Optimization of the tissue culture technology system of Mytilaria laosensis trees

doi:10.12302/j.issn.1000-2006.202303008

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2024, Vol. 48 ›› Issue (2): 69-78.doi: 10.12302/j.issn.1000-2006.202303008

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Optimization of the tissue culture technology system of Mytilaria laosensis trees

SUN Xugao(), TAO Jialu, XIE Wei, SHI Jie, ZHANG Baojin, DENG Xiaomei^*()

College of Forestry and Landscape Architectural, Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, South China Agricultural University, Guangzhou 510642, China

Received:2023-03-06 Revised:2023-04-25 Online:2024-03-30 Published:2024-04-08

Abstract

Abstract:

【Objective】 Mytilaria laosensis is an evergreen, broad-leaved tree with a wide range of suitable habitats and good economic and ecological protection. The seedling is mostly used for cultivation. Grafting and cutting techniques are still experimental, and few reports on tissue culture techniques exist. Therefore, it is of great significance to establish an efficient tissue culture technology system for M. laosensis and to provide technical support for promoting its clone in the future. 【Method】 With the semi-lignified bud at the base of the superior tree as explants, through the processes of primary axillary bud induction, secondary proliferation culture, rooting culture, the optimal explant collection time, the basic breeding and rooting culture medium, and the type and concentration ratio of plant growth regulator were studied. 【Result】 There were significant differences in the effect of different sampling times on the induction of M. laosensis. The best induction of explants was collected in October 2022, with a survival rate of 32.38%. Treatment with different hormone concentrations significantly affected the time required for bud germination and the growth status of explants. The optimal formula for axillary bud induction was MS+6-BA 0.5 mg/L+NAA 0.02 mg/L, the induction rate was 100%, and the germination time was 4.07 d. The selection of different basic mediums significantly affected the proliferation of M. laosensis. MX medium had the highest average shoot length (4.47 cm) and the largest proliferation coefficient (4.41), respectively, which were significantly higher than those of other basic media (1/2 MS, MS, DKW, WPM), and the proliferation buds grew strongly, increased quickly and had a good effect. Different hormone types and treatment concentrations significantly affected the proliferation and rooting effects of M. laosensis. The optimal breeding medium was MX (modified WPM) + 6-BA 1.0 mg/L+NAA 0.05 mg/L, and the proliferation coefficient reached 4.94. The buds grow vigorously. The optimal rooting medium was MX+NAA 0.3 mg/L, the rooting rate was 100%, the average root number was 13.18, root growth was robust, and the transplanting survival rate reached 92.3%. 【Conclusion】 The induction survival rate of M. laosensis explants was related to the location and time of material collection. October was the best time to collect explant material in Guangdong. In the case of a low proliferation coefficient of the MS basic medium, switching to an MX (modified WPM) medium could significantly increase the proliferation succession factor. Then, the induction medium, breeding medium, and rooting medium were optimized, which helped establish the tissue culture system of the superior tree of M. laosensis and improved the quality and efficiency of the tissue culture.

Key words: Mytilaria laosensis, basal medium, multiple shoots, tissue culture

CLC Number:

S722.37

SUN Xugao, TAO Jialu, XIE Wei, SHI Jie, ZHANG Baojin, DENG Xiaomei. Optimization of the tissue culture technology system of Mytilaria laosensis trees[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY, 2024, 48(2): 69-78.

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编号 No.	成分 composition	质量浓度/(mg·L^-1) concentration	编号 No.	成分 composition	质量浓度/(mg·L^-1) concentration
1	NH₄NO₃	720.00	12	CuSO₄·5H₂O	0.25
2	KNO₃	400.00	13	NiSO₄·6H2O	0.05
3	Ca(NO₃)₂·2H₂O	660.00	14	CoCl₂	0.025
4	MgSO₄·7H₂O	370.00	15	FeSO₄·7H₂O	27.80
5	KH₂PO₄	170.00	16	Na₂·EDTA·H₂O	37.30
6	KCl	65.00	17	Myo-Insitol	150.00
7	MnSO₄·4H₂O	22.30	18	Glycine	2.00
8	ZnSO₄·7H₂O	8.60	19	Thiamine·HCl	0.50
9	H₃BO₃	6.20	20	Nicotinic·acid	0.20
10	KI	0.83	21	Pyridoxine·HCl	0.20
11	Na₂MoO₄·2H₂O	0.25

指标 index	平方和 SS	自由度 df	均方 MS	F	P
污染率 pollution rate	764.584	3	254.861	17.84	0.001
褐化率 browning rate	481.057	3	160.352	5.237	0.027
存活率 survival rate	1 314.114	3	438.038	29.285	<0.001

培养基 culture medium code	生长状况 growth status of plantlets
A₀	芽生长慢,叶片大,茎伸长缓慢
A₁	芽生长快,叶绿且茎伸长明显
A₂	芽生长较快,生长良好,基部有少量愈伤
A₃	芽萌出后,基部愈伤多,苗不伸长
A₄	芽生长一般,基部愈伤明显,苗伸长慢

指标 index	平方和 SS	自由度 df	均方 MS	F	P
平均芽长 mean bud length	44.655	4	11.164	221.546	<0.001
增殖系数 growth coefficient	20.584	4	5.146	108.617	<0.001

基本培养基 basal medium	生长状况 growth status of plantlets
1/2 MS	叶色翠绿,芽较多但短小
MS	叶色深绿,芽较多但短小,出现玻璃化现象
DKW	叶色偏黄,芽细长,叶片开展
1/4 MS	叶片开展、黄绿色,芽多,茎较粗壮
MX	叶片开展,叶色翠绿,芽多且粗壮,高生长快

Optimization of the tissue culture technology system of Mytilaria laosensis trees

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试验号 test code	质量浓度/(mg·L^-1) concentration
试验号 test code	6-BA	NAA
A₀	0	0
A₁	0.5	0.02
A₂	0.5	0.05
A₃	1.0	0.05
A₄	2.0	0.10

指标 index	变异来源(因素) source of variation(factor)	平方和 SS	自由度 df	均方 MS	F	P
增殖系数 multiplication coefficient	6-BA浓度 6-BA concentration	0.855	2	0.427	6.162	0.009
	NAA浓度 NAA concentration	3.687	2	1.843	26.57	<0.001
	NAA浓度×6-BA浓度 6-BA concentration× NAA concentration	2.334	4	0.583	8.409	0.001
平均芽长 mean bud length	6-BA浓度 6-BA concentration	8.623	2	4.312	55.442	<0.001
	NAA浓度 NAA concentration	2.379	2	1.189	15.295	<0.001
	NAA浓度×6-BA浓度 6-BA concentration× NAA concentration	1.39	4	0.348	4.47	0.011

试验号 treatment cade	因素水平 factor level		评价指标 evaluating indicator
试验号 treatment cade	6-BA	NAA	增殖系数 multiplication coefficient	平均芽长/cm mean sprout length	生长状况 growth status of plantlets
B₁	1	1	4.14±0.21 b	4.07±0.25 b	良
B₂	2	1	4.32±0.37 b	3.39±0.22 c	中等
B₃	3	1	4.23±0.35 b	2.43±0.24 d	较差
B₄	1	2	4.43±0.31 b	4.05±0.33 b	较差
B₅	2	2	4.94±0.18 a	4.62±0.34 a	优
B₆	3	2	4.17±0.25 b	3.37±0.27 c	中等
B₇	1	3	4.42±0.17 b	4.15±0.33 ab	中等
B₈	2	3	3.55±0.22 c	3.83±0.17 bc	差
B₉	3	3	3.12±0.24 c	2.67±0.32 d	差
K1	4.33/4.09	4.23/3.30
K2	4.27/3.95	4.51/4.01
K3	3.84/2.82	3.70/3.55
R	0.49/1.24	0.81/0.71

指标 index	平方和 SS	自由度 df	均方 MS	F	P
平均根长 mean root length	7.852	4	1.963	49.02	0.001
生根系数 rooting coefficient	63.926	4	15.982	95.136	0.001
生根率 rooting rate	1515.305	4	378.826	31.124	0.001
苗高 plant height	3.034	4	0.759	23.654	0.001

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