Clonal in vitro multiplication of grey mangrove and assessment of genetic fidelity using single primer amplification reaction (SPAR) methods

Figures & data

Table 1. Effect of IBA on root formation from in vitro raised shoots of A. marina in the MS medium after 4 weeks of culturing.

IBA (μmol/L)	Rooting (%)	Mean number of roots/shoot	Mean shoot length (cm)
0.0	0.0	0.0^d	0.0^e
0.5	47	2.1 ± 0.11^b	3.1 ± 0.27^c
1.0	71	2.9 ± 0.24^a	3.8 ± 0.30^a
1.5	62	2.0 ± 0.23^b	3.5 ± 0.18^bc
2.0	45	1.4 ± 0.15^c	2.5 ± 0.15^d

Note: Data represent means ± standard error of the means (SEM). Means followed by the same letter within columns are not significantly different (P > 0.05) based on Tukey's test.

Table 2. RAPD primers used to evaluate the extent of genetic fidelity of micropropagated Avicennia marina plantlets.

Sl no.	Primer	Primer sequence (5′–3)	Ta (°C)	Number of bands
(1)	OPB-01	GTTTCGCTCC	35	7
(2)	OPB-02	TGATCCCTGG	35	7
(3)	OPB-03	CATCCCCCTG	35	6
(4)	OPB-04	GGACTGGAGT	35	10
(5)	OPB-05	TGCGCCCTTC	35	8
(6)	OPB-06	TGCTCTGCCC	35	10
(7)	OPB-07	GGTGACGCAG	35	11
(8)	OPB-08	GTCCACACGG	35	4
(9)	OPB-09	TGGGGGACTC	35	11
(10)	OPB-10	CTGCTGGGAC	35	3
(11)	OPB-11	GTAGACCCGT	35	10
(12)	OPB-12	CCTTGACGCA	35	6
(13)	OPB-13	TTCCCCCGCT	35	4
(14)	OPB-14	TCCGCTCTGG	35	5
(15)	OPB-15	GGAGGGTGTT	35	5
(16)	OPB-16	TTTGCCCGGA	35	4
(17)	OPB-17	AGGGAACGAG	35	7
(18)	OPB-18	CCACAGCAGT	35	6
(19)	OPB-19	ACCCCCGAAG	35	5
(20)	OPB-20	GGACCCTTAC	35	4

Note: T_a, annealing temperature.

Table 3. DAMD primers used to evaluate the extent of genetic fidelity of micropropagated Avicennia marina plantlets.

Sl no.	Primer	Primer sequence (5′–3)	Ta (°C)	Number of bands
(1)	HBV	GGTGTAGAGAGGGGT	56	0
(2)	HVR	GCTCCTCCC CTCCT	50	5
(3)	M13	GAGGGTGGCGGT TCT	57	14
(4)	33.6	GGAGGTGGGCA	52	19

Note: T_a, annealing temperature.

Table 4. ISSR primers used to evaluate the extent of genetic fidelity of micropropagated Avicennia marina plantlets.

Sl no.	Primer	Primer sequence (5′–3)	Ta (°C)	Number of bands
(1)	UBC-811	GAG AGA GAG AGA GAG AC	49	9
(2)	UBC-825	ACA CAC ACA CAC ACA CT	46	12
(3)	UBC-827	ACA CAC ACA CAC ACA CG	50	11
(4)	UBC-834	AGA GAG AGA GAG AGA GYT	50	11
(5)	UBC-841	GAG AGA GAG AGA GAG AYC	50	18
(6)	UBC-855	ACA CAC ACA CAC ACA CYT	50	8
(7)	UBC-866	CTC CTC CTC CTC CTC CTC	55	14
(8)	UBC-868	GAA GAA GAA GAA GAA GAA	46	10
(9)	UBC-880	GGG TGG GGT GGG GTG	50	18
(10)	UBC-889	DBD ACA CAC ACA CAC AC	46	5
(11)	UBC-891	HVH TGT GTG TGT GTG TG	46	7
(12)	UBC-900	ACT TCC CCA CAG GTT AAC ACA	58	0

Note: T_a, annealing temperature.

Figure 1. Effect of 6-benzyladenine (BA) on shoot regeneration (%) of A. marina in the MS medium augmented with NAA (1.0 μmol/L) + AC (3.0 g/L). Data represent means ± SEM. Values followed by the same letter within response variables are not significantly different (P < 0.05) based on Tukey's test.

Figure 2. Effect of 6-benzyladenine (BA) on in vitro shoot multiplication and shoot length of A. marina in the MS medium augmented with NAA (1.0 μmol/L) + AC (3.0 g/L). Data represent means ± SEM. Values followed by the same letter within response variables are not significantly different (P < 0.05) based on Tukey's test.

Figure 3. Representative RAPD profiles of in vitro raised and donor plants of A. marina generated using primer OPB-07. Lanes 1–10: randomly selected regenerated plants; lane D: donor plant; lane M: lambda DNA/EcoRI+HindIII marker (Cat no. SM0193; Fermentas, GmbH, Germany).

Figure 4. Representative DAMD profiles of in vitro raised and donor plants of A. marina generated using primer 33.6. Lanes 1–10: randomly selected regenerated plants; lane D: donor plant; lane M: lambda DNA/EcoRI+HindIII marker.

Figure 5. Representative ISSR profiles of in vitro raised and donor plants of A. marina generated using primer UBC-866. Lanes 1–10: randomly selected regenerated plants; lane D: donor plant; lane M: lambda DNA/EcoRI+HindIII marker.