MT Medium Research Articles

Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Calogênese, embriogênese somática e isolamento de protoplastos em variedades de laranja doce

Com o objetivo de produzir calos embriogênicos de citros, óvulos abortados de frutos maduros de 6 variedades de laranja doce (Citrus sinensis L. Osbeck), ‘Bahia Cabula’, ‘Baianinha’, ‘Hamlin’, ‘Orvalho de Mel’, ‘Rubi’ e ‘Valência’ foram introduzidos em meio de cultivo MT modificado com adição de extrato de malte, com e sem adição de 5 mg L-1 benziladenina (BA). Os calos obtidos das variedades ‘Bahia Cabula’, ‘Orvalho de Mel’, ‘Rubi’ e ‘Valência’ foram cultivados em meio basal MT, contendo os carboidratos maltose, galactose, lactose, glicose ou sacarose nas concentrações de 18; 37; 75; 110 e 150 mM para a indução da embriogênese somática. Os calos destas mesmas variedades foram submetidos ao isolamento de protoplastos, com o uso de 3 soluções enzimáticas. Os resultados revelaram que há influência do genótipo sobre a calogênese. A embriogênese somática foi estimulada na presença de galactose, para a maioria das variedades. A solução enzimática composta de 1% de celulase, 1% de macerase e 0,2% de pectoliase foi a mais adequada para o isolamento de protoplastos a partir de calos das variedades de laranja doce estudadas.

Poliembrionia e número de sementes por fruto de quatro cultivares de tangerineira

Quatro cultivares de tangerineiras foram caracterizados quanto ao número de sementes por fruto e quanto à poliembrionia, três dos quais são usados como genitores no programa de melhoramento genético do Departamento de Horticultura e Silvicultura da Universidade Federal do Rio Grande do Sul. Coletaram-se frutos de plantas da coleção de citros da Estação Experimental Agronômica, em Eldorado do Sul, RS, cujas sementes foram retiradas e contadas. Em câmara estéril, os embriões foram extraídos das sementes e inoculados em meio de cultivo MT (Murashige & Tucker, 1962), permanecendo por 49 dias a 26±3°C sob fotoperíodo de 16h. As tangerineiras Caí, Montenegrina (Citrus deliciosa Tenore), Poncã (C. reticulata Blanco) e King (C. nobilis Loureiro) produziram, em média, 16, 14, 8 e 24 sementes viáveis por fruto, respectivamente, contendo um número médio de 5,7, 5,7, 7,7, e 3,2 embriões por semente. A produção de sementes monoembriônicas foi baixa, exceto no cultivar King, que apresentou 20,4% das sementes com apenas um embrião. Registrou-se a ocorrência de embriões albinos, que constituíram uma parte ou a totalidade do conteúdo de algumas sementes. De modo geral, as espécies apresentaram diferenças significativas quanto às características estudadas. As similaridades observadas entre os dois cultivares de C. deliciosa reforçam a hipótese de Montenegrina ter se originado de Caí.

Plant regeneration from embryogenic calli of six Citrus related genera

Somatic embryos were induced from immature seeds of eight Citrus relatives including Aegle marmelos, Atalantia ceylanica, Citropsis gabunensis, Clausena excavata, Glycosmis pentaphylla, Microcitrus australasica, Murraya paniculata and Severinia buxifolia on MT medium supplemented with 0.05 mg l−1 2,4-D, 0.05 mg l−1 BA and 400 mg l−1 malt extract. Approximately 20% of somatic embryos from six of the genera underwent callogenesis during subculture on the same medium. After at least six successive subcultures on MT medium containing 10 mg l−1 BA, calli became growth regulator independent. Somatic embryos from growth regulator independent calli were obtained with MT medium containing 5% lactose. Plantlets were regenerated from somatic embryos on MT medium containing 0.02 mg l−1 NAA, and established in soil.

In vitro flowering of orange jessamine (Murraya paniculata (L.) Jack)

Branch internodes ofMurraya paniculata flowered in vitro on half-strength MT medium containing 5% sucrose supplemented with cytokinins. The cytokinins only induced vegetative shoots from the epicotyl of seedlings. The highest percentage of flowering (76.7% of the explants) was achieved from branch internodes of flowering plants close to the apex, on half-strength MT medium containing 5% sucrose and 0.001 mg l1 benzyladenine (BA) in light. Flowering required two days' exposure to BA, but shoot formation could be initiated even without exposure to BA.

Somatic hybridization between Citrus reticulata and Citropsis gabunensis through electrofusion

Protoplasts from embryogenic calli of Citrus reticulata Blanco cv. Ponkan and Citropsis gabunensis (Engl.) Swing. & M. Kell (Cabon Cherry Orange), were isolated and fused using electric current. Maximum fusion frequency was obtained with AC at 75 kV/cm (1.0 MHz) for 15 s, followed by DC square-wave pulses at 1.25 kV/cm for 40 μs. Fusion-treated protoplasts were cultured on MT medium containing no growth regulators, solidified with 0.6% Bacto Difco agar. Protoplast-derived calli were proliferated on MT medium containing 1 mg/l zeatin and 0.9% agar. A total of 31 lines of somatic hybrid calli were obtained by screening on the basis of chromosome count and isozyme analysis. The somatic hybrids were tetraploid (2n=36). Plants were regenerated from the calli via somatic embryogenesis. The somatic hybrid plants exhibited morphological characteristics intermediate to the parental plants.

SOMATIC HYBRIDIZATION BETWEEN CITRUS RETICULATA AND CITROPSIS GABUNENSIS THROUGH ELECTROFUSION

Protoplasts were isolated from embryogenic calli of Citrus reticulata cv. Ponkan and Citropsis gabunensis, and fused in electric fields. The maximal fusion efficiency was obtained by application of AC at 75 V/cm (1.0 MHz) and DC square-wave pulse at 1.125 KV/cm for 40 usec. Fusion-treated protoplasts were cultured on MT medium without phytohormone, solidified with 0.6% agar. Colonies from the protoplasts were proliferated on MT medium with zeatin 1 mg/l and 0.9% agar. Selection of somatic hybrid callus was based on chromosome count and isoenzyme analysis. The somatic hybrids were tetraploid (2n=36). C. reticulata and C. gabunensis were both homozygous at Got-1 locus, but distinguishable easily because band of the latter migrated faster than that of the former. In zymogram of somatic hybrid, both parent bands were retained and a new hybrid band was also evident between them. Embryos from somatic-hybrid callus regenerated intact plant. The hybrid plants showed intermediate morphological characteristics of the parents.

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.