Effects of auxin concentration on induction and growth of embryogenic callus from young inflorescence explants of Old World bluestem (Bothriochloa spp.) and bermuda (Cynodon spp.) grasses

Objectives of this research were to test the effects of plant genotypes and auxin 2,4-D (2,4-dichlorophenoxyacetic acid) medium concentrations on embryogenic (E) callus production of two grass species. Two Old World bluestem,Bothriochloa ischaemum, accessions (A-8793 and A-8911c) and three bermudagrass,Cynodon dactylon (L.) Pers., accessions (A-10978b, A12164, and Brazos) supplied the explant material. Immature inflorescences 9 mm in length were placed on modified Murashige-Skoog (MS) agar medium containing 0, 1, 3, or 5 mg L^-1 of 2,4-D. Explants of all genotypes produced callus by the end of a 4-week dark incubation period at 25°C. When subcultured onto fresh media and maintained at 25°C with a 16 hr photoperiod, calli became embryogenic within 8 weeks of inoculation. Three mg L^-1 of 2,4-D in the media maximized E callus production in both bluestem genotypes and in A-10978b and A-12164 bermudagrass genotypes. Maximum E callus production from Brazos bermudagrass resulted from the 1 mg L^-1 treatment. Somatic embryos developed after subculture under light. Embryos showed scutellum-like structures and coleoptile-coleorhiza bipolar organization. Plantlets were regenerated from all genotypes except Brazos, whose embryoids failed to germinate. All callus from Brazos eventually senesced. Light and scanning electron microscopy confirmed regeneration through somatic embryogenesis.     

Plant regeneration from tissue cultures initiated from immature inflorescences of a grass,Echinochloa colonum (L.) link

Organised structures develop on a white and compact callus initiated from small segments of immature inflorescences ofEchinochloa colonum cultured on Murashige and Skoog's (MS) medium supplemented with 5.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 10% coconut milk. These develop into plantlets upon subculture onto MS medium containing 0 or 0.2 mg/l 2,4-D. Twelve out of 17 plantlets regenerated grew well on transfer to soil and eleven plants produced seeds.     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid     

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     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

Comparison of developmental stages of inflorescence for high frequency plant regeneration in Triticum aestivum L. and T. durum Desf.

Summary Whole immature inflorescences at 4 different developmental stages (0.5, 1.0, 1.5, 2.0 cm in size) of different genotypes of Triticum aestivum and T. durum were cultured to see the morphogenetic responses on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l). Very young inflorescences 0.5 and 1.0cm long formed embryogenic callus from their entire surface while 1.5 and 2.0 cm long inflorescences formed embryogenic callus from the basal spikelets and rachis only. This embryogenic callus was maintained by regular subcultures on MS medium with 2,4-D (2.5 mg/l) for more than a year. Plantlets were regenerated by transferring the embryogenic callus on hormone-free MS medium. Inflorescences (0.5 and 1.0 cm long) responded best in forming callus as well as plantlets at a very high frequency. Variation in response was observed amongst the genotypes but the qualitative response of formation of embryogenic callus and later regeneration of plantlets was observed from all the genotypes. Immature young inflorescence explants could provide a suitable material for particle gun mediated genetic transformation in wheat.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962)     

In vitro regeneration of Acacia catechu Willd. from callus and mature nodal explants--an improved method

Callus was derived from cultured cotyledons on MS medium supplemented with 2,4-D (0.25 mg/l) and NAA (0.25 mg/l). Plantlets were regenerated from the callus and nodal explants on MS medium containing BAP (2.0 mg/l) and Kn (2.0 mg/l), and further multiplied on the same medium. Addition of adenine sulphate (25.0 mg/l), ascorbic acid (20.0 mg/l) and glutamine (150.0 mg/l) in the medium resulted in enhanced axillary branching. Multiple shoots formed after 6 weeks were separated and subcultured in the fresh medium of same composition. For rhizogenesis, microshoots of 2.0-2.5 cm length were dipped in sterilized IAA solution (10 mg/l) for 24 hr followed by transfer to half strength MS medium containing activated charcoal (0.02%) resulting in rooting (75%) within 8 weeks. The rooted plants were transferred to pots containing sterilized soil and sand mixture for hardening and 71% survival was recorded. Fifty true to type plantlets of A. catechu could be obtained within seven months of culture establishment.     

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2–0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA 3-indoleacetic acid - 2iPA N⁶-(²-isopentenyl) adenosine - BAP 6-benzylaminopurine - KT kinetin - CH casein hydrolysate     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997     

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     

In vitro propagation of Iris pallida

Plantlets were regenerated from callus of Iris pallida, an important perfume plant. Only the leaf base attached to the rhizome had the ability to generate yellow-colored callus on LS medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l KT in the dark. Yellow calli grew with partial differentiation into white tissue, probably embryogenic, during subculture on the same medium with a 16-h photoperiod. Only yellow-colored calli with the white tissue could differentiate into plantlets after transfer to kinetin- or gibberellin- supplemented LS medium. Regenerated plantlets which grew on the medium without growth regulators were transferred to the soil. After 2 years of cultivation in soil, the regenerated plants flowered and formed rhizomes. The components of the essential oil in the rhizome of regenerated plants were essentially the same as those in natural plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - NAA alpha-naphthaleneacetic acid - LS Linsmaier and Skoog (1965) medium     

Somatic embryogenesis and plant regeneration in an interspecific hybrid of Oryza

An embryogenic callus was obtained from immature panicle of an interspecific hybrid (Oryza sativa x O. latifolia) F₁. The medium consisted of HE salts supplemented with 2,4-D, NAA (each 2 mg/l), kinetin (3 mg/l), yeast extract (1360 mg/l) and casein hydrolyzate (300 mg/l). The callus was milk-white in colour compact and granulate in texture. Various developmental stage of embryoid, such as globular, heart-shape, scutellum-shape and mature embryoid were observed in an embryogenic callus. Plantlets were successfully regenerated from 1-month-old callus with more than 80% regenerational frequency in each subculture for 12 passages.     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2–3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10¹⁴ plantlets could be produced in a year from one expiant following the proposed protocol.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid