刺槐离体叶片高效再生体系的优化

doi:10.11707/j.1001-7488.LYKX20210980

Abstract

Abstract:

Objective: Leaves are rich in sources, easy to sample, and have little impact on the plant after collection, such they are ideal materials for plant asexual reproduction. However, the regeneration efficiency ofRobinia pseudoacacia leaves is not high enough to meet the requirements of genetic transformation. In this study, an efficient regeneration system of R. pseudoacacia leaves in vitro was established by optimizing the important factors in the regeneration process, which would lay a good foundation for R. pseudoacacia seedling reproduction, mutagenesis breeding, genetic transformation and further development of molecular breeding of R. pseudoacacia. Method: The isolated leaves of R. pseudoacacia tissue culture seedlings were used as explants, the whole process of regeneration of adventitious buds from the isolated leaves was observed to investigate the effects of genotype, sampling period, dark treatment duration as well as plant growth regulators on the regeneration of adventitious buds fromR. pseudoacacia leaves. Meanwhile, the IBA concentration suitable for rooting of adventitious buds was screened. Result: 1) Diploid genotype Pu-18-22 had the best regeneration ability; 2) Subculture of tissue culture seedlings for 45 days was the best time for selecting explants; 3) Darkness treatment for 14 days was the best darkness treatment time; 4) The optimal medium for adventitious bud regeneration was MS (culture medium) + N-(Phenylmethyl)-9H-purin-6-amine 3.0 mg·L^?1 + Kinetin 1.0 mg·L^?1 + 2,4-Dichlorophenoxyacetic acid 0.1 mg·L^?1 + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1; The adventitious bud induction rate of Pu-18-22 was 96.67%, and 7.4 adventitious buds were regenerated per leaf on average; 5) The best rooting medium for adventitious buds was 1/2MS + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1 ＋ 3-Indolebutyric acid 0.3 mg·L^?1, and the rooting rate was 100%. Conclusion: The leaves of diploid R. pseudoacacia Pu-18-22 tissue culture plantlets are used as explants that are inoculated into MS culture medium + N-(Phenylmethyl)-9H-purin-6-amine 3.0 mg·L^?1 + Kinetin 1.0 mg·L^?1 + 2,4-Dichlorophenoxyacetic acid 0.1 mg·L^?1 + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1. After 14 days of dark treatment, the leaves are transferred to normal light culture. After 45 days of the light culture, the regenerated adventitious buds are then transferred to the rooting medium of 1/2MS culture medium + Sucrose 30 g·L^?1 + Agar 6.5 g·L^?1 + 3-Indolebutyric acid 0.3 mg·L^?1, After the adventitious shoots take root, the seedlings are hardened and transplanted to obtain the complete regenerated plants.

Key words: Robinia pseudoacacia, leaf, dark treatment, plant growth regulator, regeneration system

CLC Number:

S722.3⁺7

Juan Han,Yapeng Li,Yanting Tian,Qi Guo,Yun Li,Yuhan Sun,Yongping Deng,Dongsheng Niu,Lizhuo Su,Xiuyu Li,Zuodeng Peng. Optimization of Efficient Regeneration System of Robinia pseudoacacia Leavesin vitro[J]. Scientia Silvae Sinicae, 2023, 59(4): 68-78.

Figures/Tables 14

Fig.1

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Table 1

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Table 2

Table 3

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Fig.7

Table 4

Effects of plant growth regulators on adventitious bud induction"

植物生长调节剂组合 Combination of plant growth regulators	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
SIM1	54.87±0.02f	1.5±0.1d	愈伤组织较少；不定芽分化慢，长势差，无玻璃化 Small number of calli, slow differentiation of adventitious buds, poorer growth, no vitrification
SIM2	69.63±0.05e	1.67±0.24d	愈伤组织较少；不定芽分化慢，长势较差，轻微玻璃化 Small number of calli, Slow differentiation of adventitious buds, poor growth, light vitrification
SIM3	74.86±0.03e	2.18±0.08d	愈伤组织一般；不定芽分化较慢，长势一般，轻度玻璃化 Ordinary number of calli, slow differentiation of adventitious buds, poor growth, light vitrification
SIM4	77.24±0.03cde	3.19±0.2c	愈伤组织一般；不定芽分化中等，长势良好，无玻璃化 Ordinary number of calli, medium differentiation of adventitious buds, good growth, no vitrification
SIM5	82.91±0.03bcd	4.24±0.22b	愈伤组织适中；不定芽分化较快，长势良好，轻度玻璃化 Medium number of calli, rapid differentiation of adventitious buds, good growth, light vitrification
SIM6	96.67±0.01a	7.4±0.4a	愈伤组织适中；不定芽分化快，长势旺盛，无玻璃化 Medium number of calli, faster differentiation of adventitious buds, vigorous, no vitrification
SIM7	84.24±0.01bc	4.36±0.1b	愈伤组织较多；不定芽分化较慢，长势良好，轻度玻璃化 Many number of calli, slow differentiation of adventitious buds, good growth, light vitrification
SIM8	88.64±0.02b	4.62±0.37b	愈伤组织较多；不定芽分化较慢，长势良好，轻度玻璃化 Many number of calli, slow differentiation of adventitious buds, good growth, light vitrification
SIM9	76.06±0.02de	4.23±0.17b	愈伤组织较多；不定分化慢，长势一般，中度玻璃化 Many number of calli, slow differentiation of adventitious buds, poor growth, moderate vitrification

Table 4

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Fig.9

Fig.10

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基因型 Genotype	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of Adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
普18-22 Pu-18-22	83.19±0.88a	6.26±0.15a	愈伤组织数量中等，不定芽分化数量多，长势旺盛，无玻璃化 Moderate number of calli, many differentiated adventitious buds, vigorous growth, no vitrification
普28-15 Pu-28-15	76.37±0.18b	4.4±0.15b	愈伤组织数量中等，不定芽分化数量中等，生长较慢，无玻璃化，轻度褐化 Moderate number of calli, moderate number of differentiated adventitious buds, slow growth, no vitrification, light browning
普26-8 Pu-26-8	59.44±1.06cd	3.37±0.14cd	愈伤组织数量较少，不定芽分化数量较少，生长较慢，轻度玻璃化 Small number of calli, small number of differentiated adventitious buds, slow growth, light vitrification
243	42.67±1.63e	2.27±0.2e	水渍状愈伤组织过多，不定芽分化数量少，生长较慢，中度玻璃化，无褐化 Many waterlogged calli, small number of differentiated adventitious buds, slow growth, moderate vitrification, no browning
D18-8	61.83±1.99c	3.48±0.29c	愈伤组织数量中等，不定芽分化数量较少，长势中等，轻微玻璃化 Moderate number of calli, small number of differentiated adventitious buds, moderate growth, light vitrification

取材时期 Sampling period/d	不定芽诱导率 Induction rate of adventitious buds (%)	平均不定芽诱导数 Average number of adventitious buds induced	愈伤组织、不定芽生长情况 Growth status of callus and adventitious buds
30	63.59±1.16c	1.76±0.13c	愈伤组织轻度褐化，不定芽分化较慢、长势一般 Light browning of callus, slow differentiation of adventitious buds, ordinary growth
45	87.53±0.79a	3.52±0.03a	愈伤组织无褐化，不定芽分化较快、长势旺盛 No browning of callus, rapid differentiation of adventitious buds, vigorous growth
60	75.88±1.38b	2.58±0.14b	愈伤组织生长较慢，不定芽分化较慢，长势良好 Slow growth of callus, slow differentiation of adventitious buds, moderate growth

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Optimization of Efficient Regeneration System of Robinia pseudoacacia Leavesin vitro

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处理 Treatment	植物生长调节剂 Plant growth regulators/(mg·L^?1)				接种数 Numbers of inoculations
处理 Treatment	2,4-D	6-BA	KT	TDZ	接种数 Numbers of inoculations
SIM1	0.1	1	0	0	80
SIM2	0.1	1	0.5	0.02	80
SIM3	0.1	1	1	0.04	80
SIM4	0.1	3	0	0.02	80
SIM5	0.1	3	0.5	0.04	80
SIM6	0.1	3	1	0	80
SIM7	0.1	5	0	0.04	80
SIM8	0.1	5	0.5	0	80
SIM9	0.1	5	1	0.02	80