Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

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.     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

Plant regeneration from excised hypocotyl explants of <Emphasis Type="Italic">Platanus acerifolia</Emphasis> willd

Summary A method of plant regeneration from hypocotyl segments of Platanus acerifolia Willd, has been developed. Hypocotyl slices were cultured on Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins [6-benzyladenine (BA) or kinetin] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid, α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid] for adventitious shoot induetion. The highest regeneration frequency was obtained with MS medium containing 2.0 mg l⁻¹ (8.88 μM) BA and 0.5 mg l⁻¹ (2.46 μM) IBA. Adventitious buds and shoots were differentiated from hypocotyl-derived cellus or directly from the wounded sites within 4–8 wk. The regenerated shoots were elongated and proliferated efficiently on multiplication medium. Complete plantlets were transplanted to the soil and grew normally in the greenhouse after root formation on rooting medium for 4–6 wk.     

Effects of auxins and cytokinins on formation of Catharanthus roseus G. Don multiple shoots

The effects of different combinations of plant growth regulators and light intensity on the formation of multiple shoots of Catharanthus roseus (L.) were studied. By composing three dimension surfaces and their topo views from experimental data, it was clear that Murashige-Shoog (MS) medium supplemented with 7.0 mg l^-1 BA and 1.0 mg l^-1 NAA strongly stimulated the formation of shoots, whereas medium supplemented with 2,4-d suppressed the formation of shoots or caused shoot dedifferentiated. Light intensities of 550–700 Lux were found to be beneficial to the formation of shoots when MS medium was supplemented with 2 mg l^-1 6-BA and 0–1.0mg l^-1 NAA.Abbreviations BA-6 benzyladenine - NAA -naphthalenacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

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     

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     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

Optimizing plant regeneration from seed-derived callus cultures of Kentucky bluegrass. The effect of benzyladenine

The effect of benzyladenine (BA) on the production of shoot-forming callus from seeds of two Poa pratensis cultivars was studied. Addition of low concentrations (0.1–0.3 mg l^-1) of BA to Murashige & Skoog (MS) callus induction medium containing 1 or 2 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d) stimulated somatic embryogenesis and strongly increased the percentage of seeds producing shoot-forming callus in both cultivars.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

In vitro plant regeneration from leaf callus inPiper colubrinum Link

Callus-mediated shoot regeneration from leaf explants ofPhytophthora resistant pepper (Piper colubrinum Link.) is described. The effect of basal media composition and growth regulators onin vitro response of explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 mg l^–1 BA and 0.5 mg l^–1 NAA , as well as 1.0 mg l^–1 BA and 0.5 mg l^–1 2,4-D. The shoots were rooted in half-strength MS medium with or without IAA or IBA, and then were transferred to soil with 100% survival.     

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 axillary buds of a mature tree of Ficus religiosa

Dormant axillary buds of nodal explants collected from a mature (35-year-old) tree of Ficus religiosa L. sprouted on MS medium supplemented with 6-benzyladenine (BA, 5 mg l^–1) and indole-3-butyric acid (IBA, 0.2 mg l^–1 ) within 4 days. Multiple shoots were obtained when these explants were transferred to MS medium containing 1.5 mg l^–1 BA and 1.5 mg l^–1 adenine sulphate (ADS). These multiple shoots (1 cm) were allowed to elongate to a height of 4–5 cm by maintaining them on MS medium containing 2 mg l^–1 BA, 0.5 mg l^–1 1-naphthalene acetic acid (NAA), and activated charcoal (0.3% w/v). Nodal segments taken from these in vitro-proliferated shoots yielded multiple shoots when cultured on the multiple shoot-inducing MS medium mentioned above. Root induction in these shoots (4–5 cm in height) was achieved by transferring them onto MS medium supplemented with 2 mg l^–1 IBA and 0.1 mg l^–1 NAA for 1 week; upon transfer to half-strength MS basal medium these shoots exhibited root proliferation. These rooted plantlets were successfully established in soil after a short period of acclimatization. Received: 17 April 1997 / Revision received: 2 September 1997 / Accepted: 20 September 1997     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Adventitious bud formation in Alhagi graecorum

Various parts of seedlings and in vitro propagated shoots of Alhagi graecorum Boiss were cultured on different media with different 6-benzyladenine (BA) and kinetin (KIN) concentrations to compare their potential to regenerate shoots. Murashige and Skoog (MS) medium with 2.5 μM BA and hypocotyl gave the best results. Callus was obtained from stem segments on MS medium supplemented with 2.5 μM BA, 5 μM 1-naphthaleneacetic acid (NAA) and 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Shoot formation from callus occurred upon its transfer to MS medium supplemented with 2.5 μM BA. Mature explants which showed a relatively low potential for adventitious buds or callus formation, regenerated shoots abundantly using the tiny-mature-explant method. The regenerated shoots were rooted on half strength MS medium supplemented with 5 μM 3-indolebutyric acid (IBA). This revised version was published online in June 2006 with corrections to the Cover Date.