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CAS Workshop on Ecosystem Succession Theory and Practice of Ecological Restoration 香根草体细胞胚胎发生的细胞学特点与形成条件 马镇荣
刘卫
王昌虎
夏汉平
凌定厚 (中国科学院华南植物研究所，广州
510650) 摘要：香根草已被证明是一种非常优秀的生态恢复植物，但目前使用的品种还存在一些局限性。为了选育出更优良的新品种，有必要对香根草进行离体培养。离休培养采用了两种外植体，一是从田间采集植株上的分蘖芽及带腋芽的节，二是已由器官发生方式所产生的无菌不定芽。以带腋芽的节为外植体时，先截取香根草的分蘖剪下带腋芽的节作表面消毒，再把腋芽/节切下接种到培养基上。带腋芽的节及分蘖芽在培养时污染严重，而由器官发生方式所产生的无菌不定芽在其发生初期，小块的材料上带有大量的不定芽的生长点，以这种材料作为外植体来诱导胚性愈伤组织，不仅可省去表面消毒环节，而且产生的愈伤组织质量特别好。基本培养基为MS培养基，根据不同的目的附加不同种类或配比的生长素（2，4-D、IBA、NAA等）与细胞分裂素（激动素、6BA等）。 在本试验，我们观察到香根草的外植体发育成再生植株的离体发育途径，有器官发生和体细胞胚胎发生两种，依培养基中所含细胞分裂素或生长素的种类和用量不同而异。当培养基中仅含细胞分裂素（6BA），不含生长素或仅含微量的IBA时，外植体不形成愈伤组织而直接诱导出丛生的不定芽，即由器官发生的途径形成再生植株；当培养基主要含生长素（2,4-D及NAA）而不含或仅含微量的细胞分裂素时，则外植体形成胚性愈伤组织，经由体细胞胚胎发生的途径形成再生植株。将胚性愈伤组织转入不含生长素而含细胞分裂素的培养基中，胚状体即可形成大量的再生植株。结果表明，香根草在离体培养时的这两种植株再生途径（体细胞胚胎发生与器官发生）的植株再生的能力均可以长期保持。本试验所获得的不定芽（器官发生，1999年）及胚性愈伤组织（体细胞胚胎发生，2000年）至今均保持着很高的植株再生能力。 细胞学的研究表明，香根草离体发育的启动可在外植体的表皮细胞或薄壁细胞中进行。在诱导培养基的作用下，外植体在接种初期明显膨大。从切片上观察到，膨大部分的表皮细胞或薄壁细胞的细胞质变浓（着色深），细胞核变大，并具有很大的核仁，从而发育成为胚性细胞。胚性细胞分裂活跃，经二细胞、四细胞而发育成为多细胞的胚性细胞团。在显微观察的一个视野内，可以同时观察到从单细胞、二细胞到多细胞的胚性细胞团的图象。由此可知，香根草的体细胞胚胎发生是单细胞起源的。大约在接种一个月后，这些胚性细胞团发展成为肉眼可见的胚性愈伤组织。成熟的体细胞胚具有单子叶植物典型的胚胎结构，在一个胚状体上可以清楚地观察到盾片、胚芽及胚根。在分化培养基的作用下，胚性愈伤组织上所有的胚状体可以出芽而形成大量的再生植株。所建立的香根草体细胞胚胎发生的植株再生体系，完全适用于遗传转化等生物工程方法对离体培养要求。因此，可以用生物工程的方法对香根草进行遗传改良。由于香根草不能进行有性生殖，本研究所揭示的方法对香根草的遗传改良具有重要意义。此外，还观察到一些鱼雷形体细胞胚，这是体细胞胚胎发生中的异常现象。众所周知，鱼雷形胚是双子叶植物所特有的一个胚胎发育时期。单子叶植物的胚胎发育不具有这一时期。但在香根草的体细胞胚胎发生的过程中观察到鱼雷形时期的幼胚，认为这种异常胚是离体培养所引起的。 Cytology
observation and forming condition of somatic embryogenesis in Vetiveria
zizanioides Ma Zhenrong Liu Wei
Wang Changhu Xia Hanping Ling
Dinghou (South
China Institute of Botany, Chinese Academy of Sciences, Guangzhou
510650, China) Abstract: V. zizanioides has been widely testified to be an excellent plant species with special reference to ecological restoration. However the presently used variety still has some confinements. It is necessary, therefore, to conduct in vitro culture of V. zizanioides in order to screen out more excellent new varieties. Two kinds of explants were used in this experiment for. One was tiller and the node with axillary buds of the plant from the field and the other was the asepsis adventitious buds from organogenesis of cultured materials in test tube. For the first explant, the tillers were collected from the field and the suitable node with axillary buds were cut and then sterilized finally inoculated onto the medium. In order to avoid contamination problem brought from the first explant, we found it has many excellences that the asepsis initiating adventitious buds as the explant instead of the tiller and node from the field. A small piece of initiating adventitious buds from organogenesis could be induced high quality embryogenic callus because it possessed a lot of meristem. Besides when it was as the explant, the usually sterile procedure could be left out because it per se is asepsis. The basic medium was MS medium and supplemented with different kind or/and ratio of auxin (2,4-D, NAA and IBA and so on) or cytokinin (6BA, kinetin and so on), according to different culture purpose. The two kinds of in vitro developing ways in V. zizanioides were observed in this experiment. One was organogenesis and the other, somatic embryogenesis that was dependent upon the kind of auxin or cytokinin and their dosage in the medium. When the medium contained 6BA 5 mg L-1 and IBA 0.1 mg L-1, the adventitious-buds clusters were directly induced from the explant without callus stage. When the medium contained 2,4-D 2 mg L-1 without or with very low concentration cytokinin, embryogenic callus (E-callus) was formed from the explant and than the E-callus differentiated to a large numbers of plantlets in the regenerated medium. The results showed that the differentiated ability of the two regenerated ways (organogenesis and somatic embryogenesis) in V. zizanioides could be kept for a long duration. In this experiment, the adventitious buds, which were induced in 1999, and embryogenic calli, which was obtained in 2000, were safely kept higher ability of plant regeneration until now.
The results of cytology observation proved that the
initiation action of in vitro
development in V. zizanioides
started in epidermal and parenchyma cells of the explant. By the
function of auxin/cytokinin in the medium, the explant became
swollen at the beginning stage of inoculation. By the function of
the auxin/cytokinin in the medium, some epidermal and parenchyma
cells in the swollen portion of the explant became embryogenic
cells. Under the microscope, various embryogenic cells from single
cell to multiple cell clusters could be clear observed. The
embryogenic cell possessed a thicken cytoplasm, large and darkly
stained nuclear with a big nucleolus. The division of the
embryogenic cells was very activity and in a same single scope under
microscope, different stage of embryogenic cell, from single cell,
two-four-to- multiple-cells could be observed. From these results,
it could be concluded that embryogenic callus in V.
zizanioides was single cell origin. After one or more than one
month, these E-clusters became megascopic embryogenic calli which
consisted from plenty of embryoids. A mature embryoid consisted of
scutellum, coleoptile and coleorhiza which were the typical
structure of embryo in monocotyledon. In the regenerated medium, the
embryoids on the E-calli could germinate to buds so large number
regenerated plantlets could be obtained by the somatic embryogenesis
in V. zizanioides. The
regenerated system established in this experiment was suitable to
the transformation and other biotechnology methods used in
genetic-improvement of V.
zizanioides. In the other words, the genetic-improvement of V.
zizanioides could use biotechnology methods with the technique
established by this experiment. Because sexual reproduction in V.
zizanioides was no function, the system established in this
experiment is very important in genetic-improvement of V.
zizanioides.
Besides, some abnormal phenomena that were torpedo embryoid
were observed in this experiment. As everyone known, torpedo embryo
is the special stage at the embryo development in dicotyls and all
of the monocotyledon lack this stage and instead of scutellum stage.
Obviously, this abnormal embryoid was derived from in
vitro culture.
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