has significantly improved the efficiency of micropropagation. Recent advances, including bioreactors, cryopreservation and biostimulants, have further improved plant growth and stress tolerance. In addition, studies on bioactive compounds in micropropagated blackberries highlight their potential nutritional and pharmaceutical applications. Despite progress, challenges such as microbial contamination, somaclonal variation, and response variability among cultivars remain critical areas for future research. The integration of nanotechnology, alternative culture systems (i.e., bioreactors), synthetic seed technology should represent the future research trend of blackberry micropropagation, ensuring sustainable production and conservation of genetic resources.

1. Introduction

In vitro propagation has become the dominant method for blackberry propagation [1,2,3,4]. Traditionally, blackberries have been propagated vegetatively mainly via layering and cuttings [2,5] but these methods are limited by their need for extensive plantation areas, high labor inputs, and intensive weed management [3]. In vitro propagation enables large-scale production of genetically uniform and disease-free plants, crucial aspects for commercial production and germplasm conservation [6,7,8,9].

Micropropagation protocols have been established for various blackberry cultivars but the diverse genetic background of the Rubus genus (comprising over 740 species) leads to variable micropropagation responses [2]. Therefore, optimizing culture media, nutrient composition, and plant growth regulator combinations remains crucial for the success of the micropropagation technique [8,10]. In particular, PGRs such as auxins and cytokinins influence shoot proliferation, root development, and overall plant growth [11].

First attempts to micropropagate blackberries were conducted by Broome [12] and Harper [13]. First experiment with ‘Bedford Giant’ blackberry and the ‘Tayberry’ hybrid were hindered by low multiplication index, limiting the success of the micropropagation application [2]. Despite these challenges, the micropropagation protocols for blackberries and hybrid berries have undergone significant refinement. Early research on blackberry micropropagation primarily aimed at rapid multiplication, disease elimination, and the improvement of asexually propagated varieties [2,14]. This technique has now become the preferred method for large-scale plant production in a short time frame, playing a crucial role in commercial blackberry production [2].

2. Materials and Methods

Search Strategy and Literature Handling

To perform a detailed literature review of blackberry micropropagation, a systematic search strategy was carried out using two major electronic databases: Scopus and Web of Science (WoS). The review included studies published up to January 2025. Additional relevant articles were included from Google Scholar to integrate the results.

To refine the search specifically on blackberry micropropagation, the keywords “micropropagation” and “blackberry” were used in combination in addition to the terms “in vitro culture” and “blackberry”. From the results retrieved, only scientific articles published in English were considered, studies that were not pertinent to the review were excluded. These studies appeared in the search results but did not specifically address the micropropagation of blackberry. After removing duplicates, the remaining full texts were assessed for eligibility based on predefined inclusion and exclusion criteria (e.g., study type, relevance to the review objectives, methodological quality). Selected articles were then categorized according to thematic areas emerging from the literature (e.g., establishment, multiplication, rooting, acclimatization, control of bacterial contamination, callus induction). Key information was extracted from each study, including objectives, methods, main findings, and implications. A narrative synthesis was conducted to integrate results and identify trends, gaps, and areas for future research.

3. Results

3.1. Establishment

The establishment phase of in vitro blackberry culture was mainly conducted using nodal segments [8,14,15,16,17,18,19,20], buds [11,21,22,23,24,25,26,27,28,29,30,31] or shoot tips [22,32,33,34,35,36] as explants. Murashige and Skoog medium (MS) [37] medium have been the most commonly used substrate [9,11,14,15,16,17,18,19,20,22,23,25,26,27,28,30,32,33,34,36,38,39], although with some modifications (Figure 1). For example, Isac et al. [24] used MS medium supplemented with Linsmaier and Skoog (LS) vitamins [40], while Ahmed and Abd Elaziem [34] compared MS, Murashige and Tucker (1969) (MT) [41] and Woody Plant Medium (WPM) [42]. In addition, Fathy et al. [39] evaluated MS together with WPM and Gamborg B5 (B5) medium [43]. The establishment phase lasts, for most of the cited studied, four weeks. Growth regulators applied include 6-Benzylaminopurine (BAP) used at concentrations ranging from 0.05 to 6 mg L⁻¹ [8,9,11,14,15,16,18,19,20,21,24,25,26,27,28,30,31,33,36,38]; Indole-3-Butyric Acid (IBA) used at 0.05 to 0.5 mg L⁻¹ [9,18,20,25,26,27,38]; Indole-3-Acetic Acid (IAA) used at 0.3 mg L⁻¹ [38]; Gibberellic Acid 3 (GA₃) used at 0.1 to 0.5 mg L⁻¹ [24,25,26,27,32,38] and Naphthalene Acetic Acid (NAA) used at concentrations ranging from 0.001 to 0.5 mg L⁻¹ [16,24] (Figure 1).

Table 1. Summary of the establishment phase for blackberry, detailing the basal media, cultivar, explant type, plant growth regulators (PGRs) concentration, success of establishing a sterile culture, and corresponding references.

Basal Media	Cultivar	Explant Type	PGRs (mg L−1 or Other UoM Where Specified)	Success of Establishing a Sterile Culture	References
MS	‘Black Satin’ ‘Loch Ness’	Single-node explant	2 BAP, 0.2 IBA	Not available (N/A)	[9]
Modified MS	‘Loch Ness’ ‘Chester Thornless’	Axillary and terminal bud	0.5 BAP	N/A	[21]
MS (M1) ½ MS with 8 mL Fe EDTA (M2) MS with ¼ of nitrates, 2x strength Fe salts (M3) MS with 2x strength Fe ions (M4)	‘Agavam’ (A) ‘Thornfree’ (T) ‘Darrow’ (D)	Meristem tip	(a) 1 BAP (M1) (b) 1 BAP, 0.3 IAA (M1) (c) 0.2 BAP, 0.2 IBA (M2) (d) 2 BAP, 0.05 IBA, 0.1 GA3 (M3) (e) 1 IBA (M3) (f) 1 BAP, 0.3 IAA, 0.2 GA3 (M4)	100% D (c,d)	[38]
MS WPM B5	N/A	Nodal explant	N/A	91.67% MS 91.67% WPM 33.33% B5	[39]
MS	‘Chester’	Dormant bud and cane Shoot	N/A	N/A	[22]
WPM	‘Chester Thornless’	Nodal segment	2 BAP	N/A	[8]
MS	N/A	Shoot	2 BAP	N/A	[36]
MS	‘Black Satin’	Nodal segment	6 BAP, 0.2 IBA	Almost 100%	[20]
MS	N/A	Segment with an axillary bud	N/A	N/A	[23]
MS	‘Čačanska bestrna’ ‘Chester Thornless’ ‘Driscoll’s Victoria’ ‘Loch Ness’ ‘Polar’ ‘Karaka Black’	Axillary and apical bud	0.5 BAP	N/A	[11]
MS	‘Brazos’	Nodal segment	5 μM BAP	N/A	[19]
MS with LS vitamins	‘Darrow’ ‘DAR 24’ ‘DAR 8’	Axillary bud	0.3 BAP, 0.1 GA3, 0.001 NAA	63.77%	[24]
MS	‘Čačanska bestrna’	Bud	2 BAP, 0.5 IBA, 0.1 GA3	57.2%	[25]
½ MS (liquid)	‘Navaho’ APF selections	Shoot tip	N/A	N/A	[35]
MS	‘Smoothstem’	Nodal segment	4 mM BAP, 0.25 mM IBA	N/A	[18]
MS	‘Čačanska bestrna’	Bud	2 BAP, 0.5 IBA, 0.1 GA3	N/A	[26,27]
MS ½ MS ¾ MS	‘Xavante’	Nodal segment	N/A	0% ½ MS (whitout light) 66.7% MS (whitout light) 91.7% ¾ MS (whitout light) 37.5% ½ MS (with light) 33.3% ¾ MS (with light) 54.2% MS (with light)	[17]
MS	‘Loch Ness’ (LN) ‘Smoothstem’ (S) ‘Thornless evergreen’ (TE)	Axillary and apical bud	0.7 BAP	75% LN	[28]
MS MT WPM	N/A	Shoot tip	N/A	88.33% (MS) 95% (MT) 100% (WPM)	[34]
MS	‘Čačanska bestrna’ (CB) ‘No. 7’	Shoot	1.5 BAP	100% CB 81.8% No. 7	[33]
MS	‘Chester’	Shoot tip	0.5 GA3	N/A	[32]
½ MS	‘Merton’ ‘Thornless’	Single bud	N/A	N/A	[29]
MS	‘Tupy’	Apical bud	0.05 BAP	N/A	[30]
Semi-saline MS	‘Smoothstem’ ‘Triple Crown’ ‘Karaka Black’	Apical and lateral stem bud Etiolated root bud	0.5 BAP	N/A	[31]
MS	N/A	Nodal segment	2 BAP, 0.5 NAA	N/A	[16]
MS	‘Black Satin’	Nodal segment	1 BAP	N/A	[14]
MS	‘Loch Ness’ ‘Loch Tay’	Nodal cutting	0.5 BAP	N/A	[15]

MS—Murashige and Skoog (1962) [37]; LS Linsmaier and Skoog (1965) [40]; MT—Murashige and Tucker (1969) [41]; WPM—Woody Plant Medium [42]; B5—Gamborg B5 (1986) [43]; EDTA Ethylenediaminetetraacetic acid; BAP 6-Benzylaminopurine; IBA Indole-3-Butyric Acid; IAA Indole-3-Acetic Acid; GA₃ Gibberellic Acid 3; NAA Naphthalene Acetic Acid.

provides a detailed summary of the establishment phase for the selected articles.

3.2. Multiplication

The in vitro multiplication stage of blackberry is mainly conducted using shoot explants [1,8,9,11,18,20,21,22,24,25,26,28,33,34,35,36,38,39,44,45,46,47,48,49,50,51,52], with MS medium most frequently used [1,3,9,11,18,20,21,22,23,24,25,26,28,33,34,35,36,38,39,45,46,47,48,49,50,51,52,53] (Figure 2). However, several studies have introduced modifications to the standard protocol. For example, Lepse and Laugale [38] compared different formulations of MS, including MS (full strength), MS (double strenght with half strength of nitrate), MS (full strength with Fe-EDTA) and MS (half × strength with Fe-EDTA). Similarly, Clapa et al. [11] investigated the effects of MS medium supplemented with 0.5% agar compared with MS medium containing 5% wheat starch. In addition, the use of half MS medium was considered in the studies of [24,44,52].

Variations in MS concentration have been shown to influence shoot multiplication, with some cultivars, such as ‘Black Satin’, responding positively to a doubled concentration, while others, such as ‘Loch Ness’, exhibit optimal growth at the standard concentration [9]. Furthermore, plantlets grown in a double-phase MS medium supplemented with 5 μM BAP have demonstrated increased shoot proliferation, whereas lower concentrations, such as 1.1 μM IBA, have been shown to improve rooting in cultivars like ‘Tupy’ and ‘Ébano’ [53].

The duration of subcultures varied significantly among the studies, from a minimum of 20 days [25] to a maximum of 3 months [24,28].

A wide range of growth regulators have been applied in blackberry micropropagation, with BAP being the most frequently applied cytokinin, at concentrations ranging from 0.4 to 10 mg L⁻¹ [1,3,8,9,11,18,20,21,22,23,24,25,26,28,33,34,35,36,38,39,44,45,46,47,48,49,50,51,52,53] (Figure 2). Other regulators include IBA used at 0.1 to 1.2 mg L⁻¹ [8,9,18,20,25,26,33,35,38,44,48,51]; GA₃ in the range of 0.1 to 1 mg L⁻¹ [1,8,9,18,24,25,26,33,35,36,48,51]; NAA from 0.001 to 0.5 mg L⁻¹ [1,8,22,24,25,35,39]; IAA at concentrations between 0.25 and 1 mg L⁻¹ [38,47,52]; 2-Isopentenyladenine (2iP) used at 0.25 to 2 mg L⁻¹ [34] and Thidiazuron (TDZ) also ranging from 0.25 to 2 mg L⁻¹ [34]. Each of these regulators contributes to shoot proliferation, root induction, or overall plant development depending on their concentration and the cultivar involved.

Table 2. Summary of the multiplication phase for blackberry, detailing the basal media, cultivar, explant type, plant growth regulators (PGRs) concentration, multiplication coefficient, comments and corresponding references.

Basal Media	Cultivar	Explant Type	PGRs (mg L−1 or Other UoM Where Specified)	Multiplication Coefficient	Comments	References
MS	‘Čačanska bestrna’ (CB) ‘No. 7’	Shoot	1, 2 or 4 BAP, or combination of 1 BAP, 0.1 IBA and 0.1 GA3	6.3 ± 0.5 CB (4 mg L−1 BAP) 4.2 ± 0.4 No. 7 (1 mg L−1 BAP, 0.1 mg L−1 IBA, 0.1 mg L−1 GA3)	N/A	[33]
MS	‘Black Satin’	Shoot	(a) 0.5 BAP, 0.2 IBA (b) 1 BAP, 0.2 IBA (c) 1.5 BAP, 0.2 IBA (d) 2 BAP, 0.2 IBA	(a) 4.10 with (b) 5.29 with (c) 5.30 with (d) 5.86 with	N/A	[20]
MS (0.5% agar) MS (5% wheat starch)	‘Čačanska bestrna’ (CB) ‘Chester Thornless’ (CT) ‘Driscoll’s Victoria’ (DV) ‘Loch Ness’ (LN) ‘Polar’ (P) ‘Karaka Black’ (KB)	Shoot	0.5 BAP	33.92 ± 4.44 CB (agar) 32.42 ± 4.62 CT (agar) 32.08 ± 4.70 LN (agar) 21.42 ± 2.24 DV (agar) 24.25 ± 6.08 P (agar) 54.42 ± 4.18 KB (starch) 42.58 ± 4.92 CT (starch) 26.50 ± 3.71 P (starch)	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’.	[11]
MS	‘Navaho’ APF selections	Microshoot	0.1 IBA 0.09 NAA	N/A	Incorporating IBA into a proliferation medium induced better microshoot proliferation than NAA.	[35]
MS ½ MSB Anderson WPM	‘Navaho’ APF selections	Microshoot	BAP, IBA, GA3 (1/2 MSB, Anderson, WPM) BAP, NAA, GA3 (MS)	N/A	The full-strength MS formulation was reported to be the best.	[35]
MS	‘Navaho’ APF selections	Shoot tip	(1) 0.1 GA3, 0.1 IBA (2) 1 BAP (3) 2 BAP (4) 4 BAP (5) 10 BAP (6) 1 BAP, 0.1 IBA, 0.1 GA3 (7) 2 BAP, 0.1 IBA, 0.1 GA3 (8) 4 BAP, 0.1 IBA, 0.1 GA3 (9) 10 BAP, 0.1 IBA, 0.1 GA3	N/A	The best microshoot proliferation across genotypes was observed at 4.0 mg L−1 of BA without IBA and GA3.	[35]
½ MS	‘Thornfree’ ‘Chester’	Shoot	0.1 IBA, 0.4 BAP	N/A	The obtained shoots were used for encapsulation	[44]
MS Ly de Fossard Gamborg	‘Thornfree’ (T) ‘Agawam’ (A) ‘Black Satin’ (BS) ‘Eri’ (E)	Shoot	1 BAP	5.0 ± 0.4 (T) 4.8 ± 0.4 (A) 6.1 ± 0.5 (BS) 5.2 ± 0.4 (E)	The reported results refer to the Ly de Fossard substrate, which proved to be the best.	[45]
MS solid semi-solid and liquid (TIS)	‘Čačanska bestrna’ (CB) ‘Chester Thornless’ (CT) ‘Driscoll’s Victoria’ (DV) ‘Loch Ness’ (LN) ‘Polar’ (P) ‘Karaka Black’ (KB)	Shoot	0.5 BAP	42.27 ± 4.79 LN (solid MS) 19.53 ± 4.86 DV (solid MS) 21.13 ± 3.95 CT (solid MS) 52.93 ± 2.51 LN (semi-solid MS) 48.32 ± 2.49 KB (semi-solid MS) 47.22 ± 2.13 CB (semi-solid MS) 93.90 ± 4.01 KB (TIS)	The most suitable culture system was semi-solid medium.	[46]
MS	N/A	Shoot	IAA (0.5, 0.75, 1) with BAP (2, 3)	N/A	The best results was obtained at a concentration of 2 mg L−1 BAP and 0.75 mg L−1 IAA	[47]
MS	‘Čačanska bestrna’	Encapsulated shoot	1 BAP, 0.1 IBA, 0.1 GA3	2.56	The multiplication index reported is the average of the nine subcultures.	[48]
MS	‘Black Satin’ (BS) ‘Loch Ness’ (LN)	Shoot	1 BAP, 0.5 IBA, 0.1 GA3	2.78 2nd sub vs. 2.07 1st sub (LN) 4.49 2nd sub vs. 2.89 1st sub (BS)	N/A	[9]
MS (50 g L−1 wheat starch)	‘Loch Ness’ ‘Chester Thornless’	Mini-shoot	0.5 BAP	>40%	N/A	[21]
MS	‘Guarani’ (G) ‘Caingangue’ (C) ‘Ébano’ (E) ‘Xavante’ (X)	Nodal segment	1 BAP	17.1 (G) 14.0 (C) 17.3 (E) 10.2 (X)	N/A	[3]
MS (M1) MS (2x strength, with ½ strength nitrates) (M2)	‘Agavam’	Shoot	0.5 BAP, 0.25 IAA 1 BAP	2.9 (M1) 3.1 (M2)	N/A	[38]
MS (M1) MS (2x strength, with ½ strength nitrates) (M2) MS (with Fe EDTA) (M3) MS (½ strength with Fe EDTA) (M4)	‘Agavam’ (A) ‘Thornfree’ (T) ‘Darrow’ (D)	Shoot	0.5 BAP, 0.25 IAA (M1) 1 BAP (M2) 2 BAP, 1 IBA (M3a) 1 BAP, 0.05 IBA (M3b) 1.2 IBA (M4)	1 A (M3a) 0.0 T, D (M3a) 2.7 A (M3b) 4 T (M3b) 2.5 D (M3b) 0.0 A,T, D (M4) 2 A, D (M1) 3.6 T (M1) 4 T (M2)	N/A	[38]
MS	‘Chester’	Shoot	(a) 2 BAP (b) 2 BAP, 0.1 NAA (c) 2 BAP, 0.5 NAA (d) 0.7 BAP (e) 1.4 BAP	5.2 (e)	The concentration of NAA seemed to had no effect on shoot multiplication rate.	[22]
MS	N/A	Shoot	(a) 0.2 BAP (b) 0.4 BAP (c) 0.6 BAP (d) 0.2 BAP, 0.2 NAA (e) 0.4 BAP, 0.2 NAA (f) 0.6 BAP, 0.2 NAA	1.5 2.3 2.2 1.1 1.4 1.2	N/A	[39]
WPM	‘Chester Thornless’	Shoot	Combination of BAP (1, 2, 3), NAA (0, 0.1, 0.2, 0.4) and IBA (0, 0.1, 0.2, 0.4). GA3 (0, 0.25, 0.50) IBA and NAA (0, 0.1, 0.2, 0.4)	9.66 (2 mg L−1 BAP + 0.2 mg L−1 IBA) 3.33 (3 mg L−1 BAP + 0.1 and 0.4 mg L−1 IBA) 6.33 (2 L−1 BAP without NAA) 2.21 (1.5 mg L−1 BAP + 0.4 L−1 NAA)	N/A	[8]
MS	N/A	Shoot	0.8, 1 BAP	N/A	N/A	[49]
MS	N/A	Shoot	BAP (2, 3), alone or in combination with GA3 (0.2, 0.5,1)	3.33 (2 mg L−1 BA and 0.5 mg L−1 GA3)	N/A	[36]
MS	N/A	Fragment with an axillary bud	1, 1.5 and 2 BAP	More than 3 shoots per explant, except R. adenothallus	R. floribundus produced the highest number of shoots (7.2 ± 1.9) when grown under the influence of 1.5 mg L−1 BAP	[23]
MS MS with mineral salts reduced ½	‘DAR 24’ ‘DAR 8’ ‘Darrow’	Shoot	0.5 BAP, 0.5 GA3 0.3 BAP, 0.1 GA3, 0.001 NAA	11.17 ‘Darrow’ 18.0 ‘DAR 24’ 12.83 ‘DAR 8’	MS with 0.3 BAP, 0.1 GA3, 0.001 NAA is better for all 3 cultivars	[24]
MS	‘Čačanska bestrna’	Shoot	(a) 1 BAP, 0.1 IBA, 0.1 GA3 (b) 0.5 BAP, 0.1 IBA, 0.1 GA3 (c) 1 BAP, 0.1 NAA, 0.1 GA3	2	The multiplication index is the average of the four subcultures.	[25]
MS	‘Smoothstem’	Shoot bud	(a) 2 mM BAP, 2.5 mM IBA, 0.3 mM GA3 (b) 2 mM BAP, 0.3 GA3 (c) 4 mM BAP, 0.25 mM IBA	2.3 ± 0.2 (a) 5.1 ± 0.6 (b) 6.3 ± 0.7 (c)	N/A	[18]
MS	‘Ébano’ (E) ‘Guarany’ (G) ‘Tupy’ (T)	Shoot	(a) 2 BAP (b) 2 BAP, 0.1 NAA, 0.5 GA3 (c) 1 BAP (d) 1 BAP, 0.1 NAA, 0.5 GA3	7.60 E (c) 7.02 T (c) 12.15 G (a)	N/A	[1]
MS (solid) (S) MS (liquid) (L) MS (double-phase) (DP)	‘Ébano’ (E) ‘Tupy’ (T)	N/A	4 μM BAP (S,L, DP) 0.1, 2, 3, 4, and 5 μM BAP (DP)	9.4 T (L) 11.0 E (L)	The in vitro multiplication of ‘Tupy’ and ‘Ébano’ blackberries is achievable in double-phase MS medium with 5 μM BAP.	[53]
MS	‘Kotata’	Shoot	0.4 BAP	4.0 ± 0.5	N/A	[50]
MS MS VDS MS 2x FeNaEDTA MS VDS 2x FeEDDHA	‘Black Satin’ (BS) ‘Loch Ness’ (LN)	Shoot	1 BAP, 0.5 IBA, 0.1 GA3	6.50 ± 0.27 BS (MS) 6.59 ± 0.31 BS (MS VDS) 3.84 ± 0.24 BS (MS 2x FeNaEDTA) 2.74 ± 0.15 BS (MS VDS 2x FeEDDHA) 3.13 ± 0.18 LN (MS) 2.76 ± 0.16 LN (MS VDS)	For ‘Black Satin’ it was shown that double concentration of chelates FeNaEDTA and FeEDDHA in culture media negatively affected on shoot growth and multiplication.	[51]
MS	‘Loch Ness’ (LN) ‘Smoothstem’ (S) ‘Thornless evergreen’ (TE)	Shoot	0.5 BAP	21.69 (LN) 35.89 (S) 42.32 (TE)	N/A	[28]
MS	N/A	Shoot	BAP (0.25, 0.50, 1, 1.50, 2) 2iP (0.25, 0.50, 1, 1.50, 2) TDZ (0.25, 0.50, 1, 1.50, 2)	5 (0.25 mg L−1 BA; 1.0 mg L−1 TDZ) 7 (0.50 mg L−1 BA; 0.25 mg L−1 2iP) 11 (1.0 mg L−1 BA) 17 (1.50 mg L−1 BA) 12 (2 mg L−1 BA) 9 (0.50 mg L−1 2iP) 13 (1.0 mg L−1 2iP) 15 (1.50 mg L−1 2iP) 8 (2.0 mg L−1 2iP) 2 (0.25 mg L−1 TDZ) 3 (0.50 mg L−1 TDZ) 1 (1.50, 2.0 mg L−1 TDZ)	N/A	[34]
MS	‘Čačanska bestrna’	Regenerated shoot	1 BAP, 0.1 IBA, 0.1 GA3	2.9	N/A	[26]
MS MS with mineral salts reduced ½	N/A	Shoot	1.5 BAP, 0.75 IAA	5.0 R. macrocarpus (½ MS) 6.8 R. bogotensis (½ MS)	Only the best results obtained were reported	[52]

MS Murashige and Skoog (1962) [37]; MSB Murashige and Skoog Basal medium; WPM Woody Plant Medium [42]; MS VDS [54] EDTA Ethylenediaminetetraacetic acid; FeEDDHA Ferric Ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid); BAP 6-Benzylaminopurine; IBA Indole-3-Butyric Acid; IAA Indole-3-Acetic Acid; GA₃ Gibberellic Acid 3; NAA Naphthalene Acetic Acid; 2iP 2-Isopentenyladenine; TDZ Thidiazuron.

provides a detailed summary of the multiplication phase for the selected articles.

3.3. Rooting

For the in vitro rooting stage of blackberry, shoots are the most commonly used explant type [1,8,14,15,20,24,25,26,34,36,39,45,49,50,52,55], with MS medium as the preferred substrate [14,15,20,22,36,39,49,52,53] (Figure 3). Variants of MS, such as half-strength [3,14,16,24,25,26,34,39,45,52,55] or one-third strenght MS [1] have also been employed in several studies to optimize rooting conditions. In contrast, Munoz-Concha et al. [22] compared MS, WPM and Driver and Kuniyuki (DKW) medium [56] for the cultivar ‘Chester’ and found that WPM produced more roots and longer length.

The duration of the rooting phase ranged from a minimum of 2 weeks [36] to a maximum of 60 days [53].

Growth regulators used for blackberry rooting included IBA applied at 0.1 to 2 mg L⁻¹ [1,3,8,14,15,16,20,24,25,26,34,36,39,45,49,53,55]; NAA in the range of 0.1 to 1.1 mg L⁻¹ [3,8,16,34]; GA₃ from 0.1 to 0.5 mg L⁻¹ [22,24,25,26] and IAA used at 1 mg L⁻¹ [50,55] (Figure 3).

Table 3. Summary of the rooting phase for blackberry, detailing the basal media, cultivar, explant type, plant growth regulators (PGRs) concentration, rooting success, comments and corresponding references.

Basal Media	Cultivar	Explant Type	PGRs (mg L−1 or Other UoM Where Specified)	Rooting %	Comments	References
½ MS	‘Ébano’	Nodal segment	NAA (0.55, 1.1), IBA (0.55, 1.1)	0% (NAA) 100% (IBA)	N/A	[3]
MS WPM DKW	‘Chester’	N/A	0.5 GA3	N/A	Rooting was observed on WPM medium, with more and longer roots than on MS or DKW medium	[22]
MS ½ MS ¼ MS	N/A	Shootlet	0.4 IBA	33.3% (MS) 100% (½MS) 60% (¼ MS)	N/A	[39]
WPM	‘Chester Thornless’	Shoot	NAA (0.1, 0.2, 0.4), IBA (0.1, 0.2, 0.4)	N/A	A concentration of 0.4 mg L−1 NAA gave the greatest number of roots and maximum root length.	[8]
MS	N/A	Shoot	1 IBA	N/A	N/A	[49]
MS	N/A	Shoot	IBA (0.5, 1.0, 2.0)	N/A	2.0 mg L−1 IBA has the highest root value	[36]
MS	‘Black Satin’	Shoot	1 IBA	80–100%	N/A	[20]
MS with mineral salts reduced ½	‘DAR 24’ ‘DAR 8’ ‘Darrow’	Shoot	0.1 IBA, 0.1 GA3	95% (DAR 8) 86% (DAR 24) 90% (Darrow)	N/A	[24]
MS with mineral salts reduced ½	‘Čačanska bestrna’	Shoot	1 IBA, 0.1 GA3	100%	N/A	[25]
MS with mineral salts reduced 1/3	‘Ébano’ ‘Guarani’ ‘Tupy’	Shoot	IBA (0.3, 0.5, 0.8)	100%	N/A	[1]
Solid MS	‘Ébano’ ‘Tupy’	N/A	IBA (0.5, 1.0, 1.5, 2.0 μM L−1)	N/A	An average concentration of 1.1 μM L−1 IBA resulted in improved root length and maximum root volume	[53]
½ MS	‘Brzezina Polish’ selection	Microshoot	0.5–1.5–2 µM IBA	96%	The highest rhizogenesis efficiency occurred in ½ MS + 1.5 µM IBA medium.	[45]
Macro 1/2 MS; microelements MS	‘Kotata’	Shoot	1 IAA	98 ± 0.5%	N/A	[50]
½ MS	N/A	Axillary shoot	IBA or NAA (0, 5.37 or 10.74 µM) and BA (2.22 µM)	N/A	The roots and shoots became most abundant when using the medium supplemented with 9.84 µM IBA.	[57]
½ MS	‘Čačanska bestrna’ ‘Gazda’	Shoot	1 IBA, 1 IAA	93% IBA and 100% IAA (Čačanska bestrna) 97% IBA and 73% IAA (Gazda)	N/A	[55]
½ MS salts	N/A	Shoot	IBA (0, 0.10, 0.25, 0.5, 0.75, 1, 1.25, and 1.5) in combination with NAA (0 and 0.5).	1.00 mg L−1 IBA plus 0.50 mg L−1 NAA, the highest rooting percentage of Rubus fruticosus (91%)	N/A	[34]
½ MS salts	‘Čačanska bestrna’	Shoot	1 IBA, 0.1 GA3	100%	N/A	[26]
MS ½ MS	‘Black Satin’	Shoot	IBA (0.5, 1)	100% (½ MS and 0.5 mg L−1 IBA) 87.5% (½ MS and 1.0 mg L−1 IBA) 75% (MS and 0.5 mg L−1 IBA) 83.3% (MS and 0.5 mg L−1 IBA)	N/A	[14]
MS	‘Loch Ness’ ‘Loch Tay’	Shoot	1 IBA	N/A	The obtained shoots were used for the regeneration phase	[15]
MS ½ MS	N/A	Shoot or stem cutting	N/A.	86.67% (½ MS R. macrocarpus) 100% (½ MS R. bogotensis) 46.67% (MS R. macrocarpus) 83% (MS R. macrocarpus)	N/A	[52]

MS Murashige and Skoog (1962) [37]; DKW Driver and Kuniyuki (1984) [56]; WPM Woody Plant Medium [42]; IBA Indole-3-Butyric Acid; IAA Indole-3-Acetic Acid; GA₃ Gibberellic Acid 3; NAA Naphthalene Acetic Acid.

provides a detailed summary of the rooting phase for the selected articles; where the rooting percentage was not reported, a comment has been added.

3.4. Acclimatization

The acclimatization of micropropagated blackberry plantlets is critical for successful ex vitro establishment and field transplantation. Key factors affecting survival include substrate composition, environmental conditions, and gradual adaptation protocols. Peat-based substrates, alone or mixed with sand or perlite, increase plantlet survival, with greenhouse mist systems achieving 100% survival due to well-developed root systems [18,25,26]. Hydroculture systems also produced robust plants [28,46,58]. Gradual exposure through polyethylene coverings or misting improved acclimatization, even in suboptimal conditions [3,14,22,33,34]. Peat-perlite mixtures with microelements and growth regulators accelerated adaptation [49,59].

Although plantlets rooted in vitro acclimatize faster, ex vitro rhizogenesis is more convenient [35]. The efficiency of ex vitro regeneration was achieved by soaking microsprouts in Rootone F and planting them in a 4:2:1 mixture of vermiculite, peat, and perlite, with initial maintenance of high humidity [35]. Sterile vermiculite with a solution of half-strength MS salts also provided high survival [14].

Substrate composition has a significant impact on survival. A mixture of sand and vermiculite (3:1) provided a survival of 97% compared to 70% for sand alone [45]. Coconut peat with perlite was also effective [16]. Inoculation of Bacillus thuringiensis improved growth [60]. Arbuscular mycorrhizal fungi (AMF) increased biomass, leaf area and root growth [61]. Silicon treatment improved nutrition, photosynthetic pigments and antioxidant activity [29].

Acclimatization success varied by cultivar. ‘Dar 8’ achieved 86% success under mist conditions, whereas ‘Dar 24’ had 52% [24]. Greenhouse acclimatization of R. macrocarpus and R. bogotensis resulted in 83% and 75% survival, respectively [52].

Field acclimatization depended on soil composition and planting design. Different studies highlighted the importance of soil nutrients and row orientation [27]. Gradual transition from controlled moisture conditions to greenhouse and open field conditions maximized survival [20,29,46]

Blackberry acclimatisation success generally depends on substrate selection, humidity control, and gradual hardening. Optimized protocols, including beneficial microbes, controlled environments, and suitable rooting substrates, ensure high survival and successful establishment.

3.5. Control of Bacterial Contamination

Despite advances in propagation techniques, bacterial contamination remains a significant obstacle in tissue culture systems. Bacterial contamination can go undetected in early stages and only become apparent as the shoot population increases. Biocides such as plant preservative mixture (PPM™) and Vitrofural have been tested to control bacterial growth. Both biocides effectively restricted the development of Methylobacterium lusitanum, Paenibacillus spp., Pseudomonas putida, Serratia marcescens, and Staphylococcus pasteuri in agar-diffusion assays, with PPM™ proving particularly effective against M. lusitanum for up to 21 days [62]. Despite this, the application of these biocides also impacted shoot multiplication and rooting depending on their concentration and the plant genotype. Interestingly, all concentrations of Vitrofural led to an increase in the number of axillary shoots in blackberry, suggesting its potential to enhance propagation despite certain adverse effects on root formation [62].

3.6. Callus Induction

Callus induction is a key step in plant tissue culture, influenced by the type and concentration of PGRs used in the culture medium. 2,4-dichlorophenoxyacetic acid (2,4-D) auxin has been shown to significantly increase callus formation, with an increase from 0.5 to 1.0 mg L⁻¹, in combination with 0.5 mg L⁻¹ NAA, leading to a 100% increase in callus production [39]. Similarly, callus cultures derived from leaves in vitro on MS medium supplemented with 2 mg L⁻¹ of BAP and 2 mg L⁻¹ of 2,4-D showed a specific phenolic profile, with quercetin-3-O-glucoside and quercetin-3-O-rutinoside as the primary compounds, and demonstrated lower antioxidant activity than leaf tissue [63]. The use of Yasuda medium containing 8.88 μM BAP, 10.84 μM NAA and 2% glycerol (v/v) was also shown to be effective for callus induction [57]. In addition, cytokinin TDZ showed significant potential in promoting callus formation, with 1.0 mg L⁻¹ TDZ producing the highest percentage of callus (100%) and globular embryos when cultured in complete darkness [34].

The inclusion of kinetin (KIN, 2.32 μM), NAA (2.69 μM) and BAP (8.88 μM) in the culture media for callus induction further optimized callus development in thin transverse cell layers, with antioxidant agents such as activated charcoal, cysteine and citric acid helping to minimize phenolic exudation [64].

3.7. Somatic Embryogenesis and Synthetic Seeds Production

Somatic embryogenesis (SE) holds significant potential for the efficient propagation of Rubus species, including blackberries. Different studies have explored diverse approaches to induce and optimize this process. One of the first report demonstrated successful SE in the ‘Thornfree’ cultivar, starting from pre-embryogenic determined cells that formed callus and embryos [65]. This process lasted different subsequent subcultures and a crucial role of hydrolyzed casein in the culture medium was hypothized. Furthermore, somatic embryos were obtained from ovules emphasizing the complex interplay of various factors (embryo stage, growth regulators, media supplements, photoperiod, and genotype) in somatic embryogenesis [66]. This reinforces the idea that SE protocols must be tailored to specific genotypes and optimized for a range of factors. A recent study by Sabooni and Shekafandeh [64] provides a detailed protocol for SE in blackberries using the transverse thin cell layer (tTCL) technique. This research represents a significant advancement, as it reports the first successful SE-based plant regeneration protocol for this species. Utilizing two cultivars, ‘High Prickle’ and ‘Low Prickle’, the study identified specific media formulations for different stages of SE. Optimal embryogenic callus initiation was achieved on half-strength MS medium supplemented with sucrose, KIN, and BAP. Subsequent embryo development and maturation were promoted by incorporating abscisic acid (ABA) and either malt extract or glutamine into the medium. Finally, embryo germination and plantlet development were achieved on a specific half-strength MS medium containing BAP, GA₃, and NAA.

Somatic embryos can be used for producing synthetic seeds through encapsulation. Synthetic seeds integrate the benefits of micropropagation and zygotic seeds [67]. Their small size facilitates handling and exchange between laboratories, ensures the maintenance of genetic uniformity, and permits direct sowing in field or greenhouse [68]. However, the difficulties encountered in inducing somatic embryogenesis in certain species have led to a broader definition of synthetic seeds, encompassing a range of encapsulated in vitro-derived propagules, such as shoot tips and nodal segments [68,69]. Jadán et al. [47] obtained synthetic seeds of Rubus glaucus Benth utilizing apical and nodal segments as explant sources. In particular, they achieved in vitro germination of encapsulated explants by incorporating 1 mg L⁻¹ brassinolide and 1 g L⁻¹ activated charcoal into the encapsulation matrix, demonstrating the importance of matrix components for successful germination. Regni et al. [44] compared the performance of two different encapsulated vegetative propagules: uninodal microcuttings (nodes) and the base of clumps for ‘Thornfree’ and ‘Chester’ cultivars. Both considered propagules allowed to obtain satisfactory regeneration percentages but plantlets from the encapsulated clump’s base had more shoots and roots, greater fresh and dry weights than those derived from encapsulated nodes.

The medium and long-term storage of encapsulated explants represents another critical aspect of synthetic seed technology. Ružić et al. [48] investigated the in vitro conservation of encapsulated shoot tips of blackberry ‘Čačanska Bestrna’ and raspberry ‘Meeker’ at 5 °C for up to three months. Blackberry showed greater survival and shoot formation after cold storage compared to raspberry, although both cultivars maintained normal morphology. This study suggests that encapsulated shoot tips can be successfully stored for short periods without significantly impacting subsequent shoot multiplication, rooting, or acclimatization. Expanding on this concept, Regni et al. [70] explored the effects of storage conditions and sowing substrate for synthetic blackberry seeds derived from encapsulated clump bases. In particular, the research evaluated the impact of storage duration (up to 120 days) at different temperatures (4 °C and 25 °C) and sowing substrates (agarised, perlite, and potting) on synseed viability and plantlet development. Their findings highlight the crucial role of both storage temperature and substrate. Storage at 4 °C, particularly on agarised substrate, proved superior for maintaining regeneration rates and promoting robust plantlet growth, even after extended storage periods. Conversely, storage at 25 °C significantly reduced regeneration, emphasizing its unsuitability for long-term preservation. The study also revealed that perlite and potting substrates could support regeneration, especially after storage at 4 °C. These studies contribute to a growing body of knowledge on synthetic seed technology in blackberries, emphasizing the need for optimized in vitro regeneration protocols, appropriate encapsulation matrices, and carefully controlled storage conditions for successful conservation and propagation.

3.8. Medium and Long Term Germplasm Conservation

Several strategies have been investigated to maintain blackberry genotypes under cold conditions. For example, minimal growth conditions, such as reduced temperatures (20 °C) and limited light, have proven effective in conserving blackberry cultivars for up to 15 months without significant loss in regenerative potential [3]. Cryopreservation methods, including encapsulation-dehydration, have also been successful in preserving blackberry shoot apices, achieving survival of up to 57% after cryopreservation at optimal sucrose concentrations [50]. Moreover, cold storage at approximately 4 °C under sub-optimal conditions has been demonstrated to effectively maintain blackberry microshoots, with subculturing intervals of 4 to 6 months, all while ensuring genetic stability [71]. The use of bioreactor systems, such as ElecTIS, has further improved the recovery of blackberry shoot cultures from slow growth storage (SGS), enhancing shoot proliferation and overall quality [58]. Additionally, encapsulating blackberry shoot tips in calcium alginate beads and storing them at 5 °C has proven effective in promoting successful shoot regeneration and multiplication, making this technique a valuable method for germplasm conservation [48]. Moreover, cryopreservation represents an essential techniques for the medium to long-term preservation of blackberry germplasm, ensuring the viability and regeneration potential of valuable genetic resources [50]. Together, these diverse in vitro conservation strategies offer a solid and reliable approach to preserving blackberry genetic resources, ensuring their availability for future breeding programs and sustainable use.

3.9. Bioreactors

The application of innovative bioreactor systems has been explored to optimize plant growth conditions, with some successful studies of blackberry proliferation in bioreactors based on the Temporary Immersion System (TIS). For example, a dual container system with glass bottles significantly enhanced shoot multiplication in blackberry cultures when was used with a medium volume of 175 mL and sucrose concentration of 20 g L⁻¹. However, lower sucrose levels induced hyperhydricity, compromising the quality of the plantlets [72].

As mentioned in medium and long term germplasm conservation section another promising bioreactor system, the ElecTIS bioreactor, has been shown to improve the recovery rates of shoot cultures, particularly from SGS at 4 °C. By employing an up-and-down movement, the ElecTIS ensures periodic contact between the shoot cultures and the liquid medium, promoting more robust growth. When tested on blackberry cultivars like ‘Thornfree’ and ‘Chester’, ElecTIS demonstrated superior shoot growth and quality compared to traditional gelled medium culture, while also improving chlorophyll content, stomatal functionality, and subsequent rooting and ex vitro acclimatization [58].

Another study [46] investigated the effects of three in vitro culture systems—solid medium (SM), semi-solid medium (SSM), and liquid medium using TIS on shoot proliferation and quality in six blackberry cultivars: ‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Karaka Black’, ‘Loch Ness’, and ‘Polar’. TIS, operated via a Plantform bioreactor, significantly enhanced shoot proliferation, with ‘Karaka Black’. In contrast, the longest shoots were observed in the SSM system, particularly in ‘Čačanska Bestrna’.

3.10. Light Effect

The effect of light quality on the micropropagation of blackberry has been investigated in some studies, highlighting the role of LED lighting in optimizing in vitro growth and development.

Different spectral compositions of red and blue light influence various physiological and morphogenetic responses in blackberry explants. A combination of blue and red light at a 2:1 spectral ratio significantly enhances leaf area, chlorophyll and carotenoid content, and overall vegetative growth, making it the optimal condition for in vitro rooting and shoot development [61]. The absence of light for seven days, in contrast, has been shown to reduce bacterial contamination and oxidation in ‘Xavante’ blackberry explants, improving survival when MS medium is supplemented with 75–100% salt concentrations [17]. Furthermore, studies on blackberry regeneration pathways indicate that a red:blue ratio of 2:1 and 1:1 provides the highest regeneration efficiency, while monochrome blue and red light inhibit shoot proliferation [31].

3.11. Genetic Fidelity and Stability, Breeding

Genetic fidelity and stability are critical factors when evaluating the success of micropropagation protocols. Several studies have demonstrated the efficiency of micropropagation in maintaining the genetic uniformity of blackberry cultivars, which is essential for commercial plant production. For instance, both RAPD (Random Amplified Polymorphic DNA) and SRAP (Sequence Related Amplified Polymorphism) markers have been successfully employed to assess genetic stability in micropropagated blackberry plants. In a study involving the ‘Loch Ness’ and ‘Chester Thornless’ cultivars, no genetic variations were observed between mother plants and micropropagated plantlets after multiple subcultures, thus confirming the high genetic fidelity of the plants [21,73]. Similarly, studies on Rubus hirtus using RAPD markers revealed a genetic similarity of over 86% between regenerated plants and their mother plants, indicating that micropropagation can effectively preserve genetic integrity [57]. Furthermore, the genetic stability of blackberry plants has been confirmed through cytogenetic analyses such as flow cytometry, which showed no significant differences in ploidy levels or nuclear DNA content between tissue-cultured and field-grown plants [5]. Additionally, biochemical markers like peroxidase profiles have also been utilized to examine genetic stability, with no significant phenotypic or genotypic variations detected between the micropropagated and field-grown plants [26].

Recent studies have also explored alternative culture media to enhance micropropagation efficiency and evaluated the genetic uniformity of the obtained material. Wheat starch, investigated as a gelling agent, has proven effective in maintaining genetic uniformity in micropropagated blackberry plants. The use of SRAP and start codon targeted (SCoT) molecular markers confirmed the clonal fidelity of plants grown in wheat starch-based media, validating its suitability for large-scale propagation [11]. Moreover, genetic uniformity in micropropagated blackberry shoots has been further confirmed across different in vitro culture systems, including solid, semi-solid, and temporary immersion systems. In particular, SCoT markers verified the genetic stability of regenerated shoots across all tested systems, reinforcing the reliability of these DNA-based markers in assessing clonal fidelity [46].

3.12. Effects of Substance with Biostimulant Action and Abiotic Stress

Nanotechnology has recently emerged as an innovative method for improving in vitro cultivation [74,75]. Zinc oxide nanoparticles (ZnONPs) in culture media have been shown to affect plant growth and biochemical properties. At higher concentrations, ZnONPs inhibited shoot and root development and reduced total phenolic content, chlorophyll, and carotenoid levels. However, at an optimized concentration of 10 mg L⁻¹, ZnONPs promoted optimal growth, increased antioxidant capacity, and enhanced mineral uptake in blackberry plantlets [60]. Furthermore, plantlets treated with ZnONPs exhibited better acclimatization when inoculated with Bacillus thuringiensis, emphasizing the beneficial role of microbial interactions in enhancing ex vitro adaptation [60].

In addition, the role of abiotic stressors such as lime content and salt stress in blackberry cultivation has been studied. Lime content, particularly high levels of calcium carbonate (CaCO₃), has been found to negatively affect plant growth in Rubus spp. cultivars. For example, in vitro conditions with high CaCO₃ levels reduced plant height, fresh weight, and root length in the ‘Chester’ blackberry cultivar, indicating its low tolerance to calcareous soils [32]. On the other hand, salt stress has a significant impact on photosynthetic efficiency, and different cultivars exhibit varying levels of resilience. The ‘Thornfree’ cultivar displayed higher photosynthetic efficiency and better tolerance to salt stress compared to the more susceptible ‘Boysen’ cultivar, which exhibited reduced chlorophyll content and other disruptions in photosynthetic parameters [76].

Furthermore, external treatments such as silicon application have been studied to improve blackberry tissue culture. Silicon supplementation during both adventitious root induction and adaptation stages led to better growth and increased chlorophyll and carotenoid content. The optimal silicon concentrations (1 mg L⁻¹ in the in vitro phase and 50–100 mg L⁻¹ during acclimatization) resulted in higher antioxidant activity and improved photosystem II efficiency [29]. These results suggest that silicon treatment may play an essential role in producing healthier tissue-cultured blackberry plants by enhancing overall plant quality and stress tolerance.

3.13. Bioactive Compounds

Mitrović et al. [27] have shown that in vitro propagation can produce high-quality fruit comparable to those grown through conventional methods, with only minor differences in secondary metabolites, such as 4-hydroxybenzoic acid in blackberries. This highlights the effectiveness of tissue culture in ensuring plant health and quality, a crucial consideration for sustainable farming.

Furthermore, in vitro methods, including gamma irradiation, have been used to enhance the biochemical profile of blackberry plants. Irradiation doses of 40–60 Gy have been found to improve vegetative traits, increase chlorophyll and carotenoid content, and boost phenolic and antioxidant levels, demonstrating its potential to improve both plant growth and the production of bioactive compounds [77].

Kolarević et al. [63] analyzed the phenolic profile and antioxidant activity of in vitro leaves, callus cultures, and field-grown leaves of blackberry (‘Čačanska Bestrna’). Blackberry leaves exhibited the highest antioxidant activity, while callus cultures had significantly lower phenolic content and scavenging capacity. Quercetin derivatives and phenolic acids were identified as dominant compounds. These findings highlight the potential of in vitro and field-grown blackberry leaves as valuable sources of phenolics for food and pharmaceutical applications.

Additionally, research on wild blackberry species from the Peruvian Andes has revealed the impressive bioactive potential of these plants, further supporting the relevance of in vitro propagation for conserving and exploiting genetic resources. Several species of wild blackberry, such as Rubus floribundus and Rubus weberbaueri, exhibited high antioxidant and antimicrobial properties, as well as distinct physicochemical profiles [23]. The use of plant growth regulators like BAP has been shown to significantly enhance in vitro multiplication, with optimal concentrations promoting shoot proliferation and growth [23].

4. Toward an Operational Framework for Blackberry Micropropagation

Based on over five decades of research and the findings summarized in this review, it is now possible to outline an operational framework for the micropropagation of blackberry (Rubus spp.) that may serve as a practical guide for researchers, nurseries, and biotechnology laboratories. Despite genotype-specific responses and the need for further optimization in certain cultivars, a generalized protocol can be delineated into the following key stages:

• Establishment Phase
  • Explant type: Preferably nodal segments or axillary buds.
  • Medium: MS medium supplemented with BAP (0.5–2 mg L⁻¹) and optionally low concentrations of IBA or GA₃.
  • Sterilization: Use of standard disinfection protocols; control of bacterial contamination with PPM™ or Vitrofural if necessary.

2

Multiplication Phase

• Medium: MS (solid or semi-solid), often improved by alternative gelling agents such as wheat starch.
• PGRs: BAP (2–5 mg L⁻¹) combined with IBA (0.1–0.5 mg L⁻¹); GA₃ may improve shoot elongation.
• Culture system: Semi-solid and temporary immersion systems (TIS) significantly enhance shoot proliferation.

3

Rooting Phase

• Medium: half-strenght MS or one-third strenght MS medium with IBA (0.5–1.5 mg L⁻¹); NAA can be considered in some genotypes.
• Root induction: Better rooting generally occurs ex vitro using auxin treatments.

4

Acclimatization Phase

• Substrate: Peat-perlite (2:1) or sand-vermiculite (3:1) mixtures with controlled humidity.
• Gradual transition: Polyethylene covers or mist systems; beneficial microbial inoculants (e.g., Bacillus thuringiensis, AMF) and silicon supplementation can improve survival and vigor.

5

Optional Advanced Steps

• Synthetic seed production: Use of encapsulated shoot tips or nodal segments in calcium alginate.
• Medium and long term germplasm conservation: Slow growth storage or encapsulation-dehydration of shoot apices for long-term conservation.
• Bioreactors: ElecTIS or other TIS devices for mass propagation with improved physiological outcomes.

This framework integrates classical knowledge with recent innovations (e.g., nanotechnology, LED lighting, biostimulants) and offers a flexible structured guideline for implementing efficient and scalable micropropagation of blackberry. Adapting specific parameters to the requirements of each cultivar remains essential for optimal outcomes.

5. Conclusions

Over the past decades, significant advancements have been made in the micropropagation of blackberry (Rubus spp.), refining protocols for establishment, proliferation, rooting, and acclimatization. The optimization of culture media composition, plant growth regulators, and environmental factors has contributed to improving the efficiency and reproducibility of in vitro techniques. However, challenges such as microbial contamination, somaclonal variation, and genotype-specific responses persist, requiring further investigation.

Recent innovations, including the use of bioreactors, nanotechnology applications, and synthetic seed technology, offer promising solutions for enhancing propagation efficiency and genetic conservation. Moreover, the study of abiotic stress tolerance, particularly in the context of climate change, is gaining importance to ensure the resilience of micropropagated plants in diverse environmental conditions.

Future research should focus on integrating molecular tools to assess genetic stability, exploring alternative culture systems for large-scale production, and optimizing cryopreservation techniques for long-term germplasm conservation. The continuous refinement of these methodologies will contribute to the sustainable production of high-quality blackberry plants, supporting both commercial cultivation and biodiversity preservation.