Regeneration and Agrobacterium-mediated genetic transformation of twelve <i>Eucalyptus</i> species

Tingting Zhou; Ying Lin; Yan Lin; Jianzhong Luo; Jihua Ding; Tingting Zhou; Ying Lin; Yan Lin; Jianzhong Luo; Jihua Ding

doi:10.48130/FR-2022-0015

Figures (4) Tables (4)

Figure 1.
Regeneration efficiency of different eucalypt species and genotypes using seeding hypocotyl as explants. (a) Representative image of hypocotyl explants regeneration on SIM media at different stages. Photo were taken every other week. Bar = 2 mm. (b) Regeneration efficiency was recorded based on the regeneration ratio. The regeneration rate is defined as a/b × 100%, where a is the number of explants forming shoots after four weeks of screening, and b is the number of explants before the screening. Data shown are mean values from two biological replicates. Error bars ± SE.
Figure 2.
Regeneration efficiency of different eucalyptus species and genotypes using clonal internode as explants. (a) Representative image of clonal internode explants regeneration on SIM media at different stages. Photos were taken every other week. Bar = 2 mm. (b) Regeneration efficiency was recorded based on the regeneration ratio. The regeneration rate is defined as a/b × 100%, where a is the number of explants forming shoots after four weeks of screening, and b is the number of explants before the screening. Data shown are mean values from two biological replicates. Error bars ± SE.
Figure 3.
Transformation efficiency of different eucalyptus species and genotypes. (a) T-DNA region of pCAMBIA2300::35S::DsRed2 vector for genetic transformation. The chimeric neomycin phosphotransferase II (NPT II) selection marker and the reporter gene DsRed2 were driven by cauliflower mosaic virus 35S promoter. LB and RB indicate T-DNA left and right border, respectively. Arrows indicate the direction of transcription. (b) Fluorescence observation of callus. Callus induction one month after Agrobacterium infection were observed at white light and red light with the fluorescence stereo-microscope. Bar = 1 cm. (c), (d) Transformation efficiency calculation of different eucalyptus species and genotypes using seeding hypocotyl and clonal internode as explants, respectively. Transformation efficiency was recorded based on the fluorescence callus of each explant. The transformation rate is defined as a/b × 100%, where a is the number of explants having fluorescence callus after one month of screening, and b is the total number of explants. Data shown are mean values from two biological replicates. Error bars ± SE.
Figure 4.
Agrobacterium-mediated eucalyptus genic transformation with DsRed2 as reporter gene. (a) Flow diagram for agrobacterium-mediated eucalyptus genic transformation. The yellow arrow represents the process direction of transformation. The blue circles with notes represent important transformation steps and time required (the media information of each step is listed in Supplemental Table S2). (b) DsRed2 gene expression in different tissue and organs in transgenic plants. Photos were taken at white light and red light with the fluorescence stereo-microscope. E. robusta (RO1) plants that untransformed were used as a negative control (Negative). Successful transformed plants were marked with Positive. Bar = 1 mm. (c) Positive test of DsRed2 transgenic plants using PCR amplification. M, DNA marker; (+), Positive control with transformation vector as the PCR template; (−), wild type plants; CA1 and RO1, genotypes of E. camaldulen and E. robusta respectively used for transformation donor in this study. The number represents the independent transgenic line.

Species	Clone	SIM1				SIM3
Species	Clone	Total	Shoot induction	Regeneration efficiency (%)	Differentiation amount	Total	Shoot induction	Regeneration efficiency (%)	Differentiation amount
E.urophylla	UR1	81	68	83.96 ± 0.04a	***	50	41	82.00 ± 6.00^a	***
	UR2	41	9	22.02 ± 2.98^ghi	*	56	11	18.57 ± 4.29^ijkl	*
	UR3	78	29	41.94 ± 8.06^def	***	67	28	51.04 ± 17.71^bcde	***
	UR4	35	2	5.95 ± 1.19^ijk	*	44	15	34.58 ± 5.42^efghi	**
	UR5	33	20	61.03 ± 13.97^bc	*	38	15	38.52 ± 9.10^defg	*
E.grandis	GR1	53	43	82.74 ± 7.74^a	***	49	40	81.67 ± 1.67^a	**
	GR2	87	35	40.06 ± 1.60^def	**	38	47	39.47 ± 7.89^cdef	*
	GR3	162	28	18.08 ± 3.23^hij	*	80	6	7.39 ± 2.13^kl	*
E.pellita	PE1	28	5	20.56 ± 9.44^hi	**	21	5	24.09 ± 5.91^fghijk	**
	PE2	71	40	58.97 ± 6.03^bc	**	65	22	34.25 ± 1.75^efghi	**
	PE4	43	1	1.56 ± 1.56^jk	*	66	0	0.00 ± 0.00^l
E.robusta	RO1	112	96	84.76 ± 3.81^a	***	87	43	40.42 ± 13.75^cdef	***
E.tereticornis	TE1	47	26	56.58 ± 6.58^bcd	***	44	17	39.11 ± 2.07^defg	**
	TE2	60	13	21.06 ± 2.01^hi	**	61	12	19.72 ± 0.97^hijk	*
	TE3	39	15	38.55 ± 3.55^efg	**	40	22	55.00 ± 5.00^bcd	***
E.camaldulen	CA1	102	65	64.83 ± 11.26^b	***	76	57	68.01 ± 15.63^ab	***
E.exserta	EX1	85	20	21.45 ± 9.32^hi	*	62	12	20.41 ± 6.52^ghijk	**
E.dunnii	DU1	186	88	47.13 ± 1.45^cde	**	192	67	34.74 ± 2.52^efghi	**
E.globulus	GL1	54	14	26.02 ± 0.30^fgh	*	65	8	12.67 ± 2.14^jkl	*
	GL4	107	31	28.42 ± 8.42^fgh	*	99	0	0.00 ± 0.00^l
	GL5	98	5	3.85 ± 3.85^jk	*	92	0	0.00 ± 0.00^l
E.benthamii	BE2	190	33	17.12 ± 0.45^hij	**	27	16	58.57 ± 1.43^bc	**
	BE3	79	36	45.29 ± 2.44^cde	***	88	26	28.57 ± 3.57^fghij	**
E.citriodora	CI1	122	0	0.00 ± 0.00^k		92	0	0.00 ± 0.00^l
E.variegate	VA1	123	0	0.00 ± 0.00^k		109	0	0.00 ± 0.00^l
	VA2	125	0	0.00 ± 0.00^k		116	0	0.00 ± 0.00^l
Mean values of two independent experiments ( ± ) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level. Shoots number of each explant were counted and classified (, < five shoots; five shoots < * < ten shoots; ***, > ten shoots).

Table 1.

Regeneration rate of shoots induced by hypocotyl as explants in different eucalyptus species or genotypes.

Species	Clone	SIM1				SIM3
Species	Clone	Total	Shoot induction	Regeneration efficiency (%)	Differentiation amount	Total	Shoot induction	Regeneration efficiency (%)	Differentiation amount
E.urophylla	UR1	80	40	50.51 ± 5.05^b	***	92	37	40.19 ± 0.19^bc	**
	UR3	46	4	9.58 ± 2.92^d	**	36	5	14.04 ± 0.25^d	*
	UR5	57	21	36.70 ± 8.13^bc	*	71	14	19.20 ± 4.49^cd	*
E.grandis	GR1	41	11	21.85 ± 7.56^cd	**	49	12	28.29 ± 5.04^cd	***
E.pellita	PE2	70	24	34.85 ± 1.52^bc	**	63	38	59.55 ± 16.21^ab	**
E.robusta	RO1	125	85	70.63 ± 8.91^a	**	111	64	56.33 ± 2.49^ab	***
E.camaldulen	CA1	117	97	84.01 ± 3.49^a	***	74	57	77.16 ± 0.97^a	**
Mean values of two independent experiments ( ± ) with standard errors. Values with the same letter within columns are not significantly different according to Duncan's Multiple Range Test (DMRT) at a 5% level. Shoots number of each explant were counted and classified (, < five shoots; five shoots < * < ten shoots; ; ***, > ten shoots).

Table 2.

Regeneration rate of shoots induced by stem segments as explants in different eucalypt species or genotypes.

Species	Clone	Explant	Total	Red fluorescence	Transformation efficiency (%)
E. robusta	RO1	Hypocotyl	177	89	61.48 + 4.76^a
E. camaldulen	CA1	Hypocotyl	71	31	50.55 + 2.40^a
	CA2	Hypocotyl	63	19	29.68 + 2.76^b
E. dunnii	DU1	Hypocotyl	159	45	27.30 + 6.40
E. urophylla	UR3	Hypocotyl	73	13	13.75 + 6.60^c
	UR4	Hypocotyl	93	8	8.07 + 1.62^c
E. pellita	PE2	Hypocotyl	73	6	9.19 + 1.92^c
E. grandis	GR1	Hypocotyl	41	4	9.13 + 1.98^c
E. robusta	RO1	Internode	48	11	22.78 + 5.47^a
E. camaldulen	CA1	Internode	475	124	25.46 + 4.40^a
Mean values of two independent experiments (±) with standard errors. Values with the same letter within columns are not significantly different according to Duncan’s Multiple Range Test (DMRT) at a 5% level.

Table 3.

Transformation efficiency test of different eucalypt species by monitoring the red fluorescence rate of the callus after transformed with the reporter DsRed2.

Genotype	Construct	Explant type	Explant number	Positive plants	Transformation efficiency (%)
E. robusta (RO1)	35S::DsRed	Hypocotyl	77	3	3.9
E. robusta (RO1)	35S::DsRed	Hypocotyl	67	1	1.5
E. camaldulen (CA1)	35S::DsRed	Hypocotyl	55	1	1.9
E. robusta (RO1)	35S::EgFT	Internode	51	2	3.9
E. camaldulen (CA1)	35S::EgFT	Internode	69	1	1.5

Table 4.

Transformation efficiency test of E.robusta and E.camaldulen using hypocotyl and internode as explants respectively.