Micropropagation of guava (Psidium guajava) seedlings, a plant with interest in cool subtropics, using an innovative BB culture medium

Figures & data

Table 1. Components of BB full [34] culture medium for in vitro experiments (BB basal medium and BB vitamins solution).

BB basal medium (BBb)		BB vitamins solution (vit BB)
Macronutrients	(mg L^−1)	Vitamins	(mg L^−1)
KNO3	1800	Thiamine	2.0
NH4NO3	400	Nicotinic acid	1.0
Ca(NO3)·4H2O	1200	Glycine	2.0
KH2PO4	270	Pyridoxine HCl	0.5
Magnesium		Biotin	0.1
MgSO4 · 7H2O	360	Folic acid	0.5
Micronutrients		p-aminobenzoic acid	1.0
H3BO3	4.80	Riboflabin	0.1
CuSO4·5H2O	0.25	Ca-pantothenate	0.5
MnSO4·H2O	33.50	Myo-inositol	100.0
Na2MoO4·2H2O	0.39	Ascorbic acid	75.0
ZnSO4·7H2O	17.00	Citric acid	50.0
CoCl2·6H2O	0.025
Fe-EDTA
FeSO4·7H2O	33.80
Na-EDTA·2H2O	45.40

Figure 1. Micropropagation of P. guajava. Seed germination on MS medium (A), Shoot induction on BB full medium under 4 mg L⁻¹ BA (B), Rooting (C) and Shoot growth (D) on BB full medium with 1.5 IBA + 0.5 NAA mg L⁻¹.

Table 2. 1^st experiment: Effect of different growth regulator-free basal media on Psidium guajava explants development.

Substrate	Shoot number	Shoot length (cm)	Root number	Root length (cm)
MS + vit BB	1.0 b	2.6 ± 0.55 a	1.5 ± 0.50 ab	2.8 ± 1.03 b
MS full	1.0 b	2.1 ± 0.23 b	1.1 ± 0.30 b	2.7 ± 1.40 b
WP + vit BB	1.0 b	2.4 ± 0.44 ab	1.7 ± 0.80 a	3.9 ± 1.58 a
WP full	1.0 b	2.2 ± 0.31 b	1.2 ± 0.52 bc	2.6 ± 0.87 b
BB full	1.2 ± 0.66 a	2.4 ± 0.79 ab	1.0 ± 0.75 c	2.8 ± 1.80 b

Note: MS + vit BB; MS with BB vitamins solution, MS full; MS with its vitamin), WP + vit BB; WP with BB vitamins solution, WP full; WP with its vitamins, BB full; BB with its vitamins, as in Table 1. *Mean values in the same column followed by different letter(s) are statistically different at significance level a = 0.05 (p ≤ 0.05), according to the results of the Duncans’ multiple comparisons test. Every treatment was carried out with 4 jars and 4 explants per jar. The number and length of proliferated shoots and roots were counted for each treatment. In addition, root induction was also observed during the experiment, therefore the number of the proliferated roots was noted as well.

Table 3. 2^nd experiment: Effect of different BA concentrations on the organogenesis of Psidium guajava explants, using BB full medium.

Treatments ΒΑ (mg L^−1)	Shoot number	Shoot length (cm)	Root number	Root length (cm)
0	1.00 c	2.1 ± 0.23 a	1.1 ± 0.30 b	2.7 ± 1.40 b
1	1.6 ± 0.59 b	1.4 ± 0.39 b	callus	–
2	1.9 ± 0.70 b	1.5 ± 0.51 b	callus	–
3	1.9 ± 0.70 b	1.3 ± 0.42 b	callus	–
4	2.8 ± 0.40 a	0.8 ± 0.09 c	callus	–

Note: BB full medium as in Table 1. 0 (Control), 1, 2, 3, 4 mg L⁻¹ BA. *Mean values in the same column followed by different letter(s) are statistically different at significance level α = 0.05 (p ≤ 0.05), according to the results of the Duncans’ multiple comparisons test. Every treatment was carried out with 4 jars and 4 explants/jar. The number and length of shoots was counted for each treatment. In addition, root induction was also observed in control treatment and callus formation in all the other treatments.

Table 4. 3^rd experiment: Effect of different BA concentration combined with NAA on the organogenesis of Psidium guajava explants, using BB full medium.

Treatments ΒΑ + 0.1 ΝΑΑ (mg L^−1)	Shoot number	Shoot length (cm)	Root number	Root length (cm)
0 (Control)	1.0 b	3.1 ± 1.13 a	2.2 ± 0.90	4.6 ± 2.50
0.5	1.0 b	2.1 ± 0.68 b	callus	–
1.0	1.0 b	1.7 ± 0.80 bc	callus	–
2.0	1.3 ± 0.43 a	1.5 ± 0.41 c	callus	–
4.0	1.1 ± 0.33 ab	1.5 ± 0.42 c	callus	–

Note: BB, modified basal medium as in Table 1. 0; no hormones (Control), 0.5 BA + 0.1 NAA mg L⁻¹, 1.0 BA + 0.1 NAA mg L⁻¹, 2.0 BA + 0.1 NAA mg L⁻¹, 4.0 BA + 0.1 NAA mg L⁻¹.

Table 5. 4^th experiment: Effect of different IAA or IBA concentrations in combination with 0.5 mg L⁻¹ NAA. on rooting of Psidium guajava micro-plants, established in BB full culture medium. The number and length of roots was counted for each treatment. In addition, shoot proliferation was also observed during rooting, therefore the number and height of the proliferated shoots was noted as well.

Treatments IAA or IBA + 0.5 ΝΑΑ (mg L^−1)	Shoot number	Shoot length (cm)	Root number	Root length (cm)
0 (Control)	1.0 b	4.0 ± 1.4 ab	2.3 ± 0.8 a	4.7 ± 2.6a
0.1 IAA	1.0 b	3.3 ± 1.9 abc	3.0 ± 2.5 a	3.0 ± 2.5 ab
0.5 IAA	1.4 a	3.4 ± 2.2 abc	3.0 ± 1.9 a	4.1 ± 2.5 ab
1.0 IAA	1.0 b	2.0 ± 0.1 c	2.3 ± 0.9 a	2.6 ± 0.6 b
1.5 IAΑ	1.0 b	3.2 ± 0.8 abc	2.5 ± 1.2 a	2.4 ± 1.1 b
0.1 IBA	1.0 b	3.8 ± 2.2 ab	2.5 ± 0.9 a	3.5 ± 2.7 ab
0.5 IBA	1.0 b	3.0 ± 0.8 bc	3.0 ± 1.3 a	2.4 ± 1.2 b
1.0 IBA	1.0 b	4.1 ± 1.1 ab	2.5 ± 0.5 a	3.5 ± 0.8 ab
1.5 IBΑ	1.0 b	4.8 ± 1.9 a	3.4 ± 1.6 a	5.0 ± 0.9 a

Note: 0; no hormones (Control), 0.1 IAA + 0.5 NAA mg L⁻¹, 0.5 IAA + 0.5 NAA mg L⁻¹, 1.0 IAA + 0.5 NAA mg L⁻¹, 1.5 IAA + 0.5 NAA mg L⁻¹, 0.1 IBA + 0.5 NAA mg L⁻¹, 0.5 IBA + 0.5 NAA mg L⁻¹, 1.0 IBA + 0.5 NAA mg L⁻¹, 1.5 IBA + 0.5 NAA mg L⁻¹. *Mean values in the same column followed by different letter(s) are statistically different at significance level α = 0.05 (p ≤ 0.05), according to the results of the Duncans’ multiple comparisons test. Every treatment was carried out with 4 jars and 4 explants/jar. *Mean values in the same column followed by different letter(s) are statistically different at significance level α = 0.05 (p ≤ 0.05), according to the results of the Duncans’ multiple comparisons test. Every treatment was carried out with 4 jars and 4 explants/jar. The number and length of shoots was counted for each treatment. In addition, root induction was also observed in control treatment and callus formation in all the other treatments.

Table 6. 5th experiment: Percent acclimatization success of Psidium guajava micro-plants derived from in vitro acclimatization in Magenta vessels and transferred to greenhouse.

Stages of acclimatization		Number of survived micro-plants	Percentage of survived micro-plants/Phase	Percentage of total survived micro-plants
Phase 0	Initial (in vitro)	144
Phase 1	Nylon tunnel	129	89.6	89.6
Phase 2	Greenhouse bench	120	93.0	83.3
Phase 3	Outdoor	120	100.0	83.3

The numbers indicate the survived micro-plants and the percentage (%) of success acclimatization, in a substrate of peat-moss:vermiculite:perlite (1:1:1).

Figure 2. Principal Coordinate Analysis (PCoA) plot for 9 samples (Gu1-Gu9) of P. guajava analyzed by SCoT and ISSR markers.

Figure 3. ISSR-PCR amplification in 1.5% Agarose gel. Lane M represents 1 Kb plus DNA marker and lanes G1-G9 shows P. guajava DNA samples amplified with primer UBC 835 (up) and UBC 840 (down).

Tsoktouridis G, Krigas N, Sarropoulou V, et al. Micropropagation and molecular characterization of Thymus sibthorpii benth.(lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern. In Vitro Cell Dev Biol-Plant. 2019;55(6):647–658.

 Web of Science ®Google Scholar

Data availability statement

The data that support the findings of this study are openly available in ‘figshare’ [https://figshare.com/s/3b500ba06775dfae80d8], reference number 1.