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2018 Volume 40 Issue 12
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Yun-ye WANG, Hong-yu TONG, Xiao-xue ZHOU, et al. Two-Stage Rooting of Adventitious Buds of Ficus beipeiensis in Tissue Culture[J]. Journal of Southwest University Natural Science Edition, 2018, 40(12): 46-50. doi: 10.13718/j.cnki.xdzk.2018.12.007
Citation: Yun-ye WANG, Hong-yu TONG, Xiao-xue ZHOU, et al. Two-Stage Rooting of Adventitious Buds of Ficus beipeiensis in Tissue Culture[J]. Journal of Southwest University Natural Science Edition, 2018, 40(12): 46-50. doi: 10.13718/j.cnki.xdzk.2018.12.007
shu

Two-Stage Rooting of Adventitious Buds of Ficus beipeiensis in Tissue Culture

  • 1.

    School of Life Sciences, Southwest University, Chongqing 400715, China

  • 2.

    Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing 400715, China

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  • Corresponding author: Shao-hu TANG
  • Received Date: 23/01/2018
    Available Online: 20/12/2018

  • MSC: Q948

  • The adventitious buds of Ficus beipeiensis were used as explants and a two-stage rooting method was used to generate roots (inducing adventitious roots in an agar medium and completing root development in a near-dark multi-porous matrix), and the results were compared with those of the general rooting method. The rooting rate was calculated and the growth and physiological indexes of the rooted plantlets were measured after they were cultured for 30 days. Their survival rate was counted and their growth indexes were measured 30 days after transplanting. The test results showed that the rooting rate of both methods was 100%. Compared with the general rooting method, the two-stage rooting method increased the number of root tips, total root length, total root volume and average root diameter of the rooted plantlets by 301.42%, 27.85%, 123.53% and 9.68%, respectively; enhanced plant height and leaf increment by 307.89% and 83.33%; and raised chlorophyll content and nitrate reductase activity 13.09% and 297.29%, respectively. All the rooted plantlets survived after transplanting, and the survival rate, plant height and leaf number were increased by 15.00%, 41.12% and 23.81%, respectively, and the differences between the two methods were statistically significant. In conclusion, the two-stage rooting method was significantly better than the general rooting method. The root system of the test-tube plantlets was well-developed, the survival rate after transplanting was higher, and the growth state was better. The two-stage rooting method has application value for the rooting of other plants in tissue culture.
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Two-Stage Rooting of Adventitious Buds of Ficus beipeiensis in Tissue Culture

    Corresponding author: Shao-hu TANG
  • Received Date: 23/01/2018
  • Available Online: 20/12/2018

Abstract: The adventitious buds of Ficus beipeiensis were used as explants and a two-stage rooting method was used to generate roots (inducing adventitious roots in an agar medium and completing root development in a near-dark multi-porous matrix), and the results were compared with those of the general rooting method. The rooting rate was calculated and the growth and physiological indexes of the rooted plantlets were measured after they were cultured for 30 days. Their survival rate was counted and their growth indexes were measured 30 days after transplanting. The test results showed that the rooting rate of both methods was 100%. Compared with the general rooting method, the two-stage rooting method increased the number of root tips, total root length, total root volume and average root diameter of the rooted plantlets by 301.42%, 27.85%, 123.53% and 9.68%, respectively; enhanced plant height and leaf increment by 307.89% and 83.33%; and raised chlorophyll content and nitrate reductase activity 13.09% and 297.29%, respectively. All the rooted plantlets survived after transplanting, and the survival rate, plant height and leaf number were increased by 15.00%, 41.12% and 23.81%, respectively, and the differences between the two methods were statistically significant. In conclusion, the two-stage rooting method was significantly better than the general rooting method. The root system of the test-tube plantlets was well-developed, the survival rate after transplanting was higher, and the growth state was better. The two-stage rooting method has application value for the rooting of other plants in tissue culture.

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  • 北碚榕(Ficus beipeiensis S.S Chang)属桑科榕属植物，重庆特有，现有野生植株5株；因其种群数量稀少、传粉小蜂少、结实率低、种子萌发困难，故生存现状极危[1]，2004年被列入《中国物种红色名录》[2]，2015年列入重庆市首批重点保护野生植物名录[3].

    北碚榕四季常绿，冠大浓郁[4]，是优良的园林绿化和低山造林树种[5].张艳玲等[6]开展了北碚榕的组培快繁研究，率先建立了北碚榕的离体繁殖体系，但其试管苗的再生根为棒状根，根系不发达.而榕属植物再生能力较强，小叶榕、无花果和爬藤榕等[7-14]均较易生根，有的还有气生根.所以，北碚榕组培不定芽所产生的棒状根可能主要是由环境因素导致的.在植物组织培养中，试管苗的棒状根现象极为普遍.我们认为，其原因是普遍采用琼脂固体培养基生根，通气性较差.因此，本实验中，我们对北碚榕的组培不定芽采用了两段生根法生根，即在琼脂固体培养基中诱导不定根的发生，在近黑暗、孔隙性基质中完成根系的发育，结果收到了良好的效果.

1.   实验材料与方法

    1.1.   实验材料

  • 实验材料为北碚榕‘碚榕1号’无菌苗，接种外植体为第6代丛芽的不定芽(长约3 cm).

    • 1.2.   实验方法

    1.2.1.   不定芽的两段生根

  • 不定根诱导：在无菌条件下，将不定芽接种到固体生根培养基中诱导不定根的发生，至不定芽基部刚露出白色根点为止(约10 d).

    根系的发育：将已发生不定根的不定芽转移到蛭石-珍珠岩(体积比1:1)的混合基质中完成根系的发育(约20 d).混合基质在使用前充分吸附液体生根培养基，并经高压灭菌.

    • 1.2.2.   不定芽的一般生根

  • 生根方法同上述“不定根诱导”，但不定芽一直在固体生根培养基中培养(30 d).

    • 1.2.3.   生根培养基和培养条件

  • 生根培养基为1/2 MS+0.02 IBA，其固体培养基的琼脂质量浓度为6.5 g/L.培养温度(25±1) ℃，光照强度2 500 lx(14 h/d).

    • 1.2.4.   生根苗、移栽苗的指标测定

  • 生根培养30 d后，统计不定芽生根率.炼苗后洗净根部琼脂或孔隙性基质，然后用WinRHIZO型根系分析系统(加拿大生产)测定生根苗的根系长度、直径、体积、根尖数等指标；测定和计算株高增加量、叶片增加量；采用分光光度法测定叶片叶绿素质量分数[15-17]和硝酸还原酶活性[18].

    生根苗经炼苗、洗苗后移栽到营养钵中.营养土为品氏(PINDSTRUP)营养土(丹麦生产)，移栽后常规管理. 30 d后统计移栽成活率，测定和计算株高和叶片增加量.

    • 1.2.5.   数据统计与分析

  • 实验数据利用SPSS 12.0软件进行平均数统计和水平间差异性分析，利用Microsoft Excel 2003软件对平均数和差异性作图.

2.   结果与分析

    2.1.   不定芽生根结果的比较分析

  • 30 d生根结果表明，两段生根法和一般生根法的生根率均达100%(表 1)，但两段生根法根系发达，效果显著.其根系的根尖数达1 133.33条，总根长为424.06 cm，根总体积为0.38 cm3，根平均直径为0.34 mm，比一般生根法分别提高301.42%，27.85%，123.53%和9.68%，且两者之间的差异具有统计学意义(p＜0.05).

    在固体生根培养基中，北碚榕不定芽7 d左右产生不定根，出现白色根点(图 1-A)；不论是转移到孔隙性基质中(图 1-B1)，还是继续在固体培养基中(图 1-B2)，都能全部生根(图 1-C1、图 1-C2).但最终(30 d后)两段生根法的生根效果明显优于一般生根法，前者产生的不定根数量尤多，根系庞大(图 1-D1)，而后者的不定根数量很少，且部分为棒状根(图 1-D2).

    • 2.2.   生根苗生长状况的比较分析

  • 不定芽生根30 d后，生根苗的生长状况两段生根显著优于一般生根(表 2).在两段生根中，生根苗生长良好(图 1-C1)，不定芽成苗率达100%，株高增加3.1 cm，叶片增加4.4枚，该3项指标比一般生根法分别提高38.89%，307.89%和83.33%，且二者之间的差异具有统计学意义(p＜0.05).

    • 2.3.   生根苗生理状况的比较分析

  • 由图 2a可知，在两段生根中，生根苗的叶片叶绿素质量分数(2.12 mg/g FW)比一般生根提高了13.09%，且二者间差异有统计学意义(p＜0.05).

    由图 2b可知，在两段生根中，生根苗的叶片硝酸还原酶活性(0.45 U/g FW)比一般生根显著提高297.29 %.由此可知，与一般生根法相比，两段生根法所得试管苗的生理活性较强.这与试管苗的根系体积与活力密切相关.

    • 2.4.   移栽苗生长状况的比较分析

  • 生根苗移栽30 d后，两段生根所得试管苗的移栽成活率达95.83%(表 3)，株高增加1.51 cm，叶片增加2.60枚，比一般生根分别提高15.00%，41.12%和23.81%，且二者之间的差异具有统计学意义(p＜0.05).同时，两段生根苗移栽后长势较好(图 1-E1).

3.   结论

  • 试验结果表明，两段生根法显著优于一般生根法.北碚榕组培不定芽经两段生根产生的试管苗根系发达，营养生长更旺，生理状态更佳，移栽成活率更高.两段生根法对于其它植物的组培生根有应用价值.

4.   讨论

  • 植物根系的主要功能是固着和吸收.根系将植物体固定在土壤中，以抵抗风雨等自然外力和保持直立的姿态接收阳光，进行光合作用；植物通过根系吸收土壤中的水分和矿质.因此，良好的根系是植物生长发育的重要结构基础.

    在植物组织培养中，通过愈伤组织的再分化或顶芽、腋芽等外植体的丛芽诱导所形成的不定芽，都需要通过生根培养而形成完整植株，进而移栽到田间得到生产应用.也就是说不定芽的生根是试管苗繁殖不可缺少的重要环节.

    然而，在众多植物的组培报道中，由不定芽再生的不定根许多为棒状根，侧根及根毛少，根系发育差，木本植物尤其突出.究其原因，发现众多研究普遍采用固体生根培养基生根，整个生根阶段待生根的不定芽基部始终陷入琼脂培养基中.而根系的发育与基质的透气性密切相关[19]，固体培养基通气性较差，势必影响根系发育.因此，近年来，我们开展了不定芽的两段生根法研究，在‘红阳’猕猴桃、昌州无刺花椒[20-22]不定芽生根中取得了初步效果，但未与一般生根法进行比较，且研究内容较单一，缺乏统计学结论.

    本实验采用对比实验法，以北碚榕无菌苗为材料，较为系统地研究了不定芽的两段生根，结果证明两段生根法显著优于一般生根法.在两段生根过程中，不定根发生后在多孔隙半黑暗的基质中发育，由于其发育基质的通气状况得以明显改善，且近于土壤自然环境，所以根系得到良好发育.一些实验结果与‘红阳’猕猴桃和昌州无刺花椒[20-22]不定芽的生根结果一致.本实验结果对于其它植物的组培生根有应用价值.

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