MS培养基中氮、磷无机盐对鳗草克隆幼苗生存的影响

doi:10.11923/j.issn.2095-4050.cjas20190500055

摘要/Abstract

摘要：

为了探究MS培养基中氮、磷无机营养盐对鳗草生长的影响。单独添加不同浓度KNO₃、NH₄NO₃和KH₂PO₄的天然海水中培养鳗草克隆苗,于7、14天进行植株形态学观察和叶片叶绿素含量测定。其中,KNO₃的添加量为MS培养基中的1/2倍、1倍和2倍及NO₃^-总浓度为39.4 mmol/L,KH₂PO₄和NH₄NO₃的添加量均为MS培养基中各自浓度的1/2、1和2倍。结果表明,叶绿素含量以KNO₃处理组最高(P<0.05),克隆苗培养14天无毒害现象,且有新叶发出;NH₄NO₃和KH₂PO₄处理组均对克隆苗有明显毒害作用,NH₄NO₃处理组叶片48 h后开始褐化,7天后茎尖、根尖明显变软并褐化,14天后植株死亡;KH₂PO₄处理组培养14天后叶片平均褐化率达56%。因此,1/2、MS培养基均不适用于鳗草的室内培养,其培养基的氮源应为单独添加的NO₃^--N或高浓度NO₃^--N+低浓度NH₄⁺-N。

关键词: 鳗草, 克隆幼苗, MS培养基, 氮无机盐, 磷无机盐, 室内培养

Abstract:

To explore the effects of nitrogen and phosphorus inorganic salts in MS medium on the growth of Zostera marina, the young ramets of Z.marina were cultured in natural seawater with different concentrations of KNO₃, NH₄NO₃ and KH₂PO₄, respectively, and the morphological observation was conducted and leaf chlorophyll content was determined at 7 and 14 days; among them, the addition of KNO₃ was 1/2, 1 and 2 times as much as that of MS medium, and the total concentration of NO₃^- was 39.4 mmol/L, while the addition of KH₂PO₄ and NH₄NO₃ was 1/2, 1 and 2 times as much as that of MS medium respectively. The results showed that: the chlorophyll content in KNO₃ treatment group was the highest (P<0.05), the developing ramets were cultured for 14 days without toxic phenomenon and new leaves sprouted; however, the NH₄NO₃ and KH₂PO₄ treatment groups both had obviously toxic effects on the ramets; the leaves of ramets began to brown after culturing for 48 h in NH₄NO₃ treatment group; subsequently, the tips of root and rhizome softened and began to brown after 7 days of incubation and finally the ramets’ shoots died after 14 days; while the ramets’ leaves of KH₂PO₄ treatment group had the average browning rate up to 56% after culturing 14 days. Thus, neither 1/2 MS medium nor MS medium is suitable for in vitro culture of Z.marina, and the nitrogen source of the culture medium is suggested as addition of NO₃^--N alone or a high concentration NO₃^--N with a low concentration of NH₄⁺-N.

Key words: Zostera marina, Developing Ramets, MS Medium, Nitrogen Inorganic Salt, Phosphorus Inorganic Salt, Indoor Culture

中图分类号:

Q175

张飞, 郑凤英. MS培养基中氮、磷无机盐对鳗草克隆幼苗生存的影响[J]. 农学学报, 2020, 10(8): 57-62.

Zhang Fei, Zheng Fengying. Nitrogen and Phosphorus Inorganic Salts in MS Medium: Effects on Growth and Survival of Zostera marina Developing Ramets[J]. Journal of Agriculture, 2020, 10(8): 57-62.

图/表 3

参考文献 25

[1]	Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures[J]. Physiologia plantarum, 1962,15(3):473-497. doi: 10.1111/ppl.1962.15.issue-3 URL
[2]	Trigiano R N, Gray D J. Plant tissue culture, development, and biotechnology[M].CRC Press, 2016: 186.
[3]	张翠玲, 谭乐和, 陈鹏, 等. MS培养基和IBA对糯米香茶水培生根的影响[J]. 热带农业科学, 2012,32(12):23-26.
[4]	刘峰, 施卫明. 拟南芥室内水培方法的改进[J]. 土壤, 2006,38(1):102-105.
[5]	Kiyasu H, Hirata S. Callus induction from shoot apex in eelgrass (Zostera marina)[J]. Bulletin of Ehime Prefectural Fisheries Experimental Station (Japan), 2001,9:13-16.
[6]	Loques F, Caye G, Meinesz A. Axenic culture of selected tissue of Posidonia oceanica[J]. Aquatic Botany, 1990,37(2):171-188. doi: 10.1016/0304-3770(90)90090-8 URL
[7]	Koch E W, Durako M J. In vitro studies of the submerged angiosperm Ruppia maritima: Auxin and cytokinin effects on plant growth and development[J]. Marine biology, 1991,110(1):1-6. doi: 10.1007/BF01313085 URL
[8]	刘峰. 大叶藻(Zostera marina L.)愈伤组织体系的建立及优化[D]. 青岛:中国海洋大学, 2012.
[9]	Bird K T, Brown M S, Henderson T T, et al. Culture studies of Ruppia maritima L. in bicarbonate-and sucrose-based media[J]. Journal of experimental marine biology and ecology, 1996,199(2):153-164. doi: 10.1016/0022-0981(95)00195-6 URL
[10]	Short F, Carruthers T, Dennison W, et al. Global seagrass distribution and diversity: a bioregional model[J]. Journal of Experimental Marine Biology and Ecology, 2007,350(1-2):3-20. doi: 10.1016/j.jembe.2007.06.012 URL
[11]	Bergstrom E, Silva J, Martins C, et al. Seagrass can mitigate negative ocean acidification effects on calcifying algae[J]. Scientific reports, 2019,9(1):1932. doi: 10.1038/s41598-018-35670-3 URL pmid: 30760724
[12]	Bird K T, Johnson J R, Jewett-Smith J. In vitro culture of the seagrass Halophila decipiens[J]. Aquatic Botany, 1998,60(4):377-387. doi: 10.1016/S0304-3770(97)00093-4 URL
[13]	于函. 大叶藻形态学及组织培养的研究[D]. 大连:辽宁师范大学, 2008.
[14]	于佳. 大叶藻的组织培养及盐胁迫生理响应的研究[D]. 青岛:中国海洋大学, 2013.
[15]	马欣荣. 大叶藻组织培养及其Na/H逆向转运蛋白基因的初步研究[D]. 青岛:中国海洋大学, 2005.
[16]	Jacobs R. Distribution and aspects of the production and biomass of eelgrass, Zostera marina L., at Roscoff, France[J]. Aquatic Botany, 1979,7:151-172. doi: 10.1016/0304-3770(79)90019-6 URL
[17]	昌梦雨, 魏晓楠, 王秋悦, 等. 植物叶绿素含量不同提取方法的比较研究[J]. 中国农学通报, 2016,32(27):177-180.
[18]	Romero J, Lee K S, Pérez M, et al. “Nutrient dynamics in seagrass Ecosystems” in Seagrasses: Biology, Ecology and Conservation[M]. Springer, 2006:227-254.
[19]	Touchette B W, Burkholder J A M. Review of nitrogen and phosphorus metabolism in seagrasses[J]. Journal of Experimental Marine Biology and Ecology, 2000,250(1-2):133-167. doi: 10.1016/s0022-0981(00)00195-7 URL pmid: 10969167
[20]	Burkholder J M, Mason K M, Glasgow H B. Water-column nitrate enrichment promotes decline of eelgrass Zostera marina: Evidence from seasonal mesocosm experiments[J]. Marine ecology progress series, 1992,81(2):163-178. doi: 10.3354/meps081163 URL
[21]	Peralta G, Bouma T J, van Soelen J, et al. On the use of sediment fertilization for seagrass restoration: a mesocosm study on Zostera marina L[J]. Aquatic Botany, 2003,75(2):95-110. doi: 10.1016/S0304-3770(02)00168-7 URL
[22]	Hemminga M A, Harrison P G, Van Lent F. The balance of nutrient losses and gains in seagrass meadows[J]. Marine Ecology Progress Series, 1991: 85-96.
[23]	Alexandre A, Silva J, Bouma T J, et al. Inorganic nitrogen uptake kinetics and whole-plant nitrogen budget in the seagrass Zostera noltii[J]. Journal of Experimental Marine Biology and Ecology, 2011,401(1-2):7-12. doi: 10.1016/j.jembe.2011.03.008 URL
[24]	Van Katwijk M M, Vergeer L H T, Schmitz G H W, et al. Ammonium toxicity in eelgrass Zostera marina[J]. Marine Ecology Progress Series, 1997,157:159-173. doi: 10.3354/meps157159 URL
[25]	孙伟, 汤宪春, 徐艳东, 等. 威海双岛湾海域营养盐特征、限制性分析与评价[J]. 科学技术与工程, 2016,16(25):168-173.

处理	氮、磷无机盐添加种类	浓度/(mg/L)
1/2 NH₄NO₃	NH₄NO₃	825
1 NH₄NO₃	NH₄NO₃	1650
2 NH₄NO₃	NH₄NO₃	3300
1/2 KNO₃	KNO₃	950
1 KNO₃	KNO₃	1900
2 KNO₃	KNO₃	3800
1 NO₃^--N	KNO₃	3983
1/2 KH₂PO₄	KH₂PO₄	85
1 KH₂PO₄	KH₂PO₄	170
2 KH₂PO₄	KH₂PO₄	340
对照1	-	-
对照2	NH₄NO₃+KNO₃+KH₂PO₄	1650+1900+170

处理	氮、磷无机盐添加种类	浓度/(mg/L)
1/2 NH₄NO₃	NH₄NO₃	825
1 NH₄NO₃	NH₄NO₃	1650
2 NH₄NO₃	NH₄NO₃	3300
1/2 KNO₃	KNO₃	950
1 KNO₃	KNO₃	1900
2 KNO₃	KNO₃	3800
1 NO₃^--N	KNO₃	3983
1/2 KH₂PO₄	KH₂PO₄	85
1 KH₂PO₄	KH₂PO₄	170
2 KH₂PO₄	KH₂PO₄	340
对照1	-	-
对照2	NH₄NO₃+KNO₃+KH₂PO₄	1650+1900+170

处理	叶绿素a		叶绿素b		叶绿素总量
处理	7 d	14 d	7 d	14 d	7 d	14 d
1/2 NH₄NO₃	0.10±0.03^Ba	0.03±0.01^Aa	0.05±0.01^Ba	0.03±0.01^Aa	0.15±0.02^Ba	0.06±0.01^Aa
1 NH₄NO₃	0.09±0.01^Ba	0.05±0.01^Aa	0.06±0.01^Ba	0.03±0.01^Aa	0.15±0.01^Ba	0.08±0.01^Aa
2 NH₄NO₃	0.08±0.02^Ba	0.04±0.01^Aa	0.05±0.01^Ba	0.02±0.01^Aa	0.13±0.02^Ba	0.06±0.01^Aa
1/2 KNO₃	0.44±0.03^c	0.46±0.04^c	0.20±0.02^c	0.18±0.01^c	0.64±0.05^c	0.65±0.04^c
1 KNO₃	0.42±0.04^c	0.49±0.03^c	0.17±0.01^c	0.20±0.02^c	0.59±0.06^c	0.69±0.04^c
2 KNO₃	0.46±0.05^c	0.48±0.05^c	0.16±0.02^c	0.16±0.02^c	0.62±0.05^c	0.64±0.07^c
1 NO₃^--N	0.45±0.04^c	0.50±0.06^c	0.15±0.02^c	0.16±0.02^c	0.60±0.05^c	0.62±0.07^c
1/2 KH₂PO₄	0.32±0.02^Bb	0.19±0.01^Ab	0.14±0.02^Bb	0.07±0.01^Ab	0.46±0.03^Bb	0.26±0.02^Ab
1 KH₂PO₄	0.32±0.05^Bb	0.16±0.02^Ab	0.11±0.01^Bb	0.06±0.01^Ab	0.43±0.06^Bb	0.22±0.02^Ab
2 KH₂PO₄	0.31±0.16^Bb	0.16±0.02^Ab	0.15±0.03^Bb	0.06±0.01^Ab	0.46±0.05^Bb	0.22±0.02^Ab
对照1	0.43±0.03^c	0.40±0.03^c	0.21±0.01^c	0.19±0.02^c	0.63±0.05^c	0.59±0.05^c
对照2	0.12±0.01^Ba	0.06±0.01^Aa	0.05±0.01^Ba	0.03±0.01^Aa	0.17±0.01^Ba	0.09±0.01^Aa

处理	叶绿素a		叶绿素b		叶绿素总量
处理	7 d	14 d	7 d	14 d	7 d	14 d
1/2 NH₄NO₃	0.10±0.03^Ba	0.03±0.01^Aa	0.05±0.01^Ba	0.03±0.01^Aa	0.15±0.02^Ba	0.06±0.01^Aa
1 NH₄NO₃	0.09±0.01^Ba	0.05±0.01^Aa	0.06±0.01^Ba	0.03±0.01^Aa	0.15±0.01^Ba	0.08±0.01^Aa
2 NH₄NO₃	0.08±0.02^Ba	0.04±0.01^Aa	0.05±0.01^Ba	0.02±0.01^Aa	0.13±0.02^Ba	0.06±0.01^Aa
1/2 KNO₃	0.44±0.03^c	0.46±0.04^c	0.20±0.02^c	0.18±0.01^c	0.64±0.05^c	0.65±0.04^c
1 KNO₃	0.42±0.04^c	0.49±0.03^c	0.17±0.01^c	0.20±0.02^c	0.59±0.06^c	0.69±0.04^c
2 KNO₃	0.46±0.05^c	0.48±0.05^c	0.16±0.02^c	0.16±0.02^c	0.62±0.05^c	0.64±0.07^c
1 NO₃^--N	0.45±0.04^c	0.50±0.06^c	0.15±0.02^c	0.16±0.02^c	0.60±0.05^c	0.62±0.07^c
1/2 KH₂PO₄	0.32±0.02^Bb	0.19±0.01^Ab	0.14±0.02^Bb	0.07±0.01^Ab	0.46±0.03^Bb	0.26±0.02^Ab
1 KH₂PO₄	0.32±0.05^Bb	0.16±0.02^Ab	0.11±0.01^Bb	0.06±0.01^Ab	0.43±0.06^Bb	0.22±0.02^Ab
2 KH₂PO₄	0.31±0.16^Bb	0.16±0.02^Ab	0.15±0.03^Bb	0.06±0.01^Ab	0.46±0.05^Bb	0.22±0.02^Ab
对照1	0.43±0.03^c	0.40±0.03^c	0.21±0.01^c	0.19±0.02^c	0.63±0.05^c	0.59±0.05^c
对照2	0.12±0.01^Ba	0.06±0.01^Aa	0.05±0.01^Ba	0.03±0.01^Aa	0.17±0.01^Ba	0.09±0.01^Aa

处理	根生长点状况		根状茎生长点状况		叶片褐化比例/%		新发叶片数
处理	7 d	14 d	7 d	14 d	7 d	14 d	7 d	14 d
1/2 NH₄NO₃	褐化	褐化、死亡	开始褐化	完全褐化、死亡	80	100	0	0
1 NH₄NO₃	褐化	褐化、死亡	开始褐化	完全褐化、死亡	82	100	0	0
2 NH₄NO₃	褐化	褐化、死亡	开始褐化	完全褐化、死亡	84	100	0	0
1/2 KNO₃	健康	健康	健康	健康	10	20	0	0.8
1 KNO₃	健康	健康	健康	健康	11	21	0	0.9
2 KNO₃	健康	健康	健康	健康	10	23	0	1.1
1 NO₃^--N	健康	健康	健康	健康	10	23	0	1.0
1/2 KH₂PO₄	健康	健康	健康	健康	39	60	0	0
1 KH₂PO₄	健康	健康	健康	健康	40	53	0	0
2 KH₂PO₄	健康	健康	健康	健康	40	56	0	0
对照1	健康	健康	健康	健康	10	22	0	0
对照2	开始褐化	完全褐化、死亡	开始褐化	完全褐化、死亡	56	95	0	0