Root culturing ofFaidherbia=Acacia albida as a source of explants for shoot regeneration

Root culture ofFaidherbia=Acacia albida (Del.) A. Chev. can be maintained over several months by successively subculturing excised roots in modified Bonner and Devirian medium, which was found to be the best of the three media compositions tested. Adding auxins had an inhibiting effect on root growth characteristics. Mesoinositol at 0.05 mM slightly enhanced the overall elongation rate, and sucrose at 59 mM significantly increased root elongation. The effect of sucrose could not be replaced by glucose. Subcultured roots showed progressively less elongation in successive transfers. Shoots were regenerated in vitro from root segments beginning with the first passage on 1/5 strength MS medium.The large variability in the root elongation rate indicates that this technique provides an effective way to select clones with good potential for tap root growth.     

CONTROL OF IN VITRO SEPAL DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA (RANUNCULACEAE)

Excised floral buds of Aquilegia formosa Fisch. were grown on a coconut-milk medium containing the minerals and vitamins of Murashige and Skoog, sucrose, and kinetin. The plant growth regulators indoleacetic acid (IAA, 0.5 mg/liter) and gibberellic acid (GA, 2.0 mg/liter) were added singly and in combinations; both were omitted from the control medium. The addition of GA to the basal medium was required to support sepal development on flowers at all phases of development. The formation of stomatal complexes in the epidermis of the sepals occurred only in the presence of GA. Sepals grown in the presence of GA also contained trichoblasts and developing trichomes, while none were formed in the absence of GA. The role of IAA in the development of these idioblasts was not clear but it appeared to have no effect. The hormones GA and IAA had different effects on the growth of the sepals. In the presence of GA the sepals increase in length until comparable with sepals grown in vivo. However, the sepals remained small when GA was omitted from the medium. Upon closer examination of this effect, it was determined that there was a direct proportionality between an increase in cell number in the epidermis and an increase in sepal length. The role of the two hormones in increasing epidermal cell length in sepals was distinct and separate. Exogenous IAA had no effect upon cell division but was required for cell elongation, while GA was required for cell division and had no effect on cell elongation. The GA effect in promoting cell division in the sepals was substantiated by use of autoradiography. If the buds were grown on media with GA, twice as many epidermal cells along the central file incorporated significant amounts of tritiated thymidine. The cell cycle of the epidermal cells of the GA-treated sepals was ca. 8.7 hr in duration and ca. 13.0 hr if GA was deleted from the medium.     

Factors affecting the establishment and maintenance of ‘Titan’ red raspberry root organ cultures

Root organ cultures of red raspberry (Rubus ideaus cv. Titan) were established from two cm long terminal segments of adventitious roots induced in vitro on micropropagated shoot cultures. Growth of root organ cultures was almost entirely by initiation and elongation of lateral roots rather than by terminal elongation from the apical meristem of the original root explants. Indolebutyric acid (IBA) was required for lateral root initiation and elongation. The optimal IBA concentration for lateral root initiation decreased from 0.5 to 0.1 mg/L from the first to the fifth passage in culture. Two cm root explants initiated more lateral roots per unit length than four cm explants. Liquid Anderson's medium was superior to other basal nutrient and vitamin formulations tested. Root growth in liquid culture was stimulated by aeration. Root organ cultures also grew on media solidified with gelrite or agar. A rapid transfer technique was developed for subculturing these which involved cutting and transferring 1 cm discs of roots and underlying medium. Adventitious bud formation occurred spontaneously, but sporadically in liquid cultures, and was not influenced by cytokinin, auxin (spat) light, or chilling.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl^-1 benzylaminopurine and 0.17 mgl^-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl^-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.     

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     

Tissue cultures of Artemisia pallens: Organogenesis,terpenoid production

Germinated seedlings of Artemisia pallens gave three types of cultures on MS medium supplemented with different plant growth hormones. Medium containing BA+2,4-D stimulated unorganized callus; BA+IAA medium, semi-organized tissues interspersed with shoot buds; and BA+NAA+IAA medium, multiple shoot cultures. The in vitro shoots developed roots in medium devoid of growth hormones. TLC and GLC analysis of the tissue extracts showed that linalool was present in the cultured tissues, with maximum concentration in the unorganized tissue. Although the TLC profiles of the three culture extracts were similar, the extracts did not contain the major polar compounds of the plant. The plant extracts contained more polar compounds and gave the characteristic fragrance of davana.Abbreviations MS Murashige & Skoog's basal medium - BA benzyladenine - Kn kinetin - NAA naphthaleneacetic acid - IAA indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume     

AUXIN,CYTOKININ AND GROWTH OF EXCISED ROOTS OF BRYOPHYLLUM CALYCINUM

Excised roots 1 cm long of Bryophyllum calycinum required exogenous auxin (α-naphthaleneacetic acid) for growth in still culture in a medium of sucrose, mineral salts and vitamins. Little or no growth occurred with the addition of cytokinin (6-benzylaminopurine) alone to the basal medium. Best growth was obtained with combinations of 1 μg of auxin and 1 or 2 μg of cytokinin in 50 ml of medium. Larger amounts of the two phytohormones resulted in reduced growth.     

Abscisic acid and auxin accumulation in Catasetum fimbriatum roots growing in vitro with high sucrose and mannitol content

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.     

Enhanced bud and bulblet regeneration from bulbs of<Emphasis Type="Italic"> Nerine sarniensis</Emphasis> cultured in vitro

Nerine (Nerine sarniensis) cv. Salmon Supreme in vitro-grown bulblets, 7-9 mm in diameter, were cut in half longitudinally and used for adventitious bud initiation following dissection of the roots and two-thirds of the upper part of the bulblets. The terminal apex was injured with a hot, sterile microscope dissecting needle. The highest number of buds formed (seven to nine buds per halved bulblet) on a semi-solid Murashige and Skoog (MS) basal salts medium supplemented with 3% sucrose and either 1 microM 6-benzylaminopurine (BA) and 1 microM alpha-naphthaleneacetic acid (NAA) or 0.5 microM BA and 0.1 microM NAA. Bulblet halves were cultured in the dark for 11-13 weeks with one subculture after 6 weeks. Anatomical studies indicated that the initiation of adventitious buds on the abaxial side of the inner scales of the halved bulblet was adjacent to the basal plate and started from the leaf primordium and a meristematic bulge. Buds developed directly into small bulblets after they were transferred to semi-solid MS basal salts medium supplemented with 6% sucrose, 10 microM indole-3-butyric acid (IBA) and 0.25% activated charcoal. Small bulblets cultured in liquid MS medium supplemented with additional KH(2)PO(4 )(170 mg l(-1)), 6% sucrose and 0.1 microM NAA under a 16/8-h (light/dark) photoperiod for 8 weeks grew into larger bulbs faster than those cultured on semi-solid medium. The bulbs were rooted on a semi-solid medium after 4 weeks and then transferred to the soil. As many as 18 bulblets developed and rooted from one in vitro-grown bulb after 25-27 weeks.     

Propagation of rose speciesin vitro

Summary Several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata) and interspecific hybrids were cultured to determine the appropriate concentrations of nutrients and growth regulators for shoot proliferation and root initiation. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal medium [Murashige and Skoog (MS) salts, vitamins, glycine, sucrose, and agar] supplemented with 0 μM to 17.8 μM (4 mg·l⁻¹) 6-benzyladenine (BA) and 0 μM to 0.54 μM (0.1 mg·l⁻¹) naphthalene, acetic acid (NAA). The ability of the explants to proliferate shoots and initiate roots was affected by the genotype, the nodal position of the explant, the strength of the MS basal salts, and the growth regulators used. The buds nearest the apex exhibited the slowest rate of development. Most species had the highest shoot proliferation when cultured on basal MS medium supplemented with 8.9 μM (2 mg·l⁻¹) BA, but the degree varied by species. Root development was enhanced by lowering the concentration of MS salts. With difficult-to-root species, rooting was improved by supplementing the media with 11.4 μM (2 mg·l⁻¹) indole-3-acetic acid (IAA) or by giving them a 7-d dark treatment at 10°C.     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.     

Rooting and shooting: dual function of thidiazuron in in vitro regeneration of soybean (<Emphasis Type="Italic">Glycine max.</Emphasis> L)

Thidiazuron (TDZ), primarily a cotton defoliant, has been later accepted as a plant growth regulator. In spite of extensive studies, the physiological function of TDZ is still uncertain. The aim of the present experiment was to study the activity of TDZ in in vitro regeneration of soybean. The seeds of soybean were cultured separately on MS and B5 medium supplemented with TDZ. The hypocotyls, cotyledons, cotyledonary nodes without axillary buds and cotyledonary nodes with axillary buds were used as explants and their capacity to direct regeneration was tested on both MS and B5 media containing TDZ (0.9–5.4 μM). Shoot formation was observed only on cotyledonary nodes with axillary buds cultured on MS and B5 basal media with TDZ (0.9–5.4 μM). All tested explants cultured on B5 medium with TDZ produced roots. Root formation was not observed on MS basal media supplemented with TDZ. Results show that TDZ functions as cytokinin (to produce the shoots) and auxin (to produce the roots) on various explants depending on the basal medium used.     

马尾松高效再生体系的建立(简报)

利用组织培养技术快速繁殖针叶树,不仅为植树造林所需大量苗木开辟了一条快速高效的新途径,是生产优良无性系的捷径,而且也是植物基因工程技术应用于针叶树品种改良的前提条件。它将在林木种苗产业化和林木良种化进程中发挥重要作用。针叶树的离体培养和植株再生一直是植物组织培养研究中难度较大的一个领域。近几十年来,随着世界各国对发展无性系育林业的日益重视,针叶树组织培养研究取得了很大的进展[1-3]。马尾松(Pinus massoniana Lamb.)是我国南方主要的造林及绿化树种,其木材和松脂具有重要的经济价值。然而在生产实践中,种子园种子…     

In vitro regeneration of shoot buds and plantlets from seedling root segments of Brassica napus L.

Root segments obtained from aseptically germinated seedlings of Brassica napus cv. Westar were used to optimize conditions for high-frequency shoot bud differentiation. The presence of low kinetin (0.5 M) and relatively high indole-butyric acid (1.0 M) levels facilitated optimum shoot bud differentiation. Modified MS medium (MMS) was superior to the other three basal media tested (MS, B5 and White's). Elevated sodium dihydrogen phosphate levels increased the differentiation of shoot buds. Increasing or decreasing the level of sucrose from 3% reduced the frequency of explants forming shoot buds. Addition of glutamine enhanced both the frequency of responding explants, as well as the number of shoots per responding explant. Root segments from 13-day-old seedlings produced the highest response (58%) in the presence of 100 mg l^-1 glutamine. The position of the segment on the main root, size, and the presence or absence of lateral roots altered the morphogenic response. Sealing of the donor seedling cultures with Parafilm^® instead of Stretch' n seal^® resulted in a higher production of shoot buds, although root segment cultures were not affected by the type of sealing. Spontaneous rooting occurred on all developed shoots.Dedicated to Dr. Friedrch Constabel on the occasion of his 60th birthday     

Tissue culture and plant regeneration of okra (Abelmoshus esculentus)

Explants (hypocotyl, cotyledon, cotyledonary node and leaf segment) were excised from aseptically grown okra (Abelmoschus esculentus) seedlings. The explants were cultured on a Murashige and Skoog basal nutrient medium supplemented with auxins, cytokinins and auxin-cytokinin combinations. Callus formation and root differentiation occurred in a medium containing naphthaleneacetic acid (NAA) or indoleacetic acid. There was a greater proliferation of roots on medium supplemented with NAA. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the growth medium suppressed root formation. No shoot bud or shoot development was observed at any of the auxin levels tested. Both kinetin (KN) and zeatin (Z) also proved ineffective in inducing shoot buds or shoots. Shoots were produced on cotyledon and cotyledonary node explants cultured in a medium supplemented with benzyladenine and NAA. These shoots developed roots on the same medium. The plantlets, on transfer to soil, grew normally.     

In vitro regulation of morphogenesis in peanut (Arachis hypogaea L.)

Callusing, caulogenesis, in vitro flowering and somatic embryogenesis were induced from the base of leaflets derived from mature embryos of peanut (Arachis hypogaea) by altering the hormonal composition of the Murashige and Skoog's (MS) basal medium. A combination of 4 mg/l alpha napththaleneacetic acid (NAA) and 5 mg/l 6-benylaminopurine (BAP) was optimum for inducing caulogenic buds. The caulogenic buds proliferated in medium with 3 mg/l BAP. Differentiation of these buds to shoots was achieved in MS basal medium with 0.5 mg/l each of BAP and kinetin (KN). Shoot buds and flower buds were produced when caulogenic buds were cultured on medium containing 1 mg/l BAP and 1 mg/l KN, prior to elongation. Clonally propagated plantlets derived from axillary buds elongated, formed roots and were grown to maturity in soil. Embryogenic mass formation was induced from the leaf base in the presence of 20 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos developed upon reducing 2,4-D to 3 mg/l.     

Root elongation and branching is related to local hexose concentration in Arabidopsis thaliana seedlings

Root architecture can be profoundly affected by the carbon availability in the plant. We hypothesized that this effect could be mediated by the carbon status of root cells involved in elongation and branching processes. Arabidopsis thaliana plants were grown at several photosynthetic photon flux densities (PPFD) and were supplied with various sucrose concentrations in the root medium. Hexose and sucrose concentration was estimated in individual roots in the apical growing region of the primary root and of secondary roots as well as in the zone of primordia development. Local sugar concentration was high in fast‐growing and in highly branched roots and robust relationships between root elongation rate or branching and hexose concentration (but not sucrose) were found that were common to all situations experienced. Moreover, these relationships accounted for the plant‐to‐plant variability within a treatment as well as for the variability among individual secondary roots within a plant. These results support the view that local hexose concentration integrates changes in carbon availability from several sources and acts as a signal to induce at least part of the response of the root architecture to the environment.     

Growth and Dormancy Cycles in Citrus Bud Cultures and Their Hormonal Control

An in vitro bud culture method was devised in order to better understand the control mechanism of Citrus bud development. This technique offers a new approach to the study of hormonal control of growth, dormancy and flowering cycles in perennial plants. Buds were excised from orchard trees throughout the year, cultured on defined media for prolonged periods, and their vegetative growth responses to various growth hormones were determined. The buds proceeded with their vegetative development in vitro and achieved sprouting on a basal medium. The various growth regulators affected both the time required for sprouting (TRS) and the type of growth. In summer buds, IAA delayed sprouting, while GA enhanced it and caused shoot elongation. Cytokinins specifically induced the formation of numerous adventitious buds, whereas ABA completely inhibited sprouting; this inhibition, however, was reversible. A marked decrease in total protein and in the rate of its synthesis was evident during the first 20 days of sprouting induction and early bud growth. The annual growth rhythm was determined in spring buds sampled and cultured throughout the year, and an innate dormancy of citrus buds was revealed. Both the dormancy and the sprouting periods of buds in vitro corresponded to the natural periods occurring under field conditions. The effect of exogenous IAA, GA and cytokinins on the TRS varied at different periods along the season, suggesting the concept of “critical levels” in the endogenous balance of hormones.