Review in vitro-induced flowering in bamboos

Summary The majority of bamboos (Graminae) are arborescent and perennial. The erect stems (culms) of woody bamboos are useful for a wide variety of purposes. Most bamboos flower only once at the end of very long vegetative growth phases and die afterwards. Flowering in bamboos is thought to be under genetic control, occurring somewhat like an alarm clock, going off at a preset time. The nature of this genetic clock and any interaction between the ‘internal clock’ and the environment are not yet known. Because of this ‘peculiar’ flowering behavior, bamboo seeds are available only at very long intervals. Obtaining concurrent flowering in two or more species (or varieties) in space and time is difficult, making perennial seed propagation and genetic improvement by breeding nearly impossible. Besides, this peculiar flowering behavior of bamboos is also believed to have brought the giant pandas to the verge of extinction. One of the spectacular developments in the area of bamboo genetic improvement has been the precocious in vitro induction of flowering. By this method it has been possible to rapidly accelerate the reproductive development (within 3–6 mo. versus 30–60 yr in nature). This has opened the possibility of perennial seed propagation and hybridization. In vitro induction of flowering can be obtained by diverse methods which show some similarities and differences. Induction of flowering is possible in cultures derived from both juvenile and mature explants. The proportion of seedlings induced to flower is possibly influenced by genotypic variation, though the role of methods used cannot be ruled out. A cytokinin or a shift in the auxin-cytokinin equilibrium is believed to bring about in vitro induction of flowering. The pH of the media also has an influence. Induction of flowering and rhizogenesis is considered to be an antagonistic phenomenon in vitro. A comparison between in vitro and in vivo flowering in Bambusa arundinacea has shown that though smaller, in vitro-induced florets are comparable to normal florets. There is reduced pollen fertility and some impairment in pollen wall development. Biochemical studies on the in vitro-induced flowering in bamboos have shown (1) selective expression of esterase and peroxidase isozymes during transition of nonembryogenic calluses to embryogenic calluses, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots, and (2) minimal peroxidase activity before rhizogenesis and induction of flowering in vitro. There are reports of published and comparable methods having failed to induce flowering in vitro.     

Le gisement acheuléen de plein air de La Garde (Loire). Remarques sur une série lithique acheuléenne entre Rhône et Loire

The lithic assemblage of La Garde offers the opportunity to observe a Final Acheulean series located in the Loire department (south-east France). The site was probably a multi-activity place asking large tools and flakes, according to the strategies used by humans. Three main categories of large bifacial tools mainly made of flint can be described, with a triangular or oval shape and with a transversal cutting edge. These tools are more bifacial-tools than bifaces through the kind of shaping and the retouches on the cutting edges. The Levallois flaking is associated to various other types of knapping. The site, located along a small valley, suggests a human circulating between the Saône-Rhône corridor and the interior basins of the Massif Central Mountains. The Rhodanian corridor yielded little evidence of Acheulean settlements while in the Centre of France, they are numerous. La Garde proves that systematic prospecting in this area will permit in future to complete the map of the Acheulean occupations in south-east France.     

Bud break and multiple shoot formation from tissues of mature trees ofPinus caribaea andPinus kesiya

Summary Proliferation of terminal and axillary buds of 20 yr-oldPinus caribaea andP. kesiya trees was obtained on half strength DCR medium supplemented with 0.5 mg·liter^−1 6-benzylaminopurine (BA). These sprouts further elongated with the formation of multiple shoots with the ratio of 1:3, on transfer to medium in which the 0.25 mg·liter^−1 BA of the initiation medium was replaced by 0.25 mg·liter^−1 kinetin. Rooting was obtained on the same medium. Plantlets thus formed were transferred to perlite:peat:vermiculite mixture (1:1:1) in polybags (10×5 cm) under 80±5% humidity in a polyhouse. Plantlets ofP. caribaea andP. kesiya were established with 72.5 and 83.3% survival, respectively.     

In vitro plantlet regeneration from mature zygotic embryos of Pinus wallichiana A.B. Jacks

 Plantlet regeneration was achieved in blue pine (Pinus wallichiana A.B. Jacks) by organogenesis of mature zygotic embryos. The effect of various basal media and five cytokinins on adventitious bud induction, development and elongation was investigated. Half-strength Douglas fir cotyledon revised medium (DCR) supplemented with 2.5 μm N⁶-benzyladinine (BA) and 0.025 μM thidiazuron was found to be most effective in inducing adventitious buds. The effect of a BA pulse treatment was also tested, and the bud-forming capacity of each treatment was quantified. The elongation of adventitious buds was achieved on hormone-free half-strength DCR medium containing 2% sucrose and 0.05% activated charcoal. Rooting was induced in the elongated shoots with a 6-h treatment of indoleacetic acid and indolebutyric acid solutions (1 mM each). Rooted shoots were transplanted in the greenhouse for hardening and their survival percentage was 64.4 after 5 weeks and 45.7 after 6 months. Received: 11 September 1998 / Revision received: 10 February 1999 / Accepted: 26 February 1999     

A comparison of in vitro with in vivo flowering in bamboo: Bambusa arundinacea

Seedling explants of Bambusa arundinacea were cultured in a Murashige & Skoog (MS) based liquid medium, supplemented with sucrose (2), coconut water (5) and 6-benzylaminopurine (2.2 μM). In 3–6 months about 70 of the cultures flowered. A comparison was made between in vitro and in vivo flowering. Though smaller in size, in vitro florets were morphologically comparable to the in vivo florets. Anthesis in in vivo flowering took place in the morning hours. It was more or less synchronized and was dependent on the atmospheric temperature and humidity. The lemma and palea opened to expose both androecium and gynoecium to the pollinating agent (wind). In in vitro flowering, some florets opened as in their in vivo counterparts, some did not open but the anthers protruded from the tip of the partially opened lemma and palea. Anthesis was not synchronized under in vitro conditions. Pollen fertility in in vivo and in vitro flowerings were approximately 93 and 31 respectively. Studies by scanning electron microscopy showed some discrepancies in the pollen wall development in vitro. The trifid stigmas of in vivo florets were highly feathery with many papillae and withered soon after pollination or within few hours. The stigmas of in vitro developed florets were smaller with fewer and stouter papillae. They remained turgid for relatively longer periods. Seed production in in vivo flowering was profuse whereas in in vitro flowering seeds were produced only when many florets opened at the same time, in the same culture vessel. This revised version was published online in June 2006 with corrections to the Cover Date.     

An efficient protocol for genetic transformation and shoot regeneration of turmeric (Curcuma longa L.) via particle bombardment

Turmeric (Curcuma longa L.) is an important spice crop plant that is sterile and cannot be improved by conventional breeding. An efficient method for stable transformation for turmeric, C. longa L., was developed using particle bombardment. Callus cultures initiated from shoots were bombarded with gold particles coated with plasmid pAHC25 containing the bar and gusA genes each driven by the maize ubiquitin promoter. Transformants were selected on medium containing glufosinate. Transgenic lines were established on selection medium from 50% of the bombarded calluses. Transgenic shoots regenerated from these were multiplied and stably transformed plantlets were produced. Polymerase chain reaction (PCR) and histochemical GUS assay confirmed the stable transformation. Transformed plantlets were resistant to glufosinate.     

Rapid somatic embryogenesis from the nucelli of monoembryonic mango varieties

Summary A method for rapid production of somatic embryos having normal developmental morphology, from the nucellar tissue of three monoembryonic Indian mango varieties, namely, Alphonso, Mundan and Baneshan was developed. Somatic embryogenesis could be induced using Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 5.0 mg/liter (22.62 μM, gibberellic acid (GA₃) 5.0 mg/liter (14.43 μM), sucrose 60 g/liter and activated charcoal (AC) 2.5 g/liter. Maturation of embryos was observed in a reduced concentration of basal medium supplemented with abscisic acid 1.0 mg/liter (3.78 μM) coconut water 20 percent (vol/vol) casein hydrolysate (CH) 100.0 mg/liter, sucrose 40.0 g/liter and activated charcoal 2.5 g/liter. N⁶-benzyladenine (BA) 5.0 mg/liter (22.2 μM) favored normal germination of the embryos. Plant development was complete in 75 to 80 days.     

Factors influencing bud break and rooting and mass-scale micropropagation of three Phragmites species: P. karka, P. communis and P. australis

A procedure has been described for the large-scale micropropagation of three Phragmites species, P. karka, P. communis and P. australis, from axillary buds excised from the main and side branches. Position of the buds on the branches had an effect on the bud break and establishment of the cultures under in vitro conditions. Lower buds of P. australis and middle buds of P. karka and P. communis were the most suitable. The presence of yeast extract as one of the ingredients of the sprouting medium helped in the early detection of systemic contamination. Multiple shoot formation and root initiation were obtained on Murashige and Skoog's basal medium supplemented with different concentrations of BA – 0.5 mg/l for P. karka, 0.25 mg/l for P. communis and 0.1 mg/l for P. australis– 0.5 mg/l Kn and 2% sucrose (w/v). Shoots and roots elongated on half-strength MS basal medium with 2% sucrose but without any plant growth regulators. A zone of root hair was observed in the case of P. australis. Hardening occurred on 95% of the plantlets within 30 days of transfer to the polyhouse. Over 10,000 plants were produced from three buds of each species within 9 months. The plants were supplied to a private company for their industrial waste treatment. Received: 1 June 1998 / Revision received: 28 August 1998 / Accepted: 10 October 1998     

Stable transformation of mature zygotic embryos and regeneration of transgenic plants of chir pine (Pinus roxbughii Sarg.)

A particle inflow gun was used to transfer the plasmid pAHC25 containing the bar gene conferring resistance to glufosinate and the gusA reporter gene, each driven by the maize ubiquitin promoter, to mature embryos of Pinus roxburghii (chir pine). High levels of transient expression were obtained when embryos were cultured for 6 days on 10 μM benzyl adenine-containing medium and then exposed to high osmoticum (0.5 M sucrose) before and after bombardment. Selection on medium containing Basta enabled recovery of stably transformed shoots, both from the epicotyl and from adventitious buds. The primary transformed shoots from the epicotyl were multiplied via axillary shoots. Transformation was confirmed by histochemical staining for β-glucuronidase (GUS) activity, by polymerase chain reaction (PCR) amplification of fragments of gusA and nos terminator, and by the resistance of needles to Basta.     

Cryopreservation of Embryogenic Culture of Pinus roxburghii

Embryogenic cultures of chir pine (Pinus roxburghii Sarg.) were cryopreserved successfully in liquid nitrogen. It was found that using sorbitol and dimethyl sulfoxide (DMSO) as cryoprotectants was essential for the survival of the tissue. Among the different concentrations of the cryoprotectants used, the most effective treatment was observed to be 0.3 M sorbitol and 5 % DMSO. On staining the cryopreserved tissue with fluorescein diacetate, it was observed that only a few meristematic embryo heads survived and resumed growth after a very short initial lag phase. The recovered cultures showed normal regrowth on proliferation medium and, it was also observed that washing off the cryoprotectants was necessary for the cultures to survive. The results indicate that cryopreservation can be used for conserving the germplasm, and in maintaining the embryogenic capacity of the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.