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     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

High frequency production of haploid embryos in asparagus anther culture

Summary A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l ^–1 casein hydrolysate, 800 mg l^–1 glutamine, 2 mg l ^–1 NAA, 1 mg l ^–1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l ^–1 NAA, 0.1 mg l ^–1 kinetin and 0.65 mg l ^–1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l ^–1 GA₃. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962)     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

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     

Plant regeneration from cell suspension-derived protoplasts of Saintpaulia ionantha Wendl

Summary Friable calli were induced on leaf segments of Saintpaulia ionantha Wendl. on B5 medium containing 1 mg l^–1 2,4-D and 2 g l^–1 casein hydrolysate. Cell suspension cultures were readily established from these friable calli and protoplasts could be isolated from the cells with yields of 1–3×10⁷/g f. wt.. By culturing in 0.1 % gellan gum-solidified B5 medium supplemented with 1 mg l^–1 2,4-D and 0.1 M each of sucrose and mannitol at a density of 1×10⁵/ml, the protoplasts divided within 6 days and formed macro-colonies after 2 months of culture. Shoot regeneration from protoplast-derived calli was obtained by sequential treatment of the calli with plant growth regulators: initially with 1 mg l^–1 each of NAA and BA for 2 months followed by 0.01 mg l^–1 NAA and 5 mg l^–1 BA for 4 months. Regenerated plants were established after rooting of the shoots on half-strength MS medium, and successfully transferred to the greenhouse. The regenerated plants grew into flowering stage and showed the same phenotype as the parent plant.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - f. wt fresh weight - MES 2-(N-morpholino)-ethanesulfonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency     

Stimulation of somatic embryogenesis and plant regeneration from anther culture of Vitis vinifera cv. Cabernet-Sauvignon

Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera Cabernet-Sauvignon, a cultivar so far considered as recalcitrant to in vitro regeneration. Anthers enclosing microspores near the first pollen mitosis were found to be the most responsive. However, from a practical point of view anther length proved to be an easier criterium for determining the optimal physiological anther stage. Calli derived from the anther somatic tissues produced embryoids only when cultured on a medium supplemented with casein hydrolysate. Glutamine and adenine were found to stimulate this embryoid production. Evidence is presented that early removal of cotyledons increases the frequency of normal development of embryoids into plantlets.Abbreviations MS Murashige and Skoog medium (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtaleneacetic acid - BA 6-benzylaminopurine     

Organogenesis of anther-derived calluses in long-term cultures of Oenothera hookeri de Vries

Anthers of O. hookeri containing uninucleate microspores were cultured, in vitro, at 25°C (16 hours photoperiod) on solid MS medium. After 10–15 days, on media with 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid and 6-benzylaminopurine, anthers developed friable calluses. After unsuccessful treatments on embryogenic-and/or organogenic-induction media, calluses were placed on a hormone-free MS medium for 24 months with routine transfers every 3 weeks. After this period, the calluses developed buds and subsequently plants. Ro generation plants, were morphologically distinct.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxiacetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

Somatic embryogenesis from mature zygotic embryos of Distylium chinense (Fr.) Diels and assessment of genetic fidelity of regenerated plants by SRAP markers

The objective was to establish an efficient regeneration protocol for Distylium chinense based on somatic embryogenesis and evaluate the genetic stability of plants regenerated in vitro. To induce callus mature zygotic embryos were cultured on Murashige and Skoog’s (MS) medium that was supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and N⁶-benzyladenine (BA). After 20 days, the highest rate of callus formation (88.9 %) occurred on MS medium supplemented with 0.5 mg l^?1 2,4-D and 0.1 mg l^?1 BA. It was observed that light-yellow, compact, dry, nodular embryogenic calli had formed. These calli were then subcultured on fresh MS medium supplemented with 0.1 mg l^?1 BA and 0.5 mg l^?1 α-naphthaleneacetic acid (NAA) for proliferation for an additional 30 days. To induce somatic embryos and plant regeneration, the embryogenic callus was transferred to fresh MS medium that was supplemented with different concentrations of BA and NAA. After 30 days, 0.5 mg l^?1 BA in combination with 0.5 mg l^?1 NAA produced the best result in terms of somatic embryogenesis (%), shoot differentiation (%), number of shoots per callus and shoot length. Next, the plantlets were transferred to the field for 5 weeks and a 95 % survival rate was observed. The sequence-related amplified polymorphism markers confirmed genetic stability of plants regenerated in vitro. To our knowledge, this is the first report that describes a plant regeneration protocol for D. chinense via somatic embryogenesis to be used for germplasm conservation and commercial cultivation.     

Plant regeneration from callus protoplasts of the forage legume Astragalus adsurgens Pall.

A reproducible release of viable protoplasts was obtained from friable calli of Astragalus adsurgens. Protoplasts underwent sustained divisions and formed cell colonies when cultured in either liquid or agarose-solidified Kao and Michayluk (1975) protoplast medium (KM8P) supplemented with 1.5 mg/l 2,4-D, 0.5 mg/l BA and 0.5 M glucose. Compared to liquid culture, agarose bead culture improved division frequency effectively, the two culture systems showing a plating efficiency of 0.8±0.5% and 6.5±0.7%, respectively. Upon transfer to Murashige and Skoog (1962) medium (MS) with 1–2 mg/l BA, alone or in combination with NAA or 2,4-D at 0.1 mg/l, the protoplast-derived calli produced complete plantlets through somatic embryogenesis. The maximum percentage of calli producing somatic embryos (52.5± 2.2%) occurred on MS medium containing 0.1 mg/l NAA and 1 mg/l BA, whereas the maximum number of calli regenerating plantlets (64.7±6.2) was obtained on MS medium with 0.1 mg/l NAA and 2 mg/l BA. Received: 25 April 1997 / Revision received: August 1997 / Accepted: 2 September 1997     

Rhizome induction and plantlet regeneration of Cymbidium goeringii from flower bud cultures in vitro

Apical flower buds of Cymbidium goeringli Reichenbach fil. (ca 2 mm long) exeised from infloreseences (ca 5 cm long) were explanted on modified Murashige & Skoog medium (=MS medium) supplemented with N⁶-benzyladenine (BA) and -naphthaleneacetic acid (NAA). Within 107 days of culture, swelling growth, chlorophyll synthesis, and subsequent rhizome differentiation were observed. MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA was found to be optimal for initiating rhizome development and subsequent plantlet regeneration.Explants cultured on MS medium supplemented with 1 mg l^-1 NAA alone formed a mass of rhizome branches. Multiple shoots of rhizome branches were induced from apical segments when rhizomes were transferred to MS medium containing 0.1 mg l^-1 BA and 10 mg l^-1 NAA.Abbreviations NAA -naphthaleneacetic acid - BA N⁶-benzyladenine     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Morphogenetic pathway in petiole derived callus of Amorphophallus albus in vitro

Two different morphogenetic pathways, adventitious bud and corm-like structure (CLS), were observed on organogenic calli derived from the petioles of Amorphophallus albus in vitro. The organogenic calli was established via culture of petiole segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.0 mg l⁻¹ 6-benzyladenine (BA) and subculture of the petiole-derived calli on MS medium with 0.5 mg l⁻¹ NAA and 0.5 mg l⁻¹ BA. These organogenic calli were used to induce morphogenesis via culture on MS medium with various concentrations of NAA and BA. BA alone favoured adventitious bud differentiation (57.0 ± 8.3% at maximum) from the organogenic calli but inhibited CLS formation. In the presence of NAA and BA, both adventitious bud and CLS were observed in a same culture system. The maximum CLS formation (71.2 ± 9.3%) were found on MS medium with 0.5 mg l⁻¹ NAA and 2.0 mg l⁻¹ BA, associated with 26.7 ± 8.6% adventitious bud differentiation. A small part of the adventitious buds developed into normal shoots which needed rooting culture phase to form complete plants. About 80% survival rate was obtained with these plants after transplantation to soil. More than 90% of the CLSs produced complete plants with shoots and root systems, regardless of the rooting media tested. Transplantation of the CLS-derived plants to soil gave 100% survival rate. Histological observations revealed both the two morphogenetic events originated from the meristematic cells located in superficial layers of callus tissue.     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

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     

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

Summary A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l^–1 kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l^–1 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4 dichlorophenoxyacetic acid - BA 6-benzyladenine     

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