In vitro regeneration patterns of Platycerium bifurcatum leaf cell suspension culture

A simple suspension culture system of Platycerium bifurcatum was developed where sporophytes could be regenerated directly from leaf cells or indirectly through an aposporous gametophyte stage under the same culture conditions. Single cells and aggregates of up to 100 cells developed aposporous gametophytes which later gave rise to sporophytes. Such gametophytes started apogamy when they were mostly less than 0.7 mm in length, bearing only rhizoids. In most cases, only one sporophyte was regenerated from one gametophyte. Aggregates of 500–1000 or more cells, on the other hand, regenerated sporophytes directly. Intercellular interaction was considered to be the physiological cause, and the separation of leaf cells to a certain degree drove the cells to embark on different regeneration paths. It is suggested that the possible existence of a threshold size of cell aggregates separates the two regeneration patterns. Received: 3 March 1997 / Revision received: 11 April 1997 / Accepted: 3 June 1997     

Activated charcoal affects morphogenesis and enhances sporophyte regeneration during leaf cell suspension culture of Platycerium bifurcatum

In a leaf cell suspension culture of Platycerium bifurcatum, the incorporation of activated charcoal (AC) greatly increased the number of regenerated sporophytes even in growth regulator-free medium. The degree of improvement was dependent on cell aggregate sizes and medium composition. The maximal increase was observed in medium with 5.37 μM NAA and 4.44 μM BA, from 9 to 1520 sporophytes. The qualitative improvement by AC included: (1) regeneration of single sporophytes, which was relatively less frequent in non-AC media, (2) prevention of the formation of gametophyte clusters prior to sporophyte regeneration from 30- to 60-μm cells cultured in MS basal medium, (3) prevention of the formation of bud clusters, sporophytes with multiple bud primordia, `nodule'-like bud clusters in growth regulator containing media, and (4) prevention of the occurrence of hyperhydricity of regenerated sporophytes. Received: 9 December 1996 / Revision received: 18 April 1997 / Accepted: 5 July 1997     

Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae)

Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.     

Isolation and culture of protoplasts from the marine green algaMonostroma angicava

Protoplasts were isolated enzymatically from gametophytes of the marine green algaMonostroma angicava. The protoplasts regenerated in PES medium after gradual reduction of the osmoticum. Three types of developmental process were recognized in the protoplast regeneration: an original type, in which the protoplasts regenerated into leafy gametophytes; an apogamic type, in which they regenerated into sporophytes; a callus type, in which they regenerated into callus-like tissues. The resulting gametophytes and apogamic sporophytes became fertile in successive cultures.     

Production and analysis of asymmetric hybrid plants between monocotyledon (Oryza sativa L.) and dicotyledon (Daucus carota L.)

Asymmetric hybrid plants were obtained from fused protoplasts of a monocotyledon (Oryza sativa L.) and a dicotyledon (Daucus carota L.). X-ray-irradiated protoplasts isolated from a cytoplasmic malesterile (cms) carrot suspension culture were fused with iodoacetoamide-treated protoplasts isolated from a 5-methyltryptophan (5MT)-resistant rice suspension culture by electrofusion. The complementary recovered cells divided and formed colonies, which were then cultivated on regeneration medium supplemented with 25mg/l 5MT to eliminate any escaped carrot cells. Somatic hybrids were regenerated from 5 of the 5MT-resistant colonies. The morphologies of most of the regenerated plants closely resembled that of the parental carrot plants. A cytological analysis of callus cultures induced from these plants indicated that most of the cells possessed 20–22 chromosomes and were resistant to 5MT. An isozyme analysis revealed that several regenerated plants had the peroxidase isozyme patterns of both parents. A Southern hybridization analysis with non-radioactively labelled DNA fragments of the rgp1 gene showed that regenerated plants had hybridizing bands from both rice and carrot. Chloroplast (cp) and mitochondrial (mt) DNAs were also analyzed by Southern hybridization by using several probes. CpDNA patterns of the regenerated plants were indistinguishable from those of the carrot parent. However 1 of the regenerated plants had a novel band pattern of mtDNA that was not detected in either of the parents, indicating a possible recombination of mitochondrial genomes.     

油菜游离细胞培养及易再生的体细胞无性系的建立

以芥菜型油菜(Brcssica juncea)下胚轴来源的愈伤组织为材料,进行悬浮细胞振荡培养,得到游离的单细胞。这些细胞作浅层液体静置培养,可获得高频率的愈伤组织。在附加有水解乳蛋白200毫克/升、BA 2—3毫克/升及GA,0.1—1毫克/升的MS培养基上,这些愈伤组织分化了芽。分化的芽在无激素或附加有0.01—0.1毫克/升IAA或NAA的MS培养基上长出根,从而发育成完整植株。当对游离细胞获得的再生植株的茎切段愈伤组织,再经上述程序进行细胞培养时,发现愈伤组织诱导率及愈伤组织分化频率均远远高于原供体,从而建立了B.juncea易再生的体细胞无性系。     

水稻原生质体培养及植株再生的研究

由粳稻77-170品系及籼稻品种IR-50的细胞悬浮培养物游离的原生质体,用琼脂糖包埋于RY-2培养基中,发生了持续分裂。前者植板率达2.5％以上,二者最后都再生出植株。对游离和培养方法做了如下改进:1)采用两步法,即先用果胶酶,再用果胶酶和纤维素酶的混合酶进行游离,可避免原生质体发生融合并获得高质量的原生质体;2)悬浮细胞培养基中加入ABA有利于原生质体的存活和分裂;3)琼脂糖包埋培养可大大提高植板率;4)用较高渗透压的培养基培养原生质体再生的细胞团及愈伤组织,可提高植株再生频率。由于这两个品种(系)的培养物都已继代一年半之久,再生植株均为白化苗。这是迄今第一个由籼稻原生质体再生植株的报道。     

Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum spp. hybrids)

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue cultured on modified MS medium, but only Type 3 (embryogenic) and Type 4 (organogenic) were capable of plant regeneration. Cell suspension cultures were initiated from embryogenic callus incubated in a liquid medium. In stage one the callus adapted to the liquid medium. In stage two a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture. In stage three a homogeneous cell suspension culture was developed in six cultivars after 10 to 14 weeks by selective subculturing to increase the proportion of actively dividing cells from the heterogeneous cell suspension culture. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures for up to 148 days of culture but plants could not be regenerated from homogeneous cell suspension cultures. High yields of protoplasts were obtained from homogeneous cell suspension cultures (3.4 to 5.2 × 10⁶ protoplasts per gram fresh weight of cells [gfwt^-1]) compared to heterogeneous cell suspension cultures (0.1 × 10⁶ protoplasts gfwt^-1). Higher yields of protoplasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures). This process increased protoplast yield to 9.4 × 10⁶ protoplasts gfwt^-1. Protoplasts isolated from S-cell suspension cultures were regenerated to callus and recycled to produce SP-cell suspension cultures yielding 6.4 to 13.2 × 10⁶ protoplasts gfwt^-1. This recycling of callus to produce S-cell suspension cultures allowed protoplasts to be isolated for the first time from cell lines of cultivars Q110 and Q138.     

An efficient callus suspension culture system for triploid bermudagrass (Cynodon transvaalensis × C. dactylon) and somaclonal variations

An efficient callus suspension culture and regeneration system in a triploid bermudagrass (Cynodon dactylon × C. transvaalensis cv. Tifeagle) was studied in this report. Proline improved callus proliferation, but had no effect on regeneration. 0.6–1.2 mg l⁻¹ BA improved regeneration, but higher concentrations of BA (≤1.2 mg l⁻¹) resulted in the production of rootless plantlets. The embryogenic calli were able to proliferate continuously for at least 2 years with regeneration ability through the established suspension culture system. Observations with scanning electron microscope and light microscope showed somatic embryogenesis during the regeneration. Somaclonal variations were observed in regenerated plants. More than 2000 regenerants were screened for drought tolerance in the greenhouse, from which seven lines appeared to have increased drought tolerance relative to their parental variety. It is suggested that somaclonal variation in triploid bermudagrass offers an effective tool for its breeding.     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Summary Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.     

A low-temperature method for maintaining plant regeneration activity in embryogenic callus of rice (Oryza sativa L.)

 A method was developed to maintain plant regeneration activity of rice cells (Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5  °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5  °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. Received: 7 January 1999 / Revision received: 28 April 1999 / Accepted: 26 May 1999     

REGENERATION OF LIRIODENDRON TULIPIFERA (FAMILY MAGNOLIACEAE) FROM PROTOPLAST CULTURE

Protoplasts were isolated enzymatically from suspension cultures of two embryogenic lines of yellow-poplar (Liriodendron tulipifera L.) and cultured in droplets of a regeneration medium supplemented with 1 mg/1 2,4-D and 0.25 mg/1 6BA and solidified with agarose. Suspension cultures grown in the light yielded substantial numbers of protoplasts, while insignificant numbers were isolated from cultures grown in the dark. The protoplasts reformed cell walls within three days, and 75 percent of them divided at least once within one week in culture. Embryogenic suspension or callus cultures were regenerated by placing agarose droplets containing protoplast-derived microcalli directly into liquid conditioning medium or onto plates of conditioning medium solidified with agar. Embryogenesis was obtained by transferring the callus to a hormone-free induction medium.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

Rapid production of wheat cell suspension cultures directly from immature embryos

The aim of this study was to produce suspension cultures of winter wheat directly from immature embryos bypassing the callus stage, and to determine their capacity for growth and regeneration in comparison to suspension cultures produced from callus. The study was carried out using Polish winter wheat varieties: ‘Grana’ and ‘Rosa’. Immature embryos were isolated, homogenized and transferred directly to liquid medium supplemented with 2,4-D. Actively dividing cell cultures were obtained within 2 months after the cultures were started. Suspension cultures from callus of immature embryos was also produced. With both cultivars, faster growth was observed in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Metabolic activity was higher in the suspension culture produced directly from embryos than in the suspension derived from callus only in ‘Grana’. The production of 1-amiocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, was lower in the suspension cultures produced directly from embryos than in the suspensions produced from callus. Morphogenic capacity was significantly higher in aggregates derived directly from embryos than in aggregates derived from callus. With ‘Rosa’, about one third of the aggregates derived directly from embryos regenerated shoots. Production of ACC was lower in ‘Rosa’ cell culture that regenerated then in other cell cultures that did not. Photosystem II reactions were more efficient in dark green aggregates than in light green or pale green aggregates which were unable to regenerate. With the method presented, wheat cell suspension cultures with a regeneration potential can be produced in 2 or 3 months less time than with traditional methods.     

辣椒游离小孢子细胞团培养的胚状体形成

从预培养15天后的花药中机械游离小孢子及其细胞团,经28℃液体悬浮暗培养．30天后,获得了自球形期胚到子叶期胚发育程度不等的各类胚状体。从12个花药中可以形成高达22个胚状体,且子叶期胚的比例约为23％。显微镜检表明,这些胚状体来自游离的小孢子细胞．经核的对称分裂形成多核细胞或者早期形成多细胞团,最后经细胞的分裂分化形成。胚状体体表具毛,活力有差异。在适当培养基上,具活力的鱼雷期及子叶期胚状体均能发育成正常植株。7℃、32℃、35℃8天的胁迫处理均能诱导小孢子胚状体发生。但花药培养中7℃、35℃处理下的出胚率较32℃下高,而游离小孢子细胞团培养中以35℃、32℃下较好。7℃处理下获得的胚状体数很少．对产生这种现象的原因进行了探讨。出胚率在基因型间,不同胁迫处理温度间表现明显差异。而在温度处理的不同天数间差异不明显。流式细胞仪对再生株真叶的DNA含量分析表明．获得的再生株中具有单倍体、双单倍体以及单倍一双倍嵌合体植株。本结果为进一步开展辣椒雄性生殖途径的胚状体发育研究。提高辣椒成熟胚状体的频率提供了实验体系。     

Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

Molecular cloning and characterization of calreticulin, a calcium-binding protein involved in the regeneration of rice cultured suspension cells.

A full-length cDNA clone encoding a phosphoprotein (pp56) involved in the regeneration of rice (Oryza sativa L.)-cultured suspension cells was isolated by screening a rice cultured suspension cell cDNA library. The 1558-bp cDNA sequence contains an ORF encoding an acidic (pI 4.38) protein of 424 amino acids (47.9 kDa), sharing 70-93% and 50-53% homology with other plant and mammalian calreticulins, respectively. Sequence analysis of the cDNA clone revealed several significant conserved motifs, including a calreticulin family repeat motif in the central domain and two calreticulin family motifs in the N-domain, indicating that this gene is a rice calreticulin (CRO1). The CRO1 gene in long-term rice cultured suspension cells shows constitutive expression in both suspension culture and regeneration media. In contrast, expression of the CRO1 gene in short-term rice cultured suspension cells, which possess regeneration potential, is increased dramatically when these cells are transferred to the regeneration medium. After approximately 2 weeks in the regeneration medium, the expression of the CRO1 gene reverts to constitutive levels. These results demonstrate the presence of calreticulin in rice cultured suspension cells and its developmental regulation during the regeneration of rice cultured suspension cells.