青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in <Emphasis Type="Italic">Oncidium</Emphasis>

Traditional breeding processes to genetically modify the long reproductive cycle and slow seed maturation of orchids have limits. We developed a more efficient protocol using particle bombardment to produce transgenic plants of Oncidium Sharry Baby OM8 (Orchidaceae). Pretreating protocorm-like bodies (PLBs) with 0.5 M sucrose for 2 h increased single-cell embryogenesis 3- to 4-fold; however, shoot formation was suppressed. In addition, new PLBs were regenerated from the entire sucrose-pretreated PLBs, whereas in untreated PLBs, this occurred only from the bases. Pretreated PLBs were bombarded with pSPFLP containing genes encoding a sweet pepper ferredoxin-like protein (pflp), hygromycin phosphotransferase (hpt) and -glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Pretreated PLBs showed a 14.8-fold increase in GUS expression over the untreated PLBs 40days after bombardment. The presence of pflp and hpt transgenes in the 40 putatively stably transformed lines that produced 113 clones was confirmed by PCR analysis. Six lines (eight clones) were positive for both pflp and hpt transgenes. In addition, clones derived from these lines were either all positive or all negative for the two transgenes, which suggests homogeneity in pretreated PLBs with more single-cell embryogenesis. Thus, sucrose pretreatment enhanced the regeneration of PLBs, single-cell embryogenesis and efficiency of transformation.     

Leafy cotyledon genes are essential for induction of somatic embryogenesis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

In vitro regeneration in Trifolium. 1. Direct somatic embryogenesis in T. rubens (L.)

The origin and development of zygotic and somatic embryos of Trifolium rubens L. was studied with the aid of paraffin sections and light microscopy. Zygotic embryos were collected, fixed and prepared daily from one to ten days after cross-pollination. Somatic embryos were obtained by plating petiole sections on modified L2 medium with 0.015 mgl^-1 picloram and 0.1 mgl^-1 6-BAP. Cultured petioles were collected and fixed daily from one to 25 days after plating. Two regions in the vascular bundle sheath of cultured petioles gave rise to callus. The first region was adjacent to the phloem fibers and produced friable callus. The second region gave rise to compact callus that was connected to the fascicular cambium. Somatic embryos originated from single cells in the cortex directly without intervening callus formation and from single cells in the friable callus. In addition, embryos arose from meristematic regions in compact callus. Many early stages of embryogenesis (one, two and four-celled stages) were observed in the cortex and friable callus. Zygotic embryogenesis in Trifolium differs from other legumes in that the suspensor is short and has a broad attachment. This arrangement was observed in zygotic embryos of T. rubens and in many somatic embryos. However, a continuum of somatic embryogenesis was observed where some young embryos had a Trifolium suspensor-like arrangement while others were attached to a long narrow suspensor-like structure more characteristic of Medicago.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Ethylene synthesis and growth in embryogenic tissue of Norway spruce: Effects of oxygen, 1-aminocyclopropane-1-carboxylic acid, benzyladenine and 2,4-dichlorophenoxyacetic acid

Ethylene production from an embryogenic culture of Norway spruce ( Picea abies L.) was generally low. ca 2.5 nl g⁻¹ h⁻¹, whereas 1-aminocyclopropane-1 -carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g⁻¹ during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O₂) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 μ M ) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 μl I⁻¹) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2.4-dichloro-phenoxyacetic acid (2.4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g⁻¹ h⁻¹ within the 11-day incubation period. Although 2.4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g⁻¹, was observed in tissue treated with 2.4-D (22.5 μ M ) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K_m was 50 μ M and V_max 2.7 nl g⁻¹ h⁻¹. Growth of the tissue was strongly inhibited by 2.4-D in the absence of BA, but weakly in the presence of BA (4.4 μ M ). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.