Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.     

DNA ploidy level of colchicine-treated hops (Humulus lupulus L.)

The genome doubling agent colchicine has been used effectively to obtain tetraploid plants when starting with diploid material. Since the plant meristem consists of many cells, as well as obtaining tetraploids, it is possible to obtain mixoploids (chimeras consisting of diploid and tetraploid tissue) after colchicine treatment. Flow cytometry was used to determine the DNA ploidy level of different tissues of individual hop plants following colchicine treatment of diploid hops. Plants that are shown to be mixoploid after analysis of leaf tissue can display higher levels of tetraploid nuclei in root tissue, such that they could be mistakenly classified as tetraploid. The hop variety Galena-4n, which was previously considered to be a tetraploid, is shown to be a mixoploid based on leaf material. However, Galena-4n consistently produces triploid progeny when crossed with a diploid hop variety, indicating that Galena-4n produces tetraploid reproductive tissues and is effectively a tetraploid from a breeding perspective. The results from this study indicate that care should be taken when determining the DNA ploidy level of an individual plant after exposure to genome-doubling agents based on single tissues and that leaf tissue is a choice material for flow cytometric analysis.     

Characterization of Prunus domestica L. in vitro regeneration via hypocotyls

Several important aspects of regeneration of European plum from hypocotyl explants were studied. Multiple shoots were induced and full plants were recovered for a large number of plum varieties. This indicates that European plum species is, in general, very responsive to in vitro regeneration from hypocotyls. Shoot organogenesis could be induced from both mature and immature seed explants but regeneration efficiency was higher when immature seeds were used. Rooting efficiency for varieties with low rooting tendency could be greatly increased by addition of naphthaleneacetic acid in the medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots at a high frequency and such multiplication could continue for many cycles. The secondary new shoots could be induced for various plum varieties. Plant recovery from the secondary shoots was as efficient as that from the primary shoots. This new system may be an alternative for plum transformation with a potential for increasing transformation efficiency. The system can be used for propagation of transgenic lines and other genetic clones of various varieties.     

A micropropagation system for cloning of Bambusa tulda Roxb.

The communication describes standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda. Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in winter and summer seasons, respectively facilitated optimum culture establishment and bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud break due to toxicity to explants. MS liquid medium enriched with 100 μM glutamine, 0.1 μM indole-3-acetic acid and 12 μM 6-benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The proliferated shoots were successfully rooted on MS liquid medium supplemented with 40 μM coumarin resulting in a maximum of 98% rooting. The procedure requires 45 days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30 days for root induction) and 80 days for acclimatized plantlet production.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

Molecular analysis revealed autotetraploid,diploid and tetraploid cybrid plants regenerated from an interspecific somatic fusion in Citrus

More than 150 plants were regenerated from our previous somatic hybridization between embryogenic callus line of Page tangelo (Citrus reticulata Blanco × C. paradisi Macf) and mesophyll protoplasts of rough lemon (C. jambhiri Lush) mediated by electrofusion. Preliminary screening showed that 78% of these plants were tetraploids while the rest were diploids morphologically resembling the leaf parent (rough lemon). Herein, eight plants (six tetraploids and two diploids) were selected and further analyzed by flow cytometry, simple sequence repeat (SSR), mitochondria (mt) RFLP and chloroplast (cp) SSR techniques. The results showed that four of these six tetraploids were somatic hybrids, one tetraploid was autotetraploid of Page tangelo, and the remaining one tetraploid was cybrid with nuclear and cpDNA from rough lemon and mtDNA from Page tangelo; the two diploids were verified being cybrids with nuclear DNA from rough lemon and mtDNA from Page tangelo, cpDNA was randomly inherited. The regeneration mechanism of these novel cybrids was discussed.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

Significance of explant preparation and sizing in Aloe vera L.—A highly efficient method for in vitro multiple shoot induction

The present study was carried out to assess the effect of explant preparation and sizing for in vitro micropropagation of Aloe vera L. The stem nodal explants and shoot tips were cultured on modified Murashige and Skoog's medium (1962) supplemented with different concentrations of 6-benzylaminopurine (BA), kinetin (KIN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) either singly or in combination. The best media composition was found to be MS medium supplemented with IAA (11.42 μM), IBA (9.8 μM) and BA (8.88 μM). The explants were divided into 2 sets, with and without ensheathing leaf base. Explant sizing, pruning and retention of mother tissue was highly significant in induction of multiple shoots and roots. The stem nodal explants with leaf base performed much better than those without such covering. A very high number of shoots and roots grew from these explants. The rooted plantlets were successfully acclimatized and transferred to the green house conditions and finally to field conditions.     

In vitro colchicine-induced polyploid plantlet production and regeneration from leaf explants of the diploid pear (Pyrus communis L.) cultivar, ‘Fertility’

Summary

Polyploid plantlets, including triploid, tetraploid, and mixoploid, were induced from the European pear (Pyrus communis L.) cultivar ‘Fertility’ by in vitro colchicine treatment of leaf explants. The leaf explants were incubated in 0.4% (w/v) colchicine for 24, 48, or 72 h, then transferred to adventitious shoot-induction medium. Regenerated shoots were pre-selected according to their morphological characteristics when compared to control shoots from untreated shoot proliferation cultures. Shoots with putative polyploid morphological characteristics were maintained and proliferated. The ploidy levels of all putative polyploid individuals were analysed by flow cytometry and identified by chromosome counts of shoot tip tissue squashes. Polyploid shoots were rooted, and the resulting plantlets were transferred to the field. Polyploid plantlets had a higher specific leaf mass and larger stomata than those of diploid plantlets.     

In vitro polyploidization of Mecardonia tenella, a native plant from South America

Mecardonia tenella is an herbaceous plant widely distributed in the temperate region of South America. Both plant architecture and flower size are characteristics that can be improved to become a viable new ornamental plant. Chromosome doubling by the use of agents such as colchicine is an available methodology to this end. Nodal segments from in vitro grown plants of M. tenella were submerged in the following doses of colchicine in 1% (v/v) dimethyl-sulfoxide (DMSO) solution (%, v/v): 0.0, 0.001 and 0.01 (24 and 48 h). The DNA content of the regenerated plants was measured by flow cytometer. A total of 68 tetraploid plants were detected out of 126 colchicine treated plants. The flowers and leaves of the tetraploid plants were bigger compared to those from the wild diploid type (control). Under field conditions, the selected tetraploid plants showed a more compact shape than the control plants.     

Commercial production of Cordyline terminalis (L) Kunth. from shoot apex meristem and assessment for genetic stability of somaclones by isozyme markers

Rapid multiplication of Cordyline terminalis (L) Kunth. was achieved from shoot apex explant on Murashige and Skoog's (MS) medium supplemented with 3% (w/v) sucrose and different concentration of growth regulators at various combinations. On MS medium supplemented with BA in combination with Adenine sulphate and IAA, shoot initiation and multiplication were obtained. Best elongations of shoots were found on 1/2 MS basal medium and shoots were rooted on 1/2 MS medium supplemented with IBA. Rooted plants passed through a hardening phase prior to ex vitro transfer. Clonal propagated plants established in garden soil were uniform and identical to mother plant in respect to growth characteristics and morphology. Isozymic profiles of different micropropagated clones were assessed for their genetic stability. Ten clones were tested for six isozymes. Only a few showed variation with respect to the banding pattern in esterase and superoxide dismutase. In superoxide dismutase, the two polymorphic isoforms (Rf 0.06 and 0.45) appeared in the clone C8 of the plants transferred to the field after 15 subcultural passages. Mobility and intensity of bands were monitored in other isozymes. Isozyme markers may be used as a tool for rapid screening of genetic stability in tissue cultured clones of C. terminalis.     

A practical and reliable procedure for in vitro induction of tetraploid tomato

A relatively rapid, practical and reliable procedure for in vitro production of tetraploid tomato using colchicine is described. Using this procedure 11.11% generation of tetraploids was obtained after exposure of seedling shoot meristem explants with 8 mM colchicine for 96 h. Confirmation of tetraploid production (2n = 48), was determined by flow cytometry and verified by cytogenetic analysis of root tip preparations. The results indicate that the procedure is adequate for induction and screening of tetraploid tomato plantlets.     

In vitro high frequency direct plant regeneration from whole leaves of blackberry

High frequency and direct (without callus) plant regeneration was achieved from whole leaf explants of thornless blackberry (Rubus hybrid) cv. Black Satin (EC No. 381258; PI No. 553272) in vitro. Leaf blade explants from 1-, 3- and 5-month-old mother cultures were cultured on Murashige and Skoog (MS) medium with thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), indol-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA), alone or in combination. Three-month explants cultured on 0.02 mg l⁻¹ TDZ produced a high regeneration frequency (91.7%) and the most shoots/leaf explant (17.3). The shoot primordia developed within 3 weeks from the point of detachment of the petiole from the leaf blade. The age of the explant source significantly affected the shoot regeneration potential of the leaf explants. Leaves excised from 3-month-old in vitro-cultured shoots performed better than those from 1- and 5-month-old shoots. Shoots rooted best on half-strength MS basal medium with 0.5 mg l⁻¹ IBA and 90% of the plantlets survived acclimatization. The regenerated plantlets were morphologically similar to the mother plants.     

Regeneration and characterization of Swertia chirata Buch.-Ham. ex Wall. plants from immature seed cultures

First generation immature seeds (R₁) were collected from a field transferred micropropagated plant and seeds were induced to develop organogenic calli in Swertia chirata, a traditional revenue earning medicinal plant. Half strength MS medium with different growth regulators namely, BA, Kn (2.22–4.44 μM), NAA (2.69–5.37 μM), and 2.26 μM 2,4-D were used to induce callus and organogenesis. Isolated shoots produced roots either in the same medium or in presence of NAA (2.69–10.74 μM) or IBA (2.46–9.8 μM). Fully developed plantlets were successfully transplanted to soil and the fertile seed bearing plants developed. Occasionally plants derived from more than 56 weeks old calli showed some morphological variations. Such variations in regenerated plants is not reflected in their chromosomal constitution, with normal 2n = 26 chromosomes. Likewise, no variation was observed in DNA fingerprinting patterns among the short-term raised culture regenerants, which were morphologically similar to that of the donor plant illustrating their genetical uniformity and clonal fidelity. On the contrary, variation in DNA fingerprinting patterns was observed in long-term culture raised plants.     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.     

Haploid plant production in Zantedeschia aethiopica ‘Hong Gan’ using anther culture

This report describes advances in the anther culture of Zantedeschia aethiopica. Important factors for improvement as compared to the earlier procedure were: (1) using flowers from inflorescences developed at relatively low temperature during winter, (2) high temperature stress treatment at 32 °C for 2 days in the beginning of the culture, (3) use of Gamborg B5 as anther culture medium, and (4) addition of sucrose at high concentration of 8% in the culture medium. Plants were obtained via a callus phase. Frequency of anthers producing calli was around 4–5%. About 87% of the calli gave regenerants, of which 52% were haploid, 36% were diploid and the rest had other ploidy levels. In addition to chromosome counting, cytological examination of the microspore development and amplified fragment length polymorphism (AFLP) analysis of the regenerants showed that haploid as well as diploid plants originated from the microspores. Finally, 12 doubled haploid (DH) plants could be produced from each inflorescence. One quarter of the DHs equaled the original cultivar in growth vigor, while more than one third showed good fertility, indicating that inbreeding depression was not so severe in this heterozygous species. The improved protocol now enables production of sufficient number of DHs for application of haploid technology in genetic improvement and breeding of Z. aethiopica.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

Adventitious shoot regeneration and protoplast isolation and culture were examined from leaf explants of in vitro shoot cultures of several cauliflower (Brassica oleracea var. botrytis) cultivars, sourced from Europe and Australia, was investigated with the aim to develop improved nuclear and plastid transformation protocols for this vegetable crop. Eight out of 10 cultivars regenerated shoots from at least 79% of leaf explants. Mesophyll protoplasts from leaves gave high yields and division frequencies. Growth of shoot cultures in large glass vessels with vented lids was the key factor in obtaining high protoplast division frequencies of up to 71% and at least 70% of protoplast calluses regenerating shoots.