Shoot regeneration from cultured leaves of Japanese pear (Pyrus pyrifolia)

Several experiments were conducted to investigate in vitro regeneration of adventious shoots from cultured leaves of Japanese pear (Pyrus pyrifolia). A protocol was developed and regeneration achieved from six cultivars. Leaves harvested from shoot cultures which had been preconditioned on B5 medium with 5 μM thidiazuron plus 0.25 μM gibberellic acid were placed on regeneration medium of the same composition. Frequency of regeneration per leaf was as high as 23% but cultivar and environmental factors influenced the result. More mature (basal) leaves regenerated more frequently than younger ones from the shoot tip. Leaf orientation during regeneration and photoperiod was not a strong influence but regeneration from leaf pieces was less than from uncut leaves. An alternative regeneration procedure was developed in which first, shoot cultures were grown on the preconditioning medium. Leaves of the intact shoot cultures were then induced to regenerate directly when adventitious shoots formed on leaves of the intact shoot culture leaves without excision. Adventitious shoots from both procedures developed into typical shoot cultures when transferred to shoot culture maintenance medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves

Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.     

Regeneration of Cuphea tolucana Peyr. in in vitro culture

In order to regenerate Cuphea tolucana from hypocotyl, cotyledon and root explants, a solid culture and 8 hormone combinations were used. Only the root explants did not react to any of the media. On most of the media, the other explants formed shoots, roots or callus, or their reaction was more complex. The regeneration probably occurred via direct organogenesis. The regenerants displayed a wide variety of morphological characteristics. However, their offspring did not show any differences from plants, which had not undergone culture.     

Adventitious organogenic regeneration from soybean genotypes representing nine maturity groups

In the US, soybean genotypes are classified into maturity groups (MG; total of 13) that represent areas of adaptation generally correlated with latitude bands. To determine if one regeneration procedure could regenerate representatives from diverse areas of adaptation, 18 soybean genotypes representing nine MG were compared for organogenic adventitious regeneration and plant formation from hypocotyl explants following the procedure previously tested on representatives from only three MG. Responding explants were those capable of producing shoots on the acropetal end of the explant from either the outer edge plus central region or the central region only. This enabled determination of the contribution of cotyledonary nodal tissue (outer edge) to shoot regeneration and by discounting those explants, it also enabled estimates of true adventitious regeneration. All 18 genotypes were capable of producing meristemoids/shoots solely from the central region with responses ranging from 28.5 to 64.3% after 4 weeks in culture. All genotypes were also capable of producing elongated shoots that could be successfully rooted. No morphological differences were noted among regenerants, or between them and seed-initiated plants. All regenerants produced viable seed which germinated and produced morphologically normal plants. This study confirmed the genotype- and MG-independent nature of this hypocotyl-based organogenic regeneration procedure and provided conservative estimates for responses that were truly/solely adventitious in nature.     

High frequency in vitro propagation of Phyllanthus amarus Schum. & Thom. by shoot tip culture

With the aim of micropropagation of Phyllanthus amarus, an important medicinal herb, shoot tips were cultured in Murashige and Skoog's medium supplemented with kinetin/ BAP singly or in combination with IAA. Growth regulators at lower range (0.1-1.0 mg L(-1)) stimulated direct regeneration of shoots. Kinetin was superior to BAP and kinetin-IAA combination was more suitable than kinetin alone. About 15 shoots were yielded per explant after 30 days of culture in the medium containing kinetin and IAA both at 0.1mg L(-1). The cluster of proliferated shoots elongated and rooted simultaneously under the same treatment following another subculture, thus shortening the total time schedule of micropropagation. Shoot tips of regenerated shoots were continuously used to regenerate new shoots with periodic transfer to fresh medium resulting in a steady supply of normal, healthy plants without any deviation in the production rate during a continuous one year culture. Micropropagated plants were successfully established in soil with high survivality (80%).     

Plant regeneration from shoot tips and callus of papaya

Summary Two methods of in vitro culture were employed to regenerate papaya plants. One involved regeneration of plants from callus and the other, production of multiple plants from single shoot-tip explants. Callus was induced from stem sections of papaya seedlings in a medium containing 1 mg per 1 NAA and 0.1 mg per 1 kinetin. The callus regenerated shoots and/or embryoids when transferred to a medium of lower auxin, 0 to 0.05 mg per 1 IAA, and higher cytokinin, 1 to 2 mg per 1 kinetin Multiple shoots were produced when the excised shoot-tip explants were cultured in a medium supplemented with 0.05 mg per 1 IAA and either 5 mg per 1 kinetin or 0.5 to 1.0 mg per 1 benzyladenine. Root formation of the shoots or embryoids that derived from callus or shoot tips occurred in a medium containing 5 mg per 1 IAA and in a light intensity of 3000 to 4000 Ix. The rooted plants could be established in soil and under standard greenhouse conditions after they had been acclimated by initially growing them in moist vermiculite contained in polyethylene-covered pots. This research was supported by the National Science Council, Republic of China.     

Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange

Epicotyl explants of sweet orange and citrange were infected with Agrobacterium strain EHA101 harboring binary vector pGA482GG, and factors affecting the plant regeneration and transformation efficiency were evaluated. Increasing the wounded area of explants by cutting longitudinally into two halves, and optimization of inoculation density, dramatically enhanced both regeneration and transformation frequency. Inclusion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the explant pretreatment medium and the co-culture medium improved the transformation efficiency by decreasing the escape frequency. More than 90% rooting frequency of transformed citrange shoots was achieved by two-step culture: first on media supplemented with auxins, and then on media without hormones. Inclusion of 20 mg l^–1 kanamycin in rooting medium efficiently discriminated transformed shoots from non-transgenic escaped shoots. Shoot grafting in vitro was used to regenerate transformed plants, due to the slow growth of most sweet orange shoots.     

Hormonal requirement and tissue competency for shoot organogenesis in two cultivars of Brassica napus

An investigation of the regeneration ability of explants taken from the floral stem of Brassica napus var. oleifera was performed in the winter cultivars Darmor and Bienvenu. Our purpose was to compare the regeneration ability of the two genotypes, to compare the competence of the different tissues of the stem and then to study histologically the regeneration of shoots. A strong genotypic effect was observed between the two cvs; Bienvenu had a poorer ability to produce shoots when cultured in the presence of benzyladenine: regeneration commenced later; the percentage of explants producing shoots and the number of shoots per regenerating explant were much lower. The comparison between the regeneration ability of different explants, i.e stem segments, internal stem segments, thin cell layer and peels, showed that the superficial tissues were able to regenerate roots but not shoots. Contrariwise, internal stem segments regenerated only shoots. The origin of shoots was investigated in stem segments of cv. Darmor. A kinetic histological analysis showed the basic role played by phloem and phloem-associated cells in shoot formation.     

A novel approach for efficient plant regeneration from long-term suspension culture of wheat

Summary Hexaploid wheat plants were easily regenerated from young embryo-derived callus for twelve genotypes tested. After a 2.5 years culture period, however, most of the callus cells lost their ability to regenerate into shoots, but not into roots.A novel approach was used to regenerate shoots from the long-term suspension cultured cells. In general, instead of selecting embryogenic callus as source material, this approach requires the inoculation of unselected callus into liquid medium followed by removing the free floating cell portion, selecting out non-root forming cell clumps from the root forming primary suspension culture, and growing the putative shoot-competent clumps in liquid medium with reduced auxin concentrations. We have successfully established shoot-competent wheat suspension cultures for cv. Mustang. High (>80%) frequencies of plant regeneration were observed from plating of 2.5 year suspension cultures. The suspension cultures established by this approach have been utilized to select for heat tolerant variants and will be an ideal source material for protoplast culture and transformation studies. This approach can also be applied to other cereal crops which form roots easily but are unstable in maintaining long term regenerable cultures and which are not easily adaptable to suspension culture.     

Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.     

Enhancement of in vitro organogenetic capacity of rose by preculture of donor shoots on the medium with thidiazuron

Rose genotypes can be recalcitrant in in vitro regeneration. A lack of competence for regeneration results in unsatisfactory numbers of adventitious shoots, which diminishes achievements in use of genetic engineering in rose modern breeding. Among the factors which can partially overcome this problem is the use of thidiazuron (TDZ) in the induction phase of regeneration. We studied the use of TDZ in concentrations 0.05 and 1.0 mg l⁻¹, in the medium for the culture of donor shoots with the aim of increasing the regeneration competence of five rose cultivars differing in their regeneration ability. All the tested genotypes increased their regeneration potential when the donor shoots were cultured on TDZ media for 15 or 30 days. The strongest effect, doubling to quadrupling the number of shoots, was obtained in the cultivars characterized by the lowest regeneration potential. This enhanced regeneration ability was maintained through two or three subsequent 4-week long regeneration passages.     

Comparative assessment of the efficacy of bacterial and cyanobacterial phytohormones in plant tissue culture

Efficient callus and explant regeneration medium, using microbial extract (SPE purified) or supernatant has been formulated for Brassica oleracea L. var. capitata. Two cyanobacterial strains (Anabaena sp. Ck1 and Chroococcidiopsis sp. Ck4) and two bacterial strains, (Pseudomonas spp. Am3 and Am4) known to produce a number of cytokinins, tZ, cZ, ZR, DHZR and IAA were selected for the media formulation. Supernatant from strains with high cytokinin to IAA ratio, including Pseudomonas aeruginosa Am3 (2.08) and Chroococcidiopsis sp. Ck4 (0.8) efficiently induced compact calli which were turned green upon exposure to light. The strains producing lower cytokinins to IAA ratio (0.11–0.13) on the other hand induced friable callus which were unable to regenerate on the selected media combinations. Leaf, stem and root explants of Brassica oleracea L. regenerated on MS medium supplemented with phytohormones from microbial origin with efficiency comparable to standard cytokinins and IAA. Supplements from cyanobacterial origin proved to be the best for induction of adventitious roots and shoots on internodal and petiolar segments. Hypocotyl explants however, responded well on MS supplemented with bacterial metabolites. Induction of adventitious shoots on root explants was supported by phytohormones from both origin equally well. Callus induction on the seeds and regeneration of shoots on calli was also observed. Cyanobacteria based media were more efficient to induce calli capable of regeneration upon exposure to light. Internodal explants were highly amenable to regenerate shoot and roots simultaneously. Root explants were the less successful to regenerate shoots.     

Rapid plant regeneration,validation of genetic integrity by ISSR markers and conservation of Reseda pentagyna an endemic plant growing in Saudi Arabia

Reseda pentagyna is the only endemic species among the seven species of the genera Reseda found in Saudi Arabia. Probably no information is available on regeneration by conventional method of regeneration through seeds or cuttings. Therefore, alternative method of tissue culture was attempted to regenerate and multiply the plant. High shoot regeneration (14.44 shoots/explant) was obtained after four weeks, when shoot cuttings cultured on MS containing BA at 1.0 µM. Other cytokinins e.g., Kn, 2iP and TDZ found to be less effective in bud induction and shoot multiplication. Individual shoots were rooted on MS medium supplemented with various auxins at 0.5–5.0 µM concentrations. The IBA (1.5 µM) supplemented MS media induced maximum (83.3%) rooting. The plantlets were acclimatized and hardened under greenhouse conditions in plastic pots containing soil and farm yard manure with 95.0% success. The protocol developed would help to multiply the plant as well as conserve them in natural habitat. This can also be utilized to obtain active constituents for pharmaceutics and genetic manipulations.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

Improved shoot regeneration system through leaf derived callus and nodule culture of Sansevieria cylindrica

Long-term culture establishment and efficient in vitro regeneration protocol for Sansevieria cylindrica Bojer ex Hook was developed using leaf derived callus and nodule culture. Profuse callus induction on leaf discs was achieved on Murashige and Skoog (MS) medium supplemented with 10 μM indole-3-butyric acid (IBA), while a high frequency of nodulation was induced on 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) containing media. Shoot regeneration ability from cultured tissues occurred at varying degrees on all media. Through callus culture a maximum of 17.6 ± 0.14 shoots per culture was formed on medium containing 5μM 6-benzyladenine (BA) and 2 μM α-naphthaleneacetic acid (NAA). Among nodule cultures, the 2,4-D generated nodules were more proliferative and regenerative as compared to 2,4,5-T induced nodules and a maximum of 25 ± 0.16 shoots per culture was produced on a medium containing 5 μM BA plus 1 μM NAA. The regenerated shoots were successfully rooted on a semi-solid half strength MS medium containing 5 μM IBA with an average root number 3.5 ± 0.18 and root length 6.5 ± 0.14 cm. The regenerative ability of callus tissues was steady upto one year, while the nodules retained the totipotency to regenerate on optimal medium even after 3 years of subculturing. The histological sections of nodules confirm the typical anatomy exhibiting the vascular elements in bundles with well demarcated cortex and epidermal covering.     

A rooting procedure for lentil (Lens culinaris Medik.) and other hypogeous legumes (pea,chickpea and Lathyrus) based on explant polarity

The present study assessed the rooting response of lentil nodal segments in relation to explant polarity, hormone, salt and carbohydrate concentrations of the medium. Nodal segments of lentil with an axillary bud cultured in an inverted orientation (apical end in medium) showed higher rooting frequencies than explants cultured in a normal orientation (basal end in medium). The highest rooting percentage (95.35%) and average number of shoots regenerated per explant (2.4) were obtained from explants placed in an inverted orientation on Murashige and Skoog (MS) medium salts with 3% sucrose, supplemented with 5 microM indole acetic acid (IAA) and 1 microM kinetin (KN). Reducing or increasing phytohormone concentration did not alter significantly root regeneration of inverted explants. Sucrose at 3% allowed higher root regeneration frequencies compared to 1.5% sucrose. MS full concentration permitted regeneration of longer shoots with more nodes per regenerated shoot, compared to MS half-strength, which regenerated more shoots of shorter length and with less nodes. Inverted nodal segments of other hypogeous legumes (pea, chickpea and Lathyrus) also exhibited higher rooting frequencies than explants cultured in a normal orientation on MS medium with 3% sucrose and supplemented with 5 microM IAA and 1 microM KN. The most novel application of this study is the culture of nodal segments of hypogeous legumes in an inverted orientation. This procedure is a considerable improvement over other published procedures concerning in vitro rooting of lentil, pea, chickpea and Lathyrus.     

Cost-effective in vitro propagation methods for pineapple

We have developed an efficient and cost-effective method for commercial micropropagation of Smooth Cayenne pineapple. In vitro shoots were used as starting materials, and either longitudinal sections of the shoots or leaf bases were used as the explants to regenerate shoots. When these explants were used, the axillary meristems, which usually remain quiescent during shoot multiplication, were able to form new shoots. Subsequent to the regeneration step, additional multiplication was achieved inside a 10-l Nalgene vessel with shoots immersed in liquid medium for 5-10 min/h (periodic immersion bioreactor, PIB). The shoots were then induced to form roots and transferred to soil. Using the above micropropagation method and the PIB, we produced 6,000-8,000 shoots from two initial shoots in less than 6 months. The clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.     

Rapid adventitious shoot regeneration from leaf explants of European birch

The goal of this research was to develop a rapid and efficient system for regenerating shoots from leaf explants of European birch, Betula pendula Roth. Single-node stem explants were established in culture, and microshoots were subcultured every 4 weeks through 12 subcultures. Leaves from glasshouse plants or subcultured shoots were excised from stems, cut into approximately 35-mm² pieces, and placed on Woody Plant Medium (WPM) containing different combinations of naphthaleneacetic acid (NAA) (0, 3, 6 or 9 M) and benzyladenine (BA) (0, 7.5, 15 or 22.5 M) in a 4×4 factorial design. The percentage of leaf pieces forming shoots and the number of shoots regenerated per explant were recorded after 4 weeks. Only media containing BA without NAA stimulated shoot formation on leaf explants. Fifteen micromolar BA induced the most shoots to form on leaf explants compared to 30, 45 or 60 M of this cytokinin. In addition, shoot regeneration was enhanced up to four-fold between the first and eleventh subculture. Over 90% of the leaf explants regenerated shoots with an average of 18 buds formed per explant for the eleventh subculture. Almost twice as many explants formed shoots if their adaxial side was in contact with the medium rather than oriented away from it. The ability to regenerate shoots from leaves varied among plants, regardless of stock plant age. This reliable shoot regeneration system can be used for rapid shoot proliferation and potentially for genetic engineering of European birch.     

Rapid and efficient regeneration of plants from explants of Arabidopsis thaliana

A procedure is given for the regeneration in vitro of a previously difficult to regenerate plant, Arabidopsis thaliana (L.) Heynh. Leaf explants of Arabidopsis that are given a short exposure to a callus-inducing medium prior to incubation on a shoot-inducing medium exhibit high survivability and rapidly produce shoots. Some media combinations allow shoots to form on 100% of the explants and the majority of these explants form multiple shoots. The shoots are normal in appearance and most of them can be induced to form functional roots and retain fertility. This regeneration scheme should be of use in the development of an Arabidopsis transformation procedure and may also be applicable to other recalcitrant plant species.