Somatic Embryogenesis and Plant Regeneration from Tissue Cultures of Hyoscyamus muticus L.

Somatic embryogenesis and regeneration of plantlets was achieved In callus cultures derived from cotyledonary leaf pieces of Hyoscyamus muticus L on MS medium enriched with 2 mg/l 2,4–0 and 0.5 mg/l BAP. For embryogenesis and organogenesis varying concentrations of NAA with or without BAP were added In the medium. Organogenesis was also achieved when callus was transferred to the hormone free medium.     

山黧豆(Lathyrus sativus L.)苗菜用品种的筛选与评价

针对山黧豆苗菜用特性评价体系不完善和优良品种鉴别不明确的现状,收集我国不同地区的29份山黧豆品种资源作为供试材料,模拟中农绿谷芽苗菜研究院山黧豆苗的培育工艺与条件,根据山黧豆及山黧豆苗的8项性状指标,采用基本统计量分析、K-均值聚类分析和相关性分析对山黧豆苗菜用特性进行初步评价。结果表明,各性状中苗菜产出量的变异最大,变异系数为24.42%。29份山黧豆品种资源可划分为5个类群。其中一类具有小粒、高苗菜产出量、高可溶性蛋白含量、高VC含量的特征。苗长与苗菜产出量和种皮的光滑程度极显著正相关;苗菜产出量与品种种皮光滑程度极显著正相关,与百粒重显著负相关。本研究显示,鉴定与评价山黧豆品种苗菜用特性优劣时,应重点考虑苗菜产出量并兼顾营养品质和感官品质,优先筛选苗菜产出量≥1.01的品种作为芽用备选资源。初步筛选出甘肃张掖山黧豆、白香山黧豆、阿杂山黧豆-2共3份优异资源,供进一步研究。     

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.     

Chromosomal instability in Lathyrus sativus L.

Summary In Lathyrus sativus (2n=14), variety LSD-1 shows an instability of somatic chromosome number which can be observed in root tip and shoot tip mitoses. In this variety, approximately 54% of the seedlings showed intra-individual variation in chromosome number ranging from 2n=14–3. This variability in chromosome number was recorded in approximately 60% of the dividing cells. Two seedlings were triploid with 21 chromosomes. Variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result in spindle abnormalities, chromosome degradation and minute chromosomes. The variation in chromosome number is probably responsible for the pollen polymorphism noted in this particular strain. The possible mechanism of intra-individual variability and the occurrence of the phenomenon vis-a-vis its applications are discussed.     

Efficient intergeneric fusion of pea (Pisum sativum L.) and grass pea (Lathyrus sativus L.) protoplasts

Large numbers of viable protoplasts of pea (Pisum sativum) and grass pea (Lathyrus sativus) were efficiently and reproducibly obtained and, for the first time, fused. Different procedures for fusion were compared, based either on electrofusion (750, 1000, 1250 or 1500 V cm(-1)), or on the use of macro or micromethods with a polyethylene glycol (PEG 6000 or PEG 1540), or a glycine/high pH solution. Over 10% of viable heterokaryons were obtained, with PEG as the most efficient and reproducible agent for protoplast fusion (>20% of viable heterokaryons). Both the division of heterokaryons and the formation of small calluses were observed.     

Embryogenic Suspension Culture and Plant Regeneration from Suspension-Derived Proto-plasts of Cucumber (Cucumis sativus L.)

Fast growing embryogenic cell suspension culture was established when embryogenic callus derived from cotyledon protoplasts of cucumber was transferred into a liquid culture. So far the cell line has been subcultured for two years and retained the ability of embryogenesis and plant regeneration. Experimental data showed that the concentration of ABA or sucrose had a dramatic effect on embryogenesis and synchronization of embryoid development. Low level of sucrose concentration (1%) facilitated the precocious germination of the embryoids while 1 mg/l of ABA or 7–9% of sucrose was found to be effective for reducing callusing of the cultures and synchronisticly controlling the embryoids at globular or late globular stage. Embryogenic cells taken from 3–5 days after subculture were enzymatically digested. A large amount of viable protoplasts was isolated. Protoplasts were cultured in a DPDK1 medium either by means of drop or thin layer liquid culture or by means of sodium alginate encapsulation culture. Actively dividing cells formed cell colonies and globular embryoids which were transferred onto a solidified agar medium or directly into a liquid medium to form a shaken culture. The embryoids would proliferated continuously. Embryoids eventually developed into plantlets when they were transferred onto a 1/2 MSO medium devoid of phytohormones.     

Plant regeneration from stem-derived callus of the seed legume Lathyrus sativus L.

Callus cultures from stem explants of six Lathyrus sativus L. cultivars were tested for their morphogenic capacity. Shoot-buds were formed in calli of only one cultivar. Maximum response was observed in the medium containing 10^-8 M picloram and 10^-6 M benzylaminopurin. Supplementation with adenine sulphate was required for shoot-bud formation. The greatest frequency of shoot-bud formation was detected at the second passage and complete lack of regeneration capacity was observed after the 8th passage. Cell regenerates were diploid.     

Transgenic grasspea (Lathyrus sativus L.): Factors influencing Agrobacterium-mediated transformation and regeneration

A reproducible procedure was developed for genetic transformation of grasspea using epicotyl segment co-cultivation with Agrobacterium. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the neomycin phosphotransferase II (nptII) gene and the -glucuronidase (gus)-intron, were studied as vector systems. The latter was found to have a higher transforming ability. Several key factors modifying the transformation rate were optimized. The highest transformation rate was achieved using hand-pricked explants for infection with an Agrobacterium culture corresponding to OD₆₀₀0.6 and diluted to a cell density of 10⁹ cells ml^–1 for 10 min, followed by co-cultivation for 4 days in a medium maintained at pH 5.6. Putative transformed explants capable of forming shoots were selected on regeneration medium containing kanamycin (100 g ml^–1). We achieved up to 36% transient expression based on the GUS histochemical assay. Southern hybridization of genomic DNA of the kanamycin-resistant GUS-expressive shoots to a gus-intron probe substantiated the integration of the transgene. Transformed shoots were rooted on half-strength MS containing 0.5 mg l^–1 indole-3-acetic acid, acclimated in vermi-compost and established in the experimental field. Germ-line transformation was evident through progeny analysis. Among T₁ seedlings of most transgenic plant lines, kanamycin-resistant and -sensitive plants segregated in a ratio close to 3:1.     

Differential competence for in vitro adventitous rooting of grass pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) family leguminosae is cultivated as an important food and feed crop all over the world. It is very recalcitrant and difficult to regenerate and root under in vitro conditions. In this cotext, the study was carried out in three steps to find out the effects of three auxins [naphthalene acetic acid, indole 3 butyric acid, indole-3-acetic acid (IAA)], four sucrose concentrations and six types of substrate most suited for plant growth and helpful in acclimatisation of grass pea. The results showed that 2 mg L^?1 IAA, 3 % sucrose was most suitable for rooting of grass pea. When different concentrations of sucrose were supplied to optimum concentration of IAA in Murashige and Skoog medium, 4.5 % sucrose concentration induced maximum number of 13.70 roots per explants that had positive impact on root length, fresh and dry weight of roots, plant height and morphology of the growing plants. There was 92.66 % acclimatisation and survival rate of these plants using peat moss compared to five other substrates used in this study. The developing plants were vigorous, flowered and set seed contained in pods under glass house conditions. It is concluded that rooting is affected by type and concentration of plant growth regulators and type of substrate has direct bearing on acclimatisation, flowering, pod and seed set of grass pea. As such this paper reports an efficient rooting and acclimatisation system of grass pea that will be very useful in future genetic transformation and breeding for improved characteristics.     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

树莓组织培养及植株再生

以黑莓沙泥芽为外植体,筛选培养基,建立黑莓离体再生体系。结果表明,诱导树莓侧芽的最适起始培养基为MS 6-BA1.0 mg/L NAA0.02 mg/L GA6 mg/L;芽的增殖的最适培养基为MS GA6 mg/L NAA0.02 mg/L 6-BA1.0 mg/L;最适生根培养基为MS NAA 0.5 mg/L IBA 0.1 mg/L;试管苗移栽的最适基质为蛭石:珍珠岩:营养土(1∶1∶1)。     

地中海绵枣儿的组织培养与植株再生

1植物名称地中海绵枣儿(Scilla peruviana L.)。2材料类别鳞茎,植株购于苏州花鸟市场。3培养条件(1)初代培养基:MS 6-BA 0.5mg·L-1(单位下同) NAA 0.1 GA3 0.1;(2)腋芽诱导培养     

洋甘菊组织培养及植株再生

洋甘菊种子灭菌后,置于MS 培养基上发芽形成无菌苗,发芽率可达80 %.适合洋甘菊种子的灭菌的方法是:75 %酒精30 s+3.5 %次氯酸钠15 min+2 %次氯酸钠10 min.在研究范围内,洋甘菊不定芽增殖的最佳培养基为MS+6-BA 1.5 mg/L+NAA 0.5 mg/L,碳源以蔗糖浓度3 %最佳.适宜洋甘菊生根的最佳培养基为MS+IBA 1.0 mg/L+NAA 0.5 mg/L+6-BA 0.2 mg/L.     

羽衣甘蓝的小孢子胚诱导和植株再生

以羽衣甘蓝10个品种的游离小孢子培养,研究其胚状体及其再生植株诱导方法的结果表明,琼脂糖和活性炭对诱导胚状体发生及发育有促进作用;改良MS培养基中添加0.01%的活性炭可促进植株再生;确定1/2MS NAA 0.1 mg·L-1是优化生根的培养基;小孢子再生植株成活率可达74.6%.     

Exploring the genetic diversity of Ethiopian grass pea (Lathyrus sativus L.) using EST-SSR markers

Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers for less-studied species. In this study, EST-simple sequence repeat (SSR) markers developed from Lathyrus sativus L. EST sequences and cross-transferable EST-SSRs derived from Medicago truncatula L. were utilized to investigate the genetic diversity among grass pea populations from Ethiopia. A total of 45 alleles were detected using eleven EST-SSRs with an average of four alleles per locus. The average polymorphism information content for all primers was 0.416. The average gene diversity was 0.477, ranging from 0.205 for marker Ls942 to 0.804 for MtBA32F05. F(ST) values estimated by analysis of molecular variance were 0.01, 0.15, and 0.84 for among regions, among accessions and within accessions respectively, indicating that most of the variation (84%) resides within accessions. Model-based cluster analysis grouped the accessions into three clusters, grouping accessions irrespective of their collection regions. Among the regions, high levels of diversity were observed in Gojam, Gonder, Shewa and Welo regions, with Gonder region showing a higher number of different alleles. From breeding and conservation aspects, conducting a close study on a specific population would be advisable for genetic improvement in the crop, and it would be appropriate if future collection and conservation plans give due attention to under-represented regions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9662-y) contains supplementary material, which is available to authorized users.