Production of plant proteases in vivo and in vitro — A review

A embriogenese somatica in vitro e dependente de uma serie de fatores, dentre os quais o genotipo da planta matriz doadora de explantes e a fonte de auxina para conferir competencia embriogenica sao considerados como determinativos. Uma vez obtidos, os embrioes somaticos podem ser encapsulados em alginato para a obtencao de sementes sinteticas, permitindo assim o armazenamento em baixas temperaturas e o posterior cultivo de maneira semelhante a semente verdadeira. Os aspectos citados foram estudados no presente trabalho visando elucidar pontos de controle da embriogenese somatica. Assim, embrioes zigoticos excisados de frutos maduros de cinco acessos de Feijoa sellowiana Berg, foram avaliados quanto ao seu potencial embriogenetico. Choques auxinicos de 2,4-D (20 mM) em periodos de 1, 2, 4, 8 e 16 semanas foram utilizados para estabelecer o periodo minimo de aquisicao de competencia embriogenica dos explantes e o posterior efeito deste fitorregulador sobre o desenvolvimento e qualidade dos embrioes somaticos. Diferentes composicoes de capsulas de alginato foram empregadas para avaliar a polimerizacao das capsulas e a contaminacao das sementes sinteticas. A maior percentagem de inducao da embriogenese somatica e o maior numero de embrioes ocorreram nos acessos 101 e 50-4. Choques de 2,4-D de duas semanas foram suficientes para proporcionar valores similares de inducao e producao de embrioes somaticos, aqueles obtidos com choques de 4 e 8 semanas. Estudos histologicos demonstraram a origem direta da embriogenese somatica a partir da epiderme cotiledonar. A adicao dos sais de MS e sacarose ao alginato de sodio resultou em altos valores de contaminacao das sementes sinteticas, quando comparados as capsulas isentas destas substâncias. O emprego do KNO3 (100 mM) permitiu a despolimerizacao e a abertura de 81,2% das sementes sinteticas em comparacao aos valores de 0% para o tratamento com agua.

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Somatic embryogenesis in goiabeira serrana: genotype response, auxinic shock and synthetic seeds

Distribution and potential of Cherimoya (Annona cherimola Mill.) and highland papayas (Vasconcellea spp.) in Ecuador

Food and shelter are two basic needs of life. Since the earliest times, humans have enjoyed eating fresh fruits, as indicated by the frequent mention of fruits throughout the recorded history. The cultivation of fruits such as datepalm and pomegranate, has been dated back to 7000 to 3500 BC, respectively (Singh, 1985). In today’s world fruit play a vital role in our diet due to its nutritional importance (Nagy et al., 1990). Many fruit species are trees and with a few exceptions, e.g. banana, pineapple and strawberry, are woody. Genetic improvement of fruit crops by conventional plant breeding achieved through the selection of superior genotypes from genetically variable population derived from sexual recombination and seedling propagation (Bose, 1985). Tree architecture, floral morphology, prolonged juvenile period and irregularities in bearing habit limits the improvement of fruit trees through traditional breeding methods (Nijjar, 1977). Since most important horticultural characteristic, conferred by complexes of genes, the genetic integrity of many important fruit tree cultivars has been maintained by means of vegetative propagation. Using the conventional methods of vegetative propagation such as, cutting, layering, and grafting, substantial number of plants are produced. However, many tropical fruits and spice trees (e.g. clove) can not be propagated asexually (Litz, 1984b). Due to the increasing significance of tropical fruits for food and export income (Underhill, 1993), there has been a recent expansion of efforts and resources devoted to tropical fruit cultivar improvement. It is almost imposible to keep pace with the increasing demand of tropical fruits only through the conventional methods of plant propagation. For improvement of tropical and subtropical fruits by somatic cell genetics, a highly efficient regeneration system is a prerequisite (Litz and Jaiswal, 1991). Many programs are attempting to integrate emerging biotechnologies with conventional breeding methods to facilitate and expedite the fruit tree cultivars improvement (Litz, 1997). These include in vitro culture and micropropagation (Grosser, 1994), production of homozygous parental lines by chromosome doubling of haploids (Zhang and Lespinasse, 1992), regeneration of interspecific and intergeneric hybrids for root stock development (Gmitter et al., 1992; Ochatt et al., 1992), production of artificial seeds (Akhtar and Jaiswal, 1994; Bajaj, 1995a, b, Gray, 1987a, c; Redenbaugh, 1993), in vitro selection of somaclonal variation having enhanced diseased resistance (Hammerschlag, 1992; Jain et al., 1997), genetic transformation for specific horticultural traits (Gradner, 1993; Oliveira et al., 1996)

Somatic Embryogenesis in Tropical Fruit Trees

Summary Somatic embryogenesis is a relevant micropropagation technique in Carica papaya �Goldenin view of the difficulties met in conventional seed propagation and the lack of an effective method for early sex determination in this trioecious species. Considering the interest in large-scale production of C. papaya seedlings, we adapted a somatic embryogenesis procedure associating liquid system and immature zygotic embryo explants. This protocol markedly contributed to an increase in the yield of friable embryogenic calli, resulting in a large number of normal somatic embryos at various developmental stages. From these embryos, plantlets were regenerated and evaluated for occurrence of somaclonal variation. Flow cytometry screening revealed diploid (88%), tetraploid (6%), and aneuploid (6%) plantlets. Owing to an interest in the mass propagation of diploid individuals, the karyotype of these plantlets was also analyzed. With this approach, diploidy (2X=18) was confirmed and no structural aberrations were found. Considering the results and the applicability of large-scale production of somatic embryos in C. papaya, the tissue culture protocols shown here could be used for commercial purposes. Moreover, the cytometric and cytogenetic analyses were crucial for a rapid, reliable and conclusive evaluation of the genetic stability of C. papaya plantlets regenerated by somatic embryogenesis.

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Massal Induction of Carica papaya L. ‘Golden’ Somatic Embryos and Somaclone Screening by Flow Cytometry and Cytogenetic Analysis

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenne et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.

J. Velozo

Papers

Improvement of somatic embryogenesis in highland-papaya cell suspensions