大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp. pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果。重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等。对小孢子培养及转基因研究的原理、方法和关键技术进行了总结。展望了大白菜生物技术研究的应用前景。     

大白菜生物技术研究进展

综述了国内外在大白菜(Brassica rapa ssp.pekinensis)单倍体培养、基因工程和分子标记方面的最新研究成果.重点讨论了大白菜小孢子培养的影响因素、取材要点、培养条件、植株再生和倍性鉴定以及大白菜的转基因载体选择、受体系统建立、遗传转化与转基因植株鉴定等.对小孢子培养及转基因研究的原理、方法和关键技术进行了总结.展望了大白菜生物技术研究的应用前景.     

结球白菜离体子叶不定芽再生过程中的组织学及生理变化

以日本引进品种爱知结球白菜(Brassica rapa ssp．pekinensis CV．AiehiHakusai,C1)为试材,对离体子叶不定芽再生过程中的组织学和生理变化进行了研究。结果表明,子叶在离体培养过程中,不定芽发生方式为器官直接发生。在不定芽形成前,可溶性蛋白质含量、POD和SOD的活性均呈上升趋势。随着细胞的脱分化,代谢活动逐渐旺盛,酶活性增强,可溶性蛋白质含量增加,表明不定芽形成过程中形态变化与生理变化紧密相联。培养基中添加AgNO3对酶活性有促进作用,并促进不定芽的分化。     

Natural variation in a calreticulin gene causes reduced resistance to Ca2+ deficiency‐induced tipburn in Chinese cabbage (Brassica rapa ssp. pekinensis)

Tipburn is an irreversible physiological disorder of Chinese cabbage that decreases crop value. Because of a strong environmental component, tipburn‐resistant cultivars are the only solution, although tipburn resistance genes are unknown in Chinese cabbage. We studied three populations of Chinese cabbage over four growing seasons under field conditions: (a) 194 diverse inbred lines, (b) a doubled haploid (DH100) population, and (c) an F₂ population. The 194 lines were genotyped using single nucleotide polymorphism markers, and genome‐wide‐association mapping showed that 24 gQTLs were significantly associated with tipburn disease index. Analysis of the DH100 and F₂ populations identified a shared tipburn‐associated locus, gqbTRA06, that was found to cover the region defined by one of the 24 gQTLs. Of 35 genes predicted in the 0.14‐Mb quantitative trait locus region, Bra018575 (calreticulin family protein, BrCRT2) showed higher expression levels during disease development. We cloned the two BrCRT2 alleles from tipburn‐resistant (BrCRT2^R) and tipburn‐susceptible (BrCRT2^S) lines and identified a 51‐bp deletion in BrCRT2^S. Overexpression of BrCRT2^R increased Ca²⁺ storage in the Arabidopsis crt2 mutant and also reduced cell death in leaf tips and margins under Ca²⁺‐depleted conditions. Our results suggest that BrCRT2 is a possible candidate gene for controlling tipburn in Chinese cabbage.     

Arabidopsis mutants with increased organ regeneration in tissue culture are more competent to respond to hormonal signals

Shoots and roots can be regenerated through organogenesis in tissue culture by subjecting plant explants to the appropriate regime of hormone treatments. In an effort to understand the control of shoot organogenesis, we screened for mutants in Arabidopsis thaliana (L.) Heynh. Columbia ecotype for enhanced shoot development at sub-optimal concentrations of cytokinin. Mutants in four different complementation groups were identified, one of which represents a new locus named increased organ regeneration1 (ire1) and another that is allelic to the previously identified pom1/erh2 mutant. Although the mutants were selected for their response to cytokinin, they were neither hypersensitive to, nor were they over-producers of cytokinins. The mutations identified in this study not only promote more robust shoot production in tissue culture, but also enhance green-callus and root formation. We interpret this to mean that, in tissue culture, IRE genes act before organ specification during the time when root explants acquire the competency to respond to organ formation signals. In normal plant development, IRE genes may down-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root organogenesis.     

Development of an efficient and reproducible regeneration system in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The availability of reproducible regeneration system through tissue culture is a major bottleneck in wheat improvement program. The present study has considered to develop an efficient callus induction and regeneration system using mature and immature embryos as explants in recently released agronomically superior spring wheat varieties. An efficient sterilization process was standardized using 0.1% HgCl₂ and 70% ethanol for both seeds and embryos. The maximum possible combinations of plant growth regulators (PGRs) were evaluated for their effect on different wheat regeneration processes through tissue culture starting from callus to root induction. Picloram is found as an effective auxin with 87.63–98.67% callus induction efficiency in both explants. Supplementation of CuSO₄ along with 2,4-D, zeatin in regeneration medium significantly enhanced the multiple shoot induction. The shoot development was achieved using full strength Murashige and Skoog’s (MS) medium and root induction using half MS medium without PGRs. The optimized medium and method has resulted up to 100% regeneration irrespective of the genotype used with high reproducibility. Thus, the standardized regeneration system can be used in the regeneration of healthy plants from embryos rescued from interspecies crosses, transgenic production, induced mutation breeding and recently developed genome editing techniques for the procreation of wheat plants having novel traits.     

Factors influencing plant regeneration from seedling explants of Hairy nightshade (<Emphasis Type="Italic">Solanum sarrachoides</Emphasis>)

Hairy nightshade (Solanum sarrachoides) has the potential to be a model system for the study of plant-pathogen interactions, however, the availability of tissue culture and transformation methods would strengthen its utility. For the development of tissue culture methods, we investigated, explant type (cotyledons, hypocotyls, roots), hypocotyl explant origin, cotyledon orientation (abaxial vs. adaxial) in direct contact with the medium, gelling agents (agar and agargel) and cytokinins (zeatin and 6-benzyladenine) at different concentrations. Cotyledon explants resulted in the greatest biomass as compared to root and hypocotyl. As for hypocotyl explant origin, explants proximal to the cotyledons had a significant effect on plant regeneration. However, cotyledon orientation and gelling agent had no effect on plant regeneration. Medium supplemented with either zeatin or 6-benzyladenine at 1 mg L⁻¹ resulted in significant shoot regeneration. Shoots rooted readily when cultured on a non-hormone based rooting medium.     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficient procedures for callus induction and adventitious shoot organogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Summary Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM, 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocytyl-root or hypocotyl-shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CSM 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.     

Characterization of plant phenotypes associated with loss-of-function of AtCNGC1, a plant cyclic nucleotide gated cation channel.

Of the 57 cation channel genes in the Arabidopsis genome, over a third encode cyclic nucleotide gated cation channels (CNGCs). CNGCs are ion channels regulated by cytosolic signaling molecules (cyclic nucleotides, calmodulin, and Ca(2+)), and which conduct Ca(2+) as well as K(+) and in some cases Na(+). Little is currently known about the role CNGCs may play in plant growth and development. Here, we examined the hypothesis that an Arabidopsis thaliana genotype containing a null mutation in one of the CGNC genes (AtCNGC1) would display cation uptake-related growth phenotype differences from wild type (WT) plants. We determined that AtCNGC1 protein is primarily expressed in the roots of Arabidopsis seedlings. Seedlings lacking this protein had slightly (6-22%) lower shoot Ca(2+) than WT plants. Primary roots of Atcngc1 mutant seedlings grew faster than roots of WT plants, and had larger angles of gravicurvature and less nitric oxide generation upon gravistimulation. We conclude that channels formed (at least in part) by AtCNGC1 contribute (along with other channels) to Ca(2+) uptake into plants, and that Ca(2+) uptake into roots through AtCNGC1 affects some aspects of growth in the primary root of Arabidopsis seedlings.     

壳寡糖对不结球白菜子叶离体培养再生体系的影响

以不结球白菜（Brassica campestris L．ssp．chinensis Makino）子叶为外植体,考察壳寡糖对不结球白菜子叶离体培养再生体系的影响。在添加外源激素6．BA和NAA的条件下,比较了不同浓度（0．5、1．0、2．0和10．0mg·L^-1）壳寡糖对不结球白菜子叶形成愈伤组织、再生芽和再生不定根的影响。实验结果表明,低浓度的壳寡糖能促进子叶形成愈伤组织、再生芽。壳寡糖促进子叶形成愈伤组织和再生芽的最适浓度为0．5mg·L^-1,与其他浓度壳寡糖处理组相比,该浓度壳寡糖促进了子叶愈伤组织的形成,使出愈率达到92％。此外,该浓度壳寡糖能提高子叶的芽再生频率,使再生率达到80％,同时再生芽长度、叶绿素含量及外植体总鲜重达到最大,均显著高于对照组。然而,壳寡糖对再生芽生根有抑制作用,形成的不定根数目、平均根长和最长根长度均小于对照组。     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) cotyleodon explants: responses to growth regulators and developmental aspects

Summary Adventious root and shoot formation was obtained from cotyledon fragments of chestnut (Castanea sativa Mill.) and these processes followed two phases. In a first stage after detachment of the embryonic axis, the cotyledon fragments in culture formed a cotyledon petiole, which elongated for about 6d. Thereafter, root primordia arose at the tip of the cotyledon petioles, followed by normal root development. In some cases, the cotyledon, petioles showed adventitious shoot regeneration from a nodular structure previously formed at the end of the petioles. The presence or absence of growth regulators did not significantly influence root regeneration, whereas cytokinins stimulated shoot formtion. The processes of root and shoot differentiation were studied also at the histological level. Observation with a light microscope showed that the developing root apical meristems were connected with a vascular bundle of the cotyledon petiole. Similarly, shoot bud meristem connections were observed with vascular tissue inside the nodular structure.     

Effects of antibiotics on in vitro-cultured cotyledons

Here, we report the effects of kanamycin (Km), cefotaxime (Cef), carbenicillin (Crb), and ampicillin (Amp) on morphogenesis of Chinese cabbage (Brassica rapa L. ssp. pekinensis) cultured on Murashige and Skoog medium. The four antibiotics had little effect on callus induction, but influenced shoot and root differentiation to different degrees. Even very low concentrations of Km inhibited redifferentiation of buds and roots from callus. We also found that Cef inhibited redifferentiation at a relatively low concentration and delayed organ morphogenesis. Crb had no obvious effect on bud, shoot, or root differentiation, whereas Amp stimulated root and shoot differentiation within the range 0–1,000 mg/L. The higher concentrations of Amp promoted greater stimulation of shoot differentiation. At 1,000 mg/L Amp, the shoot differentiation frequency reached 93.3% compared to 73.6% for control treatments without antibiotic supplementation. Thus, Km can be used as a selective agent for transgenic plant tissues that carry appropriate selection markers, whereas Amp (at a high concentration) or Crb may be beneficial for use in tissue culture and genetic transformation of Chinese cabbage.     

Regulation of expression and intracellular distribution of calreticulin, a major calcium binding protein of nonmuscle cells

In the present study we have demonstrated the presence of calreticulin, a major Ca(2+)-sequestering protein of nonmuscle cells, in a variety of cell types in tissue culture. The protein localizes to the endoplasmic reticulum in most cell types and also to the nuclear envelope or nucleoli-like structures in some cell types. Calreticulin is enriched in the rough endoplasmic reticulum, suggesting a possible involvement in protein synthesis. Calreticulin terminates with the KDEL-COOH sequence, which is likely responsible for its endoplasmic reticulum localization. Unlike some other KDEL proteins, calreticulin expression is neither heat-shock nor Ca(2+)-shock dependent. Using a variety of metabolic inhibitors, we have shown that the pool of calreticulin in L6 cells has a relatively slow turnover and a stable intracellular distribution. In proliferating muscle cells in culture (both L6 and human skeletal muscle) calreticulin is present in the endoplasmic reticulum, and additional intranuclear staining is observed. When fusion of the L6 cells is inhibited with either a high serum concentration or TGF-beta or TPA, the nucleolar staining by anticalreticulin antibodies is diminished, although the presence of calreticulin in the endoplasmic reticulum remains unchanged. In contrast, in differentiated (i.e., fused) muscle cells neither intranuclear nor intracellular staining for calreticulin is present. We conclude, therefore, that calreticulin is abundant in the endoplasmic reticulum in proliferating myoblasts, while it is present in only small amounts in sarcoplasmic reticulum membranes in terminally differentiated myotubes. We propose a model for the domain structure of calreticulin that may explain the differential subcellular distribution of this protein. Because of its widespread distribution in nonmuscle tissues, we postulate that calreticulin is a multifunctional protein that plays an important role in Ca(2+) sequestering and thus that it is the nonmuscle analog of calsequestrin.     

Calreticulin, a multifunctional Ca2+ binding chaperone of the endoplasmic reticulum.

Calreticulin is a ubiquitous endoplasmic reticulum Ca2+ binding chaperone. The protein has been implicated in a variety of diverse functions. Calreticulin is a lectin-like chaperone and, together with calnexin, it plays an important role in quality control during protein synthesis, folding, and posttranslational modification. Calreticulin binds Ca2+ and affects cellular Ca2+ homeostasis. The protein increases the Ca2+ storage capacity of the endoplasmic reticulum and modulates the function of endoplasmic reticulum Ca2+-ATPase. Calreticulin also plays a role in the control of cell adhesion and steroid-sensitive gene expression. Recently, the protein has been identified and characterized in higher plants but its precise role in plant cells awaits further investigation.     

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (<i>Arachis hypogaea</i> L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits. A combination of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) plant growth regulators was applied to evaluate shoot regeneration capacity whereas α-naphthalene acetic acid (NAA) combination with 6-benzylaminopurine (BAP), and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars. The result showed combination (BAP with TDZ) was found to be effective in promoting shoot. The highest shoot regeneration frequency (93%) was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency (87%) was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ. The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased. The highest rooting rate (93%) was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars. The rooting rate increased as the concentration of auxin to cytokinin ratio increased. The maximum rooting rate (83%) was obtained on MS medium supplemented with 0.3 mg/L 2, 4-D with 0.2 mg/L BAP for the cultivar N3. The result indicated that BAP with NAA was much better than BAP with 2, 4-D in rooting rate. Thus, the protocol developed was genotype independent and effective for peanut tissue culture.     

Calreticulin signaling in health and disease

Calreticulin is an endoplasmic reticulum Ca(2+) binding chaperone that has multiple functions inside and outside of the endoplasmic reticulum. It is involved in the quality control of newly synthesized proteins and glycoproteins, interacting with various other endoplasmic reticulum chaperones, specifically calnexin and ER protein of 57-kDa in the calreticulin/calnexin cycle. Calreticulin also plays a crucial role in regulating intracellular Ca(2+) homeostasis, associating calreticulin with a wide variety of signaling processes, such as cardiogenesis, adipocyte differentiation and cellular stress responses. The role of calreticulin outside of the endoplasmic reticulum is also extensive, including functions in wound healing and immunity. Therefore, calreticulin has important implications in health and disease. Signaling facts.     

Induction and origin of adventitious roots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Adventitious roots were induced from shoots and leaves of the chimera plant TCC (LI-LII-LIII = TCC; T = Tuber mustard, C = Red Cabbage), previously developed by in vitro grafting of tuber mustard (Brassica juncea) and red cabbage (B. oleracea). The regeneration frequency of adventitious roots from TCC shoots and leaf sections was markedly higher than that obtained from the parents TTT (tuber mustard) and CCC (red cabbage). Moreover, levels of α-naphthaleneacetic acid in the culture medium had lower effects on rooting efficiency of TCC chimeras compared to those of TTT and CCC. The number and fresh weight of adventitious roots per TCC shoot, 13.11 roots and 0.274 g, respectively, were also significantly higher than those of the parents. This demonstrated that replacing the histogenic LI layer (the outermost apical cell layer) with a different genotype might improve adventitious root induction capability of these vegetative tissues due to likely synergistic effects between LI and the other two histogenic layers, LII and LIII. Following polymerase chain reaction analysis and histological investigation, it was found that these adventitious roots originated from the LIII histogenic layer.