Microspore culture and observations on meiotic chromosome pairing of the haploid in Orychophragmus violaceus
YIN Jia-Ming1,2,**, ZHONG Rong-Qi1,2,**, LIN Na1,2, TANG Zhang-Lin1,2, LI Jia-Na1,2第一联系人:
收稿日期:2019-04-24接受日期:2019-08-9网络出版日期:2019-10-12
| 基金资助: |
Received:2019-04-24Accepted:2019-08-9Online:2019-10-12
| Fund supported: |
作者简介 About authors
殷家明,E-mail:1661096534@qq.com。
钟荣棋,1457139712@qq.com。
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殷家明, 钟荣棋, 林呐, 唐章林, 李加纳. 诸葛菜小孢子培养及其单倍体减数分裂染色体配对观察[J]. 作物学报, 2020, 46(2): 194-203. doi:10.3724/SP.J.1006.2020.94065
YIN Jia-Ming, ZHONG Rong-Qi, LIN Na, TANG Zhang-Lin, LI Jia-Na.
诸葛菜(Orychophragmus violaceus)为十字花科(Bassicaceae)诸葛菜属植物, 主要分布在中国, 在许多地区都有自然分布或人工引种栽培[1], 通常被用作园林、城市绿化地被植物和花坛花卉, 别称“二月蓝(兰)”。除本身具有较高的观赏价值外, 还是创造红花观光兼用型油菜品种的优良基因供体材料。油菜近缘植物芥蓝(Brassica alboglabra)与诸葛菜杂交, 杂种即开紫红或粉红花[2]。诸葛菜PAP2基因在油菜花瓣中异位表达, 得到了开红花的油菜[3]。诸葛菜具有角果长、每角粒多和粒大的特点, 在油菜育种新材料创制上有重要价值[4]。诸葛菜是极具开发前景的蔬菜和饲料种质资源[5,6], 同时也具有潜在的药用价值, 其种子提取物具有护肝和细胞保护作用[7,8]。诸葛菜可作为绿肥植物增加作物产量、减少氮肥流失以及用于土壤修复[9,10]。其种子油组份特殊, 含有特殊的双羟基脂肪酸, 高温润滑性能优于蓖麻油, 是一种十分有价值的新型工业用油料作物种质资源, 可作为生产生物柴油和高级润滑油的原材料[11,12]。
诸葛菜在我国分布广, 遗传变异大, 多样性丰富, 通过花药或花粉培养可以快速获得单倍体和纯合二倍体, 这为诸葛菜的新材料新品种选育提供了材料基础[1,13-18]。吴沿友等[19]进行诸葛菜花药培养, 得到了少量胚状体和再生植株, 而且所得植株为混倍体。贾勇炯等[20]从4℃低温处理4 d的诸葛菜花药中分离小孢子进行培养, 获得了愈伤组织和再生植株。然而, 关于诸葛菜游离小孢子培养通过胚胎发生途径获得胚状体和再生植株还未见报道。
诸葛菜属(Orychophragmus)的系统分类位置存在较大争议, 甚至在十字花科中不能被有力支持聚到任何进化枝[21]。诸葛菜属于诸葛菜复合群(Orychophragmus violaceus complex)的一种, 体细胞染色体数目2n = 24[13,15]。减数分裂染色体配对观察结果表明, 该物种可能为自然多倍体[22]。李再云等[23,24]根据甘蓝型油菜与诸葛菜属间杂种染色体的细胞学行为的观察结果提出, 诸葛菜可能为x = 6的同源四倍体。吴建国等[25]对甘蓝型油菜与诸葛菜的五倍体(AACCO)杂种及其后代的细胞遗传学研究表明, 诸葛菜可能是一天然的多倍体, 基数x = 3。李再云等[26]利用原位杂交技术, 在诸葛菜体细胞染色体中检测到8个45S rDNA位点, 暗示诸葛菜染色体基数为3。Lysak等[27]通过染色体涂染分析发现, 十字花科祖先核型的保守区段F和U在诸葛菜减数分裂粗线期染色体中检测到2个同源异源拷贝。因此, 诸葛菜被认为是一个四倍体或中四倍体(mesotetra- ploid)[27,28]。对诸葛菜单倍体减数分裂染色体配对的观察, 可为了解诸葛菜染色体组的起源和进化提供新的数据。本研究通过诸葛菜游离小孢子培养, 观察胚胎发生过程和胚状体形态, 研究热激培养时间和活性炭浓度对胚状体产量的影响, 并观察诸葛菜单倍体减数分裂染色体配对行为, 其研究结果可为诸葛菜的利用和研究提供重要参考。
1 材料与方法
1.1 材料
诸葛菜(二月兰)种子收集于重庆北碚西南大学校园内野生开放授粉植株, 由重庆市油菜工程技术研究中心繁殖保存。9月中旬播种育苗, 成苗后将幼苗移栽到玻璃温室内土壤中, 成活后每窝施复合肥约5 g, 根据需要浇水。温室光照为自然光, 温度不做特殊控制。次年2月, 植株开花后取样进行小孢子培养, 10月将小孢子植株移栽到试验地, 来年开花后进行形态观察和结实性鉴定, 并取样用于观察花粉育性和减数分裂染色体配对构型。1.2 方法
1.2.1 小孢子培养程序 花期上午8:00—9:00时, 从生长健康植株取4~5 mm长花蕾, 经5%氨替福民溶液消毒10~15 min后用无菌水清洗, 置培养皿中, 加适量B5-13培养液(pH 5.8, 含13%蔗糖), 用玻璃注射器内筒研磨挤压出花粉, 400目不锈钢筛网过滤, 将花粉悬液转移到离心管中, 160 × g离心3 min, 弃上清液, 再加入B5-13培养液摇匀离心。如此重复离心3次后, 用NLN-13培养液(pH 6.0)按1蕾花粉 mL-1密度悬浮。将小孢子悬液混匀, 分装于直径6 cm培养皿, 每皿4 mL, 在每皿中添加一定量的活性炭, 封口膜封口后在32℃暗培养数天, 然后转移到25℃下继续暗培养, 肉眼可见胚状体时转入25℃的振荡培养箱中60 r min-1培养14~20 d后统计胚状体产量。1.2.2 活性炭储存液的配制 在100 mL超纯水中加入1 g活性炭和0.5 g琼脂糖, 121℃高温灭菌后储存备用。
1.2.3 热激培养时间对胚状体产量的影响 每皿添加1 mg活性炭, 32℃下分别热激培养0、1、3、5和7 d, 研究热激培养时间对胚状体产量的影响。在初花5 d后1周内重复取材3次, 每处理每次重复5皿。
1.2.4 活性炭浓度对胚状体产量的影响 每皿添加0、1、2和3 mg活性炭, 在32℃下热激培养3 d, 研究活性炭浓度对胚状体产量的影响。在初花5 d后1周内重复取材3次进行试验, 每处理每次重复5皿。
1.2.5 胚状体发生过程和形态观察 以每皿添加1 mg活性炭、32℃热激3 d的培养物为对象, 经1、3、5、7、10和14 d培养, 分别在倒置显微镜下观察胚状体发生过程。振荡培养14~20 d后, 在体式显微镜下观察胚状体形态。
1.2.6 植株再生、移栽、育性和倍性鉴定以及单倍体染色体配对观察 将振荡培养14~20 d的子叶胚转移到1/2MS+3%蔗糖+7 g L-1琼脂培养基(pH 5.8)上, 在20℃、12.5 μmol m-2 s-1 16 h d-1光照条件下培养。出芽后将芽切下继代保存和扩繁到每个花粉植株无性系具有3~5芽。移栽前1个月, 将芽转移到1/2MS+0.5 mg L-1 IBA+3%蔗糖+7 g L-1琼脂培养基(pH 5.8)上生根。生根后的试管苗经炼苗1周后移栽到试验地, 最初1周内用白色塑料膜遮蔽。在花期, 对植株进行形态观察, 取即将开放花朵, 取出花药并挤出花粉用2%醋酸洋红染液染色后在显微镜下观察统计花粉育性。取幼嫩花蕾用卡诺氏固定液(3份95%乙醇∶1份冰乙酸)固定24 h后转入75%酒精于冰箱内4℃保存备用, 用改良卡宝品红液染色观察花粉母细胞减数分裂后期I或前期II染色体数目, 观察单倍体减数分裂终变期-中期I染色体配对构型。对加倍的植株采用人工辅助授粉自交, 以了解自交结籽率; 同时, 辅助授以天然诸葛菜混合花粉, 以了解异交结籽率。
2 结果与分析
2.1 胚状体发生过程和形态
培养1 d后, 部分小孢子体积明显增大, 呈圆球形; 另一部分小孢子体积没有明显变化(图1-A)。培养3 d后, 观察到一些小孢子发生第1次细胞分裂。分裂方式有2种, 多为对称分裂, 形成2个大小近等的细胞(图1-B); 另一种是非对称分裂, 形成2个大小明显不同的细胞(图1-C)。培养5 d后, 可观察到多细胞团(图1-D)和具有胚柄状结构的原胚(图1-E)。培养7 d后, 胚发育到球形原胚阶段(图1-F)。培养10 d后, 胚发育到球形胚时期(图1-G)。培养14 d, 肉眼可见细小的白色颗粒状培养物, 在倒置显微镜下可见球形胚状体和心形胚状体, 包括圆球形(图1-H)和卵球形(图1-I)胚状体以及桃心形(图1-J)和心脏形胚状体(图1-K)。继续振荡培养14 d后, 胚状体发育到子叶期(图1-L)。图1
新窗口打开|下载原图ZIP|生成PPT图1诸葛菜小孢子胚胎发生过程
A: 部分小孢子膨大; B: 小孢子近均等分裂; C: 小孢子不均等分裂; D: 多细胞团; E: 具胚柄状结构的原胚; F: 球形原胚; G: 球形胚; H: 具胚柄状结构的圆球形胚状体; I: 卵球形胚状体; J: 桃心形胚状体; K: 心脏形胚状体; L: 培养28 d后的胚状体。
Fig. 1Microspore embryogenesis of Orychophragmus violaceus
A: some swelled microspores; B: microsporal equal cell division; C: microsporal unequal cell division; D: multicell cluster; E: proembryo with a suspensor-like structure; F: globular proembryo; G: globular embryo; H: round sphere embryoid with a suspensor-like structure; I: oval sphere embryoid; J: heart embryoid; K: cordate embryoid; L: embryoids after 28 days of culture.
振荡培养14~20 d后在体式显微镜下观察胚状体形态。胚状体可分为子叶形胚状体(图2-A~E)、鱼雷形胚状体(图2-F)、心形胚状体(图2-G)、球形胚状体(图2-H)和不规则形胚状体(图2-I) 5种类型。同一类型的胚状体又有多种多样的形态。如子叶期胚状体就有双子叶胚状体(图2-A)、三子叶胚状体(图2-B~C)、单子叶胚状体(图2-D)和2个双子叶胚状体合生(图2-E)等多样的形态表现; 球形胚状体有橄榄球形、椭球形、卵球形和圆球形胚状体等形态差异(图2-H)。
图2
新窗口打开|下载原图ZIP|生成PPT图2诸葛菜小孢子胚状体形态
A: 双子叶胚状体; B: 三子叶胚状体; C: 具1胚根状结构三子叶胚状体; D: 单子叶胚状体; E: 合生双胚; F: 鱼雷形胚状体; G: 心形胚状体; H: 球形胚状体; I: 不规则形胚状体; 标尺 = 2 mm。
Fig. 2Morphology of the microspore-derived embryoids of O. violaceus
A: dicotyledonous embryoid; B: tricotyledonous embryoid; C: tricotyledonous embryoid with an additional radicle; D: monocotyledonous embryoid; E: twin dicotyledonous embryoids; F: torpedo-shaped embryoids; G: heart-shaped embryoids; H: globular embryoids; I: anomalous embryoids; Bar = 2 mm.
2.2 热激培养时间对胚状体产量的影响
相对于持续25℃培养, 32℃热激培养对于胚状体诱导和形成是必需的。未经热激培养不能诱导形成任何形态的胚状体, 热激培养1 d便能诱导产生胚状体。试验以热激培养3 d获得的总胚状体产量最高, 子叶形胚状体产量也最高。热激培养时间再延长, 总胚状体产量和子叶形胚状体产量都有降低的趋势(表1)。Table 1
表1
表1不同热激时间下诸葛菜小孢子胚状体产量
Table 1
| 热激时间 Heat-shock duration (d) | 子叶形胚状体 Cotyledonous embryoids (No. per bud) | 总胚状体 All embryoids (No. per bud) |
|---|---|---|
| 0 | 0 | 0 |
| 1 | 0.13±0.13 c | 0.57±0.13 c |
| 3 | 0.92±0.18 a | 1.32±0.25 a |
| 5 | 0.48±0.10 b | 0.95±0.09 b |
| 7 | 0.15±0.09 c | 0.60±0.13 c |
新窗口打开|下载CSV
2.3 活性炭浓度对胚状体产量的影响
添加活性炭对于胚状体的形成是必需的, 不添加活性炭的处理未获得任何形态的胚状体。每皿添加1 mg活性炭时, 总胚状体产量和子叶形胚状体产量最高, 但与每皿添加活性炭2 mg时的总胚状体产量和子叶形胚状体产量相比, 没有统计学意义上的显著差异。添加活性炭过多不利于胚状体的形成, 对子叶形胚状体的形成影响更大(表2)。Table 2
表2
表2不同活性炭浓度下诸葛菜小孢子胚状体产量
Table 2
| 活性炭浓度 Active charcoal content (mg dish-1) | 子叶形胚状体 Cotyledonous embryoids (No. per bud) | 总胚状体 All embryoids (No. per bud) |
|---|---|---|
| 0 | 0 | 0 |
| 1 | 0.92±0.18 a | 1.32±0.25 a |
| 2 | 0.85±0.15 a | 1.25±0.10 ab |
| 3 | 0.37±0.10 b | 0.80±0.17 b |
新窗口打开|下载CSV
2.4 植株再生、形态、育性和倍性
将子叶形胚状体转移到1/2MS固体培养基上, 有的胚状体逐渐褐化死亡; 有的胚状体胚轴伸长但不出芽; 也有的胚状体很快便开始萌发并出芽, 其下胚轴伸长, 长出白色的根, 在子叶节处长出幼芽(图3-A)。共培养子叶胚119个, 经过30 d培养, 有33个胚状体萌发出芽, 萌发率为27.73%。胚状体萌发产生的根纤弱且数量少, 将芽切下转移到1/2MS固体培养基继代保存和扩大繁殖到每个无性系有3~5芽。移栽前1个月, 将继代保存的无性系32个转移到1/2MS+IBA 0.5 mg L-1固体培养基上生根, 有22个无性系生根, 生根率为68.75%。生根后的试管苗经炼苗1周后移栽到试验地, 共移栽22个无性系, 到开花期有12个存活, 移栽存活率为54.55%。图3
新窗口打开|下载原图ZIP|生成PPT图3诸葛菜小孢子植株的再生、形态、育性和倍性
A: 胚状体萌发; B: 加倍的小孢子植株; C: 单倍体植株; D: 加倍和单倍体植株的花和花蕾; E: 加倍植株花粉; F: 单倍体植株花粉; G: 加倍植株花粉母细胞染色体(2n = 24); H: 单倍体植株花粉母细胞染色体(2n = 12); 标尺 = 10 μm。
Fig. 3Regeneration, morphology, fertility, and ploidy of the microspore-derived plantlets in O. violaceus
A: germination of embryoids; B: doubled microspore-derived plantlet; C: haploid plantlet; D: flowers and buds of double haploid and haploid plantlets; E: pollens of double haploid plantlet; F: pollens of haploid plantlet; G: chromosomes in the pollen mother cell of doubled plantlet (2n = 24); H: chromosomes in the pollen mother cell of haploid plantlet(2n = 12); bar = 10 μm.
开花期观察, 根据植株形态特别是花器官大小, 植株可分为2种类型, 即加倍的植株(图3-B)和单倍体植株(图3-C)。加倍植株生长健壮, 花蕾较多, 花朵和花蕾较大; 而单倍体植株较纤弱, 花蕾较少, 花朵和花蕾较小(图3-B~D)。在存活的12个花粉植株无性系中, 有3个为自然加倍植株, 自然加倍率为25%。加倍植株花药饱满, 内含大量可育花粉(图3-E)。不同加倍植株间花粉育性有差异, 3个加倍花粉植株可育花粉率分别为58.35%、62.68%和76.03%。单倍体植株花药干瘪, 内含花粉粒少, 在显微镜下观察全为小而不可染色的败育花粉(图3-F)。染色体数量观察表明, 加倍植株染色体为24条(图3-G), 单倍体植株染色体为12条(图3-H)。加倍植株自交能结少量种子, 表现自交高度不亲和, 3个加倍花粉植株平均每角果结籽数分别为0、0.25和0.65。对3株加倍植株辅助授以天然诸葛菜混合花粉则能结较多种子, 平均每角果结籽数分别为10.20、12.65和16.40。单倍体植株开放式授粉和人工辅助授以天然诸葛菜混合花粉均不能结籽。
2.5 单倍体减数分裂染色体配对构型
在125个花粉母细胞中, 共观察到24种染色体配对构型(表3和图4)。染色体可联会形成单价体、二价体、三价体和四价体, 单价体数变化在0~12个, 二价体数变化在0~6个, 三价体数变化在0~2个, 四价体数变化在0~3个, 平均配对构型为n = 12 = 6.352I+2.008II+0.384III+0.12IV。染色体联会形成二价体及三价体和四价体的细胞比例达到96%, 只有4%的细胞形成12个单价体(表3)。值得注意的是, 有少量细胞(0.8%)的12条染色体联会形成3个四价体(表3, 图4-H)。这说明诸葛菜具有多倍体的性质, 很可能起源于染色体基数x = 3的同源八倍体。Table 3
表3
表3诸葛菜单倍体减数分裂染色体配对构型
Table 3
| 序号 No. | 染色体构型 Chromosome configuration | 细胞数 No. of cells | 百分比 Percentage (%) | |||
|---|---|---|---|---|---|---|
| IV | III | II | I | |||
| 1 | 0 | 0 | 0 | 12 | 5 | 4.0 |
| 2 | 0 | 0 | 1 | 10 | 16 | 12.8 |
| 3 | 0 | 0 | 2 | 8 | 21 | 16.8 |
| 4 | 0 | 0 | 3 | 6 | 22 | 17.6 |
| 5 | 0 | 0 | 4 | 4 | 6 | 4.8 |
| 6 | 0 | 0 | 5 | 2 | 3 | 2.4 |
| 7 | 0 | 0 | 6 | 0 | 3 | 2.4 |
| 8 | 0 | 1 | 0 | 9 | 3 | 2.4 |
| 9 | 0 | 1 | 1 | 7 | 9 | 7.2 |
| 10 | 0 | 1 | 2 | 5 | 15 | 12.0 |
| 11 | 0 | 1 | 3 | 3 | 2 | 1.6 |
| 12 | 0 | 2 | 0 | 6 | 2 | 1.6 |
| 13 | 0 | 2 | 1 | 4 | 3 | 2.4 |
| 14 | 0 | 2 | 2 | 2 | 2 | 1.6 |
| 15 | 0 | 2 | 3 | 0 | 1 | 0.8 |
| 16 | 1 | 0 | 0 | 8 | 1 | 0.8 |
| 17 | 1 | 0 | 1 | 6 | 3 | 2.4 |
| 18 | 1 | 0 | 2 | 4 | 2 | 1.6 |
| 19 | 1 | 0 | 3 | 2 | 1 | 0.8 |
| 20 | 1 | 0 | 4 | 0 | 1 | 0.8 |
| 21 | 1 | 1 | 0 | 5 | 1 | 0.8 |
| 22 | 1 | 1 | 1 | 3 | 1 | 0.8 |
| 23 | 2 | 1 | 0 | 1 | 1 | 0.8 |
| 24 | 3 | 0 | 0 | 0 | 1 | 0.8 |
| 合计Total | 15 | 48 | 251 | 794 | 125 | 100.0 |
| 平均Average | 0.12 | 0.384 | 2.008 | 6.352 | — | — |
新窗口打开|下载CSV
图4
新窗口打开|下载原图ZIP|生成PPT图4部分诸葛菜单倍体(2n = 12)花粉母细胞减数分裂染色体配对构型(标尺 = 10 μm)
Fig. 4Some meiotic chromosome (2n = 12) pairing configurations in pollen mother cells of the haploid of O. violaceus (Bar = 10 μm)
A: 12I; B: 1II+10I; C: 6II; D: 1III+3II+3I; E: 1IV+4II; F: 1IV+1III+1II+3I; G: 2IV+1III+1I; H: 3IV.
3 讨论
小孢子胚胎发生和胚状体形态观察表明, 诸葛菜小孢子胚胎发生经历了均等或非均等细胞分裂、多细胞团、球形原胚、球形胚、心形胚、鱼雷形胚和子叶胚等时期, 这与同为十字花科的芸薹属植物的情形相似[29,30,31,32]。本试验观察到具有胚柄状结构的胚。由于具胚柄小孢子胚与合子胚更相似, 因此具胚柄小孢子胚发育途径诱导体系在研究胚胎发育机制方面有重要价值而受到重视[32,33]。形成胚柄状结构的现象在甘蓝型油菜和芥菜型油菜小孢子胚胎发生中都有报道。在甘蓝型油菜中, 小孢子培养温度和时间是影响胚柄样结构形成的关键因素。Ilic-Grubor等[34]报道在35℃热激1 h、33℃热激14~18 h后转移到24℃培养时, 甘蓝型油菜部分小孢子球形胚、心形胚和鱼雷形胚上存在胚柄状结构。Joosen等[32]和Supena等[35]研究表明, 短暂温和的32℃热激处理促进甘蓝型油菜具胚柄小孢子胚发育途径, 随着热激处理时间缩短该途径比例越高, 小孢子胚胎发育与合子胚胎发生极其相似, 胚柄结构从第1次分裂发生持续到心形胚阶段。而Prem等[33]则建立了一种新的与合子胚发生相似的小孢子胚胎发生体系, 即在持续18℃下进行甘蓝型油菜“Topas” 的小孢子培养, 小孢子的发育有3条途径, 为配子体样发育途径、不具胚柄状结构的胚胎发育途径和具有胚柄状结构的胚胎发育途径, 其中具有胚柄状结构的胚胎发育途径为主要途径, 占50%以上, 与合子胚胎发生相似, 胚柄结构从第1次分裂发生持续到心形胚阶段。在芥菜型油菜中, 基因型和供体植株生长条件影响具胚柄状结构小孢子胚的形成。Chanana等[36]对3种生长条件的6个芥菜型油菜品种进行小孢子培养, 在32℃热激3 d后转移到25℃下培养, 结果只有在昼/夜温度为10℃/5℃的生长条件下1个品种“PR-45”形成了具胚柄状结构的胚状体, 胚柄结构可持续到子叶胚。本试验观察到胚柄状结构可持续到鱼雷形胚状体(图2-F), 而子叶形胚状体上未观察到胚柄状结构。这是因为具胚柄状结构胚胎发育较缓慢还是在鱼雷期后降解消失需要进一步考察。在十字花科植物小孢子培养中, 温度胁迫是诱导小孢子胚胎发生的重要因素, 常用的处理方式是32~35℃热激处理1至数天后转移到常温(25℃)培养。本试验结果表明, 热激对诸葛菜小孢子胚的发生具有重要作用, 相对于持续25℃培养来说, 32℃热激培养对于胚状体诱导和形成是必需的。Prem等[33]在持续18℃低温胁迫下进行甘蓝型油菜“Topas”的小孢子培养获得成功, 建立了一种新的小孢子胚胎发生体系。低温胁迫和传统的高温胁迫下成熟胚状体形态相似, 胚状体萌发成苗率和花粉植株染色体自发加倍率都没有显著差异, 而低温胁迫较高温胁迫下胚状体产量要低得多, 所需培养时间也较长[33]。另外, 高温胁迫处理在十字花科植物小孢子培养中得到广泛应用, 而小孢子低温胁迫培养体系在包括诸葛菜在内的其他基因型材料上的适用性还需进一步研究。
活性炭具有吸附和减少培养基中的抑制成分和有毒代谢产物的能力, 经常用于植物组织培养中, 来改善细胞的生长和发育[37]。活性炭在十字花科植物甘蓝[38,39,40]、白菜[41,42,43,44]、黑芥[45,46]、甘蓝型油菜[47,48,49,50,51]、芥菜型油菜[52]和芝麻菜[53]的小孢子培养中都得到广泛而成功应用, 对小孢子胚胎发生和植株再生表现出促进作用。本试验也说明了活性炭在小孢子培养中的重要性, 表明添加活性炭对诸葛菜小孢子胚胎发生是必需的。
诸葛菜单倍体花粉母细胞减数分裂染色体配对观察表明, 诸葛菜很可能是一个起源于染色体基数x = 3的同源八倍体, 这与吴建国等[25]和李再云等[26]的观点一致, 而与Lysak等[27]和Franzke等[28]的观点不同。在试验中, 还观察到一定比例的花粉母细胞染色体联会形成6个二价体(表3, 图4-C), 但花粉育性和结实性观察结果都表明, 诸葛菜单倍体雌雄都不育。这说明, 即使这6个染色体构成的一组染色体, 其遗传结构和功能是不平衡的。尽管观察到形成3个四价体的细胞, 但其比例极低, 加上形成6个二价体的细胞, 总的比例也仅为3.2%; 形成12个单价体的细胞比例也仅占4%; 绝大多数细胞都是不规则的配对构型(表3)。因此, 本研究推测诸葛菜起源于x = 3的同源八倍体, 但在进化过程中染色体和染色体组的结构已发生了较大变化, 正处于向二倍化转变的过程中。Lysak等[27]的研究支持诸葛菜是四倍体, 这是否就是该多倍体二倍化过程中间环节的体现, 还需进一步研究。值得注意的是, Lysak等[27]在研究中还发现, 诸葛菜粗线期染色体中, 十字花科祖先核型F区段的1个拷贝与祖先核型的排列相似, 另1个拷贝只保留了祖先核型F区段的顶端部分, 这也说明诸葛菜染色体和染色体组结构发生了重排。
4 结论
初步建立了诸葛菜小孢子培养技术, 通过游离小孢子胚胎发生途径获得了单倍体和染色体加倍的植株。活性炭和热激培养对诸葛菜游离小孢子胚状体诱导是必需的, 在直径6 cm培养皿中培养4 mL 1花蕾花粉 mL-1的小孢子NLN悬液时, 每皿添加1 mg活性炭和32℃热激3 d的培养处理, 子叶形胚状体和总胚状体产量最高。诸葛菜单倍体减数分裂染色体配对行为的观察结果支持诸葛菜为起源于染色体基数x = 3的同源八倍体的观点。参考文献 原文顺序
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被引期刊影响因子
[本文引用: 2]
[本文引用: 2]
[本文引用: 1]
[本文引用: 1]
DOI:10.1111/pbi.12777URLPMID:28640973 [本文引用: 1]
Oilseed rape (Brassica napus L.), which has yellow flowers, is both an important oil crop and a traditional tourism resource in China, whereas the Orychophragmus violaceus, which has purple flowers, likely possesses a candidate gene or genes to alter the flower colour of oilseed rape. A previously established B.?napus line has a particular pair of O.?violaceus chromosomes (M4) and exhibits slightly red petals. In this study, the transcriptomic analysis of M4, B.?napus (H3), and O.?violaceus with purple petals (OvP) and with white petals (OvW) revealed that most anthocyanin biosynthesis genes were up-regulated in both M4 and OvP. Read assembly and sequence alignment identified a homolog of AtPAP2 in M4, which produced the O.?violaceus transcript (OvPAP2). The overexpression of OvPAP2 via the CaMV35S promoter in Arabidopsis thaliana led to different levels of anthocyanin accumulation in most organs, including the petals. However, the B.?napus overexpression plants showed anthocyanin accumulation primarily in the anthers, but not the petals. However, when OvPAP2 was driven by the petal-specific promoter XY355, the transgenic B.?napus plants produced red anthers and red petals. The results of metabolomic experiments showed that specific anthocyanins accumulated to high levels in the red petals. This study illustrates the feasibility of producing red-flowered oilseed rape, thereby enhancing its ornamental value, via the ectopic expression of the OvPAP2 gene. Moreover, the practical application of this study for insect pest management in the crop is discussed.
DOI:10.3389/fpls.2017.00001URLPMID:28220127 [本文引用: 1]
The present study investigates ameliorative effects of nitric oxide (NO) against zinc oxide nanoparticles (ZnONPs) phytotoxicity in wheat seedlings. ZnONPs exposure hampered growth of wheat seedlings, which coincided with reduced photosynthetic efficiency (Fv/Fm and qP), due to increased accumulation of zinc (Zn) in xylem and phloem saps. However, SNP supplementation partially mitigated the ZnONPs-mediated toxicity through the modulation of photosynthetic activity and Zn accumulation in xylem and phloem saps. Further, the results reveal that ZnONPs treatments enhanced levels of hydrogen peroxide and lipid peroxidation (as malondialdehyde; MDA) due to severely inhibited activities of the following ascorbate-glutatione cycle (AsA-GSH) enzymes: ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase and dehydroascorbate reductase, and its associated metabolites ascorbate and glutathione. In contrast to this, the addition of SNP together with ZnONPs maintained the cellular functioning of the AsA-GSH cycle properly, hence lesser damage was noticed in comparison to ZnONPs treatments alone. The protective effect of SNP against ZnONPs toxicity on fresh weight (growth) can be reversed by 2-(4carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, and thus suggesting that NO released from SNP ameliorates ZnONPs toxicity. Overall, the results of the present study have shown the role of NO in the reducing of ZnONPs toxicity through the regulation of accumulation of Zn as well as the functioning of the AsA-GSH cycle.
[本文引用: 1]
DOI:10.1023/A:1008668225306URL [本文引用: 1]
DOI:10.3390/ijms18061197URLPMID:28617329 [本文引用: 1]
Orychophragmus violaceus (O. violaceus) is a kind of edible wild herb in north China and its seeds have medical potential, however, the effect of O. violaceus seeds on liver injury and the mechanism of action remains poorly understood. Thus, the purpose of the present study is to investigate the effect of O. violaceus seeds on liver injury and further explore the molecular mechanism of the beneficial effects using aqueous extract from the seeds of O. violaceus (AEOV). Mice were orally administrated with saline, AEOV, and biphenyldicarboxylate for 4 days, and were then injected subcutaneously with 0.1% carbon tetrachloride (CCl?) dissolved in corn oil. Sixteen hours later, mice were sacrificed and blood samples were collected. Then, the serum was separated and used for biochemical assay. Livers were excised and were routinely processed for histological examinations. Enzyme activities and protein levels in liver homogenates were detected using commercial kits or by western blot analysis. Additionally, the hepatoprotective effect of AEOV in vitro was evaluated using epigoitrin, the major alkaloid compound isolated from AEOV. We found that AEOV attenuated liver injury induced by CCl? as evidenced by decreased levels of alanine aminotransferase (ALT) and aminotransferase (AST) in serum, improvement of liver histopathological changes, and substantial attenuation of oxidative stress and inflammation via regulation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and nuclear factor κB (NFκB) pathways. These effects of AEOV were comparable to that of biphenyldicarboxylate which was commonly used as a hepatoprotective reference. Moreover, pretreatment of HepG2 cells with epigoitrin improved cell viability, decreased lactate dehydrogenase (LDH) and malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, attenuated the NFκB pathway, and elevated the Nrf2 pathway after exposure to H?O?. These results suggest that AEOV could effectively prevent CCl?-induced liver injury in mice via regulating the Nrf2 and NFκB pathways, and reveal the cytoprotective effects of epigoitrin against H?O?-induced oxidative stress in HepG2 cells.
DOI:10.1039/C7RA02951AURL [本文引用: 1]
DOI:10.1016/S2095-3119(15)61212-6URL [本文引用: 1]
DOI:10.1007/s00253-014-5971-5URLPMID:25081560 [本文引用: 1]
Understanding of the effects of perturbation strategies on soil microorganisms is helpful in optimizing bioremediation systems and enhancing their efficiency. Four soil mesocosms were constructed for bioremediation of highly polycyclic aromatic hydrocarbon-contaminated soil using the flowering plant Orychophragmus violaceus and/or bacterium Rhodococcus ruber Em1. Bacterial community dynamics were evaluated by 454 pyrosequencing, and Em1 abundance was assessed by quantitative polymerase chain reaction. The results showed that the diversity of the bacterial community increased gradually with time; the degree of increase was in the order mesocosm PE (combination of O. violaceus and Em1), mesocosm WE (Em1), mesocosm PC (O. violaceus only), mesocosm WA (attenuation). Increased diversity may be predictive of PAH degradation. O. violaceus had a marked effect on bacterial community evolution and promoted the growth of Em1. The bacterial community of mesocosm PE gradually separated from the others, as indicated by Venn diagrams and weight-principal component analysis. Abundances of the families Cytophagaceae?+?Nocardioidaceae?+?Iamiacaeae (Actinobacteria), and Alcanivoracaceae?+?Pseodomonadaceae?+?Xanthomonadaceae (Gammaproteobacteria) were positively correlated with PAH degradation. Our findings help bridge the gap between field bioremediation and laboratory approaches, provide insight into processes of microbial ecological recovery, and will be useful in developing strategies to optimize bioremediation by modifying plant-microbe interaction patterns.
DOI:10.1016/j.enconman.2014.04.047URL [本文引用: 1]
The development of biomass on marginal lands has been investigated and proven to be feasible. Orychophragmus violaceus grows naturally in the karst region and shows a stronger bicarbonate-use capacity and adaptability than some other plants. In the present study, the systemic parameters of seed samples from four locations were determined, including the oil contents (30.59-36.81 wt%), acid values (2.76-5.68 mg KOH/g), iodine values (111.02-147.58 g I-2/100 g), and fatty acid composition. O. violaceus oil was mainly composed of palmitic acid (C16:0, 10.65-13.06 wt%), stearic acid (C18:0, 6.41-8.31 wt%), oleic acid (C18:1, 16.56-25.58 wt%), and linoleic acid (C18:2, 46.07-52.16 wt%). O. violaceus seed oil from Guiyang was converted to biodiesel by calcined porous calcite. The catalyst amount was optimised, and refined biodiesel was prepared by vacuum distillation. The fuel properties of the O. violaceus biodiesel samples all met the EN 14214 (2012) standards, except for the cetane number and oxidation stability. In summary, O. violaceus from the karst marginal land is highly recommended as a biomass feedstock. (C) 2014 Elsevier Ltd.
DOI:10.1038/s41477-018-0225-7URLPMID:30150614 [本文引用: 1]
The biosynthesis of 'unusual' fatty acids with structures that deviate from the common C16 and C18 fatty acids has evolved numerous times in the plant kingdom. Characterization of unusual fatty acid biosynthesis has enabled increased understanding of enzyme substrate properties, metabolic plasticity and oil functionality. Here, we report the identification of a novel pathway for hydroxy fatty acid biosynthesis based on the serendipitous discovery of two C24 fatty acids containing hydroxyl groups at the 7 and 18 carbon atoms as major components of the seed oil of Orychophragmus violaceus, a China-native Brassicaceae. Biochemical and genetic evidence are presented for premature or 'discontinuous' elongation of a 3-OH intermediate by a divergent 3-ketoacyl-CoA (coenzyme A) synthase during a chain extension cycle as the origin of the 7-OH group of the dihydroxy fatty acids. Tribology studies revealed superior high-temperature lubricant properties for O. violaceus seed oil compared to castor oil, a high-performance vegetable oil lubricant. These findings provide a direct pathway for designing a new class of environmentally friendly lubricants and unveil the potential of O. violaceus as a new industrial oilseed crop.
URL [本文引用: 2]
对中国种诸葛菜变种群（色括原变种及三个变种）作了大样本核型分析，结果显示：染色体相对长度从长到短呈渐缓梯度变化，次缢痕位置大多出现在第11对染色体上，一部分次缢痕变化不定。臂比指数极端变幅大，按众数求得的染色体类型是：2n=24(2st)+6sm(2st)。核型对称性分析结果是1A-2A，属于最对称性。核中有较高频率的B染色
URL [本文引用: 2]
对中国种诸葛菜变种群（色括原变种及三个变种）作了大样本核型分析，结果显示：染色体相对长度从长到短呈渐缓梯度变化，次缢痕位置大多出现在第11对染色体上，一部分次缢痕变化不定。臂比指数极端变幅大，按众数求得的染色体类型是：2n=24(2st)+6sm(2st)。核型对称性分析结果是1A-2A，属于最对称性。核中有较高频率的B染色
DOI:10.2307/3392983URL
DOI:10.1111/njb.2008.26.issue-5-6URL [本文引用: 1]
DOI:10.1007/s10722-009-9450-2URL
DOI:10.3417/2009133URL [本文引用: 1]
Two new varieties of Orychophragmus violaceus (L.) O. E. Schultz (Brassicaceae) are described from Heilongjiang Province in northeastern China. The new variety, O. violaceus var. odontopetalus Ling Wang & Chuan P. Yang, differs from the typical variety of the species in that the petal has five or more obtuse teeth (vs. smooth petals) in the upper third of the expanded petal. The clawed petals have an expanded apical section that is 10-13 mm long (vs. 7-13 mm), and the stems and pedicels are puberulent (vs. glabrous). A second new variety is O. violaceus var. variegatus Ling Wang & Chuan P. Yang, which differs from the typical variety by the marked petal variegation of white and purple (vs. no petal variegation). The uppermost leaves are often undivided, with sharply serrate margins (vs. irregularly toothed leaves). These distinctions have persisted and been stable after eight years of cultivation in field experiments in Heilongjiang. A key to the infraspecific taxa of O. violaceus is included. Both new varieties are cataloged as of Least Concern (LC), based on lUCN Red List categories and criteria.
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DOI:10.1093/molbev/msp202URLPMID:19744998 [本文引用: 1]
Brassicaceae is an important family at both the agronomic and scientific level. The family not only includes several model species, but it is also becoming an evolutionary model at the family level. However, resolving the phylogenetic relationships within the family has been problematic, and a large-scale molecular phylogeny in terms of generic sampling and number of genes is still lacking. In particular, the deeper relationships within the family, for example between the three major recognized lineages, prove particularly hard to resolve. Using a slow-evolving mitochondrial marker (nad4 intron 1), we reconstructed a comprehensive phylogeny in generic representation for the family. In addition, and because resolution was very low in previous single marker phylogenies, we adopted a supermatrix approach by concatenating all checked and reliable sequences available on GenBank as well as new sequences for a total 207 currently recognized genera and eight molecular markers representing a comprehensive coverage of all three genomes. The supermatrix was dated under an uncorrelated relaxed molecular clock using a direct fossil calibration approach. Finally, a lineage-through-time-plot and rates of diversification for the family were generated. The resulting tree, the largest in number of genera and markers sampled to date and covering the whole family in a representative way, provides important insights into the evolution of the family on a broad scale. The backbone of the tree remained largely unresolved and is interpreted as the consequence of early rapid radiation within the family. The age of the family was inferred to be 37.6 (24.2-49.4) Ma, which largely agrees with previous studies. The ages of all major lineages and tribes are also reported. Analysis of diversification suggests that Brassicaceae underwent a rapid period of diversification, after the split with the early diverging tribe Aethionemeae. Given the dates found here, the family appears to have originated under a warm and humid climate approximately 37 Ma. We suggest that the rapid radiation detected was caused by a global cooling during the Oligocene coupled with a genome duplication event. This duplication could have allowed the family to rapidly adapt to the changing climate.
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DOI:10.1007/BF00220869URLPMID:24169678 [本文引用: 1]
The intergeneric hybrid between Brassica napus and Orychophragmus violaceus was obtained by means of embryo culture technique with the latter as the pollen parent. The hybrid was morphologically intermediate between its parents, but could produce a lot of seeds when selfed. Somatic separation of the genomes from the two parental species was observed during the mitotic divisions of some of the hybrid cells. Thus, the hybrid became the mixoploid in nature, consisting of haploid and diploid cells of B. napus, and a nuclear - cytoplasmic hybrid, with the cytoplasm of B. napus and the nuclei of O. violaceus, and the hybrid cells. Pollen mother cells with 19, 12 and 6 bivalents, respectively, were produced by the hybrid. From the selfed progeny of the hybrid, mainly two kinds of plants, B. napus and the hybrid, were found. The hybrid plants of the selfed progeny again produced two kinds of plants, B. napus and the hybrid.
DOI:10.3389/fpls.2018.01557URLPMID:30450106 [本文引用: 1]
The creation of intergeneric somatic hybrids between Citrus and Poncirus is an efficient approach for citrus rootstock breeding, offering the possibility of combining beneficial traits from both genera into novel rootstock lineages. These somatic hybrids are also used as parents for further tetraploid sexual breeding. In order to optimize these latter breeding schemes, it is essential to develop knowledge on the mode of inheritance in the intergeneric tetraploid hybrids. We assessed the meiotic behavior of an intergeneric tetraploid somatic hybrid resulting from symmetric protoplast fusion of diploid Citrus reticulata and diploid Poncirus trifoliata. The analysis was based on the segregation patterns of 16 SSR markers and 9 newly developed centromeric/pericentromeric SNP markers, representing all nine linkage groups of the Citrus genetic map. We found strong but incomplete preferential pairing between homologues of the same ancestral genome. The proportion of gametes that can be explained by random meiotic chromosome associations (τ) varied significantly between chromosomes, from 0.09 ± 0.02 to 0.47 ± 0.09, respectively, in chromosome 2 and 1. This intermediate inheritance between strict disomy and tetrasomy, with global preferential disomic tendency, resulted in a high level of intergeneric heterozygosity of the diploid gametes. Although limited, intergeneric recombinations occurred, whose observed rates, ranging from 0.09 to 0.29, respectively, in chromosome 2 and 1, were significantly correlated with τ. Such inheritance is of particular interest for rootstock breeding because a large part of the multi-trait value selected at the teraploid parent level is transmitted to the progeny, while the potential for some intergeneric recombination offers opportunities for generating plants with novel allelic combinations that can be targeted by selection.
DOI:10.1111/pbr.1997.116.issue-3URL [本文引用: 2]
DOI:10.1508/cytologia.70.1URL [本文引用: 2]
DOI:10.1104/pp.107.104380URLPMID:17720758 [本文引用: 5]
The paleopolyploid character of genomes of the economically important genus Brassica and closely related species (tribe Brassiceae) is still fairly controversial. Here, we report on the comparative painting analysis of block F of the crucifer Ancestral Karyotype (AK; n = 8), consisting of 24 conserved genomic blocks, in 10 species traditionally treated as members of the tribe Brassiceae. Three homeologous copies of block F were identified per haploid chromosome complement in Brassiceae species with 2n = 14, 18, 20, 32, and 36. In high-polyploid (n >or= 30) species Crambe maritima (2n = 60), Crambe cordifolia (2n = 120), and Vella pseudocytisus (2n = 68), six, 12, and six copies of the analyzed block have been revealed, respectively. Homeologous regions resembled the ancestral structure of block F within the AK or were altered by inversions and/or translocations. In two species of the subtribe Zillineae, two of the three homeologous regions were combined via a reciprocal translocation onto one chromosome. Altogether, these findings provide compelling evidence of an ancient hexaploidization event and corresponding whole-genome triplication shared by the tribe Brassiceae. No direct relationship between chromosome number and genome size variation (1.2-2.5 pg/2C) has been found in Brassiceae species with 2n = 14 to 36. Only two homeologous copies of block F suggest a whole-genome duplication but not the triplication event in Orychophragmus violaceus (2n = 24), and confirm a phylogenetic position of this species outside the tribe Brassiceae. Chromosome duplication detected in Orychophragmus as well as chromosome rearrangements shared by Zillineae species demonstrate the usefulness of comparative cytogenetics for elucidation of phylogenetic relationships.
DOI:10.1016/j.tplants.2010.11.005URLPMID:21177137 [本文引用: 2]
Life without the mustard family (Brassicaceae) would be a world without many crop species and the model organism Arabidopsis (Arabidopsis thaliana) that has revolutionized our knowledge in almost every field of modern plant biology. Despite this importance, research breakthroughs in understanding family-wide evolutionary patterns and processes within this flowering plant family were not achieved until the past few years. In this review, we examine recent outcomes from diverse botanical disciplines (taxonomy, systematics, genomics, paleobotany and other fields) to synthesize for the first time a holistic view on the evolutionary history of the mustard family.
DOI:10.1007/978-1-4939-8944-7_5URLPMID:30460559 [本文引用: 1]
Isolated microspore culture (IMC) is the most efficient way to produce large numbers of doubled-haploid (DH) barley plants in a short time. Yet, while IMC is more cost-efficient and less labor-intensive than anther culture, it is technically more complex and requires more experienced personnel if it is to yield its full potential. In part, this is because of multiple and important interactions that exist between factors at its many different phases, including genotype effects as well. When every phase is fine-tuned, the protocol that is presented below yields a useful number of DHs with almost all genotypes and can allow the production of up to 300 DH plants from a single F1 plant in just a few months.
DOI:10.1007/BF01146783URLPMID:30727651 [本文引用: 1]
Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.
DOI:10.1007/BF03030304URL [本文引用: 1]
The ability of microspores to undergo embryo development after a successful induction treatment provides a unique experimental system to study a variety of developmental processes in plants. Recent published results focus on the cellular and molecular aspects of the early induction process. In this review, besides summarizing the current findings, the advantages of using the MDE system to study other aspects of embryo development are emphasized. The continual improvement of culturing procedures, media components, and molecular methods guarantees exciting new findings in the near future.
DOI:10.1104/pp.107.098723URLPMID:17384159 [本文引用: 3]
Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants.
DOI:10.1186/1471-2229-12-127URLPMID:22857779 [本文引用: 4]
Microspore embryogenesis represents a unique system of single cell reprogramming in plants wherein a highly specialized cell, the microspore, by specific stress treatment, switches its fate towards an embryogenesis pathway. In Brassica napus, a model species for this phenomenon, incubation of isolated microspores at 32°C is considered to be a pre-requisite for embryogenesis induction.
DOI:10.1006/anbo.1998.0661URL [本文引用: 1]
DOI:10.1093/jxb/erm358URLPMID:18272920 [本文引用: 1]
The inaccessibility of the zygote and proembryos of angiosperms within the surrounding maternal and filial tissues has hampered studies on early plant embryogenesis. Somatic and gametophytic embryo cultures are often used as alternative systems for molecular and biochemical studies on early embryogenesis, but are not widely used in developmental studies due to differences in the early cell division patterns with seed embryos. A new Brassica napus microspore embryo culture system, wherein embryogenesis highly mimics zygotic embryo development, is reported here. In this new system, the donor microspore first divides transversely to form a filamentous structure, from which the distal cell forms the embryo proper, while the lower part resembles the suspensor. In conventional microspore embryogenesis, the microspore divides randomly to form an embryonic mass that after a while establishes a protoderm and subsequently shows delayed histodifferentiation. In contrast, the embryo proper of filament-bearing microspore-derived embryos undergoes the same ordered pattern of cell division and early histodifferentiation as in the zygotic embryo. This observation suggests an important role for the suspensor in early zygotic embryo patterning and histodifferentiation. This is the first in vitro system wherein single differentiated cells in culture can efficiently regenerate embryos that are morphologically comparable to zygotic embryos. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during the early stages of plant embryogenesis.
DOI:10.1007/s00709-018-1268-3URLPMID:29922944 [本文引用: 1]
Production of doubled haploid (DH) plants is an efficient tool in genetic and plant breeding programs; however, sweet pepper (Capsicum annuum L.) is recalcitrant to microspore embryogenesis and DH production. Trying to break the barrier of DH production, three independent experiments were conducted on microspore embryogenesis of sweet pepper. In the first experiment, the effect of cold (4?°C) and heat (32?°C) pretreatments were investigated on microspore embryogenesis of three genotypes of sweet pepper including "Inspiration F1," "Maratus F1," and "Magno F1" cultivars in a factorial design with three replications. Heat shock (32?°C for 7?days), applied to mannitol-starved anthers of "Inspiration F1," showed higher multinuclear microspore percent, number of multicellular structures, total embryos, cotyledonary embryos, and regenerants. In the second experiment, the effect of different concentrations of putrescine (0, 0.5, 1, 2, and 5?mg?l-1) was evaluated on microspore embryogenesis of the three aforementioned cultivars of sweet pepper. The highest mean number of multicellular structures, cotyledonary embryos, and regenerants were achieved by applying 0.5-1?mg?l-1 putrescine during the mannitol starvation and heat shock (32?°C) treatments of isolated microspore culture of "Inspiration F1" cultivar. Significant decrease in microspore embryogenesis efficiency was observed when high levels of putrescine (2 and 5?mg?l-1) were used. Microspore embryogenesis was prevented completely at 5.0?mg?l-1 putrescine. In the third experiment, the effect of different concentrations of ascorbic acid (0, 20, 50, 100, and 200?mg?l-1) was investigated and the results showed that the application of ascorbic acid (20 and 50?mg?l-1) during mannitol starvation and heat shock treatment (32?°C) caused remarkable improvement in the number of produced cotyledonary embryos and their regeneration ability compared to control treatment. However, the application of higher levels of ascorbic acid (100 and 200?mg?l-1) inhibited microspore cell divisions and embryogenesis. In conclusion, the results indicated that both putrescine and ascorbic acid have significant effect on microspore embryogenesis efficiency of sweet pepper when they are used in appropriate concentrations.
DOI:10.1016/j.biotechadv.2008.08.003URL [本文引用: 1]
Abstract
Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.URLPMID:26415277 [本文引用: 1]
The influence of different factors on microspore embryogenesis in Chinese cabbage (B. rapa ssp. chinensis) was studied. A genotype dependence for embryo formation was observed. The majority of embryos and plants were obtained from microspores isolated from flower buds (2-2.9 mm in length) and cultured in the NLN liquid medium with 13% sucrose (w/v) supplemented with 24-epibrassinolide and 1% activated charcoal. Embryos cultured on the 1/2 Murashige-Skoog culture medium with 2% sucrose (w/v), 0.1 mg/L benzylaminopurine, and 3g/L Phytagel stimulated the formation of secondary embryos that resulted in development of large number of doubled haploid plants.
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以结球甘蓝的双交种和四交种为材料, 研究了影响甘蓝游离小孢子培养的若干因素。结果表明: 基因型是影响小孢子胚状体产生的最关键因素之一; 用高出胚的材料与不易出胚的材料杂交后, 能明显提高不易出胚材料的出胚能力; 双交种比单交种容易培养出健壮的胚, 成苗率也高; 高糖培养4 d后添加低糖培养液可显著提高胚产量。
URL [本文引用: 1]
以结球甘蓝的双交种和四交种为材料, 研究了影响甘蓝游离小孢子培养的若干因素。结果表明: 基因型是影响小孢子胚状体产生的最关键因素之一; 用高出胚的材料与不易出胚的材料杂交后, 能明显提高不易出胚材料的出胚能力; 双交种比单交种容易培养出健壮的胚, 成苗率也高; 高糖培养4 d后添加低糖培养液可显著提高胚产量。
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DOI:10.1023/B:EUPH.0000004945.01455.6dURL [本文引用: 1]
Brassica rapa (syn. Brassica campestris) ssp. chinensis is an important vegetable crop, but it is relatively recalcitrant to microspore culture. One genotype each of B. rapa ssp. chinensis var. communisand var. utilis were used formicrospore culture. Embryo production of3.8–42.4 embryos/bud was obtained. A high rate of plant regeneration directly from microspore-derived embryos without subculture was achieved by an improved protocol involving replacement of culture media and reduction of sucrose concentrations after 48 h of induction,among other modifications. More than 70%of regenerated plants were spontaneous diploids. Some spontaneous tetraploid plants were also obtained from isolated microspores of both genotypes tested. These tetraploids may be directly exploited a snew varieties in a Brassica rapabreeding programme.
URL [本文引用: 1]
利用Leica体视显微镜, DAPI荧光染色观察比较大白菜小孢子正常发育为成熟花粉粒与游离小孢子培养胚胎发生细胞核的分裂方式, 探讨大白菜游离小孢子培养胚胎发生及其自然加倍的机理。观察结果显示以B途径为主要发育途径的大白菜小孢子, 胚胎发生的启动机制是热激诱导下单倍体小孢子体积膨大, 染色体发生自然加倍, 从而激发小孢子进入孢子体发育途径; 大白菜小孢子胚再生植株具有较高的自然加倍率, 这与小孢子培养热激诱导激发小孢子单核自然加倍为二倍体密切相关。
URL [本文引用: 1]
利用Leica体视显微镜, DAPI荧光染色观察比较大白菜小孢子正常发育为成熟花粉粒与游离小孢子培养胚胎发生细胞核的分裂方式, 探讨大白菜游离小孢子培养胚胎发生及其自然加倍的机理。观察结果显示以B途径为主要发育途径的大白菜小孢子, 胚胎发生的启动机制是热激诱导下单倍体小孢子体积膨大, 染色体发生自然加倍, 从而激发小孢子进入孢子体发育途径; 大白菜小孢子胚再生植株具有较高的自然加倍率, 这与小孢子培养热激诱导激发小孢子单核自然加倍为二倍体密切相关。
URL [本文引用: 1]
对影响白菜游离小孢子培养的关键因素进行分析研究。单因素研究结果表明: 54个基因型之
间的小孢子胚诱导率差异显著; 对供体材料低温处理, 在0～5 d范围内小孢子胚诱导率差异不大, 超过
5 d则明显降低; 高温诱导在12～60 h范围内差异不大, 超过此范围则小孢子胚诱导率明显降低; NLN培
养基中添加生长素(NAA) 和细胞分裂素(6-BA) 对小孢子胚诱导率影响不大, 添加的浓度过大时小孢子
胚诱导率反而降低; 活性炭的有无和浓度大小对小孢子胚诱导率影响极大。通过基因型、生长素、细胞分
裂素、活性炭4因素分析表明: 基因型之间差异显著, 活性炭不同浓度之间差异显著; 基因型与活性炭不
同浓度之间的互作差异显著; 其它的5个一级互作、4个二级互作和1个三级互作差异均不显著。
URL [本文引用: 1]
对影响白菜游离小孢子培养的关键因素进行分析研究。单因素研究结果表明: 54个基因型之
间的小孢子胚诱导率差异显著; 对供体材料低温处理, 在0～5 d范围内小孢子胚诱导率差异不大, 超过
5 d则明显降低; 高温诱导在12～60 h范围内差异不大, 超过此范围则小孢子胚诱导率明显降低; NLN培
养基中添加生长素(NAA) 和细胞分裂素(6-BA) 对小孢子胚诱导率影响不大, 添加的浓度过大时小孢子
胚诱导率反而降低; 活性炭的有无和浓度大小对小孢子胚诱导率影响极大。通过基因型、生长素、细胞分
裂素、活性炭4因素分析表明: 基因型之间差异显著, 活性炭不同浓度之间差异显著; 基因型与活性炭不
同浓度之间的互作差异显著; 其它的5个一级互作、4个二级互作和1个三级互作差异均不显著。
DOI:10.1007/BF00730666URL [本文引用: 1]
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DOI:10.1016/S0176-1617(88)80264-5URL [本文引用: 1]
DOI:10.1023/A:1020687517888URL [本文引用: 1]
The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF1 hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 °C after being transferred to solid regeneration mediumand an initial period of low temperature (2 °C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.
DOI:10.1023/B:GROW.0000017488.29181.faURL [本文引用: 1]
Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.
DOI:10.1007/s10535-006-0004-6URL [本文引用: 1]
DOI:10.1007/s11240-007-9313-5URL [本文引用: 1]
DOI:10.1007/s11240-008-9373-1URL [本文引用: 1]
DOI:10.1007/s11240-008-9359-zURL [本文引用: 1]
