Establishing an Efficient Regeneration Protocol in vitro for Rapid Micro-propagation of Two New Varieties of Ilex verticillata L.

doi:10.7525/j.issn.1673-5102.2021.02.010

Abstract

Abstract:

An efficient and rapid micro-propagation system using tissue culture for two new varieties “Red Sprite” and “Winter Gold” of Ilex verticillata L. was established with the young shoots as explants， and an optimum method produced large numbers of genetically uniform seedlings and maintained its excellent character for I.verticillata L was offered. The optimal medium for axillary bud induction was MS+1.0 mg·L^-1 6-BA+0.2 mg·L^-1 NAA， and the induction rate of both varieties were 100% respectively. The preferred medium for cluster bud proliferation was MS+0.1 mg·L^-1 NAA+0.5 mg·L^-1 6-BA+0.5 mg·L^-1 CPPU. The highest proliferation coefficient was 5.7 for “Red Sprite”， and 4.5 for “Winter Gold” for 25 d culturing respectively. The optimal seedling medium for “Red Sprite” was MS+0.1 mg·L^-1 NAA+0.5 mg·L^-1 6-BA+1.0 mg·L^-1 TDZ+1.0 mg·L^-1 GA， and that of “Winter Gold” was MS+0.1 mg·L^-1 NAA+0.5 mg·L^-1 6-BA+0.05 mg·L^-1 TDZ+1.0 mg·L^-1 GA， the seedling height was up to 6.80 and 4.38 cm respectively. The optimal rooting induction medium was 1/2MS+0.4 mg·L^-1 NAA+0.4 mg·L^-1 IBA+1.0 mg?L^-1 activated carbon， the rooting rate of “Red Sprite” and “Winter Gold” were 95.0%， or 97.0% respectively. The best transplanting substrates were peat∶perlite=3∶2， with a survival rate of 95.7% for “Red Sprite” and 96.8% for “Winter Gold” respectively.

Key words: Ilex verticillata L., varieties of “Red Sprite”, varieties of “Winter Gold”, micropropagation, an efficient regeneration protocol

CLC Number:

Rui LIU, Jia-Yi ZHANG, Chao-Qi GE, Lu-Lu XIE, Ying CHEN, Can ZHANG, Xi CHEN. Establishing an Efficient Regeneration Protocol in vitro for Rapid Micro-propagation of Two New Varieties of Ilex verticillata L.[J]. Bulletin of Botanical Research, 2021, 41(2): 221-231.

Figures/Tables 9

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编号 No.	品种 Variety	6-BA (mg·L^-1)	NAA (mg·L^-1)	CPPU (mg·L^-1)	芽数(个)/瓶 Shoots of per bottle	增殖系数 Proliferation coefficient
1	“Red Sprite”	0.1	0.1	0.0	21.0±2.6d	2.7±0.1c
2		0.1	0.5	0.5	27.7±2.5c	3.3±0.2c
3		0.1	1.0	1.0	40.7±2.1b	4.2±0.2b
4		0.5	0.1	0.5	57.0±2.6a	5.7±0.3a
5		0.5	0.5	1.0	42.3±1.5b	4.8±0.5b
6		0.5	1.0	0.0	27.3±2.1c	3.2±0.4c
7		1.0	0.1	1.0	30.0±1.7c	4.3±0.7b
8		1.0	0.5	0.0	32.0±1.0cd	4.3±0.2b
9		1.0	1.0	0.5	28.7±1.5c	3.2±0.2c
水平1平均Average of level 1		3.4	4.23	3.4
水平2平均Average of level 2		4.57	4.13	4.07
水平3平均Average of level 3		3.93	3.53	4.4
极差Range(R)		1.17	0.7	1.03
10	“Winter Gold”	0.1	0.1	0.0	9.0±1.0cd	1.1±0.0e
11		0.1	0.5	0.5	9.0±1.0cd	1.5±0.3d
12		0.1	1.0	1.0	17.7±0.4b	2.9±0.3b
13		0.5	0.1	0.5	28.3±1.54a	4.5±0.4a
14		0.5	0.5	1.0	18.0±0.8b	3.0±0.3b
15		0.5	1.0	0.0	8.0±1.0d	1.8±0.3c
16		1.0	0.1	1.0	29.0±1.0a	3.5±0.3b
17		1.0	0.5	0.0	9.3±0.6cd	1.4±0.1d
18		1.0	1.0	0.5	6.7±0.6e	1.3±0.1d
水平1平均Average of level 1		1.83	3.03	1.43
水平2平均Average of level 2		3.10	1.97	2.43
水平3平均Average of level 3		2.07	2.00	3.13
极差Range(R)		1.27	1.07	1.70

编号 No.	品种 Variety	TDZ (mg·L^-1)	GA (mg·L^-1)	苗高 High of shoot(cm)	增殖系数 Proliferation coefficient	生长状况 Growth states
1	“Red Sprite”	0.05	1.0	5.8±0.6b	3.4±0.5c	茎干略有增粗和伸长，叶尖略有枯黄，部分叶片明显枯黄 The stems in the treatment were slightly thickening and elongation,the leaf tip or whole leaves obviously yellowed
2		0.25	1.0	5.4±0.8b	3.0±0.5c	茎干略有增粗和伸长，叶尖略有枯黄 The stems in the treatment were slightly thickening and elongation,the leaf tip slightly yellowed
3		0.50	1.0	6.2±0.6ab	4.1±0.6b	茎干的增粗和伸长较为明显，叶片鲜绿 The stems in the treatment were observed significant thickening and elongation,the leaves were observed bright green
4		1.00	1.0	6.8±0.7a	4.6±0.3a	茎干明显增粗和伸长，叶片浓绿，新生叶片多于前3种 The stems in the treatment were significantly thickening and elongation,and more leaves than the first three teatments, leaves were thick green
5	“Winter Gold”	0.05	1.0	4.4±0.7a	3.1±0.4a	茎干明显增粗和伸长，叶片鲜绿 The stems in the treatment were observed significant thickening and elongation,the leaves were observed bright green
6		0.25	1.0	3.7±0.1b	1.9±0.3b	茎干略有增粗，伸长明显，叶片鲜绿 The stems in the treatment were observed significant thickening and elongation,the leaves were observed bright green
7		0.50	1.0	3.5±0.2b	1.8±0.4b	茎干明显增粗，无明显伸长，叶缘发黄 The stems in the treatment were observed significant thickening, but not significant elongation, the leaf tip slightly yellowed
8		1.00	1.0	3.4±0.3b	1.6±0.2b	茎干无明显增粗和伸长，叶缘发红 The stems in the treatment were observed no significant thickening and elongation,the leaf tip slightly reded

编号 No.	品种 Variety	NAA (mg·L^-1)	AC (g·L^-1)	IBA (mg·L^-1)	芽苗高度 High of shoot(cm)	根数(条) Roots number	生根率 Rate of rooting(%)
1	“Red Sprite”	0.4	0.0	0.0	3.1±0.3d	4.0±1.4d	25.0e
2		0.4	0.5	0.0	2.9±0.5d	2.9±0.8e	66.7cd
3		0.4	1.0	0.0	4.3±0.4cd	6.3±0.9bc	66.7cd
4		0.4	2.0	0.0	2.6±0.5e	3.4±0.9d	56.4d
5		0.1	1.0	0.0	4.6±0.2c	6.9±1.2bc	75.0c
6		1.0	1.0	0.0	4.0±0.4cd	3.8±0.5d	50.0d
7		0.0	1.0	0.1	2.8±0.1de	11.3±1.0a	48.3d
8		0.0	1.0	0.4	3.0±0.3d	5.7±0.6c	56.7d
9		0.0	1.0	1.0	2.8±0.2d	4.7±0.6d	75.7c
10		0.4	1.0	0.4	5.5±1.0b	7.0±1.6bc	95.0a
11		0.1	1.0	1.0	6.6±1.0a	3.5±0.6d	80.0b
12		1.0	1.0	1.0	3.6±0.8cd	8.6±1.1b	100.0a

编号 No.	品种 variety	NAA (mg·L^-1)	AC (g·L^-1)	IBA (mg·L^-1)	芽苗高度 High of shoot(cm)	根数(条) Number of root	生根率 Rate of rooting(%)
1	“Winter Gold”	0.4	0	0	2.0±0.1d	1.8±0.3e	25.0e
2		0	1.0	0.1	3.8±0.3c	11.8±0.9a	33.3e
3		0	1.0	0.4	4.2±0.2b	9.9±1.6b	66.7c
4		0	1.0	1.0	4.0±0.4b	9.3±1.8b	69.2c
5		0.1	1.0	0	3.3±0.3c	3.5±0.6d	50.0d
6		0.4	1.0	0	5.2±0.5a	7.0±1.3c	87.5b
7		1.0	1.0	0	3.1±0.1c	3.8±0.5d	50.0d
8		0.4	1.0	0.4	5.0±1.4a	7.0±1.4c	97.0a
9		0.1	1.0	0.4	4.9±1.2a	5.5±0.6d	90.0b
10		1.0	1.0	1.0	3.9±0.9b	9.0±1.2b	67.0c

编号 No.	品种 Variety	栽培基质 Media	移栽成活率 Rate of survival(%)
1	“Red Sprite”	泥炭土∶花园土∶珍珠岩∶蛭石=3∶1∶3∶3 Peat∶garden soil∶perlite=3∶1∶3∶3	82.7±5.9b
2		泥炭土∶花园土∶珍珠岩∶蛭石=4∶1∶2∶3 Peat∶garden soil∶perlite∶vermiculite=4∶1∶2∶3	85.7±6.8a
3		泥炭土∶花园土∶珍珠岩∶蛭石=5∶1∶1∶3 Peat∶garden soil∶perlite∶vermiculite=5∶1∶1∶3	73.3±4.0c
4		泥炭土∶花园土∶珍珠岩=3∶1∶2 Peat∶garden soil∶perlite=3∶1∶2	92.3±5.1b
5		泥炭∶珍珠岩=3∶2 Peat∶perlite=3∶2	95.7±3.2b
6	“Winter Gold”	泥炭土∶花园土∶珍珠岩∶蛭石=3∶1∶3∶3 Peat∶garden soil∶perlite∶vermiculite=3∶1∶3∶3	74.3±2.9b
7		泥炭土∶花园土∶珍珠岩∶蛭石=4∶1∶2∶3 Peat∶garden soil∶perlite∶vermiculite=4∶1∶2∶3	80.0±2.1a
8		泥炭土∶花园土∶珍珠岩∶蛭石=5∶1∶1∶3 Peat∶garden soil∶perlite∶vermiculite=5∶1∶1∶3	63.3±43.c
9		泥炭土∶花园土∶珍珠岩=3∶1∶2 Peat∶garden soil∶perlite=3∶1∶2	88.2±6.1b
10		泥炭土∶珍珠岩=3∶2 Peat∶perlite=3∶2	96.8±3.1b