Reliable plantlet formation from embryos and seedling shoot tips of radiata pine

A method has been devised for the reliable production of plantlets from embryos and seedling shoot tips of Pinus radiata D.Don (radiata pine). Buds were induced on an agar or liquid Schenk and Hildebrandt (SH) medium containing 5.0 mg/l benzylaminopurine (BAP). Except for some abnormal buds, the buds grew into elongated shoots on an agar SH medium without cytokinin. The transfer of shoots from a SH medium to a Gresshoff and Doy (GD) medium was found to be an important pretreatment which increased the survival of the shoots when they were placed in a peat and pumice mix for root formation. Elongated shoots were induced to form roots under non-sterile conditions in a humid environment with occasional misting. An intervening 5-day treatment of shoots in an agar medium containing 2.0 mg/l indolebutyric acid (IBA) and 0.5 mg/l napthaleneacetic acid (NAA) significantly increased the percentage of shoots forming roots and the number of roots formed per shoot over control shoots placed directly in the peat:pumice mix. An enhanced level of CO₂ during root formation had no effect on the time of root formation or on the percentage of shoots forming roots. These results concerning the elongation, growth and rooting of adventitious shoots are now being applied to the development of very large numbers of plantlets starting from cotyledons from partially germinated seeds.     

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N ⁶ -benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA). This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue culture and plant regeneration of watermelon (Citrullus vulgaris Schrad. cv. Melitopolski)

Plants were regenerated from cotyledon and hypocotyl explants of watermelon (Citrullus vulgaris). The explants were cultured on a Murashige and Skoog's basal nutrient medium supplemented with auxin, cytokinin and auxin-cytokinin combinations. Green healthy nodular and compact callus was obtained in medium containing naphthalene acetic acid and benzylaminopurine. Shoot differentiation and root differentiation from the cotyledon and hypocotyl after callus formation in different media containing benzylaminopurine or naphthalene acetic acid, respectively. Shoot formation required benzylaminopurine. Kinetin proved ineffective in inducing shoot buds or shoots. Root differentiation occurred in a medium containing naphthalene acetic acid or indole acetic acid. There was a greater proliferation of roots on medium supplemented with naphthalene acetic acid. The regenerated shoots developed roots when transferred to medium containing naphthalene acetic acid and complete plantlets could be transferred to soil for further growth.Abbreviations BAP 6 Benzylaminopurine - NAA -Naphthalene acetic acid - MS Murashige and Skoog's medium - IAA Indole acetic acid - KN Kinetin     

Establishment of Plantlets and Evaluation of Differentiated Roots for Alkaloids in Rauwolfia serpentha

Different explants of Rauwolfia serpentina were tested for their capacity and root differentiation. MS sedium containing 2.0 mg I^-1 BAP or 1.5 mg I^-1 BAP with 0.5 mg I-* NAA gave the best shoot proliferation from shoot apices (84.12%). Multiple shoots subcultured on the same media gave higher number of shoots (6–9). Callus formed at the cut ends of explants produced shoots when subcultured on the above media. Rooting was achieved after transfer of shoots either to hormone free or half strength or full strength MS medium with different concentrations of IAA (0.5,1.0 mg I^-1 and IBA (0.5,1.0 mg I^-1).The complete regenerated plantlets have been successfully transferred to earthen pots in green house. Maximum root differentiation from leaves, stem and nodal cuttings derived calli was obtained on MS medium supplemented with NAA (2.0 mg I^-1) and BAP (1.5 mg I^-1). Identification of reserpine and other alkaloids from differentiated roots holds an interesting alternative for controlled production of alkaloids from this endangered, overexploited medicinal plant species.     

Influence of cytokinins, auxins and polyamines on in vitro mass multiplication of cotton (Gossypium hirsutum L. cv. SVPR2)

In the present investigation, the influence of different forms of cytokinins, auxins and polyamines were tested for mass multiplication and regeneration of cotton. Initially, for the identification of effective concentration for multiple shoot induction, various concentrations of BAP, Kin and 2iP along with IAA and NAA were tested. Among tested concentrations, media fortified with MS salts; B5 vitamins; 30 g/l, glucose; 2.0 mg/l, 2iP; 2.0 mg/l, IAA and 0.7 % agar showed best response for multiplication of shoot tip explants (20 shoots per shoot tip explants). In nodal explants, maximum of 18.6 shoots were obtained in the media fortified with MS salts, B5 vitamins, 30 g/l, glucose, 2.0 mg/l, 2iP, 1.0 mg/l, NAA and 0.7 % agar. Effect of different concentrations of polyamines like spermidine and putrescine were also tested along with the above said multiplication media. Among the various treatments, 20 mg/l of putrescine showed best response and the multiple of shoots were increased to 26.5 shoots per shoot tip explants and 24.5 shoots per nodal explants. Elongation of shoots was achieved on multiple shoot induction medium. Significant number of roots were initiated in the medium supplemented with MS salts, vitamin B5 and IBA (2.0 mg/l). The frequency of root induction was increased by addition of, PVP (10 mg/l) along with root induction medium and after 2 weeks, the roots reached the maximum length of 22 cm. Further, these plantlets were hardened by using sand, soil and vermiculate in 1:1:1 ratio. The hardened plants were transferred to the environmental growth chamber for proper acclimatization. The hardened plants were then transferred to field for boll yielding and they exhibited 100% survival.     

In vitro morphogenesis from cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc], an under-utilised grain legume

The morphogenetic competence of Bambara groundnut was assessed for different landraces, explant sources and media compositions. With cotyledon explants, the best callusing occurred on a medium containing 3 mg/l BAP + 0.5 mg/l NAA, while roots were produced with 3–5 mg/l BAP + 0.5 mg/l NAA. Shoots regenerated (∼6%) from cotyledons on media with BAP alone (3–5 mg/l) or combined with 0.01–0.1 mg/l NAA. Flowers were regenerated on 5 mg/l BAP + 0.5 mg/l NAA, without any intervening callus phase. With epicotyls, the highest callusing was on 3 mg/l BAP + 0.5 mg/l NAA, and shoots regenerated (15–20%) on 3 mg/l BAP alone or with NAA at concentrations that depended on the landrace studied. Regenerated shoots rooted on hormone-free medium, and plants transferred to the greenhouse were all morphologically normal and fertile. Flow cytometry showed that most regenerants were diploid and in addition permitted to distinguish between landraces according to their relative nuclear DNA content. This is the first report on de novo regeneration in vitro of Bambara groundnut, an important yet neglected legume crop.     

An efficient in vitro regeneration system of fieldpea (Pisum sativum L.) via. shoot organogenesis

In-vitro regeneration in fieldpea was achieved from immature embryonic axes and cotyledonary node explants of six genotypes on modified MS media supplemented with different concentration of plant growth regulators, 6-Benzylamino purine (BAP) and Naphthalene acetic acid (NAA). The best regeneration response, leading to multiple shoot formation efficiency (22.34 shoots/explant) was observed in the medium supplemented with 1.0 mg/L BAP and 0.2 mg/L NAA and best frequency (67.55?±?4.74) was achieved on medium containing 2.0 mg/L BAP and 0.4 mg/L NAA. The shoots were subcultured on a medium supplemented with a combination of 1.0 mg/L GA₃, 2.0 mg/L BAP and 0.4 mg/L NAA, which resulted in elongation of 85 % of shoots. Rooting attempted from the elongated shoots, on half strength MS medium and supplemented with three different auxins IBA, IAA and NAA separately, exhibited similar results. Alternatively, micro-grafting of in vitro regenerated shoots onto pre-germinated root stocks raised in green house facility was attempted with high success rate (75 %). The grafted plants could be successfully hardened, fertigated with Hoagland solution and distilled water in a ratio of (1:10) for acclimatization and further development. All the genotypes tested, produced multiple shoots that could be established to mature fertile plant, hence, the medium combinations used were found to be genotype neutral.     

Efficient in vitro regeneration of fireweed,a medicinal plant

Epilobium angustifolium L. (fireweed) is a medicinal plant that has been used to treat diarrhea, mucous colitis, irritable-bowel syndrome, skin problems, prostate problems, menstrual disorders, asthma, whooping cough, and hiccups. A highly efficient and rapid regeneration system via multiple shoot formation was developed for fireweed. Explants (leaf, petiole, root, and stem segments) excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. Explant browning, a major problem for regeneration, was overcome by adding 100 mg/l ascorbic acid to all prepared media containing growth regulator combinations. Root explants formed more shoots than other explants. Best shoot proliferation was obtained from root explants cultured on media with 0.1 mg/l BA and 0.5 mg/l IAA. Regenerated shoots were transferred to rooting media containing different concentrations of IAA, IBA, NAA or 2,4-D. Most shoots developed roots on medium with 0.5 mg/l IAA. Rooted explants were transferred to vermiculate in Magenta containers for acclimatization and after 3 weeks they were planted in to plastic pots containing potting soil and maintained in the plant growth room.     

毛白杨非转化组培根及Ri质粒转化植株的根切段在培养中分化的比较(简报)

毛白杨(Populus tomentosa)是我国特有的树种,适应性强,枝叶茂盛,防护性能好,且材质轻软,纹理细致。既是重要的速生用材树种,又是防护林和行道绿化的重要树种,它也是造纸、火柴、纤维工业的重要原料。虽然毛白杨能通过扦插等技术进行繁殖,但毛白杨扦插时生根比较困难。通过导入外源的与激素合成有关的基因来调节再生植株在组织培养中的分化在草本植物中已有不少报道,见许智宏等。本研究试图通过比较正常植株与发根农杆菌Ri质粒转化的再生植株的根切段在培养中分化的差异,以期能建立一种有效的毛白杨的遗传转化系统。     

Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.)

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.     

Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard

Rapid shoot proliferation was established by adventitious shoot formation on internodal segments. Cross sections of the shoot initiation area were observed microscopically and adventitious shoots were studied under the scanning electron microscope. Shoots were directly formed on the epidermis of internodal segments in vitro without callusing, but not on that of nodal segments with axillary buds. The use of media containing 0.01 – 0.1 mg/l 6-benzyladenine or 0.1 mg /l kinetin and culture under 16 h light increased the number of shoots per segment. The shoots thus obtained were rooted on phytohormone-free Woody Plant or Gamborg B5 solid medium, and were then transferred to soil. When potted, these grew well in a greenhouse. The emetic alkaloid content of adventitious shoots and regenerated plants was determined by HPLC. In vitro shoots cultured in Woody Plant liquid medium supplemented with 0.01 – 0.1 mg/l 6-benzyladenine contained 0.04 – 0.07 % dry wt. emetine and 0.4 – 0.5 % dry wt. cephaeline. One-year old regenerated plants cultivated in a greenhouse demonstrated the same alkaloid content (roots contained 0.82 % dry wt. emetine and 2.16 % dry wt. cephaeline) as the parental plant.Abbreviations MS Murashige — Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - B5 Gamborg B5 (Gamborg et al. 1968)] - WP woody plant (Lloyd and McCown 1980) - RC root culture (Thomas and Davey 1982) - HF phytohormone free - BA 6-benzyladenine - Kin kinetin - SEM scanning electron microscopy - RDF rotating drum fermenter     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)

Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B₅) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and 0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter) before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of nodulelike structures on basal medium (B₅) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios.     

Micropropagation of an endangered plant species: Coronopus navasii (Brassicaceae)

Plantlets of Coronopus navasii, an endangered species from SE Spain, were successfully regenerated from shoot and root segments excised from young seedlings. Initiation of multiple buds and development of leaves were obtained in MS modified medium plus l mg/l BAP and 0.1 mg/l NAA. Rooting was achieved by transfer of the isolated shoots to fresh MS medium without plant growth regulators. Plant survival of 47% was obtained six weeks after removal from in vitro culture conditions.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - MS medium see Murashige and Skoog 1962     

Comparative study of different cytokinins in the induction of morphogenesis in lentil (Lens culinaris Medik)

Summary This study presents the first comparative analysis of the effect of four different cytokinins, applied to different lentil (Lens culinaris Medik.) explants in four different concentrations. with regard to the regeneration of shoots and roots of the pulse crop. The variables explant, phytohormone and concentration were all found to be highly significant. Mature seed explants showed the highest shoot regeneration over all the phytohormones and concentrations tested. Thidiazuron (TDZ) and benzyladenine (BA) showed a higher number of regenerated shoots than kinetin (KIN) and zeatin (ZEA); an increase from 1.25 μM to 10 μM of any cytokinin in general doubled the number of shoots regenerated. The average length of regenerated shoots was found to be inversely proportional to the number of shoots regenerated. TDZ and BA were found to inhibit root development more than KIN and ZEA. The highest root regeneration frequency was obtained from shoots regenerated on media containing 1.25 μM ZEA. The study concludes that in order to obtain whole plants it is best to regenerate shoots on media containing the cytokinins KIN or ZEA at low concentrations, in order to be able subsequently to regenerate roots.     

Highly efficient shoot regeneration of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) using a two-stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD

A two-stage culture procedure has been developed for highly efficient shoot regeneration from leaf and internode explants of Bacopa monnieri. Adventitious shoot buds were obtained on the shoot induction medium containing Murashige and Skoog’s (MS) basal salt supplemented with 1.5 mg/l thidiazuron and 0.5 mg/l naphthalene acetic acid; these shoot buds were subcultured on the multiplication (second) medium amended with BAP (benzyl amino purine). Multiplication medium containing 0.5 mg/l BAP produced more shoots (135) and longer shoots (7.8 cm) with more nodes (6). Best response of root induction with more number of roots (16.5) and longer roots (8.7 cm) was observed in half strength MS basal medium supplemented with 1.0 mg/l IBA (indole-3-butyric acid) and 0.5 mg/l phloroglucinol. In vitro obtained plants were transferred to the field after hardening with a 100% survival rate. Random amplified polymorphic DNA analysis was carried out using five random primers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic integrity of micropropagated plants.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

In Vitro Clonal Propagation Through Bud Culture of Hemidesmus indicus (L) R Br: An Important Medicinal Herb

The present study involves in vitro propagation of Hemidesmus indicus (L) R Br through bud multiplication and subsequent plant regeneration. The buds multiplied to produce numerous shoots at variable rates in presence of a-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) as well as NAA and kinetin. The best response in bud multiplication was obtained in Murashige and Skoog’s (MS) basal medium supplemented with 0.1 mg I^-1 NAA and 2.0 mg I^-1 BAP (7-8 shoots per explant) and the bud break time was only 4 days after inoculation. The multiplication rate was low when the buds were cultured in NAA and kinetin media and the shootlets regenerated were very thin, weak and elongated. The shoots regenerated were further cultured on MS and half strength MS basal media with variable levels of indole-3-butyric acid (IBA) for initiation of roots. Culture of shootlets for 34 weeks in one half strength of MS medium followed by culturing in the same medium with 1.5 mg 1-1 IBA induced highest production of roots (3-5 roots per shoot) within 2 weeks. Chromosome number stability with no detectable structural changes was observed in the regenerates. The rooted plants were successfully established in the soil with 85% survival rate.     

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     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.