High frequency plant regeneration in Indian Siris via cyclic somatic embryogenesis with biochemical, histological and SEM investigations

Cassava is a key food security staple and a competitive feedstock for multiple industrial processes and end uses. Farmers grow hybrids which are reproduced vegetatively. Several programs have used the same breeding scheme for the last 40 years. Significant progress has been made, particularly with the first improved varieties released in the 1980s and 1990s. However, gains slowed down since then. Biotechnology tools, after more than two decades, have not yet had impact on increasing yields. Results from ongoing genomic selection show promising results for high-heritability traits, but not for fresh root yield (FRY). Key challenges to increase FRY are the strong influence of nonadditive genetic effects and the heterozygous nature of breeding parents. There is large within-family variation masking the true breeding value of each progenitor. To improve yields, breeding must shift from making crosses where breeders hope to find hybrids that are superior to those already available (a strategy that has made only slow progress in the last decade or two) to develop and improve inbred progenitors that can produce more reliably better hybrids. The use of inbred progenitors and implementation of reciprocal recurrent selection should be an efficient way to exploit heterosis and epistasis, which are large components in the determination of FRY. Induction of flowering would also accelerate genetic gains. In the near-term future, available molecular markers can be used to improve the breeding value of progenitors rather than in selection of segregating progenies. They can also be used to screen germplasm collections in search of useful traits.

Cassava (Manihot esculenta Crantz)

The uncommon ability of monoembryonic seeds of Eugenia to develop seedlings after cutting or even after the removal of more than half of their storage tissue has recently been reported. Some Eugenia species are included among those at risk of disappearing, so the regeneration of plantlets from fragments of seeds could be an important mechanism both for the production of more than one normal seedling per seed and to understand the survival strategies of these species. Because the period in which this ability is present has not been studied, we analyzed the regenerative capacity of seeds of five Brazilian native species of Eugenia, considering two levels of maturity and three germination phases. Mature seeds have a greater potential to regenerate than immature ones; germinating seeds showed that the longer the root protrusion, the smaller the potential to regenerate new seedlings. However, our results clearly demonstrate that the ability to regenerate new roots or complete seedlings is present during a long period from the beginning of development until the late phases of germination. Cutting is potentially important to obtain plants on a larger scale. Because of intense predation of these seeds, this ability could be linked to an essential strategy for survival even after the removal of more than half of the storage tissues. As an alternative strategy to orthodox seeds, whose preservation is based on tolerance to desiccation, the recalcitrant seeds of Eugenia probably adopted the great reserve quantity and the regenerative capacity, allowing them to undergo successive germinations.

The development of seedlings from fragments of monoembryonic seeds as an important survival strategy for Eugenia (Myrtaceae) tree species

Wild populations of Paphiopedilum niveum (Rchb.f.) Stein, an endangered orchid species, have been threatened with extinction due to climate change and overcollection. Conventional methods of propagation are unable to meet market demands. The current study reports the production of genetically stable, protocorm-derived protocorm-like bodies (PLBs) of P. niveum that can be used for commercial production. These PLBs are referred to hereafter as somatic embryos (SEs). Factors affecting SE induction and SE proliferation were examined. The highest percentage of SE formation (68.33 ± 11.77%) and the maximum number of SEs per explant (5.19 ± 0.67) were obtained on modified Vacin and Went (MVW) medium containing 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The highest increase in fresh weight (FW) of proliferated SEs (183.33 ± 28.93 mg per 100 mg initial FW) was gained on hormone-free MVW. These SEs eventually developed into vigorous plantlets after culturing on MVW supplemented with 0.2% (w/v) activated charcoal and 50 g L−1 banana homogenate for 12 wk. A histological study revealed that SEs originated from both the epidermal and the sub-epidermal layers of the original protocorm via direct somatic embryogenesis. The genetic homogeneity between the mother generation (V1; the original protocorm) and subsequent generations (V2 and V3; the primary and secondary somatic embryos, respectively) was shown to be identical via random amplification of polymorphic DNA (RAPD) assessment. All regenerated plants grew well under greenhouse conditions.

In vitro cloning via direct somatic embryogenesis and genetic stability assessment of Paphiopedilum niveum (Rchb.f.) Stein: the endangered Venus’s slipper orchid

Stevia rebaudiana (Asteraceae) is a plant of economic importance because of its medicinal properties and the presence of sweetener compounds on its leaves. These compounds can be a substitute for sucrose in a wide variety of products used by persons with diabetes and obesity problems. To standardize an efficient and effective propagation method for the different Stevia genotypes grown in Colombia, this study evaluated the effect of different combinations of the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 6-(gamma, gamma-dimethylallylamino) purine (2iP) and Zeatin on the induction and development of somatic embryos. Adenine and coconut water were also evaluated as supplements in the basal culture medium Murashige and Skoog Basal Salt Mixture (MS) with glutamine. The combination of 2,4-D (18.09 µM) and 2iP (7.38 µM) produced the highest number of somatic embryos per explant, which had well-defined characteristics. The genotype showed a significant effect on the embryogenic response. In the "SRQ-93" genotype, the formation and development of somatic embryos was achieved, whereas the genotypes "Bertoni" and "Morita II" only yielded embryogenic and non-embryogenic calli, respectively. The conversion to seedlings was achieved on the regeneration medium containing gibberellic acid (GA 3 ) (0.29 µM) and activated charcoal.

EFFECT OF GENOTYPE ON THE in vitro REGENERATION OF Stevia rebaudiana VIA SOMATIC EMBRYOGENESIS Efecto del genotipo sobre la regeneración in vitro de Stevia rebaudiana a través de embriogénesis somática

In 2005, the Rayong-FCRC released a new high-yielding cassava cultivar designated Rayong 9 (R9) for ethanol production. However, the rate of distribution to farmers has been limited by the traditional vegetative propagation method of this crop which does not always satisfy the needs in planting material. The objective was to improve secondary somatic embryogenesis of the cassava clone Rayong 9 (R9) selected in Thailand by the Rayong Field Crops Research Center (Rayong-FCRC) for its suitability to produce ethanol. Fragments of cotyledon-stage somatic embryos were subcultured onto MS medium supplemented with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D) at 4 mg/l in sight of inducing secondary somatic embryogenesis. Five different cytokinins, 6-benzylaminopurine (BAP), kinetin, zeatin, isopentenyladenine (2-iP) and adenine were added at 1 mg/l to the induction and maturation media to test their efficiency. Onto cytokinin-free media, 70 to 80% of explants produced embryoids, each explant giving 2 to 4 new embryoids within 7 weeks. The conversion rate of the embryoids into plantlets ranged from 11 to 26% depending on the type of cytokinin. With the exception of adenine, the other cytokinins inhibited the intensity of somatic embryogenesis, by 75% in the case of zeatin and 30% in the case of kinetin. Addition of adenine did not significantly improve the number of embryoids per explant. However, at 10, 20 and 40 mg/l adenine tended to improve the process relatively to embryoid sizes and plantlet survival rates in the greenhouse. 

	 

	Key words: Adenine, biofuel, cassava, cotyledonary-stage, embryoids, icrocuttings, micropropagation.

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P Wongtiem

Papers

Effects of cytokinins on secondary somatic embryogenesis of selected clone Rayong 9 of Manihot esculenta Crantz for ethanol production