Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

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.     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

Somatic embryogenesis,plant regeneration,and the evaluation of camptothecin content in <Emphasis Type="Italic">Nothapodytes foetida</Emphasis>

Summary Somatic embryogenesis and plant regeneration have been achieved in Nothapodytes foetida, which is known for its rich source of anti-cancer and anti-AIDS alkaloids. Callus cultures were initiated from immature zygotic embryos cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), and kinetin. MS medium devoid of plant growth regulators favored the development of globular somatic embryos that differentiated further into plantlets. Plantlet regeneration efficiency was effectively increased on MS medium supplemented with BA. Over 90% of the in vitro plantlets survived when transferred to the soil. Alkaloids were detected in different stages of somatic embryos, regenerated plantlets, and different parts of the 2-yr-old regenerated plants. The somatic embryos contains camptothecin (0.011% dry weight. DW) and 9-methoxycamptothecin (0.0028% DW). Two-yearold field-grown plants obtained from somatic embryos were analyzed and contained higher levels of camptothecin (0.20% DW) and 9-methoxycamptothecin. (0.097% DW) accumulated in roots, followed by stem and leaves. Alkaloids were quantified and identified by TLC and HPLC.     

High frequency somatic embryogenesis and regeneration in different genotypes of <Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench from immature inflorescence explants

This study describes a protocol for the induction of high frequency somatic embryogenesis directly from immature inflorescence explants in three sorghum genotypes (SPV-462, SPV-839, and M35-1). The effect of various growth regulators on somatic embryogenesis was investigated. High frequency somatic embrogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and addition of 0.5 mg l⁻¹ kinetin (KN) in the medium further improved the formation of somatic embryos per explant in all genotypes. The presence of 1.5 mg l⁻¹ 6-benzylaminopurine plus 1.0 mg l⁻¹ KN in MS medium was most efficient for maturation and germination of somatic embryos. The genotype SPV-462 performed better than SPV-839 and M35-1 in terms of induction and germination of somatic embryos. Organogenesis also occurred in callus of all genotypes at the frequency of 20–25%. Regenerated plants from somatic embryos were successfully acclimatized in soil in the greenhouse where plants were grown to maturity, flowered, and set seeds. Regenerated plants appeared normal like that of the seed-raised plants.     

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.)

Summary Pepper (cv. New Mexico — 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 M), thidiazuron (10 M) and a high concentration of sucrose (6–10%). The best response was observed on MS medium containing 2,4-D (9 M), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA₃ or TDZ, alone and in combination, and following transfer to pots developed into normal plants.Abbreviations CM Coconut milk - 2,4-D 2,4-dichlorophonoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - NAA napthaleneacetic acid - TDZ thidiazuron     

Primary and repetitive secondary somatic embryogenesis of <Emphasis Type="Italic">Lepidosperma drummondii</Emphasis> (Cyperaceae) and <Emphasis Type="Italic">Baloskion tetraphyllum</Emphasis> (Restionaceae) for land restoration and horticulture

Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity, in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii.     

Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan)

Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos. The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis thaliana

A simple, rapid and effective system to regenerate Arabidopsis plants via direct somatic embryogenesis has been established. Somatic embryogenesis was induced directly during culture of immature zygotic embryos. The frequency of somatic embryogenesis was strongly influenced by the stage of development of the explants. Explants in different developmental stages were cultured on B5 agar medium containing 5 M 2,4-dichlorophenoxyacetic acid and the highest frequency (up to 90%) of somatic embryogenesis was observed in zygotic embryos with fully-developed cotyledons. The first somatic embryos developing directly from explant tissue were noticed after 8 days of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all six genotypes tested (Columbia, C-24, RLD, Wassilewskaja, Landsberg erecta and Wilna). Subculture of somatic embryos onto auxin-free medium resulted in their conversion into plants with an average frequency of 79.5%. The regenerates showed normal morphological characteristics and were fertile. All 56 analysed plants displayed a diploid number of chromosomes and two out of 96 (2.1%) tested plants carried a chlorophyll or embryo-lethal mutation.     

Somatic embryogenesis from immature zygotic embryos of Rosa bourboniana desp

Summary A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l⁻¹) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and September. IHBT Publication No: 0447     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

蔷薇科植物体细胞胚胎发生及影响因素研究进展

总结了近30年来蔷薇科植物体细胞胚胎发生及影响因素的研究进展。蔷薇科植物体胚发生多数是直接发生途径和间接发生途径同时存在,但以间接发生途径为主。合子胚作为外植体明显好于营养器官作为外植体。诱导体胚发生的植物生长素类调节剂以NAA、2,4-D为主,细胞分裂素类调节剂以6-BA为主,少数植物种类的体胚诱导需要添加KT。冷处理对蔷薇科植物的体胚分化有效。光照对蔷薇科植物的体胚发生没有显著的影响,有时光照会抑制体胚发生。今后应逐步开展对蔷薇科植物体细胞胚胎发生的生理、生化及分子机理的研究,这在蔷薇科植物的新品种培育、遗传改良、优良单株的离体扩殖等具有重要意义。     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.Abbreviations BAP 6-benzylaminopurine - Kinetin 6-furfurylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog