Molecular identification and high fidelity micropropagation of shell ginger (Alpinia zerumbet)

Figures & data

Table 1. Effects of different concentrations and combinations of BAP, Kin, and NAA on multiple shoot induction from the rhizome bud explant in Alpinia zerumbet grown in MS medium using 3% w/v sucrose. Shoot number and length were counted after 12 weeks of the respondents’ culture, while induction frequency was recorded in a separate experiment after four weeks of culture. Values are the means ± standard error. Different superscript in the same column indicates significant differences (P < 0.05) within treatments.

Plant growth regulator (mg/L)			Shoot induction rate (%)	Mean number of shoots/culture (±SE)	Mean length (cm) ±SE
BAP	NAA	Kin
0.5			25.0	2.50 ± 0.32^e-h	2.06 ± 0.26^ef
1.0			54.2	2.90 ± 0.34^d-h	2.12 ± 0.25^ef
2.0			54.2	3.08 ± 0.29^d-h	2.79 ± 0.33^b-f
3.0			79.2	3.17 ± 0.33^d-g	2.96 ± 0.28^b-f
4.0			83.3	4.29 ± 0.45^c-e	3.74 ± 0.21^a-c
5.0			83.3	7.29 ± 0.75^b	3.67 ± 0.34^a-d
6.0			91.7	11.9 ± 0.82^a	3.19 ± 0.14^a-f
7.0			75.0	5.71 ± 0.56^bc	2.63 ± 0.28^c-f
8.0			50.0	4.40 ± 0.40^c-e	2.73 ± 0.34^c-f
6.0	0.5		50.0	4.75 ± 0.34^cd	2.30 ± 0.29^d-f
6.0	1.0		41.7	3.88 ± 0.32^c-f	2.48 ± 0.30^c-f
5.0	0.5		45.8	3.04 ± 0.32^d-h	2.35 ± 0.30^c-f
5.0	1.0		37.5	2.54 ± 0.25^e-h	2.01 ± 0.30^f
		2.0	41.7	1.08 ± 0.06^h	4.15 ± 0.23^ab
		3.0	41.7	1.58 ± 0.25^gh	4.48 ± 0.24^a
		4.0	58.3	2.13 ± 0.41^f-h	4.20 ± 0.24^ab
		5.0	38.0	1.40 ± 0.30^gh	3.43 ± 0.39^a-e
	0.5	4.0	50.0	1.29 ± 0.30^gh	3.23 ± 0.17^a-f
	1.0	4.0	50.0	1.17 ± 0.17^gh	3.48 ± 0.34^a-e

Table 2. Effects of different concentrations of IBA and NAA on root induction of in vitro-grown micro-shoots of A. zerumbet. Data were recorded after four weeks of culture on the MS medium using 3% (w/v) sucrose. Values are the means ± standard error. Different superscript in the same column indicates significant differences (P < 0.05) within treatments.

Plant growth regulator (mg/L)		Root induction rate (%)	Mean number of roots/culture (±SE)	Mean length (cm) ±SE
IBA	NAA
0.5		79.2	5.25 ± 0.39^d	3.63 ± 0.64^a
1.0		91.7	10.54 ± 0.75^ab	5.54 ± 0.66^a
1.5		100	8.63 ± 0.66^bc	4.83 ± 0.68^a
2.0		100	7.21 ± 0.56^cd	5.58 ± 0.72^a
3.0		70.8	6.13 ± 0.52^cd	5.25 ± 0.68^a
	0.5	91.7	4.83 ± 0.59^d	4.75 ± 0.58^a
	1.0	91.7	6.71 ± 0.63^cd	4.42 ± 0.59^a
	1.5	100	13.04 ± 0.85^a	5.79 ± 0.46^a
	2.0	95.8	10.46 ± 0.59^ab	4.08 ± 0.45^a
	3.0	95.8	7.17 ± 0.68^cd	3.38 ± 0.53^a
	4.0	83.3	5.46 ± 0.61^d	3.54 ± 0.50^a

Figure 1. Morphological traits of A. zerumbet (a) portion of a stand at its full blossom (b) closer look of a flower (c) plant bearing immature fruits (d) different floral parts (e) enlarged view of stamen and stigma (f) cross-section of the ovary. Images are taken from the plant grown at the experimental field.

Figure 2. PCR amplification of matK and rbcL. Lanes: L (1 kb plus ladder), A (Negative control), B MatK, C rbcL.

Figure 3. Experimental results of multiple shoot induction and plant regeneration from rhizome buds explants of A. zerumbet (a) rhizome buds used as a source of explants, (b) shoot initiation from the inoculated rhizome buds in MS medium, (c-d) shoot proliferation in MS medium supplemented with 6.0 mg L⁻¹ BAP, (e) shoot multiplication, (f) plantlets rooted on MS medium amended with 1.5 mg L⁻¹ NAA (g) hardened plants of A. zerumbet in plastic pot.

Figure 4. (a) Representative agarose gel electrophoresis image of an amplified sequence from a RAPD reaction directed by primer OPA-13. Lane L: 1 kb plus ladder. Lane M: PCR amplification of mother plant DNA. Lane 1-5: PCR amplification of in vitro regenerated plants DNA. (b) Agarose gel electrophoresis of an amplified sequence from an ISSR reaction directed by primer UBC 822. Lane L: 1 kb plus ladder. Lane M: PCR amplification of mother plant DNA. Lane 1-5: PCR amplification of in vitro regenerated plants DNA.

Table 3. List of random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) primers, their resultant amplicon sizes, and numbers.

S. No.	Primer code	Primer sequence	PCR products (bp)	Bands/primer
1	OPA-01	CAGGCCCTT	300–900	6
2	OPA-02	TGCCGAGCTG	200–700	8
3	OPA-13	CAGCACCCA	200–1500	10
4	OPA-16	AGCCAGCGA	210–1700	7
5	OPC-12	TGTCATCCCC	390–1200	8
6	OPD-10	TGTCTGGGTG	380–1160	6
7	OPE-12	TTATCGCCCC	150–900	4
8	OPE-15	ACGCACAAC	250–1200	4
Total				53
1	UBC-822	(TC)8A	290–1200	7
2	UBC-823	(TC)8C	400–900	5
3	UBC-835	(AG)8YC	250–1500	7
4	UBC-845	(CT)8RG	180–2990	10
5	UM11-C9	(CAC)3GC	250–2000	11
6	UM14-C6	(CA)6GC	210–950	10
7	UM14-T4	(CA)6GT	220–800	7
8	UM18-C1	(CT)8AC	350–500	3
Total				60