Sprouted Sorghum Extract Elicits Coleoptile Emergence, Enhances Shoot and Root Acclimatization, and Maintains Genetic Fidelity in indica Rice

doi:10.1016/j.rsci.2017.08.005

Abstract

Abstract:

The high growth-stimulating effect of plant extract has urged the plant biotechnologists to use natural supplements in the culture media instead of synthetic phytohormones. We advocated the effect of sprouted sorghum extract (SSE) on emergence, in vitro acclimatization, and genetic fidelity in coleoptile derived callus of indica rice variety ADT36. The use of SSE with Murashige Skoog medium efficiently acclimatized the root and shoot apical systems. A higher mat and seminal roots (3.4 g biomass) with an efficient shoot primordium elongation were observed with an increase in the concentration of SSE. Seeds treated with SSE medium showed higher germination and earlier coleoptile maturation about 48 h compared to untreated seeds, and there was a higher expression of eEF-1α with an increase in coleoptile length. B5 medium was effective on inducing embryogenic and nodular callus from 3-day-old coleoptile with 3.0 mg/L 2,4-dichlorophenoxyacetic acid and further proliferated effectively with 0.8 mg/L kinetin with a fresh weight of 180 mg. Highly significant regeneration was observed with combination of 2.5 mg/L 6-benzylamino purine and 3.0 mg/L α-naphthaleneacetic acid. The metabolic and genetic profiles of in vitro and directly cultivated plants were the same, examined through Fourier-transform infrared spectroscopy, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and R-ISSR (combination of RAPD and ISSR) markers, respectively, and thus confirming the significant efficacy of the SSE incorporated medium. Disarmed T-DNA was transformed to coleoptile derived callus through Agrobacterium tumefaciens LBA4404 and confirmed by GUS assay. The T-DNA integration was confirmed by DNA blot analysis using DNA from transient GUS-expressed explants. Thus, SSE can be used as a natural and organic supplement for organogenesis and efficient acclimatizations of shoot and root apical meristems in regenerated plants.

Key words: callus induction, coleoptile, Oryza sativa, random amplified polymorphic DNA, inter-simple sequence repeat, regeneration, tissue culture

Radhesh Krishnan Subramanian, Muthuramalingam Pandiyan, Pandian Subramani, Banupriya Ramachandradoss, Chithra Gunasekar, Ramesh Manikandan. Sprouted Sorghum Extract Elicits Coleoptile Emergence, Enhances Shoot and Root Acclimatization, and Maintains Genetic Fidelity in indica Rice[J]. Rice Science, 2018, 25(2): 61-72.

Figures/Tables 12

Table 1 Different types of media for germination and elongation.

Germination medium	Basal medium	Additive	Hormone
MS	MS	-	-
HMS	½ MS	-	-
MIBA-1	MS	-	0.5 mol/L IBA
MIBA-2	MS	-	1.5 mol/L IBA
MSSE-10	MS	10% SE	-
MSSE-20	MS	20% SE	-
MSSE-30	MS	30% SE	-
MSSE-40	MS	40% SE	-

Fig. 1. Coleoptile emergence on different percentages of sorghum extract in medium.A, Germinated sorghum seeds in distilled water; B, Germinated ADT36 rice seeds on HMS medium; C, Germinated ADT36 rice seeds on MS medium; D, Germinated ADT36 rice seeds on MIBA-1 (1) and MIBA-2 (2) media; E, Coleoptile formation on MSSE-10 (1), MSSE-20 (2), MSSE-30 (3) and MSSE-40 (4) media; F, Dissected germinated coleoptile (1), leaf base (2) and seed (3) media. MS, Murashige and Skoog; HMS, Half of MS; MIBA, MS with indole-3-butyric acid; MSSE, MS with sorghum extract. The lengths of bars for A to F are 1 cm.

Fig. 2. Effect of different types of medium on callus induction.B5, Gamborg; LS, Linsmaier and Skoog; MS, Murashige and Skoog; N6, Chu’s N-6.

Fig. 3. Callus induction.A, Calli from mature seed; B, Calli from leaf base; C, Calli from coleoptile with 0.5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid); D, Calli from coleoptile with 1.0 mg/L 2,4-D; E, Calli from coleoptile with 1.5 mg/L 2,4-D; F, Calli from coleoptile with 2.0 mg/L 2,4-D; G, Calli from coleoptile with 2.5 mg/L 2,4-D; H, Proliferated calli; I, The closer view of proliferated calli; J, Effect of synthetic kinetin and 6-beniylamino purine (BAP) on callus proliferation. The lengths of bars for A to I are 1 cm. Bars for J represent the standard deviations.

Fig. 4. Callus regeneration.A, Three-day-old regenerated callus (incubated at dark); B, Regenerated callus with SAMs (shoot apical meristems); C, Upper view of regenerated callus with shoot primordium; D, Bright field microscopic-cross sectional view of torpedo shaped callus; E, Globular callus; F, Regenerating globular callus; G, Regenerated callus with SAM. The lengths of scare bars for A to C are 1 cm.

Table 2 Effect of plant growth hormones on callus regeneration initiation of shoot primordium.

	BAP	NAA (mg/L)	Average regeneration (%)	Shoot height
	(mg/L)	NAA (mg/L)	Average regeneration (%)	(cm)
Initiation of shoot primordium
	0	-	5.67 ± 0.58	-
	0.1	-	3.33 ± 1.53	-
	0.2	-	11.33 ± 0.58	-
	0.3	-	12.00 ± 3.61	-
	0.4	-	15.33 ± 1.53	-
	0.5	-	13.00 ± 1.00*	-
	0.6	-	14.00 ± 1.73	-
	0.7	-	14.33 ± 1.53	-
	0.8	-	16.00 ± 2.00	-
	0.9	-	20.00 ± 1.00*	-
	1	-	21.67 ± 1.53	-
	1.5	-	25.00 ± 1.00*	-
	2	-	26.67 ± 1.53	-
	2.5	-	35.67 ± 1.53	-
	3	-	33.67 ± 2.52	-
	3.5	-	38.67 ± 1.53	-
	4	-	35.67 ± 4.58	-
Development of SAMs
	2.5	0.1	37.00 ± 1.00*	8.20 ± 0.09*
	2.5	0.2	36.67 ± 1.15*	8.77 ± 0.66*
	2.5	0.3	37.67 ± 1.53	10.07 ± 0.47*
	2.5	0.4	38.67 ± 1.53	10.87 ± 0.36*
	2.5	0.5	36.67 ± 2.08	10.30 ± 1.25
	2.5	0.6	43.67 ± 1.53	13.32 ± 0.36*
	2.5	0.7	43.33 ± 1.53	13.43 ± 0.43*
	2.5	0.8	47.00 ± 2.65	13.39 ± 1.02
	2.5	0.9	50.00 ± 1.00*	14.50 ± 0.42*
	2.5	1	49.67 ± 4.16	13.45 ± 1.84
	2.5	1.5	56.00 ± 2.65	15.58 ± 2.06
	2.5	2	56.00 ± 3.60	15.70 ± 0.48*
	2.5	2.5	62.33 ± 1.53	15.72 ± 0.46*
	2.5	3	62.00 ± 1.00	16.24 ± 0.64*
	2.5	3.5	57.67 ± 1.53	14.92 ± 0.70*
	2.5	4	53.00 ± 2.65	12.55 ± 0.67

Fig. 5. Plantlet acclimatization and root and shoot establishment on medium with different plant growth hormones (PGHs) or sprouted sorghum extracts (SSEs).A, HMS; B, MS; C, MIBA-1; D, MIBA-2; E, MSSE-10; F, MSSE-20; G, MSSE-30; H, MSSE-40; I and J, Effect of PGHs or SSEs on seed sprouting and coleoptile induction.MS, Murashige and Skoog; HMS, Half of MS; MIBA, MS with indole-3-butyric acid; MSSE, MS with sorghum extract. Bar for I and J represents standard deviation.

Fig. 6. Fourier-transform infrared spectroscopy spectrum from seeds of direct germinated (A), in vitro germinated (B), regenerated (C) and hardened plants (D).Dashed box represents the cellular component.

Fig. 7. Representative gel picture depicting genetic profile of random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and R-ISSR (combination of RAPD and ISSR) primers of direct germinated (lanes 2, 6 and 10), in vitro germinated (lanes 3, 7 and 11), regenerated (lanes 4, 8 and 12), and hardened plants (lanes 5, 9 and 13).The arrow represents monomorphic band.

Table 3 Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) primers used to check genetic profile.

No.	Primer	Annealing temperature		No.	Primer	Annealing temperature		No.	Primer	Annealing temperature
No.	Primer	37 ºC	55 ºC	No.	Primer	37 ºC	55 ºC	No.	Primer	37 ºC	55 ºC
1	OPD3	+	+	10	ISSR46	-	+	18	OPD3-ISSR35	-	+
2	OPD8	+	+	11	ISSR67	+	+	19	OPD3-ISSR42	+	-
3	OPD18	+	-	12	ISSRA8	-	+	20	OPD3-ISSR67	-	-
4	OPF4	+	-	13	ISSRA10	-	+	21	OPD3-ISSRA16	-	-
5	OPG5	+	-	14	ISSRA16	+	+	22	OPD8-ISSR35	+	-
6	OPH3	+	-	15	ISSRA33	-	+	23	OPD8-ISSR42	+	+
7	ISSR15	-	+	16	ISSRY5	-	+	24	OPD8-ISSR67	-	+
8	ISSR35	+	+	17	ISSRY11	-	+	25	OPD8-ISSRA16	+	-
9	ISSR42	+	+

Fig. 8. eEF1-α expression profile of coleoptile induced from different media.

Fig. 9. T-DNA gene transfer and validation in different explants.A, Checking hygromycin resistance by coleoptile derived callus. 1 to 6 means control, 10, 20, 30, 40 and 50 μg/L hygromycin; B, GUS expression in callus; C, GUS expression in mature seed; D, GUS expression in root; E, GUS expression in leaf blade; F, GUS expression in leaf base; G, GUS expression in coleoptile; H, Cross-section of GUS expressed in leaf base; I-J, Cross-section of GUS expressed in coleoptile; K, DNA blot hybridization of hpt gene integration in different explants (1, Control-wild type leaf; 2, Callus; 3, Mature seed; 4, Root; 5, Leaf blade; 6, Leaf base; 7, Coleoptile).The lengths of bars for A to G are 1 cm.

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