In vitro regeneration of Ruscus hypophyllum L. plants

Callus cultures were established from stem explants of Ruscus hypophyllum on a modified basal medium of Murashige and Skoog (1962) supplemented with 1 mg l^-1 2,4-D+0.1 mg l^-1 BAP. The optimal 2,4-D concentration for promoting shoot bud formation and growth was 0.05 mg l^-1 along with 0.5 mg l^-1 BAP. Sixty percent of rootless shoots produced flowers on the regenerating medium. Rooting was induced when shoots were transferred to half strength MS inorganic salts supplemented with 2 mg l^-1 IBA. Eighty percent of plants transferred to soil have survived.     

Plant regeneration from shoot apical meristems of Melia azedarach L. (Meliaceae)

A protocol was developed for plant regeneration of Melia azedarach L. by in vitro culture of apical meristem (0.5 mm in length). The influence of six clones was investigated. The culture procedure comprised two sequential steps: 1) Induction of shoots by in vitro culture of axillary buds from adult trees (10–15 years old) by culture on Murashige and Skoog (1962) medium (MS) supplemented with 0.5 mg·dm⁻³ BAP (6-benzylaminopurine), 0.1 mg·dm⁻³ IBA (indolebutyric acid), and 0.1 mg·dm⁻³ GA₃ (gibberellic acid). The Multiplication of the regenerated shoots was achieved in MS + 0.5 mg·dm⁻³ BAP + 0.1 mg·dm⁻³ GA₃. 2) In vitro culture of the apical meristems from the regenerated shoots in MS medium (0.7 %) supplemented with various combinations of BAP and IBA. Maximum shoot proliferation was obtained on MS medium supplemented with 0.5 mg·dm⁻³ BAP and 0.1 mg·dm⁻³ IBA. Regenerated shoots were rooted on MS + 3.5 mg·dm⁻³ IBA (4 days) followed by subculture on MS lacking growth regulators (30 days). Complete plants were transferred to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Chimonanthus praecox</Emphasis> (L.), a winter flowering ornamental shrub

A procedure for the micropropagation of Chimonanthus praecox (L) Link, wintersweet, has been developed using buds from adult trees excised in spring. Shoot cultures established on Murashige and Skoog (1962) medium supplemented with 0.5 mg l⁻¹ 6-benzyladenine (BAP) and 0.1 mg l⁻¹ indole-3-butyric acid (IBA) were difficult to maintain in vitro through extended periods of time due to browning of the medium, shoot and leaf necrosis, and hyperhydricity. A treatment combining the use of 0.1% w/v activated charcoal and addition of a double phase agar-solidified/liquid medium improved propagation, enabling a successful in vitro propagation scheme to be developed. Optimal shoot multiplication occurred on medium containing 0.5 mg l⁻¹ BAP, and rooting on medium with 2.0 mg l⁻¹ IBA for 7 d, followed by transfer to hormone-free medium. Rooted plantlets were easily acclimated in a glasshouse and replanted and cultured outdoors.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kao's liquid medium containing 7.2% glucose, 2,4-d (1 mg 1^-1), NAA (0.1 mg 1^-1) and zeatin riboside (0.5 mg 1^-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1^-1) and BAP (1 mg 1^-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1^-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1^-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - 2 IP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - Kn kinetin     

An efficient protoplast-to-plant system for the hybrid ornamental shrub,Weigela ×florida cv. Bristol Ruby (Caprifoliaceae)

Strategies were developed for the successful isolation of large numbers of highly viable protoplasts from the leaves, stems and roots of axenic plants of the hybrid ornamental shrubWeigela ×florida cv Bristol Ruby. Protoplasts, of all sources, were cultured on different media, leading to the establishment of sustained divisions, and coupled with the production of multi-celled (>50 cells) colonies. However, those colonies derived from mesophyll protoplasts only were capable of a further proliferation to the callus stage. Upon transfer to a regeneration medium consisting of MS salts and organics plus a range of concentrations of NAA and BAP, such calli underwent caulogenesis, with optimum responses for a medium with 1.0 mg l⁻¹ NAA and 1.0 mg l⁻¹ BAP. The protoplast-derived shoots thus obtained were multiplied on MS medium with 0.1 mg l⁻¹ IBA, 0.5 mg l⁻¹ BAP and 0.1 mg l⁻¹ GA₃. Individual shoots were subsequently rooted on a half-strength MS medium plus 3.0 mg l⁻¹ IBA, and complete protoplast-derived plants were finally transferred to the glasshouse for acclimatization.     

菜心组织培养技术初探

为建立菜心(Brassica campestris ssp.chinensis var.utilis)的快繁技术体系,以花药和子叶-子叶柄为外植体进行组织培养研究。结果表明,花药培养以选取未开放的花蕾为宜,且花柱略高于花瓣,此时小孢子多数处于单核靠边期。菜心花粉的萌发率不高,且秋冬季的花粉比夏季的萌发率高。菜心花药愈伤组织诱导培养基为:MS+1.0 mg L–1 KT+1.0 mg L–1 2,4-D+3%糖+6 g L–1琼脂+8%椰乳,不定芽诱导培养基为:MS+2.0 mg L–1 6-BA+0.5 mg L–1 NAA+1.0 g L–1活性炭+2%糖+6 g L–1琼脂或MS+2.0 mg L–1 ZT+0.5 mg L–1 IAA+0.5 g L–1 AgNO3+1.0 g L–1活性炭+2%糖+6 g L–1琼脂。花药培养的不定芽诱导率为36.7%,不定芽培养出现褐化现象,不能形成再生植株;而以子叶-子叶柄为外植体培养获得的植株再生率可达80%。     

Factors affecting in vitro clonal propagation of Prosopis cineraria

Genotype, age of tree, nature of explant and size (length and diameter), season of explant collection, explant position on medium, plant growth regulators and certain additives (ascorbic and citric acids, adenine sulphate, L-arginine, glutamine and ammonium citrate), incubation conditions, and subculturing period greatly influenced the in vitro clonal propagation of P. cineraria. The maximum number of 10–12 shoots were induced from the nodal shoot segment from pruned thorny adult trees on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole- 3-acetic acid (IAA)+2.5 mgl^-1 benzylaminopurine (BAP)+additives. Higher temperature (31+-2°C) and mixed (fluorescent and incandescent) light of 50 mol m^-2 s^-1 photon flux density for 12 h per day photoperiod favoured shoot induction and subsequent growth. Explants from thornless trees produced 6–8 shoots per explant on MS medium containing 0.1 mgl^-1 IAA+5.0 mgl^-1 BAP + additives. Nodal shoot segments obtained from root and stump sprouts produced multiple shoots. Root segments differentiated into multiple shoots on MS medium containing 0.5 mgl^-1 indolebutyric acid (IBA)+2.5 mgl^-1 BAP.Differentiated shoots multiplied best on MS medium containing 0.1 mgl^-1 naphthalene acetic acid (NAA)+1.0 mgl^-1 BAP + additives. To yield multiple shoots the original explant was transferred 6 times on fresh medium after harvesting the differentiated shoots. Shoots were rooted by pulsing with 100 mgl^-1 IBA for 4 h and then culturing on hormone-free half strength MS medium. Initial dark incubation for 5 days at high temperature (33±2°C) was found essential for root induction from shoots which was 63% within two weeks. The rooted plantlets contained a consistent number of chromosomes (2n=28). It is suggested that the protocol developed could be useful for cloning of mature and tested trees of P. cineraria.     

Organogenesis and embryogenesis from callus cultures of Azadirachta excelsa

ABSTRACT

Callus production from leaf explants of Azadirachta excelsa (Jack) Jacobs was favoured by Murashige and Skoog medium supplemented with 4 mg l^-1 indole-3-butyric acid (IBA) and 1 mg l^-1 6-benzyladenine (BAP). Increasing the concentration of BAP to 2 mg l^-1 induced shoot regeneration. Adding polyvinylpyrrolidone (PVP) to the medium resulted in a significantly increased number of shoots. Transfer onto medium containing 0.5 mg l^-1 BAP, 0.4 mg l^-1 gibberellic acid (GA₃) and 2% sucrose stimulated elongation of the internodes; subsequent transfer onto medium containing 1 mg l^-1 IBA induced root formation. The histological analysis demonstrated that organogenesis and embryogenesis occurred in the same callus. However, shoots originated inside the callus mass, whereas the embryos originated on the surface. Given that the embryos did not develop beyond the globular or heart-shaped stage, we concluded that the plants regenerated from callus were derived only from organogenesis.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

速生白榆的组织培养与快速繁殖

该文以速生白榆半木质化枝条为外植体,使用75%的酒精和0.1%HgCl_2消毒处理,外植体经过启动培养后,在增殖培养基中进行丛生芽诱导,将丛生芽切成单株进行生根诱导,最终建立起成熟的速生白榆组培快繁体系。结果表明:外植体最佳消毒处理组合为75%的酒精处理50 s+0.1%HgCl_2处理8 min,外植体污染率为17.3%,成活率为78%;将消毒处理过的外植体接种到启动培养基中,培养25 d,最终筛选出最适白榆外植体启动的培养基为MS+1.0 mg·L~(-1)6-BA+0.1 mg·L~(-1)IBA+30 g·L~(-1)蔗糖+6.5 g·L~(-1)琼脂,启动率高达87.5%;将经过启动培养后的外植体腋芽切下,接种到增殖培养基中进行丛生芽诱导,最终筛选出最佳增殖培养基为MS+0.5 mg·L~(-1)6-BA+0.1 mg·L~(-1)KT+0.1 mg·L~(-1)IBA+30 g·L~(-1)蔗糖+6.5 g·L~(-1)琼脂,继代周期25 d,增殖系数达6.2;将丛生芽切成单株,接种到生根诱导培养基中,筛选出最佳生根培养基为1/2 MS+0.1 mg·L~(-1)IBA+0.1 mg·L~(-1)IAA+30 g·L~(-1)蔗糖+6.5 g·L~(-1)琼脂,生根诱导30 d,生根率达97%。将生根苗在室外炼苗后,移栽到珍珠岩∶蛭石∶泥炭土体积比为1∶1∶1的混合基质中,成活率在90%以上。较高的增殖系数、生根率和移栽成活率可以降低生产成本,进而实现工厂化育苗。     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Clonal propagation ofBougainvillea glabra ‘Magnifica’ through shoot apex culture

Shoot apices ofBougainvillea glabra ‘Magnifica’ were induced to regenerate an average of ten shoots from their bases in response to BAP (0.5 mg/l) plus IAA (1.5 mg/l). All the isolated shoots from such cultures were rooted in a medium containing 0.1 mg/l each of IBA and 2,4,5-T and lacking BAP. Plantlets were then successfully grown in potted soil where they flowered normally. NBRI, research publication no. 32/80.     

红花草莓的组织培养与快繁技术研究

以红花草莓叶片为外植体,通过筛选诱导愈伤组织、不定芽及壮苗、生根的培养基,建立一套实用且易推广的红花草莓组培快繁技术体系。结果表明:在愈伤组织的诱导过程中TDZ的诱导效果优于6-BA,TDZ与NAA配合使用效果优于与IBA的组合。6-BA浓度为0.5 mg·L~(-1)时不定芽诱导率高达86.6%。低浓度的6-BA和8 g·L~(-1)的琼脂更有利于壮苗培养,NAA比IBA更有利诱导生根。综上述,最适红花草莓愈伤组织的诱导培养基为MS+1.0 mg·L~(-1)TDZ+0.5 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+7 g·L~(-1)琼脂;最适不定芽分化的培养基为MS+0.5 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+7 g·L~(-1)琼脂;最适壮苗培养基为MS+0.1 mg·L~(-1)6-BA+0.1 mg·L~(-1)NAA+30 g·L~(-1)蔗糖+8g·L~(-1)琼脂;最适生根培养基为MS+0.5mg·L~(-1)NAA+30 g·L~(-1)蔗糖+8 g·L~(-1)琼脂。试管苗移栽生长20 d后,成活率高达93%,且后期草莓苗生长壮健。此体系的建立为优质红花草莓种苗大规模生产提供了科学依据和技术支持。     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.     

In vitro propagation of the nitrogen-fixing tree-legume Acacia mangium Willd.

In vitro propagation was initiated from 2-week-old and 7-month-old explants of Acacia mangium. Juvenile explants (2 week-old) of 5- to 10-mm lengths composed of two leaves were cultured on Murashige and Skoog (MS) medium containing 1.0 or 2.0 mg L^-1 6-benzyladenine (BAP). After 6 weeks, most explants had formed a large cluster of 14–18 axillary shoots produced by prolific branching of the primary axillary shoot after elongation. The maximum multiplication rate (40) was obtained in the first subculture; the rate decreased to 10–20 in the second one. The mean length of shoots was not significantly affected by BAP concentrations during the subsequent cultures. Rooting ability of juvenile explants was greatly affected by BAP concentrations used in the multiplication medium. When both types of explants were multiplied on a MS medium containing 1.0 mg L^-1 BAP and transferred to a half-strength MS medium containing 0.05 mg L^-1 IBA, only 10% of the juvenile explants were rooted versus 70% of the 7-month-old explants. Rooted plants transferred onto artificial substrate were all nodulated, when inoculated with a specific Bradyrhizobium sp. strain.     

Plant regeneration from leaf-derived callus cultures of the tropical pasture legume Stylosanthes scabra Vog.

Callus cultures of 5 genotypes of S. scabra Vog. were optimally established from leaf tissue on Murashige and Skoog (MS) basal medium supplemented with 0.5–2.0 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 1.0–2.0 mg l^-1 6-benzylaminopurine (BAP). On media containing 2, 4-D only, calli were soft, and rhizogenesis occurred on calli of 4 genotypes. Calli formed on media containing BAP only, but not with kinetin only. In the presence of 2, 4-D, BAP inhibited rhizogenesis and promoted better callus growth than kinetin. High frequency shoot induction was achieved for 3 genotypes on MS +2.0 mg l^-1 BAP. Roots formed on shoots when sub-cultured on half-strenght MS without growth regulators. The form of cytokinin used in the callus induction media appeared to affect subsequent shoot organogenesis. Genotypic differences were observed for shoot organogenesis. There was some morphological variation evident among regenerants.     

Rapid clonal multiplication of Angelonia salicariefolia through tissue culture

Nodal explants of Angelonia salicariefolia were cultured on MS basal medium and induced to form shoots when supplemented with either Kn (1.0 mg/l) or BAP (1.0 mg/l). Rooted shoots were formed in response to Kn+NAA (1.0 mg/l+0.5 mg/l). Subcultures of the shoots of these cultures grown on the same medium supplemented with 0.5 mg/l of NAA, IAA or IBA, together with lowered concentrations of inorganic salts, induced root formation in 20–30 days. Up to 18×10³ plants were produced from one plant in less than a month. Successful transfer of regenerants into soil has been accomplished.