Establishment of an effective protocol for cultivation of tissue cultured seedlings in Pinus massoniana superior provenance
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摘要:目的马尾松是我国南方生态建设和造林用材的主要树种，受种苗良种化限制，人工林生产力水平整体不高，产业发展缓慢。因此，构建高效马尾松优良种质繁育技术体系对推动良种推广利用，加快产业发展，提升行业竞争力很有必要。方法以马尾松骨干育种资源桐棉种源为研究对象，未成熟球果合子胚为外植体，通过体胚发生技术获取成熟胚状体作为本试验供试材料，针对马尾松体胚萌发率低、芽苗活性差、生根成苗困难等技术瓶颈，系统分析了活性炭（AC）、基本培养基、植物激素对体胚萌发及萌发体胚芽苗复壮与不定根诱导的影响。结果（1）AC能显著提升成熟胚状体萌发培养效果，但过高浓度AC会导致培养效果减弱，其中以0.83 mol/L AC效果最佳。在附加AC基础上，利用高N、低NH4+/NO3− 比，K、Ca量适中的1/2MMS基本培养基能进一步提升萌发效果，萌发率可达94.1%。（2）0.42 mol/L AC能有效促进萌发体胚芽苗伸长，在4 μmol/L TDZ作用下，腋芽诱导效果好，有效芽增殖系数5.6/35 d，芽高9.2 cm/50 d。（3）经1.2 μmol/L NAA + 2 μmol/L PBZ处理60 d，单芽生根率达94.3%、根条数6.4，移栽3个月后成活率为95.8%。结论本研究首次通过体胚发生与器官发生途径相结合的技术构建了高效组培繁育体系，可用于马尾松优良种质的快速繁殖以及遗传转化方面的研究，为马尾松良种产业化以及进一步开展基因工程分子育种方面的研究奠定了坚实基础。Abstract:ObjectivePinus massoniana is a main tree species for ecological construction and timber in southern areas of China. However, the productivity of plantation is generally low resulting from the limitation of improved varieties, which leads to the slow development of industrialization in P. massoniana. It is necessary to develop an effective propagation system of elite germplasm for P. massoniana in order to promote the use of improved varieties, accelerate the industrial development, and enhance the competitiveness of industry.MethodIn this study, ‘Tongmiansong’ (TM), the backbone of P. massoniana breeding resources was used as the research object, zygotic embryos excised from immature cones were applied to explants, and mature somatic embryos (SE) obtained via somatic embryogenesis were used for testing materials. Concerning the technical bottlenecks of P. massoniana tissue culture, including low germination rate, poor shoot growth, and recalcitrance to rooting, effects of active charcoal (AC), basal media, and plant hormones on SE germination as well as reinvigoration and adventitious rooting of shoots were investigated in the present study.Result(1) AC significantly improved germination of TM mature SE, while a high level of AC was able to weaken their germination effects, and the best effect was observed at the 0.83 mol/L AC treatment. Based on the application of AC in the medium, the basal medium composed of high N, low ratio of NH4+/NO3− , and moderate K and Ca furtherly enhanced the germination of SE, reaching 94.1% of germinating rate. (2) 0.42 mol/L AC effectively promoted the elongation of germinated SE. Under the treatment of 4 μmol/L TDZ, induction of axillary buds was better, achieving effective bud proliferation coefficient of 5.6/35 d, and shoot height of 9.2 cm/50 d. (3) After 60 days of 1.2 μmol/L NAA + 2 μmol/L PBZ application in the rooting medium, rooting rate was 94.3%, root number was 6.4, and survival rate was 95.8% after 3-month transplanting.ConclusionThe effective breeding system by tissue culture for TM was firstly established via a combined approach of somatic embryogenesis and organogeneis in this study, which would be used for the rapid propagation of elite germplasm for P. massoniana as well as for the research on genetic transformation, providing solid foundation for industrialization of improved varieties and molecular breeding in P. massoniana.
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Keywords:
- Pinus massoniana /
- tissue culture /
- somatic embryo /
- adventitious rooting /
- plant regeneration
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松属（Pinus）是针叶树各属中最大且最为重要的一个属[1]。马尾松（Pinus massoniana）综合利用价值高、推广应用前景广阔，是我国南方生态建设和工业用材的主要造林树种[2]。广西多年来一直从事马尾松良种选育方面的工作，在全国处于领先地位，先后选育了宁明桐棉、忻城古蓬、容县浪水等速生、优质、高产优良种源11个。其中，宁明桐棉种源（桐棉松）在全国17个省（区）的马尾松全分布地理种源试验中表现优异，通过了国家良种审定，是我国最为优良的马尾松地理种源之一。由于桐棉松干形通直、皮薄、生长快、产量高，是马尾松骨干育种资源[3]。然而，由于马尾松育种周期长，加之近年来种子园母树林老化、结实量低等问题，导致良种匮乏，人工林生产力水平整体不高，严重限制和阻碍了马尾松产业的发展[4]。
无性快繁技术能有效提升良种选育效率，同时还能保持亲本的优良性状，是解决良种匮乏，改善人工林生产力低的有效途径。受插穗母株与季节影响，扦插育苗效率较低[5]，而组培技术在速生、优质、高抗林木优良种质创制与利用方面发挥了重要作用，为多目标品种林业的发展提供了有力的生物技术工具[6-7]。体胚发生（somatic embryogenesis，SE）和器官发生（organogenesis）是植物组培育苗中两种主要途径[6]。其中，通过SE途径的植株再生，繁殖速度快、育苗效率高，但程序繁琐、技术难度大；而通过器官发生途径的植株再生，作为一种传统的组培育苗技术，虽繁殖系数低于前者，但具有高度保持亲本遗传稳定性的优势，且操作较为简便，在生产上应用广泛[8-10]，二者各有利弊。
马尾松属于组织培养顽拗型的树种[11]。在马尾松体胚育苗方面，众多学者先后以成熟或未成熟种子合子胚为材料诱导了体胚发生，但由于体胚萌发率低，且经萌发形成的体胚苗生长势弱、根系质量差、成活率低，植株再生困难[4, 12]。而以茎芽、种子等为外植体，通过器官发生途径，先后获得了马尾松的再生植株[13-15]，但受繁殖效率的影响，生产成本高昂，尚未实现组培苗规模化生产与应用。基于此，本研究旨在优化马尾松体胚萌发效率，并通过芽苗复壮与不定根诱导，创建一种体胚发生与器官发生途径相结合的高效组培育苗技术，为实现马尾松优良种质无性育苗产业化奠定坚实基础。
1. 材料与方法
1.1 试验材料
以2013—2016年6月中下旬，于广西宁明县国有派阳山林场桐棉松优良林分中采集的未成熟球果为体胚培养材料，参照杨模华等[4]方法，无菌条件下剥取带胚乳的幼胚为外植体，经过体胚诱导（图1A）和维持增殖（图1B）培养获得了大量活力旺盛的桐棉松胚性细胞系。根据胚性愈伤组织的增殖速率，选择培养时间 ≤ 1年，增殖系数 ≥ 25.0/14 d，色泽透明，无水渍化现象的17个胚性细胞系作为研究对象，以LP[16]为基本培养基，附加37.8 µmol/L ABA，55.5 mmol/L 麦芽糖，15 mmol/LPEG 8000，23.8 mmol/L 琼脂（> 900 g/cm2），经成熟培养（图1C、G）4 ~ 5个月后，将分化成型的胚状体（图1H）作为本试验研究材料。
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图 1 桐棉松体胚发生途径植株再生A.体胚诱导；B. 体胚增殖；C. 成熟培养；D. 诱导阶段胚性细胞显微观察；E. 增殖阶段胚性细胞显微观察；F. 成熟培养胚性细胞显微观察；G. 形成优势、成熟胚的胚性细胞团；H. 成熟体细胞胚；I. 体胚萌发培养；J. 生长中的萌发体胚；K. 再生体萌苗；L. 体胚苗移栽。标尺: 2 cm（A）, 1cm（B, I−L）, 0.5 cm（C）, 2 mm（G, H）, 500 μm（D, E）, 200 μm（F）. A, induction of somatic embryos; B, proliferation of somatic embryos; C, maturation culture; D, microscopic observation on induced somatic embryos; E, microscopic observation on proliferated somatic embryos; F, microscopic observation on somatic embryos during mature culture; G, embryogenic cells with dominative and mature somatic embryos; H, mature somatic embryos; I, germination of somatic embryos; J, growth of germinated somatic embryos; K, regenerated somatic seedlings; L, transplanting of somatic seedlings. Scale bars: 2 cm (A), 1cm (B, I−L), 0.5 cm (C), 2 mm (G, H), 500 μm (D, E), 200 μm (F).Figure 1. Plant regeneration via somatic embryogenesis in Pinus massoniana ‘Tongmiansong’1.2 试验设计
1.2.1 体胚萌发
（1）活性炭(AC)处理：将成熟胚状体直接转入1/2LP培养基中，附加0 ~ 1 mol/L AC ，73.1 mmol/L蔗糖，23.8 mmol/L琼脂（> 900 g/cm2），在无光照、（4 ± 0.5）℃条件下预培养1周，然后于80 µmol/（m2·s）光照强度下培养4 ~ 5周，光照时间12 h/d，培养温度（25 ± 0.5）℃。该试验设13个AC处理，每处理3重复，每重复60个胚状体，共接种2 340个胚状体。
（2）基本培养基处理：将胚状体接种在附加0.83 mol/L AC的1/2LP、1/2DCR[17]、1/2GD[18]、1/2MS[19]、1/2MMS[11]5种不同基本培养基中，其余培养条件同上。本试验共设置5个处理，每处理3重复，每重复60个胚状体，共接种900个胚状体。
待萌发培养40 d左右，将上胚轴部位形成绿色叶片的体胚计为萌发体胚（图1I）。
体胚萌发率 = 萌发体胚数/接种胚状体总数 × 100%。
1.2.2 芽苗复壮
将萌发体胚下胚轴靠胚根部分2 ~ 3 mm切除（图2A），然后转入MMS并附加0、0.42、0.83 mol/L AC，87.7 mmol/L蔗糖，23.8 mmol/L琼脂（> 900 g/cm2）培养基中培养40 d（图2B），待萌发体胚伸长形成健壮芽苗（图2C），截取3 ~ 5 cm顶芽直接转入MMS + 87.7 mmol/L蔗糖+ 17.8 mmol/L 琼脂（> 900 g/cm2）和 0、2、4、6、8 µmol/L 6-BA 或TDZ 的培养基中处理35 d诱导腋芽（图2D），最后转入MMS + 0.42 mol/L AC的无激素培养基中培养50 d促进健壮丛芽形成（图2E）。培养光照强度40 µmol/（m2·s），光照时间12 h/d，培养温度同上。将丛生芽中芽高 ≥ 3 cm的单芽计为有效芽，有效芽增殖系数 = 有效芽个数/接种顶芽总数。本阶段在促萌发体胚伸长生长的试验中，共接种1 632个生长良好的萌发体胚（包括0、0.42、0.83 mol/L 3个AC处理，每处理5重复，每重复80 ~ 144个体胚）；从伸长的萌发体胚中，则截取了1 280个顶芽（包括0、2、4、6、8 µmol/L 5个6-BA和0、2、4、6、8 µmol/L 5个TDZ浓度处理，每处理4重复，每重复28 ~ 40个顶芽）进行腋芽诱导与复壮。
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图 2 通过芽苗复壮与不定根诱导的桐棉松萌发体胚离体植株再生A.切除胚根的萌发体胚；B. 切根处理后的萌发体胚复壮培养；C. 复壮的体胚苗；D. 复壮体胚苗腋芽诱导；E. 形成丛生芽的复壮体胚苗；F. 复壮体胚苗的大量增殖与扩繁；G. 形成发达根系的生根苗；F. 生根苗苗圃移栽；I. 移栽3个月后成活的生根苗。标尺: 10 cm（F）, 5 cm（I）, 2 cm（H）, 1cm（A−E, G）. A, germinated somatic embryos with radicle cutting; B, reinvigoration of germinated somatic embryos after radicle cutting; C, reinvigorated shoots originated from somatic embryos; D, induction of axillary buds for reinvigorated shoots originated from somatic embryos; E, clustered shoots from reinvigorated shoots originated from somatic embryos; F, massive proliferation of reinvigorated shoots originated from somatic embryos; G, rooted shoots with robust root system; H, transplanting of rooted shoot in the nursery; I, survival seedlings after 3-month transplanting. Scale bars: 10 cm (F), 5 cm (I), 2 cm (H), 1 cm (A−E, G).Figure 2. In vitro regeneration of plants originated from germinated somatic embryos of Pinus massoniana ‘Tongmiansong’ through shoot reinvigoration and adventitious root induction1.2.3 不定根诱导
在前期研究中，笔者已报道培养基中添加NAA对马尾松不定根诱导是有效的，并在1.2 µmol/L NAA处理下生根效果最佳，生根率为80%左右[20]。为进一步改善生根效果，利用激素协同作用，在开展1.2 µmol/L NAA + 0 ~ 16 µmol/L IAA、IBA或多效唑（PBZ）生根诱导预实验前提下，本试验单个截取丛芽中2 ~ 3 cm高的顶芽接种在1/2MMS，附加1.2 µmol/L NAA和/或2 µmol/L IAA、IBA、PBZ，以及58.5 mmol/L蔗糖、14.9 mmol/L琼脂（> 900 g/cm2）的培养基中进行生根诱导60 d（图2G），培养光照、温度条件同1.2.2。将不定根长度 ≥ 0.5 cm的芽苗计为生根苗，并以接种单芽中50%以上为生根苗的生根处理时间作为生根时间，统计长度 ≥ 1 cm的不定根数作为根条数。其中，生根率 = 生根苗数/接种单芽数 × 100%。在不定根诱导试验中，共接种了1 040个单芽（包括无激素对照、1.2 µmol/L NAA、1.2 µmol/L NAA + 2 µmol/L IAA、1.2 µmol/L NAA + 2 µmol/L IBA、1.2 µmol/L NAA + 2 µmol/L PBZ 5种激素处理，每处理5重复，每重复40 ~ 48个单芽）。
1.3 移栽与管护
将生根瓶苗移至自然环境条件下炼苗棚中，按照从99%至70%逐渐降低的湿度条件进行炼苗。两周后，将苗木从瓶中取出，洗净培养基，用0.1%多菌灵浸泡根部10 min后移至装有体积比为3∶2∶3∶2的黄心土、泥炭土、椰糠、珍珠岩混合基质的育苗杯中（图2H）。然后，根据常规育苗管理方法，进行水肥、光照和病虫害管护。移栽3个月后统计苗高 ≥ 15 cm的株数并计为成活株数（图2I），成活率 = 成活株数/移栽生根苗数 × 100%。
1.4 数据分析
通过单因素方差分析（One-way ANOVA，P < 0.05）对不同AC浓度、培养基类型与激素处理间的均值进行差异显著性检验；利用Duncan’s multiple range test进行多重比较，t-test进行两个处理间差异性分析。数据处理采用SPSS 19.0统计分析软件，用Excel软件绘图。
2. 结果与分析
2.1 AC、基本培养基对体胚萌发与生长的影响
在前期研究中，笔者以桐棉松未成熟球果合子胚为外植体，经过体胚发生途径，完成了体胚苗的再生（图1）。但整体上，体胚萌发率偏低，胚根发育不完善，导致移栽成活困难，体胚育苗效率低。然而，在获取成熟胚状体的基础上，通过器官发生途径，对萌发体胚芽苗进行复壮与不定根诱导，育苗效果显著提升（图2）。
从图3可以看出，培养基中不添加AC时，体胚萌发率为0。随着培养基中附加的AC浓度的增加，体胚萌发率呈上升趋势，在AC浓度为0.75 ~ 0.83 mol/L时，体胚萌发率最高，但AC浓度为0.92 ~ 1 mol/L时，体胚萌发率降低。这说明，AC在桐棉松胚状体萌发中很有必要，但较高浓度AC对体胚萌发可能会产生一定的抑制性。
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图 3 活性炭对桐棉松体胚萌发的影响不同小写字母表示不同AC浓度处理间差异显著（P < 0.05）。Different lowercase letters indicate significant differences among varied AC concentration treatments at P < 0.05 level.Figure 3. Effects of active charcoal on germination of somatic embryos in Pinus massoniana ‘Tongmiansong’在培养基中均附加0.83 mol/L AC的基础之上，不同基本培养基上所接种成熟胚状体的萌发效果也不同（表1）。从培养效果来看，以1/2MMS的培养效果最佳，萌发率高达94.1%，且萌发体胚生长健壮、活性好；其次是1/2LP培养基，萌发率68.5%，但芽苗生理活性较弱，有一定的褐化现象；在1/2DCR培养基上，萌发率最低（10.7%），且萌发芽苗生长缓慢，有严重的褐化问题，体胚萌发困难。这些结果表明，培养基中矿质元素组成对体胚萌发的影响显著。
表 1 基本培养基对桐棉松体胚萌发的影响Table 1. Effects of basal media on germination of somatic embryos in Pinus massoniana ‘Tongmiansong’基本培养基 Basal media 萌发率 Germination rate/% 萌发芽苗生长情况 Growth of shoots from germinated somatic embryos 1/2LP 68.5 ± 2.1b 叶片颜色深绿，顶芽形成缓慢，有褐化现象
Leaves are dark green, the formation of top shoots is slow, and the browning of shoots is occasionally observed1/2DCR 10.7 ± 2.2e 生长缓慢，叶片颜色发黄，褐化严重
Growth of shoots is slow, their leaves are yellow, and the browning of shoots is serious1/2GD 37.7 ± 2.6d 叶片短小、颜色发黄，顶芽形成困难，褐化现象明显
Leaves are short and yellow, the formation of top shoots is difficult, and the browning of shoots is obvious1/2MS 42.4 ± 3.2c 叶片呈浅绿色，形成顶芽水渍化现象明显
Leaves are light green, and the formed top shoots are easy to be water-soaking1/2MMS 94.1 ± 2.8a 叶片颜色翠绿，顶芽形成快、生长健壮，无褐化、玻璃化现象
Leaves are green, the formation of top shoots is efficient, and the shoots are robust without browning and vitrification注：同一列中不同小写字母表示不同基本培养基间差异显著（P < 0.05）。Note: different lowercase letters from the same column indicate significant differences among varied basal media at P < 0.05 level. 2.2 AC、细胞分裂素对芽苗复壮的影响
为实现萌发体胚芽苗的复壮，首先对萌发体胚进行切根处理，然后将上胚轴接种在含不同AC浓度的MMS培养基中。由图4显示结果来看，与不添加AC的对照相比，在0.42 mol/L AC处理下，萌发体胚芽苗生长最快，40 d伸长培养后即可形成健壮芽苗（图2C），平均芽高为7.1 cm。在0.83 mol/L AC处理下，芽苗高度虽高于对照，但明显低于0.42 mol/L AC处理。
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图 4 活性炭对桐棉松萌发体胚芽苗生长的影响不同小写字母表示不同AC浓度处理间差异显著（P < 0.05）。Different lowercase letters indicate significant differences among varied AC concentration treatments at P < 0.05 level.Figure 4. Effects of active charcoal on growth of shoots from germinated somatic embryos in Pinus massoniana ‘Tongmiansong’从伸长芽苗上截取顶芽接种在添加不同浓度的细胞分裂素（6-BA和TDZ）培养基上进行增殖。表2结果表明，细胞分裂素是实现腋芽诱导的关键。从细胞分裂素处理浓度来看，在6 µmol/L 6-BA和4 µmol/L TDZ处理下，有效芽增殖系数较高，但丛芽高生长和芽苗生长情况均以4 µmol/L 6-BA或TDZ处理下的效果佳。在同一浓度处理条件下，有效芽增殖系数仅在4 µmol/L 6-BA和TDZ间差异显著，分别为2.9和5.6；而丛芽高度在2 ~ 6 µmol/L浓度处理范围内，TDZ作用下丛芽高度显著高于6-BA，但在8 µmol/L浓度处理下，丛芽高度在6-BA和TDZ作用下无明显差异。从6-BA和TDZ对芽苗生长影响来看，在较高浓度（6 ~ 8 µmol/L）作用下，与TDZ处理相比，6-BA处理下芽苗针叶较为卷曲、密集，茎节间距短，芽苗褐化与玻璃化问题更为严重。
表 2 细胞分裂素对桐棉松萌发体胚芽苗增殖和生长的影响Table 2. Effects of cytokinins on proliferation and growth of shoots originated from germinated somatic embryos in Pinus massoniana ‘Tongmiansong’激素浓度
Hormone concentration/
（µmol·L− 1）有效芽增殖系数
Proliferation coefficient of effective shoots丛芽高度
Height of clustered shoots/cm芽生长情况
Shoot growth6-BA TDZ 6-BA TDZ 6-BA TDZ 0 0dA 0eA — — 未形成丛芽，针叶短小、颜色深绿，茎节间短，植株矮小
Cluster shoots are not found, leaves are short and dark green, internodes of shoots are short, and the whole plant is dwarf2 0.9 ± 0.2cA 1.1 ± 0.5dA 4.8 ± 0.4bB 8.1 ± 0.8bA 多呈单芽，大部分芽节间短、针叶密且呈深绿色
Most of shoots are single, their internodes are short, and leaves are compact and dark green丛生芽少，针叶细长，叶片为浅绿色
Cluster shoots are rare, leaves are tenuous and light green4 2.9 ± 0.7bB 5.6 ± 0.7aA 7.8 ± 0.8aB 9.2 ± 0.8aA 丛芽多，芽苗节间长，叶片颜色翠绿，生长健壮
Plenty of cluster shoots are observed, with long internodes, green leaves, and they grow well丛芽多，针叶长，叶片颜色翠绿，茎节间长，生长健壮
Plenty of cluster shoots are investigated, with long and green leaves, long internodes, and the whole shoots are thriving6 4.1 ± 0.5aA 3.3 ± 1.0bA 5.0 ± 0.8bB 6.7 ± 0.9cA 短簇状丛生芽较多，茎节间短，叶片颜色发黄，有褐化现象
Short cluster shoots are usually found, with short internodes, yellow leaves, and the browning of shoots is observed丛芽多，顶稍针叶较短，有部分叶片颜色发黄
Cluster shoots are sufficient, leaves of top shoots are short, and part of leaves are yellow8 2.5 ± 0.8bA 2.1 ± 0.7cA 3.6 ± 0.8cA 3.7 ± 0.4dA 丛芽呈短簇状，叶子卷曲，短小，褐化、玻璃化现象明显
Cluster shoots are short and small, leaves are cured and short, and the vitrification of shoots is remarkable丛芽矮小，叶片颜色发黄，有玻璃化现象
Cluster shoots are shorts, leaves are yellow, and the vitrification of shoots is occasionally found注：同一列中不同小写字母表示不同激素浓度处理间差异显著，同一行中不同大写字母表示两种激素处理间差异显著（P < 0.05）。Notes: different lowercase letters from the same column indicate significant differences among varied hormone concentration treatments, while different capital letters from the same row indicate significant differences between two hormone treatments at P < 0.05 level. 2.3 生长调节剂对不定根形成的影响
从表3可以看出，生长调节剂（PGR）影响桐棉松不定根形成。在无PGR添加培养基上，单芽生根率为0。从几种PGR组合来看，以NAA + PBZ处理下生根效果最好，生根率高，生根时间短，根条数多，根系长，移栽成活率高；其次是NAA + IAA处理，除根条数和移栽成活率外，其单芽生根率、生根时间、根长与NAA + PBZ处理均无明显差异。
表 3 生长调节剂对桐棉松萌发体胚复壮芽苗生根能力的影响Table 3. Effects of plant growth regulators on rooting capacity of reinvigorated shoots originated fromgerminated somatic embryos in Pinus massoniana ‘Tongmiansong’激素处理
Hormone treatment生根率
Rooting rate/%生根时间
Rooting time/d根条数
Number of roots根长
Root length/cm成活率
Survival rate/%对照 Control 0c — — — — NAA 87.7 ± 4.5b 28.7 ± 2.1a 2.3 ± 0.4d 0.6 ± 0.2b 50.3 ± 3.6d NAA + IAA 91.4 ± 2.7ab 20.8 ± 1.5c 4.2 ± 0.7b 2.1 ± 0.4a 83.4 ± 3.2b NAA + IBA 90.5 ± 2.2ab 24.0 ± 2.3b 3.5 ± 0.8c 2.7 ± 0.4a 70.8 ± 4.6c NAA + PBZ 94.3 ± 3.8a 18.6 ± 1.2c 6.4 ± 0.7a 2.3 ± 0.6a 95.8 ± 2.4a 注：同一列中不同小写字母表示不同激素处理间差异显著（P < 0.05）。Note: different lowercase letters from the same column indicate significant differences among varied hormone treatments at P < 0.05 level. 3. 结论与讨论
研究发现，活性炭能改善繁殖材料褐化，促进植株伸长生长，因此被广泛应用于植物组培中[8]。以往研究证明，培养基中附加的活性炭能影响植物对培养基中金属元素的获取[9]。Van Winkle等[21]认为，活性炭在松树组培中所发挥的作用主要在于其对酚酸物质及残留激素的吸收。而Pan等[10]认为，受树种和培养材料的影响，活性炭在植物组培中存在促进或抑制性的效果。对马尾松而言，活性炭在组培中的促进作用在笔者以往研究中已证实[11, 15]。本研究在无活性炭培养基中未发现萌发体胚，而通过在培养基中添加活性炭，体胚萌发率、芽苗生长高度显著增加，但过高浓度活性炭导致体胚萌发率与苗高增长量下降。这说明，活性炭对提升桐棉松体胚萌发率、促进芽苗伸长生长起到关键性的作用，但过量使用会降低培养效果。因此，根据培养材料及其生长情况的差异，应对活性碳使用量进行优化，根据本试验研究结果，建议胚状体萌发0.83 mol/L、芽苗伸长0.42 mol/L。
一般情况下，因树种、繁殖材料、基因型的不同，所适用的培养基也不同[22]。其中，培养基中矿质营养元素（大量元素）组成对植物组培效果影响最大[1]。在探讨了活性炭对马尾松体胚萌发作用的基础上，为进一步改善马尾松体胚萌发效果，本试验对DCR、GD、LP、MS、MMS 5种不同类型基本培养基上胚状体萌发情况进行了研究。通过观测发现，与低矿质元素含量的DCR、GD相比，在高矿质元素含量的LP、MS、MMS上体胚萌发率普遍偏高。LP、MS、MMS均属于高N培养基，但LP、MMS中铵态氮含量较低，此外LP中K、Ca含量高于MS和MMS。马尾松属高N需求树种[23]，高N培养基更适于马尾松体胚萌发可能与此有关。从LP、MS、MMS上萌发体胚生长情况来看，LP体胚芽生长活性差，可能是受到高K、Ca影响。K、Ca参与植物光合、呼吸等多种生理代谢活动，在细胞壁、韧皮部、木质部发育中起重要作用[24-25]。LP上芽苗长势弱可能是高浓度K、Ca引起的生理代谢活动紊乱，细胞壁加厚，木质化加剧，芽苗出现生理老化所致，这有待从解剖构造和生理生化角度进一步验证。整体而言，5种供试基本培养基中以MMS体胚萌发培养效果最好，与MS相比，MMS减少了铵态氮用量，提高了硝态氮/铵态氮比值，从而有效避免了因芽苗细弱出现的水渍化问题。初步认为，高N但低铵态/硝态氮比，K、Ca量适中的培养基更利于桐棉松体胚萌发。
细胞分裂素能调节植物细胞生长发育，促进组织分化与生长，是实现培养材料复壮的重要调控因子[26]。然而研究发现，高浓度细胞分裂素的应用会导致培养材料中毒，进而发生水渍玻璃化、褐化，伸长生长困难等问题[27-28]。因此，挖掘开发一种高活性细胞分裂素很有必要。为进一步开展桐棉松萌发体胚芽的生理复壮，本研究以常规使用的6-BA作为参照，分析了TDZ对萌发体胚芽苗腋芽诱导与生长的影响。从研究结果来看，在无激素培养基上未发现丛芽，说明受马尾松萌蘖能力差的影响，芽增殖培养基中细胞分裂素的添加必不可少。在2 ~ 8 µmol/L的测试浓度范围内，TDZ以4 µmol/L条件下的芽苗复壮效果好，而6-BA在6 µmol/L时芽增殖最多（4.1），在4 µmol/L时丛芽最高。6-BA与TDZ的分子量均为220，但TDZ在较低浓度时获得了较高的增殖系数（5.6），且芽苗伸长快。此外，在6 ~ 8 µmol/L较高浓度条件下，6-BA作用下的芽苗褐化、玻璃化现象明显较TDZ严重，暗示了6-BA对培养材料的毒害性大于TDZ。这些说明，TDZ可作为一种高活性的高效细胞分裂素应用于植物组培芽苗复壮培养中。
马尾松属诱生根原基树种，不定根形成困难[13]。生长素是诱导马尾松不定根形成的关键，通过本试验无生长素对照处理中未发现生根苗这点可以证实。NAA能促进马尾松不定根发生与形成，但生根效果不稳定，生根率波动范围较大[11, 13, 15, 20, 29]。本研究利用NAA与IAA、IBA或PBZ进行组合，生根效果得到了不同程度的改善，其中以NAA + PBZ效果最佳。PBZ作为赤霉素（GA）的一种生物合成抑制剂，被应用于多种植物组培生根培养中[30-31]。GA有类似于IAA的生物活性，能协同IAA促进植物细胞生长，加快不定根发育[32]。然而有学者提出，GA通过影响IAA的极性运输，抑制根系径向发育，对不定根形成具有抑制作用[33-34]。我们推测，PBZ的促根性效果可能是由于繁殖材料中较高的内源GA水平所致，这需进一步开展有关生根能力与内源激素水平相关性方面的研究。此外，与NAA相比，NAA + IAA、NAA + IBA作用下生根率无明显变化，但生根时间、根条数、根长与移栽效果显著提升，其中NAA + IAA效果优于NAA + IBA。从不定根发育过程来看，包括不定根发生（生根率）和形态建成（根条数、根长）两个阶段[35]。其中，IAA对不定根形态建成的促进性在一些国外研究中已证实[36-37]，这点与本试验研究结果一致。因此，对不定根发生容易，而根系发育缓慢、根条数少的树种，可考虑在培养基中添加IAA促进根系质量的改善。
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图 1 桐棉松体胚发生途径植株再生
A.体胚诱导；B. 体胚增殖；C. 成熟培养；D. 诱导阶段胚性细胞显微观察；E. 增殖阶段胚性细胞显微观察；F. 成熟培养胚性细胞显微观察；G. 形成优势、成熟胚的胚性细胞团；H. 成熟体细胞胚；I. 体胚萌发培养；J. 生长中的萌发体胚；K. 再生体萌苗；L. 体胚苗移栽。标尺: 2 cm（A）, 1cm（B, I−L）, 0.5 cm（C）, 2 mm（G, H）, 500 μm（D, E）, 200 μm（F）. A, induction of somatic embryos; B, proliferation of somatic embryos; C, maturation culture; D, microscopic observation on induced somatic embryos; E, microscopic observation on proliferated somatic embryos; F, microscopic observation on somatic embryos during mature culture; G, embryogenic cells with dominative and mature somatic embryos; H, mature somatic embryos; I, germination of somatic embryos; J, growth of germinated somatic embryos; K, regenerated somatic seedlings; L, transplanting of somatic seedlings. Scale bars: 2 cm (A), 1cm (B, I−L), 0.5 cm (C), 2 mm (G, H), 500 μm (D, E), 200 μm (F).
Figure 1. Plant regeneration via somatic embryogenesis in Pinus massoniana ‘Tongmiansong’
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图 2 通过芽苗复壮与不定根诱导的桐棉松萌发体胚离体植株再生
A.切除胚根的萌发体胚；B. 切根处理后的萌发体胚复壮培养；C. 复壮的体胚苗；D. 复壮体胚苗腋芽诱导；E. 形成丛生芽的复壮体胚苗；F. 复壮体胚苗的大量增殖与扩繁；G. 形成发达根系的生根苗；F. 生根苗苗圃移栽；I. 移栽3个月后成活的生根苗。标尺: 10 cm（F）, 5 cm（I）, 2 cm（H）, 1cm（A−E, G）. A, germinated somatic embryos with radicle cutting; B, reinvigoration of germinated somatic embryos after radicle cutting; C, reinvigorated shoots originated from somatic embryos; D, induction of axillary buds for reinvigorated shoots originated from somatic embryos; E, clustered shoots from reinvigorated shoots originated from somatic embryos; F, massive proliferation of reinvigorated shoots originated from somatic embryos; G, rooted shoots with robust root system; H, transplanting of rooted shoot in the nursery; I, survival seedlings after 3-month transplanting. Scale bars: 10 cm (F), 5 cm (I), 2 cm (H), 1 cm (A−E, G).
Figure 2. In vitro regeneration of plants originated from germinated somatic embryos of Pinus massoniana ‘Tongmiansong’ through shoot reinvigoration and adventitious root induction
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图 3 活性炭对桐棉松体胚萌发的影响
不同小写字母表示不同AC浓度处理间差异显著（P < 0.05）。Different lowercase letters indicate significant differences among varied AC concentration treatments at P < 0.05 level.
Figure 3. Effects of active charcoal on germination of somatic embryos in Pinus massoniana ‘Tongmiansong’
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图 4 活性炭对桐棉松萌发体胚芽苗生长的影响
不同小写字母表示不同AC浓度处理间差异显著（P < 0.05）。Different lowercase letters indicate significant differences among varied AC concentration treatments at P < 0.05 level.
Figure 4. Effects of active charcoal on growth of shoots from germinated somatic embryos in Pinus massoniana ‘Tongmiansong’
表 1 基本培养基对桐棉松体胚萌发的影响
Table 1 Effects of basal media on germination of somatic embryos in Pinus massoniana ‘Tongmiansong’
基本培养基 Basal media 萌发率 Germination rate/% 萌发芽苗生长情况 Growth of shoots from germinated somatic embryos 1/2LP 68.5 ± 2.1b 叶片颜色深绿，顶芽形成缓慢，有褐化现象
Leaves are dark green, the formation of top shoots is slow, and the browning of shoots is occasionally observed1/2DCR 10.7 ± 2.2e 生长缓慢，叶片颜色发黄，褐化严重
Growth of shoots is slow, their leaves are yellow, and the browning of shoots is serious1/2GD 37.7 ± 2.6d 叶片短小、颜色发黄，顶芽形成困难，褐化现象明显
Leaves are short and yellow, the formation of top shoots is difficult, and the browning of shoots is obvious1/2MS 42.4 ± 3.2c 叶片呈浅绿色，形成顶芽水渍化现象明显
Leaves are light green, and the formed top shoots are easy to be water-soaking1/2MMS 94.1 ± 2.8a 叶片颜色翠绿，顶芽形成快、生长健壮，无褐化、玻璃化现象
Leaves are green, the formation of top shoots is efficient, and the shoots are robust without browning and vitrification注：同一列中不同小写字母表示不同基本培养基间差异显著（P < 0.05）。Note: different lowercase letters from the same column indicate significant differences among varied basal media at P < 0.05 level. 
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表 2 细胞分裂素对桐棉松萌发体胚芽苗增殖和生长的影响
Table 2 Effects of cytokinins on proliferation and growth of shoots originated from germinated somatic embryos in Pinus massoniana ‘Tongmiansong’
激素浓度
Hormone concentration/
（µmol·L− 1）有效芽增殖系数
Proliferation coefficient of effective shoots丛芽高度
Height of clustered shoots/cm芽生长情况
Shoot growth6-BA TDZ 6-BA TDZ 6-BA TDZ 0 0dA 0eA — — 未形成丛芽，针叶短小、颜色深绿，茎节间短，植株矮小
Cluster shoots are not found, leaves are short and dark green, internodes of shoots are short, and the whole plant is dwarf2 0.9 ± 0.2cA 1.1 ± 0.5dA 4.8 ± 0.4bB 8.1 ± 0.8bA 多呈单芽，大部分芽节间短、针叶密且呈深绿色
Most of shoots are single, their internodes are short, and leaves are compact and dark green丛生芽少，针叶细长，叶片为浅绿色
Cluster shoots are rare, leaves are tenuous and light green4 2.9 ± 0.7bB 5.6 ± 0.7aA 7.8 ± 0.8aB 9.2 ± 0.8aA 丛芽多，芽苗节间长，叶片颜色翠绿，生长健壮
Plenty of cluster shoots are observed, with long internodes, green leaves, and they grow well丛芽多，针叶长，叶片颜色翠绿，茎节间长，生长健壮
Plenty of cluster shoots are investigated, with long and green leaves, long internodes, and the whole shoots are thriving6 4.1 ± 0.5aA 3.3 ± 1.0bA 5.0 ± 0.8bB 6.7 ± 0.9cA 短簇状丛生芽较多，茎节间短，叶片颜色发黄，有褐化现象
Short cluster shoots are usually found, with short internodes, yellow leaves, and the browning of shoots is observed丛芽多，顶稍针叶较短，有部分叶片颜色发黄
Cluster shoots are sufficient, leaves of top shoots are short, and part of leaves are yellow8 2.5 ± 0.8bA 2.1 ± 0.7cA 3.6 ± 0.8cA 3.7 ± 0.4dA 丛芽呈短簇状，叶子卷曲，短小，褐化、玻璃化现象明显
Cluster shoots are short and small, leaves are cured and short, and the vitrification of shoots is remarkable丛芽矮小，叶片颜色发黄，有玻璃化现象
Cluster shoots are shorts, leaves are yellow, and the vitrification of shoots is occasionally found注：同一列中不同小写字母表示不同激素浓度处理间差异显著，同一行中不同大写字母表示两种激素处理间差异显著（P < 0.05）。Notes: different lowercase letters from the same column indicate significant differences among varied hormone concentration treatments, while different capital letters from the same row indicate significant differences between two hormone treatments at P < 0.05 level.
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表 3 生长调节剂对桐棉松萌发体胚复壮芽苗生根能力的影响
Table 3 Effects of plant growth regulators on rooting capacity of reinvigorated shoots originated fromgerminated somatic embryos in Pinus massoniana ‘Tongmiansong’
激素处理
Hormone treatment生根率
Rooting rate/%生根时间
Rooting time/d根条数
Number of roots根长
Root length/cm成活率
Survival rate/%对照 Control 0c — — — — NAA 87.7 ± 4.5b 28.7 ± 2.1a 2.3 ± 0.4d 0.6 ± 0.2b 50.3 ± 3.6d NAA + IAA 91.4 ± 2.7ab 20.8 ± 1.5c 4.2 ± 0.7b 2.1 ± 0.4a 83.4 ± 3.2b NAA + IBA 90.5 ± 2.2ab 24.0 ± 2.3b 3.5 ± 0.8c 2.7 ± 0.4a 70.8 ± 4.6c NAA + PBZ 94.3 ± 3.8a 18.6 ± 1.2c 6.4 ± 0.7a 2.3 ± 0.6a 95.8 ± 2.4a 注：同一列中不同小写字母表示不同激素处理间差异显著（P < 0.05）。Note: different lowercase letters from the same column indicate significant differences among varied hormone treatments at P < 0.05 level.
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