水稻离体授粉的胚胎学研究

采用2个水稻（Oryza sativa L.)品种“春江05”（早粳）和“95046”（晚粳）,对离体授粉过程中的胚胎学进行了详细的研究。结果表明：（1）2个品种均胡离体授粉结实,平均结实率为52．1％,其中28．4％胚胎发育正常,2．2％胚胎发育异常,21．5％愈伤组织化;（2）离体授粉时的胚胎发育途径和体内自然发育基本相同,中是合子和初生胚乳核首次启动分裂及以后的发育均较延缓,但最终也能萌发成幼苗。（3）观察到具细长胚柄的原胚及液泡化原胚等异常的胚胎类型;（4）子房内形成的愈伤组织分为致密和松散两种类型,二均可化出不定芽和不定根,还对离体授粉的方法,离体授粉中正常和异常的胚胎发育途径进行了讨论。     

Embryological Studies on In Vitro Pollination in Rice

n vitro pollination and its embryological studies were carried out in two japonica cultivars of rice (Oryza sativa L.), “Chunjiang 05" and “95046". N6 basic medium supplemented with different exogenous hormones was used for ovary culture after in vitro pollination. The main results were as follows: (1) both cultivars were induced to set kernels after in vitro pollination. The frequency of seedset was 52.1%, including 28.4%normal embryo development, 2.2% abnormal embryo development and 21.5% callus formation. (2) The processes of embryo and endosperm development after in vitro pollination were basically as normal as those in vivo , except there was some retardation in the first division of zygotes and primary endosperm nuclei as well as in their subsequent development. However, both kernels and plantlets could be produced finally. (3) A few abnormal embryos were observed, for instance, proembryos with elongated suspensor and vacuolated proembryos. (4) Two types of calli in the cultured ovaries appeared, namely, the compact callus and the fragile callus, which were able to differentiate into adventitious buds and roots.     

蝴蝶兰愈伤组织诱导研究

对蝴蝶兰愈伤组织诱导试验结果表明,授粉后30d的蝴蝶兰子房适宜诱导愈伤组织,在1/4MS+2,4-D1.0mg/L+6-BA 0.1mg/L+蔗糖3.0%培养基上,蝴蝶兰子房切段愈伤组织的诱导率达到90.0%。     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Isolation of tomatine from cultured excised roots and callus tissues of tomato

Tomatine was present in cultured excised tomato roots but in lower concentrations than in seedling radicles of the same age. The alkaloid was not detected in 'spent' root medium. Newly-initiated callus cultures of hypocotyl, radicle and cotyledon origin produced roots, and tomatine was isolated from both roots and callus. Roots contained more tomatine than callus, but neither contained as much as the organ explants from which the cultures were initiated. The number of roots produced decreased with time, as did also the tomatine content of the callus tissues. After 447 days, when no organized structures were produced by callus cultures, tomatine was not detected. An established hypocotyl callus contained small amounts of tomatine when grown on certain nutrient media, but a chlorophyllous sub-isolate of this callus did not produce detectable quantities of the alkaloid. Tomatine was not detected in an established root callus isolate or in suspension cultures initiated from established, tomatine-containing hypocotyl callus.     

Plantlet differentiation in leaf and root cultures of birch (Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl-trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.     

In vitro propagation of Clianthus formosus (Sturt's desert pea) an Australian native legume

Hypocotyl and cotyledon segments of Clianthus formosus were cultured on a modified deFossard medium M supplemented with cytokinins. 62% of cultures on medium with 20 μM BA produced callus which subsequently gave rise to shoots. 40% of shoots excised from callus produced roots after transfer to auxin-rich media (20 μM NAA or 10 μM IBA+10 μM NAA). Root production was enhanced following a 7-day dark treatment. 32% of nodes from mature plants produced multiple shoots on 2 μM BA+2 μM KIN. 30% of these shoots rooted on medium without hormones. 70% of rooted plantlets were successfully transferred to potting medium and glasshouse conditions. A period of cold treatment (10 days at 5°C) reduced vitrification from 68 to 22% of cultures.     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

Callus Induction and Organ Formation from Young Leaf of Wheat Triticum aestivum

Callus was obtained on modified PRL-4 medium supplemented with 2, 4-D from the segments of the first leaf of wheat seedling which had been germinated for 4 days. Roots and shoots were initiated and complated plantlets thus regenerated. The frequency of callus formation and its growth rate depended upon the concentration of 2, 4-D and the locations of segments on the leaf. When the concentration was 4 ppm or less, the callus could be produced only within the region from the leaf base to about 1cra apart. Both KT and 6-BA were inhibitory to callus formation. Histological examination showed that callus originated from the cells located at the place of vascular bundle sheath. The roots were frequently induced from leaf callus. During the process of callus induction, after 10–14 days incubation, the roots began to appear in the medium containing 2, 4-D of 2 ppm or less. The lower the concentration, the nearer to the base of the leaf the root-forming place was. In contrast, the higher concentration induced rooting at the higher part of the leaf but inhibited at the lower place. After 3 weeks incubation, the highest frequency of root differentiation was about 60%. The callus failed to differntiate in short-period incubation in higher concentration of 2, 4-D, but when it was transferred to lower level, roots could be initiated. Shoot and plantlets were regenerated from callus cultured in several phytohormone combinations and under different conditions. But the frequency of shooting was very low, and some seedling were morphologically abnormal. The nature of shooting is not yet clear and further studies should be carried on. In this paper, causes of failure in the protoplas culture of mesophyll tissue of wheat leaf were also discussed.     

The study of the conditions for the fertilizationin vitro in maize

Suitable conditions for the fertilizationin vitro in maize have been studied. The germinating capacity of pollen in synthetic media was low; it was confirmed that it might be stimulated by supplementing agar with egg yolk. Application of pollen onto styles overhanging from the culture medium of excised ovaries was examined. After 5 days the styles could be cut off the ovaries, for the pollen tubes had already penetrated the embryo sacs. However, better results were obtained when cultivating ovaries along with segments of the maize cob. Solid media were more suitable for the development of kernels. Some of them germinatedin situ and gave rise to normal plants. From nucellar meristems of young kernels a callus could be derived which, on further cultivation, became green and regenerated shoots and roots. The cells of meristems exhibited a varying number of chromosomes.     

In vitro plantlet regeneration from seedling nodal explants of Acacia catechu

Multiple shoots were initiated after 20 days in stem nodes excised from in vitro grown seedlings of Acacia catechu, on Murashige and Skoog's medium adjuvanted with 1 to 100 microM of N6-benzyladenine (BA). Explants were subcultured on the same medium augmented with 1.5 g l(-1) of polyvinylpyrrolidone (PVP) after 30 days. In the second subculture, after 30 days, the explants were transferred to a medium lacking PVP, but containing 10 microM of BA, where nine or ten shoots differentiated per explant within next 30 days. If individual shoots along with some callus were subcultured on BA (10 microM), nearly 15 shoots per explant regenerated in 90 days. Thus, the average number of shoots obtained from each node was 142 after 180 days. Since a seedling develops four nodes after 20 days, theoretically an average of 568 shoots can be obtained from a single seed. If shoots were individually subcultured on 1/2-strength MS medium with 14.7 microM of indole-3-butyric acid (IBA), roots developed in 20 days. Addition of 40 mg l(-1) of glutamic acid to the rooting medium prevented leaf senescence. These plantlets thrived well in garden soil, sand and silica (1:1:1).     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

Interspecific Hybridization of Trifolium repens with T. hybridum Using In Ovulo Embryo and Embryo Culture

In ovulo embryo culture followed by culture of excised immatureembryos produced interspecific hybrids between Trifolium repensL. (white clover) and autotetraploid T. hybridum L. (alsikeclover). Ovules containing hybrid embryos were excised 12–14 dafter pollination and cultured on Nitsch (1951) medium supplementedwith 15% young cucumber juice for 5–6 d. Embryos weresubsequently excised and transferred to hormone-free EG medium,a medium suitable for the culture of immature embryos. A total of 118 hybrid seedlings were obtained from 1978 reciprocalpollinations. All seedlings produced showed various chlorophylldeficiencies, either totally albino or albino with green sectors.Transmission electron microscope studies were carried out toinvestigate plastid development in embryos and seedlings. Someembryos produced only callus. Plants were regenerated from sevencalli. Two semi-albino plants survived transfer to soil, andone plant produced flowers. Backcrosses to T. repens producedone green plant. Hybridity is supported by analysis of morphological characters,karyotype and the gel electrophoretic separation of leaf isozymes. Pollen irradiated with 40 Gy of gamma rays was also used forpollinations. Results indicate that in certain cases ionizingradiation might be useful in overcoming hybrid inviability. Trifolium repens, Trifolium hybridum, clover, interspecific hybridization, in ovulo embryo culture, irradiation     

In vitro development of parthenocarpic fruits of Crocus sativus L.

In vitro development of parthenocarpic fruits of Crocus sativus L. was induced by culturing ovaries on MS agar medium supplemented with growth-regulators (2,4-D, GA₃ and BAP). Amongh these, 2,4-D was the most effective in promoting fructification. The fructigenic activity was independent of both the stage at which the ovaries were excised (before, during or after anthesis) and pollination of the stigmas. Unlike the above compounds, abscisic acid inhibited fructification.     

Production of plantlets from the ovary wall of Haworthia turgida var. pallidifolia

Summary In a modified culture medium, excised ovaries of Haworthia turgida var. pallidifolia can be grown to the normal looking capsules; the ovary wall can produce leafy shoots and callus tissue in this medium. The callus tissue in turn is found to differentiate into plantlets.     

Study on the Hybrid Endosperm Culture of Wheat-Rye In Vitro

The endosperm culture of wheat-rye hybrid was studied in order to explore a new pathway of chromosome engineering. The preliminary results were obtained to show that the endosperm callus formation could be induced from the young endosperm within 7–14 days after crossing on the medium supplemented with 2 ppm 2,4-D, 0.5 ppm kinetin and 3%–8% sucrose. The induction frequency of callus amounts to 35.3%. When the calli were transfered onto an auxin step-down medium containing 0.5 ppm IAA and 1 ppm kinetin, both shoots and roots were formed. 4 endosperm plantlets were obtained. The chromosome number in somatic cells of endosperm plantlets was very unstable. The numbers varied from 6—42, but there is no 49 to be found. The chromosome number with 1—4 times of 7 can be found in higher percentage.     

Studies on Morphological Differentiation of Endosperm Plantlets of Chinese Gooseberry in Vitro

The present report deals with the process of embryoid induction and plantlet formation from cell-type endosperm of Actinidia chinensis var. chinensis and A. chinensis var. hispida. The callus was induced from endosperm on MS basic medium supplemented with zeatin 3 ppm, 2,4-D 0.5 ppm and CH 400 ppm and then transferred to differentiation medium of MS supplemented with zeatin 1 ppm and CH 400 ppm. After about half a month, the embryoid appeared from callus and then developed into plantlets. It could be seen from the histological figure 14—15, the embryoid of Chinese gooseberry is linear-shaped and consists of cells arranged in a long line in callus. When the cells regenerated at botk ends of the linear-shaped embryoid, the polarity of the embryoid is easily distinguished. The plantlets produced from embryoid appear rather stout at first and after some time, they changes gradually into normal plantlets.     

条叶龙胆根培养物中龙胆苦苷的产生和含量

条对龙胆愈伤组织中龙胆苦苷含量较低,根的分化可以提高龙胆苦苷的含量。培养的离休根中龙胆苦苷的含量则更高。随着继代次数的增加,愈伤组织中龙胆苦苷含量下降快,而分化根的愈伤组织和离体培养根中龙胆苦苷含量下降较慢。在愈伤组织的液体培养中,1/2MS培养基附加低浓度的植物激素,龙胆苦苷含量有所提高。植物激素配比促进愈伤组织中分化根的形成,也促进龙胆苦苷的产生;但促进愈伤组织的生长时,不利于龙胆苦苷产生。在离体根培养中,添加 1mg/L的金雀花碱,促进龙胆苦苷的产生,含量可达1.48％,明显高于愈伤组织(0.52％)和分化根的愈伤组织(0.65％),也高于试管苗根的龙胆苦苷含量(1.01％)。     

An improved protocol for carrot haploid and doubled haploid plant production using induced parthenogenesis and ovule excision in vitro

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.     

5,6-二氯-吲哚乙酸对烟草愈伤组织衰老的延缓作用

5,6-二氯-吲哚乙酸对革新烟草愈伤组织生长有影响。当愈伤组织在MS_0(对照)和MS 2,4-D培养基上培养22d时,生长停滞,细胞已呈空泡状,正常的超微结构完全破坏,细胞器不复存在,愈伤组织明显褐化。但在MS 5,6-Gl_2-IAA培养基上的愈伤组织仍能正常生长,鲜重和干重下降亦明显延缓,细胞含有原生质内含物,各种细胞器的超微结构仍保持正常。此外,后者的SOD同工酶也明显不同于其它培养基上的愈伤组织,暗示5,6-Gl_2-IAA对烟草愈伤组织衰老的延缓作用可能与SOD同工酶的调节作用有关。