Multiple Shoot Regeneration from Young Shoots of Kenaf (Hibiscus cannabinus)

A method was developed to initiate multiple shoots from the young shoot of kenaf. Young shoots along with the cotyledons were excised from ten-day old aseptically germinated seeds and pre-cultured for two weeks in Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) or a combination of BA and kinetin. After two weeks in culture, elongated shoots were excised above the cotyledonary nodes and cultured on fresh medium of the same composition. Multiple shoots were initiated within eight weeks. The number of shoots varied among cultivars. The highest number of shoots (11/explant) occured in cultivar Tainung 2 (T2) cultured in MS medium supplemented with 8.8 μM BA. Concentrations of BA higher than 8.8 μM had a negative effect on the number of shoots. Furthermore, callus growth was initiated from which morphologically abnormal shoots were induced. Kinetin had a significant effect only on cultivar Everglades 41 (E41). Shoot elongation and rooting were obtained simultaneously in half strength MS basal medium with no plant growth regulators. About 98% of the rooted plants were grown to maturity under greenhouse conditions. This method was successful with all four genotypes tested. However, significant genotypic variations were observed among the genotypes.     

Efficient shoot and root formation from cotton shoot apices

Successful shoot and root induction were obtained from shoot apices of two cotton (Gossypium hirsutum L.) genotypes, Nazilli 84S and Çukurova 1518, which are widely planted in Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) medium supplemented with various plant growth regulators using seven-day-old shoot apices as explants. The shoot apex size was of 2–3 mm; it contained the meristem and unexpanded leaves. Shoot apices were placed on MS plus vitamins and combinations of various plant hormones. The best regeneration responses were obtained for cv. Nazilli 84S (98%) on MS + 0.1 mg/l kinetin (KIN) + 1 g/l polyvinylpyrrolidone (PVP) and for Çukurova 1518 (94%) on MS + 0.1 mg/l KIN + 2 mg/l NAA + 1 g/l PVP. Including germination, all regeneration and rooting processes lasted only 5 weeks. The shoot apices of both genotypes developed successfully without intervening callus formation, and no significant differences between cultivars were found. All regenerated plants of both genotypes were phenotypically normal and set seeds. This shoot meristem-based rapid regeneration method can also be used in the cases of biolistic and Agrobacterium-mediated transformation.     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid     

Tissue culture propagation of running buffalo clover (Trifolium stoloniferum Muhl. ex A. Eaton)

Rapid propagation of running buffalo clover (Trifolium stoloniferum) was achieved on Murashige & Skoog (MS) medium. Excellent shoot proliferation and shoot growth were obtained on medium containing 0.5 or 1 mg l^-1 BA. In vitro proliferated shoots were rooted on MS or half-strength MS medium containing 0 to 0.4 mg l^-1 IAA. Both the number of roots initiated and the length of the longest root were significantly higher on MS medium than on half-strength MS medium. Rooted plantlets were successfully transferred to soil.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

In vitro callus induction and regeneration studies in Withania somnifera

Callus cultures were initiated from axillary leaves, axillary shoots, hypocotyls, and root segments on Murashige and Skoog (MS) (1962) medium supplemented with 2,4-D (2 mg l⁻¹) and KN (0.2 mg l⁻¹). Shoots differentiated best from axillary shoot base callus on MS medium containing BA (2 mg l⁻¹). Regenerated shoots rooted best on MS medium containing IBA (2 mg l⁻¹) alone, and IBA (2 mg l⁻¹) with IAA (2 mg l⁻¹). Plantlets were transferred to pots containing sand and soil mixture, acclimatized in a culture room and afterwards transferred to the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

In vitro propagation ofHancornia speciosa,a tropical fruit tree

Summary Hancornia speciosa fruit is highly desired for the juice and ice cream industry in tropical regions. A rapid reduction in germination ability ofH. speciosa seeds has been a problem for its large-scale cultivation. This paper describes anin vitro technique that may lead to an alternative propagation method forH. speciosa. Shoot apices and nodal segments from aseptically germinated young embryos were cultivatedin vitro on. Murashige and Skoog (1962) medium supplemented with growth regulators. Shoot multiplication was maintained by sequential subculture of shoot tips and nodal segments. N⁶-benzyladenine was the most effective cytokinin for the induction of shoot growth. N⁶-furfurylamino-purine, at various concentrations, yielded multiplication rates sevenfold lower than the highest multiplication rate found with N⁶-benzyladenine. Increased root initiation rate and root elongation was observed with the presence of γ-(indole-3) butyric acid in the half-strength Murashige and Skoog culture medium, especially at 10μM. N⁶-benzyladenine strongly inhibited rooting, even in the presence of γ-(indole-3) butyric acid. Thein-vitro-raised rooted plantlets were acclimatized to greenhouse environment through progressive reduction in relative humidity and later transplanted to the field.     

Micropropagation ofHesperaloe parviflora

Summary We successfully micropropagatedHesperaloe parviflora from mature plants. Shoot cultures were directly initiated from mature plants using pedicel bud explants on a modified Murashige and Skoog medium containing Nitsch and Nitsch vitamins and 1 μM zeatin riboside. Axillary shoot multiplication from established cultures was most responsive to changing concentrations of N⁶-benzyladenine (BA) with the greatest production of 6 μM BA. Growing shoots on a medium supplemented with 6 μM BA for 6 wk and then transferring cultures to a 1 μM BA medium for 6 more wk increased the number of transferable shoots, but not significantly. However, our data predicts that the maximum number of transferable shoots produced from a single microshoot would occur on media with 5.4 μM zeatin riboside. Shoots rooted easilyin vitro orex vitro and rooted shoots were easily acclimatized. The methods described in this paper are being used to commercially micropropagateH. parviflora.     

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.     

Whole plant regeneration from the shoot apex ofArachis hypogaea L.

Summary Arachis hypogaea L. peanut, has been a difficult species to manipulate in tissue culture. Lack of a reliable and quick regeneration method for peanuts has proven to be one of the hindrances in the application of transformation protocols to the crop. A protocol to initiate shoot apex elongation and rooting of these shoots is described. This protocol was successful with two peanut cultivars. Shoot apices were isolated from germinated seedlings and placed on Murashige and Skoog salts containing N⁶-benzyladenine for shoot initiation. Once shoot elongation occurred, the explant was transferred to a rooting medium containing Murashige and Skoog salts and only one plant growth regulator, α-naphthalene acetic acid. In as few as 3 weeks, the explants began to root and could be transferred to soil. Forty-five percent of explants isolated from germinating peanut seeds would root on this medium. Elongation and rooting of the shoot apices were not hindered by the addition of an antibiotic to the medium, indicating that the regeneration method could be useful inAgrobacterium tume-faciens-mediated transformation protocols.     

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     

Micropropagation of Three Varieties of Caralluma adscendens via Nodal Explants

A procedure for in vitro propagation of pharmaceutically valuable varieties of Caralluma adscendens from nodal explant, is described. The highest shoot multiplication with 80% frequency was achieved within one month on Murashige and Skoog’s medium supplemented with 8.87 μM BA. Shoot multiplication occurred in subsequent subcultures in culture bottles on MS medium. Regenerated shoots were rooted on half strength MS medium supplemented with NAA (0.54 μM) in all the three varieties. The rooted plants were hardened for establishment in soil.     

Micropropagation of mature siberian elm in two steps

A simple and reliable two-step micropropagation system was developed for mature Siberian elm (Ulmus pumila L.). Shoot-tip cultures were initiated and multiplied on a modified Murashige and Skoog medium supplemented with 1 M benzyladenine, B5 vitamins, solidified with 0.22% Gelrite, and subcultured weekly. Proliferated shoots 2–3 cm in length rooted successfully in sterile and non-sterile soil mix at 100% efficiency. Healthy plants were acclimatized to the ambient environment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IBA indole-3-butyric acid - MS Murashige & Skoog medium (1962)     

In vitro shoot proliferation and production of sets from garlic and shallot

A procedure is described to regenerate shoots and bulbs in vitro with high frequency from shoot tips of garlic and shallot plants using benzyladenine or thidiazuron. Regenerated shoots were induced to form bulbs in Murashige and Skoog medium (1962) containing 5 g l^-1 activated charcoal and 120 g l^-1 sucrose under a long-day photoperiod. Bulbs formed in vitro were transferred to soil without acclimatization and produced viable plants. This method could be useful to produce low-cost bulbs, which are easy to handle and store until needed.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog's (1962) medium - NAA naphthaleneacetic acid - TDZ thidiazuron     

In vitro regeneration from bulb explants of Indian squill,Urginea indica Kunth

Callus cultures were established from bulb explants of diploid Urginea indica Kunth (Indian squill) on a modified basal medium of Murashige and Skoog (1962) supplemented with either 2 mg/l^-1 2,4-D+15% (v/v) CM or 4 mg/l^-1 2,4-D+2 mg/l^-1 NAA+2 mg/l^-1 KN+1 g/l^-1 YE. Shoot primordia developed after 2–3 subcultures in that medium. Increased growth of shoot primordia was obtained in media containing less auxins and vitamins. Rooted bulbous plantlets obtained were maintained in MS medium with 0.5% sucrose.Adventitious shoots were induced from adaxial epidermal cells of outer scales of regenerated bulbs used as secondary expiants in presence of 1 mg/l^-1 of 2,4-D with slightly higher concentration of the three vitamins of MS medium. From each scale leaf, approximately 400 bulblets were produced in 18 weeks in liquid culture. 90% of the plants transferred to potted soil have survived.     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. I. Protoplast culture and organogenesis

High yields of viable protoplasts were obtained from callus cultures derived from shoot apices of Vigna aconitifolia (JACQ) Marechal. The protoplasts divided and formed cell clusters on modified MS medium. The protoplast-derived callus formed multiple shoot buds on MS and B5 basal media without supplements, on MS medium containing supplements and on B5 medium containing charcoal (0.25%). Shoot formation occurred.Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - CM coconut milk - NAA 1-napthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - CB Cellulase from Penicillium funiculosum - C Cellulase from Trichoderma reesei - CRIO Cellulase R10 - MR10 Macerozyme R10 - PDS Potassium dextran sulphate NCL communication No. 3376     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962