Development of the CELLOP optimisation model for plant cell cultivation

A mathematical computer-aided model CELLOP was constructed in which the desirability functions in a three-dimensional experimental design can be used to find the optimal growing conditions for plant cells. CELLOP is intended for the optimisation of 3 to 6 physical, chemical, or biological variables in the cultivation conditions of plant cell cultures. The model was used to optimise the culturing conditions (calcium, inorganic nitrogen, and sucrose concentrations) for coumarin-producing, spontaneously embryogenic cell lines of angelica Angelica archangelica L. subsp. archangelica and hogs fennel Peucedanum palustre (L.) Moench. For A. archangelica the overall optimum concentrations were 0.47 mM Ca²⁺, 5.06 mM NO₃ ⁻, 0.40 mM NH₄ ⁺, and 96.25 mM sucrose. The dry mass was 24.7 % higher and the coumarin content 40.5 % higher than those achieved in the standard 75 % Gamborg B5 medium. For A. archangelica the highest embryogenic activity was reached in the media containing 1.25 mM Ca²⁺. In the case of P. palustre the overall optimum concentrations were 1.60 mM Ca²⁺, 2.84 mM NO³ ₋, 0.23 mM NH₄ ⁺, and 85.10 mM sucrose. For P. palustre the dry mass production increased by 61.8 % and the coumarin content by 58.1 % compared to the values achieved in the Gamborg B5 medium. For P. palustre the highest embryogenic activity was reached in the presence of 50.0 mM NO₃ ⁻ and 4.01 mM NH₄ ⁺.     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Long-term suspension cultures of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) with high embryogenic potential

Cucumber (Cucumis sativus L.) cytokinin-independent embryogenic cell suspension cultures were derived and maintained for more than 3.5 years without losing the embryogenic potential. The preparation and the characteristics of the cucumber embryogenic cell suspension possess many similarities to that of carrot. The cultures were induced from hypocotyl explants of in vitro grown cucumber plants in liquid MS media containing 2,4-dichlorophenoxyacetic acid as the sole growth regulator during 6 weeks and they contained a heterogeneous array of several different types of single cells and cell clusters (PEMs). The established cell suspensions were subcultured in 1-week interval, while the inoculation density was optimized to 2.0 × 10⁵ cells ml⁻¹ using cell viability as a marker. Somatic embryos were obtained after the transfer of the proembryogenic masses to a hormone-free semisolid MS medium with a frequency of 388 ± 57 somatic embryos per 1 ml of packed cell volume of the established cucumber embryogenic culture within 7 days. The frequency of normal somatic embryos with two cotyledons was found to be 78%. Such embryos possessed the potential of spontaneous maturation and the embryo conversion rates were 87%. The yield of normally growing plants was much higher compared with that previously described for cucumber systems. Somatic embryo-derived plants were successfully transferred to the greenhouse where they flowered and fruited.     

Establishment and maintenance of friable,embryogenic maize callus and the involvement of L-proline

Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659–668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473–497, plus 0.5 mg 1^-1 thiamine hydrochloride and 150 mg 1^-1 L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium containing inorganic elements of Murashige and Skoog (1962), plus 0.5 ml 1^-1 thiamine hydrochloride and 150 mg 1^-1 L-asparagine monohydrate - N6 medium of Chu et al. (1975) Paper No. 13,904, Scientific Journal Series Minnesota Agricultural Experiment Station     

CHARACTERIZATION OF AN EMBRYOGENIC CELL SUSPENSION CULTURE DERIVED FROM CULTURED INFLORESCENCES OF PENNISETUM AMERICANUM (PEARL MILLET,GRAMINEAE)

An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Summary Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

Plant development from isolated microspores of Zea mays L.

This paper reports the development of plants from mechanically isolated microspores of corn (Zea mays). Large populations of corn microspores were isolated using technology previously developed for rapeseed. Embryos and callus were developed from microspores in the late uninucleate stage. Scutellar-type embryos developed after two weeks and these could be transferred and germinated on a hormone free medium. However, the large majority of plants recovered from embryos developed only upon transfer to a corn embryogenic callus medium. These embryos produced shoots through organogenesis, and subsequently could be induced to form roots. Plants were developed from these colonies and grown in the greenhouse. The frequency of mature plants developed from the embryos was approximately 5 %. Non embryogenic callus which developed from some microspores have thus far either failed to develop or have developed only roots. Seed set has been obtained on some of the regenerated plants.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).     

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.     

A comparative study of somatic embryogenesis in Secale vavilovii

Summary The ability of immature embryos, inflorescences and leaves of Secale vavilovii to form embryogenic callus was tested on Murashige and Skoog (1962) medium supplemented with different concentrations of 2,4-D. All cultured immature embryos formed calluses. The highest percentage of embryogenic callus production was from 1–2 mm embryos. Young leaves also formed calluses, mainly from the 10–15 mm basal segment, the percentages of embryogenic calluses being higher when cultures were maintained in darkness. Embryogenic calluses were obtained also from all the cultured immature inflorescences, in the three cases, rooted green plants were obtained and grown in soil. Comparison of the responses of the three explants used indicates that immature inflorescence is the most useful explant for obtaining regenerated plants in Secale vavilovii.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV) was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.     

Micropropagation of <Emphasis Type="Italic">Arnebia hispidissima</Emphasis> (Lehm). DC. and production of alkannin from callus and cell suspension culture

Alkannin, a red-purple dye and bioactive compound found in the roots of Arnebia hispidissima has antibiotic and anti-inflammatory properties and is also used in cosmetic and textile industries at a large-scale. In the present communication, we demonstrate the establishment of callus and cell suspension culture of A. hispidissima with the aim of optimizing the production of alkannin. Highest alkannin content was recorded in cell suspension and callus culture established on M-9 medium. Production of alkannin was influenced by the different culture medium. Evaluation of alkannin content of roots of field-grown plants and in vitro grown cell, tissue and organ showed that alkannin production was higher in all in vitro grown culture systems (cell suspension, callus and roots) than the roots of field-grown plants. The present investigation may be applicable in designing systems for the large-scale cultivation of A. hispidissima cell suspensions for the production of alkannin.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Production of fertile somatic hybrid plants of sunflower and Helianthus giganteus L. by protoplast fusion

Summary Protoplasts of Helianthus giganteus and Helianthus annuus were fused using polyethylene glycol. Before fusion H.giganteus protoplasts were subjected to iodoacetic acid treatment to render them unable to divide. Fused protoplasts were cultured in V-KM medium containing benzylaminopurine and naphtaleneacetic acid. Hybrid calli were identified on the basis of their ability of embryogenic development contributed by the Helianthus giganteus parent. Fifty embryogenic calli were cultured on MS based medium without growth regulators to induce further development of somatic embryos. Elongated shoots were removed, rooted and transferred into growth chambers. Overall morphology of the plants was intermediate between the two parents. Their hybrid nature was confirmed by chromosome counting and by the analysis of esterase isozymes. The plants flowered within two to three months and later died. Thus the perennial nature of H.giganteus is a recessive trait in this interspecific hybrid. Seeds were obtained from two of the regenerated plants. From these seeds normal fertile F₂ plants could be grown.Abbreviations PEG polyethylene glycol - NAA naphtaleneacetic acid - BA benzyladenine - MS Murashige and Skoog medium - V-KM protoplast culture medium of Binding and Nehls     

High-frequency plant regeneration from coleoptile tissue of indica rice (Oryza sativa L.)

Summary An efficient method was established for high-frequency embryogenic callus induction and plant regeneration from 3-,4-, 5- and 7-d-old coleoptile segments of Indica rice (Oryza sativa L. cv. Kasturi), Compact and friable callus developed from the cut ends and also on the entire length of the coleoptile segments cultured on Murashige and Skoog (MS) basal medium (1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 4.50–18.0 μM), kinetin (2.32 μM) and sucrose (3%, w/v). High frequency embryogenic callus induction and somatic embryo development was achieved when embryogenic calluses were transferred to MS medium supplemented with 2.25 μM 2,4-D, 2.32 μM kinetin, 490 μM L-tryptophan and 3% (w/v) sucrose. Plant regeneration was achieved by transferring clumps of embryogenic callus onto MS medium containing 2.85 μM indole-3-acetic acid (IAA), 17.77 μM 6-benzylaminopurine (BA) and 3% (w/v) sucrose. Histological observations of embryogenic calluses revealed the presence of somatic embryos and also plant regeneration via multiple shoot bud formation. Three, 4- and 5-d-old coleoptile segments showed a significantly (P<0.05) higher frequency of plant regeneration and mean number of plantlets per explant in comparison to 7-d-old coleoptile segments. The highest frequency (73.5%) of plant regeneration and mean number of plantlets (11.9±1.0) was obtained from 4-d-old coleoptile segments. Regenerated shoots were rooted on MS basal medium containing 4.92 μM indole-3-butyric acid (IBA) and plants were successfully transferred to soil and grown to maturity.