Establishment of juvenile-like shoot cultures and plantlets from 4–16 year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees

A method was developed for the establishment of shoot cultures from Douglas-fir trees selected for outstanding growth and form in a 12-year-old genetic test. Vegetative buds from the lower crown were sterilized and grafted in vitro onto juvenile clonal rootstock. The rootstocks were produced from adventitious buds induced on cotyledons, and were maintained through micropropagation. Buds that established grafts slowly elongated into shoots, which were harvested and multiplied through micropropagation. Grafts often grew several new shoots which in turn could be harvested. In 1987, 2830 buds were grafted from 18 superior trees. Twenty nine grafts (1%) produced shoots which established 11 of the 18 trees in culture. Their appearance and behavior in vitro became more juvenile over 1–3 years, as indicated by shoot and needle morphology, disappearance of episodic growth pattern, increase in multiplication rates, and ability of needles to produce adventitious buds.The five most prolific of the 11 clones were given a pre-rooting treatment and planted in soil under fog. The success of rooting and subsequent establishment in soil varied from 5 to 17% depending on clone. In contrast, trees multiplied in vitro for 1–2 years longer showed soil establishment rates from 8–60%. This technique allows establishment, multiplication, and maintenance in vitro of cultures from high value Douglas-fir genotypes. Such cultures may serve as a starting point for further research on rejuvenation and cloning.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Müll. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.     

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     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Shoot organogenesis from petiole explants in the aquatic plant Nymphoides indica

A protocol for rapid shoot organogenesis from petiole explants of the ornamental aquatic plantNymphoides indica L. Thwaites O. Kuntze was developed for use in future mutation breeding and cultivar selection studies. Optimum culture conditions for shoot organogenesis were determined. Effects of factorial combinations of 2-iP, BA or kinetin (0–25 μM) in factorial combination with IAA or NAA (0–25 μM) were examined. On the basis of regeneration frequency (80%) and adventitious shoot number (11.5 shoots per explant), most efficient shoot organogenesis occurred on petiole explants cultured on a basal medium consisting of full-strength MS inorganic salts, 0.56 mM myo-inositol, 1.2 μM thiamine-HCl, 116.8 mM sucrose supplemented with 10 μM BA and 20 μM IAA and solidified with 0.8% TC agar. Formation of adventitious shoots by direct and indirect shoot organogenesis from the same explant was verified by histological sectioning. With the exception of variegated leaf production on a single adventitious shoot produced in the presence of 25 μM kinetin and 15 μM NAA, no visible phenotypic abnormalities were observedin vitro in any of the shoots generated. Solid achlorophyllous adventitious shoots were recovered following culture of this variegated leaf tissue. Plantlets were easily acclimatized toex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phenolic Content of Microcuttings of Adult Chestnut along Rooting Induction

From the same adult 80-year-old tree of chestnut (Castanea sativa Mill.) 2 types of material have been taken and micropropagated. This resulted in in vitro easy-to-root microshoots (basal shoot origin - BS), and hard-to-root microshoots (crown shoot origin - CR). In these shoots, the phenolic contents were analysed at 0, 2, 5 and 8 days after in vitro rooting induction by 2 minute-dipping into an indole-3-butyric acid (IBA) solution (4.9 mM) and subsequent culture in a hormone-free rooting medium. The variation of the phenolic content along the adventitious rooting process differs between CR and BS microshoots for tannin, flavonol and elagic acid concentrations, which could be related to their differential rooting capacity.     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

In vitro organogenesis from leaf explants of Annona squamosa Linn

Multiple shoot formation was induced from excised leaf explants of Annona squamosa Linn. (custard apple) seedlings on a Murashige and Skoog basal medium containing benzylaminopurine and kinetin. Various auxins in combination with the above medium produced callusing of the explants. In an investigation of environmental factors affecting shoot induction it was seen that the maximum number of shoots were obtained using the leaf base with petiole at a temperature of 27°C and a light intensity of 1000 lux. Roots were initiated erratically when individual shoots were treated with an auxin and then transferred to an auxin free medium. The process of the development of adventitious buds in leaf culture was analysed histologically.NCL Communication No. 3104.     

Adventitious shoot regeneration in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch]

A method for adventitious shoot regeneration from leaves of micropropagated peach shoots has been developed. Apices were excised from in vitro shoot cultures of a seed-derived (juvenile) genotype (P16Cl5) and mature genotypes (Babygold 6, 842 Standard, San Giorgio and Yumyeong). Apices were cultured 21 days in the dark on a medium supplemented with 6-benzyladenine and α-naphthaleneacetic acid and then transferred to an auxin-free medium and incubated in the light for 21 days. The first four apical leaves were excised from these apices and cultured in the same way. During the dark incubation period, leaves developed a callus at the petiolar base. Adventitious shoots appeared on this callus after transfer to auxin-free medium and culture under light conditions. The morphogenic ability of the callus was maintained for several months.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

Somatic embryogenesis inAesculus

Summary Somatic embryogenesis was observed with explants taken from four types ofAesculus tissue: (a) shoots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (b) roots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (c) shoots from newly forced 3-yr-old seedlings (A. glabra), and (d) newly forced shoots from a 30-yr-old tree (A.×arnoldiana “Autumn Splendor”). Shoots provided three types of explants, single node, shoot apex, and internodal section, and all exhibited embryogenesis. Proembryogenic masses developed in a few cases after 6 wk in culture but were more commonly seen after 3 mo. The yellow, friable proembryogenic masses emerged from proximal cut ends of explants. Almost all cultures that formed embryos possessed leaves, either from developing apical or axillary buds or from adventitious buds, prior to the emergence of proembryogenic masses. Only tissues that had begun to senesce and had been exposed to cytokinin (benzyladenine at 5 or 25 μM) formed somatic embryos. Embryos with distinct cotyledonlike structures and root/shoot axes developed during the 10 to 16 wk following the inital emergence of proembryogenic masses. Enhanced frequency of embryogenesis was obtained by dark culture of root and shoot explants from 4-wk-old germinated embryos (A.×arnoldiana) and by dark and cold (5°C) treatment of shoot tissue cultures derived from 3-yr-old seedlings (A. glabra). Embryogenic potential was greatest in the most juvenile tissue and least in the mature tissue. Five percent of shoot explants taken from the 30-yr-old select treeA.×arnoldiana “Autumn Splendor” produced somatic embryos.     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

Reduction of hyperhydricity in sunflower tissue culture

In order to reduce the occurrence of hyperhydrated shoots, the response of three sunflower inbred lines was examined on regeneration media containing various concentrations of kinetin, silver nitrate, and casein hydrolysate, calcium nitrate and cobalt nitrate. There were differences among the inbred lines for all the parameters taken into account to outline the in vitro efficiency. Percentage of hyperhydrated primordia, average number of shoots and of primordia per total explants, and percentage of hyperhydrated shoot traits differed among all media. The genotype × culture media interaction was significant for average number of shoots and primordia per total explants, regeneration percentage and percentage of hyperhydrated primordia. Among all media tested, those containing silver nitrate significantly reduced hyperhydricity in a dose-dependent way. The addition of silver nitrate showed to be useful in improving the quality of sunflower micropropagated plants by reducing this undesirable phenomenon.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Development of an in vitro culture technique for conservation of Saccharum spp. hybrid germplasm

An in vitro method for the establishment and storage of over 200 Saccharum spp. hybrid clones was developed that involved only 1 medium for shoot development and multiplication, and no decontamination procedures. Apical buds, from the leaf axils of developing leaves surrounding the apical meristem, were cultured on medium containing the plant growth regulators 6-benzylaminopurine (BAP) and 6-furfurylaminopurine (kinetin), and regenerated multiple shoots. Shoots transferred to medium containing naphthaleneacetic acid (NAA) developed roots. In vitro plants transferred to a medium containing half strength salts and vitamins without plant growth regulators were placed in storage at 18°C. After 12 months of storage plants were transferred to fresh medium and returned to storage. The genetic integrity of clones (based on phenotype assessment) was not affected by the in vitro culture method and up to 14 months of low-maintenance storage conditions. These in vitro plants will be further tested for genetic stability using biochemical and molecular techniques.