Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple

Plantlets were obtained from usually dormant axillary buds, excised from the crown of pineapple (Ananas comosus L. Merr.) and grown in culture. Multiple shoots arose from single buds grown on Murashige and Skoog medium supplemented with auxins and kinetin. Shaking culture-flasks during growth increased the number of multiple shoots formed, when compared with stationary liquid cultures. Leaf explants excised from in vitro plantlets developed into a callus capable of plantlet regeneration. Subjecting developing buds to surgical segmentation also resulted in multiple shoot formation. Such shoots, when excised and grown on Murashige and Skoog medium supplemented with auxins, developed roots and grew into complete plantlets capable of being grown in soil.     

Propagation of Lilium hybrids. II. Production of plantlets from bulb-scale callus cultures for increased propagation rates

A method has been developed for the large-scale propagation of lilies from callus cultures. Bulb scales, being available at all times, are an ideal source of explants. Callus was induced throughout the annual cycle of all the 12 Oriental hybrid cultivars tested, by treating the bulb scales with a combination of 5 μM BA and 5 μM 2,4-D. Once initiated, this callus grew vigorously on media lacking exogenous growth substances. Callus induction by BA and 2,4-D, and the subsequent growth of such callus in the absence of exogenous growth substances, occurs within a broad range of cultivars and species within the genus. The callus could be maintained on a medium lacking ammonium salts, but the maximum growth rate was obtained at 10 mM NH₄NO₃. At low (0.2 mM) or zero levels of NH₄NO₃, light as compared with darkness was inhibitory to growth.The callus maintained its capacity for organogenesis. Maximum production of plantlets was obtained in continuous light on agar medium with 0.5 μM NAA.Potentially, the successive use of liquid and agar cultures can produce 6 × 10¹² plants per year from 1 g (dry wt.) of callus. Plantlets derived from callus were consistently diploid and could be readily transferred to soil.     

Plantlet regeneration and stability from callus cultures of Freesia × hybrida Bailey cultivar ‘Royal’

True-to-type plantlets of Freesia × hybrida Bailey cultivar ‘Royal’ were generated from callus after 27 months of sub-culture in liquid medium. Callus was initiated from young flower pedicels cultured on semi-solid Linsmaier—Skoog (LS) medium supplemented with 5 mg/l of 2,4-D and 0.5 mg/l kinetin, and transferred to the same medium in liquid form without hormones and thereafter sub-cultured every 7–10 days. Liquid cultures with 2.4–4.3 g of callus per 25 ml medium produced largest increases in callus fresh weight. Callus generated the most shoots when cultured on LS medium supplemented with 5 mg/l of kinetin and incubated in the light, while fewer plantlets were produced when no growth regulator or GA₃ or PBA were used. Callus cultures incubated in continuous darkness did not form shoots.     

Shoot production from onion callus tissue cultures

Shoots have been produced on callus derived from onion set and from seedling radicle tissue. Whilst callus of set origin responded optimally to medium containing the cytokinin 6-(3-methyl-2-buten-1-ylamino)-purine (2iP) at 2.00 mg l^?1 and naphthaleneacetic acid (NAA) at 0.06 mg l^?1, seedling radicle callus showed a range of response. The medium used for callus initiation, the age of callus, and the provision of a dark period following inoculation on to the organogenesis medium have been shown to be critical for shoot formation. A limited number of embryoids have been produced from cultures; their occurrence was usually associated with an increase in shoot numbers for the generative callus tissue. Meristemoid areas have been observed in light micrographs of callus cultured upon organogenesis media.     

中国李Nubiana胚性愈伤组织诱导及植株再生

以中国李Nubiana试管苗幼嫩叶片为外植体,探讨激素浓度配比、暗培养时间、琼脂浓度等对胚性愈伤组织诱导及TDZ浓度对胚性愈伤组织不定芽再生的影响,建立其愈伤组织诱导及不定芽再生体系。结果表明,外源激素对中国李Nubiana胚性愈伤组织诱导影响显著,TDZ不适合胚性愈伤组织的诱导;暗培养28 d,产生的愈伤组织质地较好,诱导愈伤组织的效果最佳。胚性愈伤组织在WPM+NAA 0.5 mg·L-1+6-BA 2.0 mg·L-1+TDZ 2.0 mg·L-1+蔗糖30g·L-1+琼脂6.0 g·L-1培养基上光培养可再生出不定芽,其再生率为6.7%。     

Continuous propagation of tomato plants by means of callus cultures

Callus obtained in vitro from stem internode tissue was used to investigate the possibilities for accelerated vegetative propagation of Lycopersicum esculentum Mill. and Lycopersicum peruvianum (L). The results of different tests with phytohormones pointed to a high endogenous auxin level in the original stem explants of L. peruvianum. In L. esculentum shoots were obtained when high amounts of zeatin or coconut-milk were applied.Explants of CCC-pretreated (2 chloroethyl trimethyl-ammonium chloride) tomato plantlets showed a significant increase in shoot formation as compared to the untreated ones.Callus tissue that was more than 2 years old and had been used in 30 transfers still had the capacity to produce normal shoots and roots.More than 200 resulting plants were observed in glasshouse conditions for possible genetic variations. No striking deviations from the original phenotype occurred. Seeds harvested from the fruits of self-fertilized flowers on these plants were sown under normal growth conditions. Some plants of one particular cultivar showed signs of accentuated vegetative development.     

红掌不同品种对愈伤组织诱导和植株再生的影响

红掌不同品种在相同培养条件下,无论外植体是哪种种类,其在培养基上的出愈率和分化生根能力均存在差异。我们对所取试验品种材料根据出愈率和生根能力的难易程度做了初步排序。     

天女木兰嫩茎愈伤组织诱导及再生体系建立研究

为了保护濒危植物天女木兰,采用植物组织培养方法,以嫩茎为材料,进行愈伤组织诱导与分化、不定芽生根与试管苗生根继代增殖的培养,以及试管苗移栽与定植的研究,建立起天女木兰再生体系技术.结果表明:MS+ZT 0.4 mg/L+2,4-D 2.5 mg/L是嫩茎愈伤组织诱导培养和继代增殖培养的理想培养基;MS+GA30.5 mg/L+BA 0.6 mg/L是愈伤组织分化培养的理想培养基;把不定芽用10 mg/L的IAA溶液处理24 h后,接种到1/3 MS+IBA 0.6 mg/L+0.8 DA-60.5 mg/L+NAA 0.5 mg/L培养基上的生根培养方法是不定芽生根培养和试管苗生根继代增殖培养的理想方法;在温室中试管苗易移栽成活,定植的试管苗保持了野生天女木兰的所有植物学性状.     

石龙芮毛茛愈伤组织诱导及再生体系建立研究

为保护野生资源、实现人工栽培,以石龙芮毛茛的下胚轴为材料,进行愈伤组织的诱导和分化、不定芽分化、试管苗生根和生根继代、试管苗移栽和定植的研究,建立起石龙芮毛茛的下胚轴再生体系.结果表明:MS+KT 0.4 mg/L+2,4-D 1.8 mg/L是愈伤组织诱导培养和增殖继代培养的理想培养基;MS 0是愈伤组织分化培养的理想培养基;采用向培养瓶中加入约5 ml浓度为2 mg/L的NAA溶液处理24 h,再把不定芽切下接种到1/3 MS+IAA 0.3 mg/L培养基的方法,是不定芽生根培养和生根继代培增殖培养的理想方法.试管苗移栽、定植成活率较高;定植的试管苗保持石龙芮毛茛的所有植物学性状.     

Plantlet regeneration from root callus of different Prunus species

Caulogenesis was obtained in vitro on the calli of Prunus sp., P. dawyckensis, P. canescens and hybrid P. incisa x serrula. These calli were formed on the roots of plantlets derived from meristem culture on a medium containing 6-benzylaminopurine (BAP) (10^?3 g/l) and gibberellic acid (GA₃) (10^?4 g/l). The first buds appeared after a month. After transfer of the calli on the same medium, the caulogenesis continued. The degree of regenation varied according to the species.     

Vegetative propagation of Anthurium scherzerianum Schott through callus cultures

A tissue culture method is described for the vegetative propagation of Anthurium scherzerianum Schott through callus induction, callus subculture, adventitious bud formation in callus and rooting of excised shoot cuttings. One genotype which has been propagated vegetatively in vitro, is already growing in the greenhouse.     

枇杷属植物野生种的茎段愈伤组织诱导植株再生研究

【目的】建立野生种枇杷茎段愈伤组织诱导植株再生的离体培养技术体系。【方法】以枇杷属植物的2个野生种、1个属间杂交后代为试材,采用茎段为外植体,在不同植物生长调节剂的组合配比培养条件下,开展野生枇杷茎段诱导愈伤组织以及愈伤组织诱导出不定芽的植株再生研究。【结果】最适合野生种枇杷茎段愈伤组织诱导植株再生的培养基配方为:MS+6-BA 1.0 mg·L~(-1)+TDZ 0.1 mg·L~(-1)+NAA 0.1 mg·L~(-1),如栎叶枇杷茎段愈伤组织诱导植株再生,其愈伤率、丛芽率和成苗率均达到100%,不定芽数最多可达38.75个;但对于属间杂交后代的石斑木×台湾枇杷最适合茎段愈伤组织诱导植株再生的培养基配方为:MS+6-BA 1.0 mg·L~(-1)+NAA 0.1 mg·L~(-1),其丛芽率和芽苗数量分别为100%和48个,成苗率也为100%;在众多的生长调节剂中,TDZ对野生枇杷的愈伤组织诱导再分化或脱分化是比较敏感的,高浓度的TDZ虽有利于愈伤组织诱导再分化不定芽,但不利于成苗。【结论】研究得出枇杷属植物以及属间杂交后代茎段愈伤诱导植株再生的共性规律和种间的差异性,建立起枇杷属植物野生种离体再生系统,简化植株再生诱导的步骤和缩短植株再生诱导的时间,能在短时间内产生一定数量的组培苗,为枇杷属种质资源研究积累了新的资料。     

几种抗生素对梨叶片愈伤组织和不定梢诱导的影响

研究了氨苄青霉素(Amp)、羧苄青霉素(Carb)、头孢霉素(Cef)和卡那霉素(Km)对梨叶片愈伤组织和不定梢诱导的影响。结果表明,Amp对巴梨叶片分化影响较小,当浓度达500mg/L时,其出愈率和不定梢再生率分别为96.48%和56.17%,与对照差异不显著;Cef浓度在50-300mg/L时对巴梨和身不知叶片再生均无显著影响,只有当Cef浓度高于400mg/L时,巴梨的不定梢再生率才受到显著抑制;巴梨和身不知叶片对Carb的反应不相同,低浓度(≤200mg/L)的Carb对巴梨的出愈率和不定梢再生率影响较小,却显著抑制身不知的不定梢再生率,高浓度(≥300mg/L)的Carb不仅极显著抑制巴梨和身不知的出愈率,而且完全抑制了2者的不定梢再生;Km浓度大于10mg/L时,明显抑制愈伤组织的生长,且随着浓度的增加,死亡率逐渐增大,当Km浓度达100mg/L时,巴梨和身不知叶片死亡率达100%和96.30%。     

枣和酸枣田间愈伤组织途径芽再生技术的应用与优化

【目的】检验建立的枣田间愈伤组织途径芽再生技术的通用性并对其进一步优化。【方法】将其在115个枣和4个酸枣基因型上进行应用,进而分析基因型、愈伤组织状态、枝条粗度和位置等对田间愈伤途径芽再生的影响。【结果】发现80.67%的基因型能够实现芽再生,其中‘北京酸枣’‘龙壶枣’‘北京马牙枣’的平均单枝截面出芽数分别高达10.33、9.40、8.00个;芽再生能力随着愈伤发育等级的增加而升高,以5级愈伤的芽再生能力最强;直径为1.4~1.9 cm的枝条平均单枝截面出芽数达2.16个,显著高于其他粗度枝条的出芽数;树冠上部枝条的单枝截面出芽数显著高于中下部枝条的出芽数。【结论】枣田间愈伤组织途径芽再生技术在不同枣和酸枣基因型上有良好的通用性;影响田间愈伤组织途径芽再生的最主要因素为基因型,其次是愈伤组织状态及枝条粗度和垂直位置,枝条角度及水平位置对于芽再生的影响不显著。进一步优化了枣田间愈伤组织途径芽再生技术体系。     

In vitro stable regeneration of onion and garlic from suspension culture and chromosomal instability in solid callus culture

An efficient regeneration system from bulb-derived callus tissues in suspension of onion (Allium cepa L.) and garlic (A. sativum L.) was established. Callus culture was induced in Gelrite^®-solidified Murashige and Skoog's (MS) modified basal medium with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.93 μM kinetin supplements. After 4 weeks of induction, the callus tissue was partly transferred to liquid MS media containing different levels of α-naphthaleneacetic acid (NAA) and kinetin for plant regeneration and the rest was maintained in the same medium for chromosome analysis and nuclear DNA quantification following in situ microspectrophotometry. The cultures in suspension, maintained in agitated condition for 8 weeks, showed a high frequency of rapidly regenerated plants after transferring to Gelrite^®-solidified one half strength of MS basal medium. Chromosome analysis of the regenerated plants, transferred to the field with 90% survival rate, revealed stable chromosome number (2n = 16) in both species. On the other hand, callus tissues maintained in solid induction medium for long period showed abnormality in chromosome behavior leading to the formation of both hypo- and hyper-diploid cells along with the diploid cells. The frequency of aneuploid cells (2.2–48.9%) increased with callus age in both species with high and statistically significant number of hyperdiploid cells. The role of endoreduplication as well as non-disjunction of chromosomes resulting in instability in chromosome number has been suggested. This was also supported by the nuclear DNA value in successive passages with statistically significant increase.     

Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.     

High frequency embryogenic callus induction and plant regeneration from mature caryposis of big bluestem and little bluestem

Big bluestem (Andropogon gerardii Vitman) and little bluestem [Schizachyrium scoparium (Michaux) Nash.] are native to the North America and are important forage grasses and ornamental grasses. Both grasses are proposed as ideal biomass producers for cellulosic ethanol production. To apply genetic transformation, which is an important tool for incorporating desirable agronomic traits into plants to both species, however requires an efficient and reproducible regeneration protocol. We used mature caryopses from big and little bluestem as explants and tested the effect of various combinations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) (1, 2, 3, 4 or 5 mg l⁻¹) and kinetin (KT) (0, 0.1 or 0.2 mg l⁻¹) on embryogenic callus induction with LS as the basal medium. The highest percentage of embryogenic calli induction occurred on medium containing 2, 4-D alone at 2 mg l⁻¹ for ‘Bison’ and on medium containing 4 mg l⁻¹ 2, 4-D alone for ‘Bonilla’ big bluestem. For little bluestem, the highest percentage of embryogenic callus induction occurred on medium containing 3 mg l⁻¹ 2, 4-D plus 0.1 mg l⁻¹ kinetin, suggesting that addition of KT is beneficial. Shoot regeneration took place on LS basal medium without any plant growth regulator for both species, although the addition of KT increased both regeneration frequency and the number of shoots produced per callus. Rooting of shoots reaching about 2 cm long occurred readily with or without α-naphthaleneacetic acid (NAA). Rooted plantlets were all successfully established in the soil.     

High frequency in vitro embryogenic callus induction and plant regeneration from indiangrass mature caryposis

Indiangrass [Sorghastrum nutans (L.) Nash.] is native to the North America and is an important component of the original tall grass prairie. It is also an important ornamental and forage grass. Recently, it has been proposed as an ideal biomass producer for cellulosic ethanol production. Genetic transformation is an important tool for introducing important agronomic traits into plants, but an efficient and reproducible in vitro regeneration protocol is a prerequisite for successful genetic transformation. In this report, we used mature caryopses as explants and tested the effect of various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (1–5) and kinetin (KT) (0, 0.1, and 0.2) on embryogenic callus induction using LS basal medium. Caryopses cultured on media supplemented with 2,4-D alone generally outperformed those cultured on media supplemented with both 2,4-D and kinetin for embryogenic callus induction. The best treatment is LS basal medium supplemented with 3 mg l⁻¹ 2,4-D. LS basal medium supplemented with KT of 0, 0.5, 1, 2 or 5 mg l⁻¹ were tested for regeneration efficiency which was shown to increase as the KT concentration increased. The quality of the shoots produced on the medium containing KT at 5 mg l⁻¹, which produced the highest regeneration frequency appeared to be lower as leaves become vitrified. Shoots were moved to a rooting medium containing either 0 or 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA). Rooted plantlets were then transferred to soil-containing pots and were placed in a mist room for 1 week before they are transferred to a normal greenhouse where they all survived. The reported regeneration protocol is very efficient and highly reproducible in spite of the heterogeneous nature of the tested cultivar; thus it should be suitable for genetic transformation.     

Regeneration of different Cyclamen species via somatic embryogenesis from callus,suspension cultures and protoplasts

The present study is the first report of the establishment of embryogenic callus cultures from seedling tissue, the regeneration of plants via somatic embryogenesis and the development of a regeneration system from protoplast to plant, using three wild species of Cyclamen, Cyclamen graecum Link, Cyclamen mirabile Hildebrand, Cyclamen trochopteranthum Schwarz (syn. Cyclamen alpinum hort. Dammann ex Sprenger). The ability to form embryogenic callus and to regenerate via somatic embryogenesis was strongly genotype-dependent for each species. From 0.5 g callus, up to 1461 somatic embryos were formed in the case of C. mirabile. Culture media with different concentrations of plant growth regulators, CaCl₂ and activated charcoal significantly influenced embryo formation in this species. Up to 1.4 × 10⁶ protoplasts were isolated from 1 g of C. graecum cell suspension. Diverse growth responses of the protoplasts in two embedding agents, agarose and alginate, were observed for the different Cyclamen species. These specific growth characteristics could be used as a selection marker for future fusion experiments. From both protoplast culture systems, somatic embryos were regenerated, grown to plantlets and acclimatised to greenhouse conditions.     

Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA₃ (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions.