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.     

In vitro propagation of Agave fourcroydes Lem. (Henequen)

In vitro plant regeneration of Agave fourcroydes Lem. (Agavaceae) is described. Results suggest that the NO₃ ^-:NH₄ ⁺ balance in the culture medium is a key factor controlling callus growth and organogenesis in rhizome cultures. Stem callus showed limited organogenic capacity, but high cytokinin concentrations induced adventitious shoot formation on stem explants. When these shoots were excised and subcultured, new callus formed at their base from which new shoots arose. The shoots from stem explants and rhizome callus formed extensive root systems in vitro and were transferred to pot culture with a 90% survival rate.     

Plant regeneration of Solanum lycopersicoides Dun. from stem explants,callus and suspension cultures

Protocols were established for achieving plant regeneration from stem internode, callus, and cell suspension cultures of Solanum lycopersicoides Dun. Two accessions of S. lycopersicoides exhibited different responses as to callus formation on various media, requirement of gibberellic acid for shoot regeneration, and ability to grow in suspension culture. The optimum medium for initiation and maintenance of cell suspension cultures was Murashige and Skoog [9] medium with 15 mg l^– NAA. For shoot regeneration, of three cytokinins tested, zeatin was found most effective relative to number, rapidity of response and overall quality of shoots. Shoot regeneration from stem explants, callus and suspension cultures was optimum on MS + 3.0 mg l^–1 zeatin + 0.1 mg l^–1 gibberellic acid.Michigan Agricultural Experiment Station Journal Article No. 11589.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Inheritance of somatic embryogenesis in red clover (Trifolium pratense L.)

 Red clover genotypes capable of regenerating plantlets in vitro from non-meristematic tissue-derived callus are rare. Selection for genotypes capable of somatic embryogenesis identified a clone comprised of a group of plantlets regenerated from a hypocotyl-derived callus culture on L2-based media and another group of plantlets originating from crown divisions of the epicotyl-derived plant. The callus-derived plants of this clone were highly regenerative when reintroduced to callus culture, but the epicotyl-derived plants produced nonregenerative callus cultures. F₁, F₂ and BC₁ populations were evaluated to determine the mode of inheritance of the regeneration trait. Reciprocal crosses did not differ, indicating a lack of maternal effects. Results were compatible with genetic control of regeneration by two complementary genes. We propose the genotype Rn1-Rn2- for regenerative plants. Three petiole segment explants were sufficient to evaluate regenerative ability in seedlings. Regenerative ability was often associated with abnormal leaf morphology in a few to several leaves. Received: 17 February 1998 / Accepted: 7 April 1998     

Somatic embryogenesis in Canary Island date palm

Shoot regeneration was obtained from leaves of in vitro cultures of wild pear genotypes. The highest regeneration rates, ranging from 40% to 64%, depending on the genotype, were obtained using leaves wounded by three cuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l^-1 cefotaxime and maintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-free medium in light (expression phase). A concentration of 8.8 μM BA induced the highest number of explants to produce adventitious shoots. TDZ was less effective than BA and induced hyperhydricity in regenerated shoots. The histological studies revealed that the regenerated shoots originated mainly from callus formed by epidermal and sub-epidermal cells and by cells of the vascular tissue. The regenerated shoots were micropropagated, rooted and transplanted to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation

Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation. Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration. Mode of regeneration was found to be the most significant of the three factors. Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase. The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success. Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low. The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants). The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5–6 weeks after inoculation with A. tumefaciens. This line was also self-compatible, enabling the production of seed without the need for hand-pollination. A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B. oleracea genotype for studying gene function. The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

Regeneration and transformation in adult plants of Campanula species

Adult plants are known for recalcitrance when it comes to adventitious organ formation and regeneration. Methods used for regeneration in explants from seedlings of Campanula carpatica failed to work for explants from adult plants of the same species. The present investigation generated efficient regeneration methods for mature specimens of four species of Campanula, C. carpatica, C. haylodgensis, C. portenschlagiana and C. poscharskyana. Petiole explants from dark-grown in vitro shoot cultures grown from nodal cuttings of adult plants regenerated successfully (95%), while explants from light-grown in vitro shoot cultures and greenhouse-grown plants regenerated at 12% and zero percentage, respectively. Dark-treatment, along with media manipulation with plant growth regulators, further enhanced regenerative capacity of the explants. A MS-based medium containing 10mg l ⁻¹ TDZ and 0.25 mg l⁻¹ NAA was the most efficient regeneration medium. Transgenic shoots from C. carpatica (3%) and C. haylodgensis (1%) and transgenic callus from all species were produced using Agrobacterium tumefaciens, and transformation was confirmed by histochemical and Southern blot analyses. Protocols developed in this study may be useful for achieving efficient regeneration and transformation of recalcitrant adult plants.     

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, <Emphasis Type="Italic">Tylophora Indica</Emphasis> (Burm. F.) merrill

Summary Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.     

A method for increased plant regeneration from immature F1 and BC1 embryos of Cucurbita maxima Duch. x C. pepo L. hybrids

Plants have been regenerated from abnormal embryos with spongy cotyledons and albino sectors, derived from Cucurbita maxima and C. pepo F₁ and BC₁ hybrids. Shoot regeneration was induced directly from the cotyledons without an intervening callus phase on the medium without hormones. On the rooting medium, shoots continued to proliferate, which allowed for further multiplication in vitro. The number of plants obtained varied with genotype and ranged up to 65 plants per embryo.     

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.     

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA₃ within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.     

Improvement of embryogenic callus induction and shoot regeneration of buffalograss by silver nitrate

Addition of the ethylene antagonist, silver nitrate (AgNO₃), into callus induction medium significantly enhanced embryogenic callus production (both induction frequency and callus growth) of field-collected male immature inflorescence cultures of buffalograss NE84-45-3 and 'Texoka'. No stimulatory effect of AgNO₃ was observed on embryogenic callus induction for female immature inflorescence culture of a female genotype `609' and `Texoka'. Calli initiated on AgNO₃-containing media had more shoot-regenerating calli than those initiated on AgNO₃-free media, when they were transferred to the regeneration media. Benzyladenine at 2.2 μM gave the best response for regeneration, regardless of the callus source. Although average number of shoots regenerated per callus was lower for calli initiated on AgNO₃-containing media, total number of shoots regenerated was higher. The stimulatory effect, however, was environment and genotype dependent. While the addition of AgNO₃ significantly stimulated embryogenic callus induction of NE84-45-3 immature inflorescences collected in Fall 1995 and May 1997, it only slightly increased the embryogenic callus induction frequencies in May 1996 when rainy conditions occurred. For male inflorescences of `Texoka' collected in early May, AgNO₃ significantly enhanced embryogenic callus production consistently over the two-year period (1996, 1997). Published as Journal Series No. 1351, Agricultural Research Division, University of Nebraska. This revised version was published online in June 2006 with corrections to the Cover Date.     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

Biosynthesis and accumulation of a medicinal compound,Picroside-I,in cultures of <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> Royle ex Benth

Establishment of callus cultures and plant regeneration from different explants coupled with estimation of Picrosides in morphogenetically different developmental stages showed that Picroside-I accumulates in shoot cultures of Picrorhiza kurroa with no detection of Picroside-II. The Picroside-I content was 1.9, 1.5, and 0.04 mg/g in leaf discs, stem and root segments, respectively. The Picroside-I content declined to almost non- detectable levels in callus cultures derived from leaf discs, stem segments with no change in Picroside-I content in root segments or calli derived thereof. The biosynthesis and accumulation of Picroside-I started in callus cultures differentiating into shoot primordia and reached to the concentrations comparable to original explants of leaf discs and stem segments in fully developed shoots with contents of 2.0 and 1.5 mg/g, respectively. The shoots formed from root-derived callus cultures were relatively slow in growth as well as the amount of Picroside-I content was comparatively low (1.0 mg/g) compared to shoots derived from callus cultures of leaf and stem segments, respectively. The current study concludes that the biosynthesis and accumulation of Picroside-I is developmentally regulated in different morphogenetic stages of P. kurroa tissue cultures.     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

Genetic factors influencing regeneration ability in rye (Secale cereale L.). I. Immature inflorescences

Immature inflorescences of ten rye inbred lines (inbred degree S10 and S11) were cultured on solidified MS medium supplemented with 3.0 mg/dm³ of 2,4-D. According to their capability for callus production explants were classified into two groups : responsive (giving weak or intensive callus production) and non responsive (lack of callus formation). After transferring responsive material into hormone-free medium the regeneration of roots or shoots from the intensive growing callus was observed. Consistent differences between lines in the portion of explants with a certain response were found. They were divided into five groups reacting in the same way. Lines with different in-vitro response were crossed in an incomplete diallel. F₁, F₂ and F₃ generations were analyzed and the following conclusions drawn: the ability for plant regeneration from immature inflorescences in rye is determined by numerous loci, has a recessive character, and both callus production and regeneration suppression may be controlled by complementary genes.