Plant regeneration from callus culture in potato

Summary A procedure for plant regeneration from callus culture of potato, Solanum tuberosum L. is described. Calli were induced from 1–2 mm long shoot apices of potato cultivars Cara and A25/19 on half-strength Murashige and Skoog's medium (half-MS) supplemented with 3.2 mg IAA (indole-3-acetic acid), 1.0 mg kinetin (6-furfurylamino)purine], and 0.5 mg 2,4-D [2,4-dichlorophenoxy)acetic acid]/1. Sixty percent explants produced nodular calli on this medium within 30 days. Calli differentiated into shoot-primordia when subcultured on half-MS medium supplemented with 0.5 mg 2,4-D and 1.0 mg zeatin [6-(4-hydroxy-3-methybut-2 enylamino)amino purine]/1. Differentiated calli on half-MS medium without growth hormones produced complete plantlets which were cloned on the same medium and transferred into soil.     

Genotypic differences in organogenesis from callus of ten triticale lines

Summary Callus was obtained from immature excised embryos of triticale using MS medium supplemented with 3 mg/l 2,4-D and 1 mg/l kinetin. The presence of 2,4-D was essential for continued callus proliferation. Plantlets were induced from the calli by sub-culturing on medium either devoid of auxin or containing 0.1 mg/l 2,4-D. The capacity to produce callus and to form organs and plantlets differed markedly among the genotypes used. Lines also had distinct response to presence and absence of 2,4-D in the regeneration media. The callus of most triticale lines used differentiated into organs more readily on MS medium supplemented with 0.1 mg/l 2,4-D than on medium without growth regulators. Very high frequencies (up to 75%) of plantlet regeneration were observed in several of the triticale lines studied.     

甘薯胚性细胞悬浮培养系的建立

地甘薯胚性细胞悬浮增减系的进行了研究。将１２个基因的长约０．５ｍｍ的茎尖培养在含有０．２ｍｇ／Ｌ或２．０ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基上，形成了胚性愈伤组织。胚性愈伤组织的形成率因基因型和２，４－Ｄ深度不同而很大差异，为０－７５．７％。一方面，将胚性愈伤组织继续增减在含有２，４－Ｄ的ＭＳ培养基上，它们形成了处于各发育时期的体细胞胚。将具有体细胞胚的胚性愈伤组织转移到ＭＳ基本培养基上，体细胞胚发育成     

Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes

Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.     

In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.)

A protocol for in vitro induction of tetraploids via colchicine-treated somatic embryos from immature zygotic embryos of diploid grapevine (Vitis vinifera L.) is reported. Embryogenic callus was initiated from immature zygotic embryos cultured on Nitsch and Nitsch (NN) medium supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was transferred to NN medium containing 1.0 mg/l α-naphthalene acetic acid (NAA) and 0.5 mg/l benzyladenine (BA) to establish somatic embryogenesis. The vigorously growing globular embryos were selected and treated by 0, 10 or 20 mg/l colchicine for 1, 2 or 3 days, and then immediately transferred to NN medium supplemented with 0.03 mg/l NAA and 0.5 mg/l BA, for somatic embryo conversion and plant regeneration. The number of surviving embryos and regenerated plantlets following colchicine treatment decreased with increasing colchicine concentration and treatment time. Among 29 randomly investigated plantlets regenerated from colchicine-treated somatic embryos, five solid tetraploids (2n = 4× = 76) were identified by chromosome counting analysis; all others were diploid (2n = 2× = 38). Ploidy level of plant regenerated was also determined from leaves using flow cytometry. No chimeras with both 2C and 4C nuclei was produced from colchicine-treated somatic embryos. Significant differences in leaf stomata parameters were observed between diploid and induced tetraploid plantlets.     

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)

Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin     

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Summary Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N₆) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N₆ medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.     

Regeneration of dihaploid chicory (Cichorium intybus L. var. foliosum Hegi) via microspore culture

In order to develop fully inbred chicory plants, dihaploid plants were raised from callus derived from microspores of three selected Witloof, Robin and Treviso types. Microspores were isolated from florets containing pollen at the uninuclear state and cultured in a modified MS medium plus 0.5mg/l 2,4-D, 0.5mg/l IAA and 2.0mg/l zeatin. During culture periods of up to 6 months, gametoplasts emerged from pollen grains, divided and started to form colonies and calli. These were subcultured on the same basal medium supplemented with 0.5mg/l BA and 0.5mg/l IAA. Shoot growth was enhanced on a low salt-containing medium supplemented with 0.4mg/l kinetin and 0.2mg/l IAA. Shoots were rooted on a half-strength Lepoivre medium plus 0.2mg/l IBA and finally transferred to soil. Florets were excised from 34 capitula, but only microspores from four of them developed into plants via callus. More than 450 plants were raised in the greenhouse and the field. Leaves from these plants were subjected to DNA fluorescence analysis via flow cytometry: a range of ploidy levels was detected. The cell composition of 44 of these plants was predominantly haploid, with a diploid background. Regenerant plant phenotypes were compared with the parent genotypes. The value of such haploids in commercial chicory breeding is discussed.     

Continuous Plant Regeneration from Established Embryogenic Cell Suspension Cultures of Italian Ryegrass and Tall Fescue

The suitability of different protocols was compared for entire plant regeneration by somatic embryogenesis, of the forage plants Lolium multiflorum Lam. (Italian ryegrass) and Festuca arundinacea Schreb. (tall fescue). In the first protocol, miniature embryos were used as starting material, while mature seeds were retained in the other two. Whichever the considered protocol, undifferentiated calli were produced on Murashige and Skoog MS medium supplemented with 2,4-D. The calli were subcultured in the dark on solid MS agar medium, containing 5 mg/1 2,4-D (protocol 2) or on solid MS medium followed by transfer to a rotated liquid MS medium with 2 mg/1 2,4-D (protocol 1). In these conditions, induction of somatic embryogenesis occurred, and whole plants were regenerated during a limited lapse of time, upon transfer in the light, to MS medium supplemented with BAP but devoid of 2,4-D. The simultaneous elimination of 2,4-D and transfer to light appeared essential for full regeneration of the plants. Using this characteristic, an additional step was added to a new protocol (protocol 3) in which microcalli, cultured on liquid MS medium containing 5 mg/1 2,4-D, were transferred to the same medium with 2 mg/1 2,4-D, in the dark. In these conditions, the suspensions kept their embryogenic potential for months. In all cases, plantlets were successfully transferred into the soil. An evaluation of the somaclonal variation potential of the plants issued from each protocol is now underway.     

甘薯（Ipomoea batatas (L.) Lam.）及其近缘野生种原生质体的植株再生

对甘薯品种高系14号及其近缘野生种I.triloba L、和I.lacunosa L,进行原生质体植株再生研究。从离体培养植株的叶柄分离出原生质体,将其培养在含有0.05mg/L 2,4-D和0.5mg/L激动素(KT)的MS培养基中,从原生质体获得了高频率的愈伤组织。培养8-12周后,将直径达2—3mm的小愈伤组织转移到添加0.05mg/L 2,4-D的MS培养基上。转移3-6周后,将愈伤组织进一步转移到添加吲哚乙酸(IAA)和6-苄基嘌呤(BAP)的MS培养基上,一些愈伤组织再生出植株。未再生植株的愈伤组织进一步在MS基本培养基上培养,它们也再生出植株。本研究从I.triloba原生质体获得高频率的植株再生;首次从I.lacunosa原生质体再生出植株;从高系14号原生质体也再生出完整植株。     

Genetic factors influencing the regeneration ability of rye (Secale cereale L.). II. Immature embryos

Summary Immature embryos of seven rye inbred lines were cultured on modified MS medium containing 3 mg/dm^–3 2,4-D. According to thein vitro response lines were divided into four groups: (1) those producing non-embryogenic callus (NEC) from above 30% of the embryos and having a high rate of non-responding (NR) embryos, (2) those producing non-embryogenic callus (NEC) from the majority of embryos, (3) those producing NEC by the majority of embryos with a high percentage of calli regenerating roots, (4) those producing embryogenic callus (EC) and regenerating plants by above 50% of the embryos. The inheritance of these response types was analysed in F₁, F₂, and F₃ generations of crosses of some lines. The results obtained indicate that EC production and both plant and root regeneration are determined by recessive genes whereas the reduced ability for NEC production most probably by dominant genes. The lack of response is controlled by at least two interacting genes.     

Indirect Organogenesis through Seedling-Derived Leaf Segments of <Emphasis Type="Italic">Ficus Religiosa</Emphasis> - a Multipurpose Woody Medicinal Plant

The aim of this study is to introduce the suitable protocol for indirect regeneration from seedling-derived leaf segment of Ficus religiosa. The leaf explant successfully produced callus on MS medium containing various concentrations of auxin in combination with BAP. The maximum callus induction (100%) was achieved in MS medium containing 0.5 mg/l 2,4-D plus 0.05 mg/l BAP and MS medium containing 1.5 mg/l NAA plus 0.15 mg/l BAP as well. MS medium consisting of 2,4-D produced yellow-brownish and friable callus (type I) while the yellowish and compact calli (type II) were obtained in MS medium consisting of NAA. On the other hand, MS medium supplemented with IBA formed greenish and compact calli (type Ш). The regeneration rate in type II callus was less than the type I, and there was no shoot induction observed on type Ш calli. MS medium supplemented with 1.5 mg/l BAP in combination with 0.15 mg/l IBA had the highest regeneration frequency (100%) and maximum shoot numbers (5.16) as well as shoot length (2.56 cm) in type I callus. A maximum of 93.33% root induction was observed in MS medium supplemented with 2.0 mg/l IBA plus 0.1mg/l NAA. The plantlets were successfully transferred to the greenhouse. This system could be utilized for large-scale multiplication of Ficus religiosa.     

Intergeneric hybridization between Brassica species and Crambe abyssinica

A protocol for high frequency callus induction and plant regeneration from sunflower (Helianthus annuus L.) anthers is described. Different variables using Murashige & Skoog (MS) basal medium supplemented with 2.0 mg/l α-naphthaleneacetic acid (NAA) and 1.0 mg/l N₆-benzyladenine (BA) were tested for their ability to enhance the frequency of anther callusing and subsequent embryogenesis. Of these, agar concentration, sucrose concentration, carbohydrate source had significant effect on callusing, while differences due to incubation under dark vs light conditions, cold pretreatment of capitula for 1 to 6 days prior to anther inoculation and genotype on callusing were non-significant. However, all these factors exerted highly significant influence on embryogenesis when calli from the various media were transferred to medium supplemented with 0.1 mg/l NAA and 0.5 mg/l BA. With the procedure developed, callusing as high as 100% and embryo formation at a frequency of 44% was achieved. Although complete embryos were formed the frequency of their conversion to whole plantlets was low (14.3%). Hence, the embryogenic pathway was bypassed to obtain multiple shoots by transferring embryogenic calli with developing embryos to MS medium supplemented with 0.5 mg/l BA. Elongated shoots rooted on half-strength MS medium supplemented with 0.5 mg/l NAA. Cytological analysis of embryogenic callus and somatic embryos revealed haploids at a frequency of 30% while that of rooted plants showed haploid regenerants at a frequency of 8.3%. Nevertheless, the frequency of putative haploid plants could be enhanced through mass multiplication using nodal explants of the regenerants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of a technique for in vitro unpollinated ovary culture in rice, Oryza sativa L.

A simple and efficient technique for in vitro unpollinated ovary culture in rice which is also applicable for indica genotypes was developed for breeding and genetic studies. Sampling explants at the auricle distance of 7–12 cm between the two uppermost leaves of a tiller, providing a chilling pretreatment and ovaries with 1/3 of the hulls intact gave optimum response to culture. For callus induction with the spontaneous breaking of ovaries, N₆ media supplemented with NAA (2 mg/l) and DMSO (0.6–0.8%) gave a mean PCI value of 3.8% and range of 0.8–12.5% among genotypes. Media combining 2,4,5-T or 2,4-D with NAA in N₆ medium also has reasonably good callus induction. For calli induced inside, 2,4-D (0.2–0.5 mg/l), NAA (2 mg/l) and KT (1 mg/l) contained media were superior. The maximum green plant regeneration (PPR) of 77.3% was found with the medium containing NAA 0.25 mg/l, IAA 0.5 mg/l and KT 2.0 mg/l. Significant genotype, medium and their interaction effects for per cent ovary survival and callus induction were observed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of growth regulators on callus cell growth,plant regeneration,and somaclonal variation of smooth bromegrass (Bromus inermis Leyss.)

Summary Immature inflorescences of smooth bromegrass were cultured on MS agar media supplemented with varying combinations of 2,4-D and kinetin. Callus was initiated from segments of young inflorescences on each medium. All of the calli were subcultured monthly for 5–6 times and transferred onto hormone-free MS medium for plant regeneration. Addition of kinetin to the basal medium stimulated shoot initiation in the callus cultures. Plantlets were regenerated only from calli grown on media containing 2 and 6 mg I^-1 2,4-D with a supplement of 0.2 mg I^-1 kinetin. No albino plantlets were produced. Morphological characteristics and dry matter yield of ten somaclones and the parental plant (SBG7) were studied in the greenhouse in a randomized complete block experiment with five replications. There was significant variation (P>0.01) among genotypes for all morphological characteristics studied. Although all somaclone heights and leaf widths were lower than those of the parental plant, the somaclone F9A, F10A, and F10B had larger tiller numbers, and leaf/stem ratio by dry weight than the parental plant. Only somaclone F9B gave higher specific leaf area and leaf area ratio than the parental plant. Almost all somaclones had the same leaf length, total dry weight, and specific leaf weight as the parental plant. The variation found in somaclones should permit selection for desirable agronomic traits.     

Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos

Summary Experiments upon in vitro culture of immature durum wheat embryos, harvested at different growth stages, were made in two consecutive years. Callus formation and plantlet regeneration were obtained. The ability to form callus and the degree of morphogenetic processes varied with the different hormonal treatments used and with the age of the embryos. In the first year the best response for callus growth was observed with 2,4-D 2 mg l^-1 plus adenine 50 mg l^-1 or 2,4-D 5 mg l^-1 alone in the more mature embryos (15 and 20 days after anthesis). On the contrary, NAA 5 mg l^-1 had a greater shoot regeneration effect. In the next year, at all 2,4-D concentrations and for the two different ages of the embryos tested, all embryos formed callus. Regeneration of plantlets was obtained in higher percentage in calli originated from the more developed embryos. The effect of changed media upon plantlet regeneration was studied after callus transplant.Investigation by cytophotometry and chromosome counts on different calli showed, practically in all cells, a diploid condition. A histological analysis demonstrated embryogenic somatic characteristics in many samples of callus. The pattern of organogenesis seemed to be via adventitious bud formation but structures resembling embryoids were also observed in the callus.     

Genotype and hormonal effects on callus formation and regeneration in mulberry

Summary Twenty-five mulberry genotypes were studied for callus induction, to evaluate the effectiveness of hormones in promoting callus growth and to identify genotypes capable of regenerating plants. Fifteen genotypes showed callus initiation. Genotypic variation was also noted for longevity and rate of growth of callus cultures. Calli of different genotypes were maintained for more than one year. Frequency of callus initiation was high on Murashige & Skoog's modified medium incorporated with 2.0 mg/l 2,4-D, 100 mg/l casein acid hydrolysate and 150 ml/l coconut water. Regeneration through organogenesis was achieved in six genotypes indicating genotypic specificity.     

Long-term callus cultures of diploid Barley (Hordeum vulgare). I. Auxin effects on culture initiation and maintenance

Summary Use of 2,4-D was superior to NAA or IAA for embryogenic callus initiation or maintenance in barley cultivar Bruce. A concentration of at least 2.0 mg/l 2,4-D was desirable for culture initiation. The developmental size of the embryo was more important than embryo age for obtaining embryogenic calli. Even brief exposures (20–40 days) of calli to concentrations of higher than 5.0 mg/l 2,4-D or 10.0 mg/l NAA resulted in inhibition of subsequent plant regeneration and therefore, concentrations above these could not be used for maintenance cultures. In the long-term maintenance cultures, the best production of embryogenic calli was with 0.1 mg/l and 1.0 mg/l 2,4-D.     

Production of interspecific hybrids between Glycine max and G. tomentella through embryo culture

Summary Interspecific hybrids have been obtained in an incompatible cross between Glycine max and G. tomentella through the in vitro culture of hybrid embryos. The percentage of successful pod setting in the crosses averaged 12.8% but there were marked differences depending on the soybean cultivar used as the female parent. Hybrid embryos at globular to heart stages were extracted from the embryo sac 15–25 days after pollination and cultured in vitro. Hybrid plants were successfully obtained by culturing the embryos on B5 medium supplemented with 0.1 mg/l IBA followed by culture on B5 medium supplemented with 0.1 mg/l TBA plus 0.25 mg/l 2-iP. The F1 plants resembled the wild male parent in growth form, but had an intermediate leaf shape between that of the parents.     

Studies on somaclonal variants for resistance to scab in bread wheat (Triticum aestivum L.) through in vitro selection for tolerance to deoxynivalenol

This study was conducted to develop an efficient in vitro selection system for scab resistance by using in vitro screening for tolerance to deoxynivalenol (DON). Immature embryos of two wheat varieties, a scab-resistant variety Sumai 3 and a susceptible variety Mianyang 11, and their reciprocal F₁ hybrids were cultured on MS medium supplemented with 2,4-D 2 mg/l and 0.6 × 10^-4 M DON for callus induction. The responses of callus induction and plant regeneration to 0.6 × 10^-4 M DON differed significantly between resistant and susceptible varieties, according to observed scab resistance levels at the plant level in the field. The percentage of callus formation of resistant variety Sumai 3 on induction medium containing DON was higher than that of susceptible variety Mianyang 11. Regeneration of DON-tolerant calli on DON-containing differentiation medium differed significantly between Sumai 3 and Mianyang 11. Averaged across the DON-tolerant calli of two varieties and their reciprocals, regeneration of DON-tolerant calli was decreased 3-fold on DON-containing medium. By an inoculation test with conidiospores of Fusarium graminearum Schw, we obtained several resistant lines from progenies of regenerated plants from DON-tolerant calli. These somaclonal lines had lower disease scoring (reaction index, infected spikelets and disease incidence), shorter plants and better yield components than Sumai 3, a famous Chinese resistant variety. This revised version was published online in July 2006 with corrections to the Cover Date.