Efficient organogenesis and taxifolin production system from mature zygotic embryos and needles in larch

Xuetong Yan; Kejia Wang; Keyuan Zheng; Lifeng Zhang; Yang Ye; Liwang Qi; Mulan Zhu; Xuetong Yan; Kejia Wang; Keyuan Zheng; Lifeng Zhang; Yang Ye; Liwang Qi; Mulan Zhu

doi:10.48130/FR-2023-0004

Figures (3) Tables (4)

Figure 1.
Callus induction from needle explants and mature zygotic embyro explants in L. olgensis. (a) Sterilized stem sections for obtaining needle explants, bar = 0.7 cm. (b) Needle explants operated in DCR medium augmented with 3 mg·L⁻¹ 6-BA and 0.3 mg·L⁻¹ NAA, bar = 1 cm. (c) Calli from needle explants generated in 4 w, bar = 1 cm. (d) Calli from needle explants generated in 8 w, bar = 0.6 cm. (e) Sterilized mature zygotic embryo as explants, bar = 1 cm. (f) Calli from mature zygotic embryo explants generated in 3.0 mg·L⁻¹ 6-BA combined with 0.3 mg·L⁻¹ NAA, bar = 1 cm. (g) Calli from mature zygotic embryo explants generated in 4 w, bar = 1 cm. (h) Calli from mature zygotic embryo explants generated in 8 w, bar = 0.6 cm.
Figure 2.
Characteristic nature of Larix olgensis callus from different treatments. (a) DCR + 3 mg∙L⁻¹ 6-BA + 0.3 mg∙L⁻¹ NAA (90%+). (b) DCR+2 mg∙L⁻¹ 6-BA+0.2 mg∙L⁻¹ NAA. (c) DCR + 1 mg∙L⁻¹ 6-BA + 0.1 mg∙L⁻¹ NAA. (d) DCR + 3 mg∙L⁻¹ 6-BA + 0.3 mg∙L⁻¹ NAA, a genotype different from that in (a). (e) DCR + 1 mg∙L⁻¹ 6-BA + 1 mg∙L⁻¹ NAA. (f) DCR + 0.3 mg∙L⁻¹ 6-BA + 3 mg∙L⁻¹ NAA. (g) MS + 0.5 mg∙L⁻¹ 6-BA + 0.05 mg∙L⁻¹ NAA. (h) DCR + 0.5 mg∙L⁻¹ 6-BA + 0.05 mg∙L⁻¹ NAA. (i) DCR + 0.5 mg∙L⁻¹ 6-BA + 0.05 mg∙L⁻¹ NAA transfer to DCR + 2 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA. (j) DCR + 0.3 mg∙L⁻¹ 6-BA + 3 mg∙L⁻¹ NAA (in dark). (k) MS+0.05 mg∙L⁻¹ 6-BA + 0.5 mg∙L⁻¹ NAA (in dark). (l) MS + 3 mg∙L⁻¹ 6-BA + 0.3 mg∙L⁻¹ NAA (in dark). (m), (n) Modified high auxin culture, the yellow callus turn red. (o) After subculture over five times, the callus turned brown. The bar in the pictures is 0.32 cm except in (n) which is 0.43 cm.
Figure 3.
Summary of in vitro propagation from callus of Larix olgensis. (a) New shoots developed from callus placed in the DCR medium supplemented with 1.0 mg∙L⁻¹ BA and 0.1 mg∙L⁻¹ NAA, bar = 0.6 cm. (b) Shoot organogenesis occurring on callus after 8 weeks in regeneration medium, bar = 0.6 cm. (c) Developed shoots from callus, bar = 0.6 cm. (d) Adventitious shoots elongation, bar = 0.6 cm. (e) Further shoot elongation, bar = 0.68 cm. (f) Rooting of in vitro regenerated shoots in DCR medium supplemented with 3 mg∙L⁻¹ IBA and 100 mg∙L⁻¹ PG, bar = 0.83 cm. (g) Roots of fully developed plantlets, bar = 0.61 cm. (h) Acclimatized potted plants, bar = 2 cm.

Order	Basic medium	Plant growth regulators (mg∙L⁻¹)	Mature zygotic embryo explants			Needle explants
Order	Basic medium	Plant growth regulators (mg∙L⁻¹)	Callus induction rate (%)	Color	Texture	Callus induction rate (%)	Color	Texture
1	MS	6-BA 3:NAA 0.3	90.2 ± 0.11^a	Rose red	Compact	43.3 ± 1.91^bc	Brown	Compact
2		6-BA 2:NAA 0.2	90.1 ± 1.73^a	Pink	Compact	38.9 ± 1.13^bcd	Brown	Compact
3		6-BA 1:NAA 0.1	78.2 ± 4.45^b	Red and white	friable	35.6 ± 2.94^cdc	brown	Compact
4		6-BA 0.5:NAA0.05	52.1 ± 1.41^cd	Red and white	Friable	27.8 ± 1.11^fg	Brown	Friable
5		6-BA 1: NAA 1	51.6 ± 1.86^cd	Cream	Compact	43.3 ± 0^bc	Cream	Friable
6		6-BA 0.05:NAA 0.5	32.2 ± 1.68^f	Cream	Friable	38.9 ± 111^bcd	Yellow and green	Friable
7		6-BA 0.1:NAA 1	43.4 ± 3.41^de	Yellow and green	Friable	27.8 ± 1.11^fg	Yellow and green	Friable
8		6-BA 0.3:NAA 3	44.5 ± 3.26^de	Yellow and green	Friable	13.3 ± 1.93^h	Yellow and green	Compact
9	DCR	6-BA 3:NAA 0.3	96.8 ± 1.86^a	Rose red	Compact	86.7 ± 1.93^a	Rose red	Compact
10		6-BA 2:NAA 0.2	92.2 ± 0.97^a	Rose red	Compact	46.7 ± 1.93^b	Green	Friable
11		6-BA 1:NAA 0.1	76.1 ± 1.94^b	Pink	Friable	22.2 ± 1.11^gh	Yellow and green	Friable
12		6-BA 0.5:NAA0.05	49.0 ± 5.35^cd	Pink	Friable	14.55 ± 2.22^h	Dark brown	Friable
13		6-BA 1: NAA 1	51.2 ± 2.46^cd	Red and white	Compact	32.2 ± 1.11^efg	Green	Compact
14		6-BA 0.05:NAA 0.5	36.6 ± 3.51^ef	Red and white	Friable	34.5 ± 7.78^cde	Cream	Friable
15		6-BA 0.1:NAA 1	50.1 ± 2.50^cd	Red and white	Compact	32.2 ± 8.89^efg	Yellow and green	Compact
16		6-BA 0.3:NAA 3	55.7 ± 4.93^c	Cream	Compact	23.3 ± 0^gh	Yellow and green	Compact
Means ( ± standard error) within a column followed by the same superscript letter are not significantly different using Tukey’s multiple comparison test and p ≤ 0.05.

Table 1.

Induction percentage and characteristics of callus from mature zygotic explants and needle explants of Larix olgensis.

			Mature zygotic embryo explants		Needle explants
	Plant growth regulators (mg∙L⁻¹)		Percentage shoot regeneration (%)	Average number of adventitious shoots	Percentage shoot regeneration (%)	Average number of adventitious shoots
	6-BA	NAA	Percentage shoot regeneration (%)	Average number of adventitious shoots	Percentage shoot regeneration (%)	Average number of adventitious shoots
1	0.5	0.05	41.1 ± 2.94^fg	304 ± 7.5^d	32.22 ± 1.11^de	120 ± 6.1^h
2	0.5	0.1	42.2 ± 1.11^fg	315 ± 4.5^d	32.22 ± 2.22^de	111 ± 3.8^h
3	0.5	0.2	32.2 ± 1.11^h	150 ± 14.8^f	28.89 ± 1.11^e	66 ± 2.3ⁱ
4	0.5	0.5	40.0 ± 1.93^g	211 ± 5.5^e	34.44 ± 1.11^d	51 ± 1.8^g
5	1	0.05	57.8 ± 1.11^d	426 ± 13.9^c	68.89 ± 1.11^a	161 ± 6.2^f
6	1	0.1	83.3 ± 1.93^a	528 ± 11.5^b	73.33 ± 1.93^a	307 ± 1.8^b
7	1	0.2	76.7 ± 1.93^b	636 ± 21.7^a	72.22 ± 1.11^a	323 ± 3.8^a
8	1	0.5	65.6 ± 2.22^c	460 ± 22.8^c	73.33 ± 1.93^a	221 ± 6.4^c
9	2	0.05	53.3 ± 3.85^de	322 ± 11.7^d	50.00 ± 1.93^c	176 ± 5.5^e
10	2	0.1	47.8 ± 2.94^ef	237 ± 11.5^d	51.11 ± 1.11^c	141 ± 7.0^g
11	2	0.2	46.7 ± 1.93^efg	423 ± 23.5^c	57.78 ± 2.22^b	203 ± 3.9^d
12	2	0.5	47.8 ± 1.11^ef	200 ± 6.1^e	33.33 ± 1.93^de	57 ± 1.2^ig
Means (± standard error) within a column followed by the same superscript letter are not significantly different using Tukey’s multiple comparison test and p ≤ 0.05.

Table 2.

Percentage shoot regeneration from calli of Larix olgensis.

	Plant growth regulators and AC (mg∙L⁻¹)	Adventitious shoot elongation percentage (%)	Average shoot length (cm)
1	6-BA 1:NAA 0.1	21.1 ± 2.22^e	0.9 ± 0.03^e
2	6-BA 0.5:NAA 0.05	65.6 ± 1.11^b	1.6 ± 0.06^d
3	6-BA 0.5:NAA 0.05:AC 2000	75.6 ± 2.94^a	3.5 ± 0.11^a
4	6-BA 0.3:NAA 0.03	46.7 ± 1.93^c	1.3 ± 0.03^d
5	6-BA 0.3:NAA 0.03:AC 2000	61.1 ± 1.11^b	2.8 ± 0.12^b
6	6-BA 0.1:NAA 0.01	21.1 ± 1.11^e	1.4 ± 0.01^d
7	6-BA 0.1:NAA 0.01:AC 2000	35.6 ± 2.22^d	2.2 ± 0.10^c
Means (± standard error) within a column followed by the same superscript letter are not significantly different using Tukey’s multiple comparison test and p ≤ 0.05.

Table 3.

Effect of different concentrations of 6-BA and NAA on adventitious bud elongation of Larix olgensis.

Basic medium	Exogenous additives (mg∙L⁻¹)	Adventitious root induction percentage (%)	Rooting start time (d)
DCR	NAA 3	8.9 ± 2.22^e	70
	IBA 2:NAA 2	15.6 ± 2.22^de	64
	IBA 3:AC 2000	46.7 ± 3.85^b	52
	IBA 3	13.3 ± 0.00^e	63
	IBA 3:PG 50	33.3 ± 3.85^c	45
	IBA 3:PG 100	62.2 ± 5.88^a	38
	IBA 3:PG 150	57.8 ± 4.44^ab	36
	IBA 3:PG 100:AC 2000	53.3 ± 3.85^b	32
1/2DCR	NAA 3	15.56 ± 2.22^de	70
	IBA 2:NAA 2	22.22 ± 2.22^d	63
	IBA 3:AC 2000	35.56 ± 2.22^c	60
	IBA 3	11.11 ± 2.22^e	60
	IBA 3:PG 50	31.11 ± 4.44^c	50
	IBA 3:PG 100	48.89 ± 2.22^b	41
	IBA 3:PG 150	46.67 ± 0^b	40
	IBA 3:PG 100:AC 2000	37.78 ± 2.22^c	36
Means (± standard error) within a column followed by the same superscript letter are not significantly different using Tukey’s multiple comparison test and p ≤ 0.05.

Table 4.

Rooting of regenerated shoots in DCR media supplemented with auxin, AC and PG strength.