Proline and Glutamine Improve in vitro Callus Induction and Subsequent Shooting in Rice

doi:10.1016/S1672-6308(14)60300-0

Abstract

Abstract:

This study was conducted to evaluate the effects of proline and glutamine on in vitro callus induction and subsequent regeneration and to develop a reproducible and highly efficient plant regeneration protocol in four rice genotypes, viz. Pawana, Jaya, Indrayani and Ambemohar. Considerable variation in response to plant growth regulators and amino acid supplements used was observed in all the four genotypes. Medium supplemented with proline and glutamine was shown to be superior to medium without proline and glutamine. The best callusing from mature embryo was observed on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 500 mg/L proline and 500 mg/L glutamine. Shoot induction was higher in the callus obtained from medium supplemented with 500 mg/L proline and 500 mg/L glutamine. The highest shoot regeneration frequency (83.2%) was observed on MS medium with 2.0 mg/L benzylaminopurine, 0.5 mg/L 1-naphthaleneacetic acid, 500 mg/L proline, and 500 mg/L glutamine in the callus obtained from MS medium supplemented with 2.0 mg/L 2,4-D, 500 mg/L proline and 500 mg/L glutamine. Among the four genotypes, Pawana has the highest regeneration efficiency (83.2%), whereas the regeneration efficiency of the rest three rice genotypes was in the range of 32.0% to 72.3%. This optimized regeneration protocol can be efficiently used for Agrobacterium mediated genetic transformation in rice.

Key words: callus induction, glutamine, proline, rice, Agrobacterium mediated genetic transformation, 2, 4-dichlorophenoxyacetic acid, Murashige and Skoog medium

Pawar Bhausaheb, Kale Prashant, Bahurupe Jyoti, Jadhav Ashok, Kale Anil, Pawar Sharad. Proline and Glutamine Improve in vitro Callus Induction and Subsequent Shooting in Rice[J]. Rice Science, 2015, 22(6): 283-289.

Figures/Tables 5

References 16

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Medium number	Callus induction				Medium number	Shoot regeneration
Medium number	MS	2,4-D (mg/L)	Proline (mg/L)	Glutamine (mg/L)	Medium number	MS	Kinetin (mg/L)	BAP (mg/L)	TDZ (mg/L)	NAA (mg/L)	Proline (mg/L)	Glutamine (mg/L)
C₁	1	0.5	0	0	S₁	1	2	0	0	0	0	0
C₂	1	1	0	0	S₂	1	0	2	0	0	0	0
C₃	1	1.5	0	0	S₃	1	0	0	2	0	0	0
C₄	1	2	0	0	S₄	1	2	0	0	0.5	0	0
C₅	1	2.5	0	0	S₅	1	0	2	0	0.5	0	0
C₆	1	2	500	0	S₆	1	0	0	2	0.5	0	0
C₇	1	2	0	500	S₇	1	2	0	0	0.5	500	500
C₈	1	2	500	500	S₈	1	0	2	0	0.5	500	500
					S₉	1	0	0	2	0.5	500	500

Medium number	Callus induction				Medium number	Shoot regeneration
Medium number	MS	2,4-D (mg/L)	Proline (mg/L)	Glutamine (mg/L)	Medium number	MS	Kinetin (mg/L)	BAP (mg/L)	TDZ (mg/L)	NAA (mg/L)	Proline (mg/L)	Glutamine (mg/L)
C₁	1	0.5	0	0	S₁	1	2	0	0	0	0	0
C₂	1	1	0	0	S₂	1	0	2	0	0	0	0
C₃	1	1.5	0	0	S₃	1	0	0	2	0	0	0
C₄	1	2	0	0	S₄	1	2	0	0	0.5	0	0
C₅	1	2.5	0	0	S₅	1	0	2	0	0.5	0	0
C₆	1	2	500	0	S₆	1	0	0	2	0.5	0	0
C₇	1	2	0	500	S₇	1	2	0	0	0.5	500	500
C₈	1	2	500	500	S₈	1	0	2	0	0.5	500	500
					S₉	1	0	0	2	0.5	500	500

Callusing medium	Ambemohar	Indrayani	Jaya	Pawana	Average (Medium)
C₁	40.0 ± 0.48 a	65.2 ± 0.56 e	68.1 ± 0.52 f	78.1 ± 0.42 i	62.9
C₂	52.1 ± 0.42 b	72.3 ± 0.48 g	72.9 ± 0.49 g	83.7 ± 0.62 j	70.3
C₃	56.2 ± 0.62 c	74.0 ± 0.58 g	77.3 ± 0.78 h	87.2 ± 0.74 k	73.7
C₄	60.3 ± 0.54 d	79.2 ± 0.48 i	82.1 ± 0.62 j	91.9 ± 0.57 l	78.4
C₅	56.0 ± 0.62 c	74.8 ± 0.68 h	75.4 ± 0.56 h	90.4 ± 0.64 l	74.2
C₆	64.8 ± 0.69 e	83.8 ± 0.72 j	86.2 ± 0.48 j	94.8 ± 0.75 m	82.4
C₇	64.1 ± 0.79 e	84.1 ± 0.68 j	84.3 ± 0.62 j	95.2 ± 0.81 m	81.9
C₈	68.7 ± 0.58 f	86.2 ± 0.64 j	89.2 ± 0.72 k	97.0 ± 0.64 m	85.3
Average (Genotype)	57.8	77.5	79.4	89.8	76.1
Significance of two-way ANOVA
Medium (M)		P < 0.001
Genotype (G)		P < 0.001
M × G		P < 0.001

Callusing medium	Ambemohar	Indrayani	Jaya	Pawana	Average (Medium)
C₁	40.0 ± 0.48 a	65.2 ± 0.56 e	68.1 ± 0.52 f	78.1 ± 0.42 i	62.9
C₂	52.1 ± 0.42 b	72.3 ± 0.48 g	72.9 ± 0.49 g	83.7 ± 0.62 j	70.3
C₃	56.2 ± 0.62 c	74.0 ± 0.58 g	77.3 ± 0.78 h	87.2 ± 0.74 k	73.7
C₄	60.3 ± 0.54 d	79.2 ± 0.48 i	82.1 ± 0.62 j	91.9 ± 0.57 l	78.4
C₅	56.0 ± 0.62 c	74.8 ± 0.68 h	75.4 ± 0.56 h	90.4 ± 0.64 l	74.2
C₆	64.8 ± 0.69 e	83.8 ± 0.72 j	86.2 ± 0.48 j	94.8 ± 0.75 m	82.4
C₇	64.1 ± 0.79 e	84.1 ± 0.68 j	84.3 ± 0.62 j	95.2 ± 0.81 m	81.9
C₈	68.7 ± 0.58 f	86.2 ± 0.64 j	89.2 ± 0.72 k	97.0 ± 0.64 m	85.3
Average (Genotype)	57.8	77.5	79.4	89.8	76.1
Significance of two-way ANOVA
Medium (M)		P < 0.001
Genotype (G)		P < 0.001
M × G		P < 0.001

Callusing medium	Ambemohar	Indrayani	Jaya	Pawana
C₁	34.7 ± 0.35 a	54.6 ± 0.54 f	43.9 ± 0.42 c	81.9 ± 0.56 i
C₂	36.8 ± 0.44 a	56.3 ± 0.49 f	45.8 ± 0.58 c	85.5 ± 0.45 j
C₃	38.6 ± 0.56 b	58.6 ± 0.71 g	47.5 ± 0.52 d	87.8 ± 0.67 k
C₄	43.9 ± 0.54 c	60.9 ± 0.57 h	51.5 ± 0.44 e	90.7 ± 0.73 l
C₅	39.9 ± 0.48 b	59.6 ± 0.54 g	48.7 ± 0.48 d	88.8 ± 0.59 k
C₆	47.9 ± 0.57 d	63.8 ± 0.36 h	56.1 ± 0.63 f	93.5 ± 0.52 m
C₇	47.0 ± 0.48 d	62.8 ± 0.55 h	55.0 ± 0.57 f	92.0 ± 0.45 l
C₈	49.4 ± 0.36 e	65.9 ± 0.46 i	59.6 ± 0.53 g	94.9 ± 0.67 m
Average (Genotype)	42.3	60.3	51	89.4
Significance of two-way ANOVA
Medium (M)		P < 0.01
Genotype (G)		P < 0.001
M × G		P < 0.001