从烟草单倍体植株未传粉子房又培养出单倍体植株

离体培养未传粉的子房或胚珠,诱导大孢子发育,以期获得单倍体植株,是国际、国内正在探索的新课题。 几十年来,国内外科研工作者培养未传粉的子房或胚珠,希望得到单倍体植株。如Nishi等曾培养未传粉的水稻子房,得到的是二倍体和四倍体的植株,没有得到单倍体的植株。内宫博文等曾培养未传粉的玉米子房和茄子的胚珠,也没有获得单倍体植株。其他学者也作了一些工作,但都没有得到单倍体植株。我们于1978年从烟草和小麦未传粉子房培养中获得了单倍体植株,1979年进行了首次报道。继之,又得到了一些新的结果,现报道如下:     

从未受精的玉米子房培养出单倍体植株

本实验用Ms和N_6两种不同培养基,对未受精的成熟的玉米子房(已抽出花丝)和未受精的未成熟的玉米子房(雄穗的花粉发育到单核靠边期)进行了离体培养,不经愈伤组织直接获得了单倍体植株,成苗率达2％。实验结果表明,未受精的玉米子房的发育时期对培养出单倍体植株比培养基的选择显得更为重要。     

从未授粉的小麦及烟草子房培养出单倍体植株

在子房和胚珠培养方面国内外已做了许多工作,但离体培养未授粉的子房获得单倍体植株迄今国内外尚未见报道。从未授粉的子房长出单倍体植株,不但具有育种工作上的实用价值,同时也具有遗传学、胚胎学及细胞学等基础学科的理论意义。我们用未授粉的小麦和烟草子房离体培养,从胚珠中长出了单倍体植株。现将主要的试验结果报道如下:     

离体培养未传粉烟草子房的胚状体发育

近几十年来,许多科研工作者把培养活细胞的技术,用于诱导离体未传粉的子房和胚珠的培养,除大麦外,其他的试验结果都未获得单倍体植株。我国科学工作者,近年来在离体子房和胚珠培养方面有了新的进展,分别从小麦、水稻、烟草未传粉子房,通过离体培养,获得了单倍体植株。离体培养未传粉子房所获得的单倍体植株是来源于胚珠中的哪一部分,以及通过什么样的发育过程而长出来的,本文将报道这方面的试验结果。     

亚麻未授粉子房单倍体植株的诱导

通过未授粉子房培养诱导单倍体植株,已在近10种作物上取得成功。我们以亚麻为材料,接种未授粉子房,诱导出单倍体植株。现将结果报道如下。     

未传粉烟草子房离体培养胚状体的发育

关于离体培养未传粉子房获得的单倍体植 株,是从胚珠中的哪一部分长出来的,以及通过 什么样的发育过程而长成单倍体植株的,迄今 在国内外尚未见报道。我们在烟草和小麦方面 培养未传粉的子房获得了单倍体植株,并有重 复试验的结果。     

从水稻未授粉的幼嫩子房培养出单倍体小植株

通过子房或胚珠的离体培养,诱导大孢子或雌配子体产生单倍体植株是国际、国内正在探索的一项新课题,但由于技术原因迄今未取得如同花药培养那样广泛的进展。Nishi等最先从水稻子房培养产生了二倍体与四倍体植株,但未获得单倍体。Uchimiya等在茄子未受精胚珠的培养中观察到单倍体细胞的分裂;在玉米的子房培养中由愈伤组织分化了根。Mullins等进行葡萄未受精胚珠的液体培养,由珠心产生的愈伤组织再生了小植株。Hsu等用2-chloroethylpho-     

从薏苡的未受精子房培养出单倍体植株

通过子房或胚珠的离体培养,诱导大抱子 或雌配子产生单倍体植株,无论在遗传学、胚胎 学、细胞学等基础科学理论方面或在育种实践 方面都具有一定意义。据我们所知,迄今仅在 水稻‘刃、大麦〔,,、小麦〔‘〕、烟草〔‘,、玉米t,,‘,、百合〔们 等植物上获得了单倍体植株。我们继着茵花药 培养获得单倍体植株〔3,之后,最近在未授粉的 昔茵子房培养上,亦获得了单倍体植株。现将 试验结果报道如下：     

从未传粉的烟草不育系子房培养出单倍体植株

我们用未传粉的烟草不育系子房离体培养 得到了单倍体植株,现将试验结果简报如下：     

百合未授粉子房的培养及其胚胎学观察

取未授粉的百合[Lilium davidii var.willmottiae(Wilson)Roffill]子房,放入改良 MS和 N固体培养基中,在25—28℃的弱光下培养,10天后子房显著膨大,40天后可产生6—12mm 大小的愈伤组织。转移到分化培养基上40—50天可形成完整的植株。经根尖染色体倍性观察有65.71%的再生植株为单倍体植株,具12条染色体;其余34.29%的再生植株为二倍体植株。在于房培养过程中进行了胚胎学观察,发现在离体培养条件下百合子房出现了非正常发育途径,单倍体植株来源于大孢子四分体,而不是来源于卵细胞。同时在珠心细胞中观察到旺盛的分裂活动和细胞团的形成,这可能是二倍体植株的来源。     

韭菜胚囊发育与胚胎发生

韭菜胚囊发育为葱型,胚胎发生属柳叶菜型。成熟胚囊中,三个反足细胞形态上常类似卵器,其中二个呈助细胞状,一个呈卵细胞状。卵状反足细胞可分裂成多细胞原胚,但随着胚乳的发育而退化。在未受精胚囊中,卵细胞和卵状反足细胞均可分裂,它们的发生过程与合子胚相似,但因无胚乳哺育,均不能继续发育。论证了反足细胞胚的性质,初步探讨了胚乳与反足细胞无配子生殖的关系。     

Embryological Studies on Narcissus tazetta var. chinensis

Chinese narcissus (Narcissus tazetta var.chinensis Roem) blooms but has no seeds.Embryological studies on the species were conducted to discover the causes of its sterility.Its anther wall is composed of four layers of cells,and its tapetum is of the secretory type.The cytokinesis of microspore mother cells is of the successive type,and the tetrad is tetrahedral.During meiosis of microspore mother cells,some chromosomes lagged,and several micronuclei were found in tetrads.Only 27.7% of the pollen grains contained full cytoplasm,and 1.3% of them germinated in culture medium.No pollen grain,however,could germinate on the stigma.The ovary is trilocular with axile placenta,and the ovules are bitegmic,tenuinucellate,and anatropous.Its embryo sac is of the polygonum type.Most embryo sacs degenerated,and only about 4.5% of the ovules contained a normal embryo sac with an egg cell,two synergids,three antipodal,and a central cell containing two polar nuclei.One reason for the sterility of Chinese narcissus is the abnormality of microsporogenesis and megasporogenesis,in which only a few functional pollen grains and embryo sacs are produced.The other reason is that the pollen grains cannot germinate on the stigma.     

In Vitro Divisions of Unfertilized Central Cells and Other Embryo Sac Cells in Nicotiana tabacum var macrophylla

The embryo sacs and female cells could be isolated from the unfertilized ovules of Nicotiana tabacum L. var. macrophylla which were treated in a solution containing 1.5 % cellulase R- 1O, 1% macerozyme R-10, 10% mannitol, 10 mmol/L CaCI:, pH 5.8 for 3 h followed by given slight pressure with a micropipette. The central cells could be kept viable for 10 h and the egg cells for 3 h in 10% mannital. Sometimes, the in situ fusion products of egg cell and synergid protoplasts could be obtained and kept viable for at least 5 h. The high concentration (20 mg/L) of 2, 4-D was used in enzyme solution to induce the division of the unfertilized central cells and other megagametophytic cells in subsequent culture. Treatment of 2,4-D together with enzymatic maceration of ovules was proved to be better than its direct treatment of isolated embryo sac or its component cells. Isolated embryo sacs were cultured in microchambers (Millicell-CM PICM 012 50 MILLIPORE) feeded with divided mesophyll protoplasts of Nicotiana rustica L. The medium was KMSp medium supple- mented with 1% glucose, 0.1 mol/L mannitol, 0.1 mol/L sorbitol, 0.25 mol/L sucrose, 1 mg/L BA, 6% to 10% coconut water, and 0.15% low gelling agarose. Division of central cells, antipodal cells and the in situ fusion products of egg cell and synergid protoplasts were induced. The unfertilized central cell was for the first time to be induced in vitro to develop into small cell clusters.     

烟草未受精中央细胞及其它胚囊细胞的离体分裂

自70年代中期以来,未传粉子房和胚珠的离体培养已在多种植物中取得成功,得到的单倍体植株来源于胶囊中的卵细胞、助细胞以及反足细胞。而分离的未受精胚囊及其成员细胞的离体培养虽屡经尝试,迄今只有Kranz等诱导了玉米未受精卵细胞分裂形成小愈伤组织,至于中央细胞与其它雌配子体细胞则无离体分裂的报道。本文报道大叶烟草未受精中央细胞首次培养成细胞团及其它胚囊细胞启动离体分裂的实验结果。     

Embryological Studies on Narcissus tazetta var. chinensis

Chinese narcissus (Narcissus tazetta var. chinensis Roem) blooms but has no seeds. Embryological studies on the species were conducted to discover the causes of its sterility. Its anther wall is composed of four layers of cells, and its tapetum is of the secretory type. The cytokinesis of microspore mother cells is of the successive type, and the tetrad is tetrahedral. During meiosis of microspore mother cells, some chromosomes lagged, and several micronuclei were found in tetrads. Only 27.7% of the pollen grains contained full cytoplasm, and 1.3% of them germinated in culture medium. No pollen grain, however, could germinate on the stigma. The ovary is trilocular with axile placenta, and the ovules are bitegmic, tenuinucellate, and anatropous. Its embryo sac is of the polygonum type. Most embryo sacs degenerated, and only about 4.5% of the ovules contained a normal embryo sac with an egg cell, two synergids, three antipodal, and a central cell containing two polar nuclei. One reason for the sterility of Chinese narcissus is the abnormality of microsporogenesis and megasporogenesis, in which only a few functional pollen grains and embryo sacs are produced. The other reason is that the pollen grains cannot germinate on the stigma. This paper was translated from Journal of Xiamen University (Natural Science), 2005, 44(1) (in Chinese)     

甜菜无融合生殖单体附加系M14 成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14(Beta vulgaris, 2n=18+1)为实验材料, 利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明: M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态: (1)极性正常的卵细胞, 细胞核位于合点端, 细胞质含有大量核糖体、线粒体、内质网等细胞器; (2)细胞核位于细胞中央; (3)细胞核位于珠孔端, 且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近; 未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显, 细胞器的种类与数量多, 呈现旺盛代谢活动特征, 成为二倍体孢子无融合生殖过程中, 卵细胞与次生核自发分裂的细胞学标志。     

甜菜无融合生殖单体附加系M14成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14（Betavulgaris,2n=18＋1）为实验材料,利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明：M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态：（1）极性正常的卵细胞,细胞核位于合点端,细胞质含有大量核糖体、线粒体、内质网等细胞器;（2）细胞核位于细胞中央;（3）细胞核位于珠孔端,且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近;未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显,细胞器的种类与数量多,呈现旺盛代谢活动特征,成为二倍体孢子无融合生殖过程中,卵细胞与次生核自发分裂的细胞学标志。     

Isolation of Viable Embryo Sacs and Their Protoplasts of Nicotiana tabacum

Isolation of fixed and fresh embryo sacs has been reported. However,the isolation of protoplasts of embryo sac elements is reported here for the first time.The protoplasts of egg cell, synergids, central cell and antipodal cells have been isolated with the retaining of their viability. Though this is a preliminary work, it indicatesthe potentiality of isolation of naked female gametes of angiosperms, which may beused in genetic manipulation and plant biotechnology. Nicotiana tabacum was grown in the greenhouse of the Department of Biology,Peking University. From opened and unpollinated flowers, the ovaries were removedand sterilized with 70% alcohol. The ovules were dissected out from those ovaries andfollowed by incubation (4–8 hrs. 28℃) in anenzyme solution containing 2% driselase, 0.65 M mannitol and 0.25% potassium dextran sulfate. Ovules from 3 4 ovariescould be incubated with 1 ml of enzyme solution in a 3 cm petri dish. All these manipulations and the following procedures were carried out under sterile conditions. Afterincubation, ovules were washed 3 times with a washing solution of 0.65 M mannitol.The isolated embryo, sacs and their protoplasts were obtained by gently squashing digested ovules in a small volume of washing solution on a slide. When the fresh ovules were incubated 3–3.5 hrs in the enzyme solution, the embryosacs may be successfully isolated in an intact manner, either for mature or immatureembryo sacs. The isolated embryo sac looked plump, viable and very distinct in itsstructure. If the isolated embryo sacs were incubated in 0.01% fluorescein diacetate(FDA) used as a test for the viability of the embryo sac, and observed under fluorescein microscope, the cytoplasm of all embryo sac elements, including egg cell, synergids,central cell and antipodal cells, showed strong fluorescence. It is proved that these iso-lated embryo sacs are still viable. When the incubation of ovules was prolonged as to 8 hrs in certain cases, theboundary wall of the embryo sac may be partially digested and the protoplasts of embryo sac elements came out from micropylar or chalazal end after squashing. The difference of the protoplasts derived from different embryo sac elements could be recognized by their relative size and other characteristics. The egg protoplast is smallerthan that of the synergid. However, the protoplasts of antipodal cells were. obviouslysmaller than that of egg. But the central cell protoplast was the largest among theseprotoplasts and possessed two polar nuclei and a very large central vacuole. All theseisolated protoplasts of embryo sac elements were also proved viable with FDA method. The importance of isolated protoplasts of embryo sac elements is discussed withrespect to genetic manipulations.     

韭菜未传粉子房培养中单倍体的胚胎发生和植株再生

在自然生长条件下,韭菜反足细胞可与合子同时分裂形成多细胞原胚,但很快退化,种子成熟时仅含有合子胚。在未传粉子房培养条件下,反足细胞和卵细胞可经相似的早期胚胎发生过程再生单倍体植株。高频率的胚胎发生为韭菜单倍体育种提供了一条新途径。文中讨论了反足细胞无配子生殖的有关问题。     

Embryogenesis of Polyembryonic Rice ApⅢ: Structural and Histochemical Studies of Egg Apparatus Around Fertilization

The structural and histochemical changes of the egg apparatus in the polyembryonic rice (Oryza sativa L.), ApⅢ with the highest frequence of additional embryos among the polyembryonic rice investigated, before and after fertilization were studied and compared with those of normal and other polyembryonic rices in a similar developmental period. A total of 2 932 ovules were observed and each of them contained only asingle embryo sac with a set of egg apparatus. Among 1 655 embryo sacs, there were 1 643 embryo sacs (99.27%) with one normal egg apparatus in each embryo sac, and only 12 embryo sacs (0.73%) from the remainder with 4 celled egg apparatus, i.e. two eggs and two synergids. Neither the numerous poly egg apparatus and egg like cells, nor the double set of embryo sacs each containing one egg apparatus and other abnormal egg apparatus in single ovary, which were reported by earlier investigators to have high frequency of embryo production in SB 1 and ApⅣ, were observed. The egg cell was located at the subterminal site of the micropylar end of embryo sac. The cytoplasm of egg cell was rich in protein materials and poly saccharide grains, which did not disappear until the division of zygote. The prominent nucleus was closely surrounded by protein and polysaccharide grains, which did not disappear until the division of zygote. No cytological difference was found between egg cells from the normal and abnormal egg apparatus. The two synergids were fully developed and situated at the upper most part of the micropylar end of the mature embryo sac. In most embryo sacs, the synergids were flask shaped with longer necks, and a widened cap shaped top, in close contact with the micropyle. The synergids had a well developed filiform apparatus. The characteristic appearance of the filiform apparatus as well as the cap neck region of synergids before and after pollen tube penetration were easily distinguishable from the egg cell. The structure, the stainability with Coomassie Brilliant Blue and PAS reaction, the process of accumulation, distribution and disapperance of the cytoplasmic protein materials and polysaccharide grains of the two synergids, the persistent and rarely the receptive synergids before and after pollen tube penetration, were closely similar to those of egg cell of the same developmental stage. In comparison with normal and other polyembryonic rice reported, the size of nucleus and nucleolus and their stainability also strongly resembled those of egg cell. Based on the results observed, the main conclusions are summarized as follows: (1) the additional embryos very frequently developed in the young and mature seed of polyembryonic rice ApⅢ were produced by one or two synergids of normal egg apparatus, rarely by 4 celled egg apparatus; (2) during fertilization, the synergids, in addition to the natural specific function of introducing pollen tube and transferring sperms to egg cell and central cell, could be closely associated with the potentiality to breed one or two additional embryos; and (3) as compared with that of normal or other polyembryonic rice it is firstly disclosed that in a few embryo sacs of ApⅢ, the cytoplasmic and nuclear structure, the active anabolism and catabolism of protein and polysaccharide materials and the delayed disorganization at the mid basal region of the receptive and persistent synergid still remained unchanged before the division of zygote. Such salient features could be the predisposition for the origin of additional embryos in ApⅢ.