RESUMO
Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2-3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.
Assuntos
Edição de Genes , Gossypium , Edição de Genes/métodos , Gossypium/genética , Sistemas CRISPR-Cas/genética , Plantas Geneticamente Modificadas/genética , Genótipo , Transformação GenéticaRESUMO
This study aims to highlight the major effects of biochar incorporation into potting soil substrate on plant growth and performance in early growth stages of five elite Italian varieties of durum wheat (Triticum durum). The biochars used were obtained from two contrasting feedstocks, namely wood chips and wheat straw, by gasification under high temperature conditions, and were applied in a greenhouse experiment either as pure or as nutrient-activated biochar obtained by incubation with digestate. The results of the experiment showed that specific genotypes as well as different treatments with biochar have significant effects on plant response when looking at shoot traits related to growth. The evaluated genotypes could be clustered in two main distinct groups presenting, respectively, significantly increasing (Duilio, Iride, and Saragolla varieties) and decreasing (Marco Aurelio and Grecale varieties) values of projected shoot system area (PSSA), fresh weight (FW), dry weight (DW), and plant water loss by evapotranspiration (ET). All these traits were correlated with Pearson correlation coefficients ranging from 0.74 to 0.98. Concerning the treatment effect, a significant alteration of the mentioned plant traits was observed when applying biochar from wheat straw, characterized by very high electrical conductivity (EC), resulting in a reduction of 34.6% PSSA, 43.2% FW, 66.9% DW, and 36.0% ET, when compared to the control. Interestingly, the application of the same biochar after nutrient spiking with digestate determined about a 15-30% relief from the abovementioned reduction induced by the application of the sole pure wheat straw biochar. Our results reinforce the current basic knowledge available on biological soil amendments as biochar and digestate.
RESUMO
BACKGROUND: Biotechnological breeding of elite sugarcane cultivars is currently limited because of the difficulty of regenerating plants by tissue culture. Here, we report that commercially elite sugarcane genotypes, which are adapted to Argentinian agro-ecological conditions, are capable of being regenerated via indirect somatic embryogenesis. Leaf rolls of five elite genotypes were cultured following two callus induction protocols using different concentrations of 2,4-D as the growth regulator. Embryogenic calluses were regenerated under light conditions. Regenerated plants were subsequently acclimatized in the greenhouse under two acclimatization procedures before being transplanted to the field. RESULTS: Four of the five genotypes were able to form somatic embryos following the two induction protocols. The variables related to embryogenic callus production were influenced by the interaction between genotype and culture conditions. For plant regeneration, the embryogenic calluses were further cultured on an IBA-supplemented medium, where we observed a high genotype dependence. Calluses from the four cultivars regenerated a good number of plants. With the procedures described here, we obtained more than 90% of well-acclimatized plants both in the greenhouse and in the field. CONCLUSIONS: This protocol provides a simple way to regenerate sugarcane plants through indirect somatic embryogenesis. Also, the results confirm that tissue culture ability is highly genotype-dependent in sugarcane. Our findings suggest that these elite cultivars could be good candidates for biotechnological breeding.
RESUMO
La yuca (Manihot esculenta Crantz) es un cultivo de alta importancia en países tropicales. La transformación genética de yuca ha sido posible desde hace 15 años mediante la producción que callo embriogénico friable (CEF) a partir de embriones somáticos. En el presente trabajo se evalúan la inducción de embriones somáticos usando tres diferentes auxinas sintéticas y la producción de CEF a partir de éstos en los cultivares de yuca SG107-35 y BRA685. Estos cultivares son resistentes a la bacteriosis vascular de yuca cuyo agente causal es Xanthomonas axonopodis pv. manihotis, una de las principales limitantes del cultivo. Los resultados obtenidos muestran que en ambos cultivares la hormona Picloram a una concentración de 12 mg/l fue más eficiente que 2,4-D y Dicamba para producir embriones somáticos. Adicionalmente se consiguió la producción de CEF y la regeneración de plantas mediante embriogénesis somática en el cultivar BRA685. Los resultados del presente trabajo son importantes para evaluar la transformabilidad de distintos cultivares de yuca. Actualmente este número es bastante reducido principalmente porque la producción de CEF es fuertemente influenciada por el genotipo. Por tal razón solo se transforma de manera rutinaria y eficiente en el cultivar 60444. La posibilidad de transformación de distintos cultivares de yuca permitirá explotar la enorme variabilidad del cultivo, invitándonos a aumentar los esfuerzos para mejorar y universalizar los protocolos de transformación de yuca.
The cassava (Manihot esculenta Crantz) crop has a very important role as a food, feed and a raw material in developing countries; therefore it is a priority to develop technologies oriented to the solution of problems and agronomic improvement of the crop. The genetic transformation of cassava was developed 15 years ago by producing friable embryogenic callus (FEC) from somatic embryos as target tissue for transformation. In the present work we evaluated the induction of somatic embryos by using three different synthetic auxins and the production of FEC from both SG107-35 and BRA685 cassava cultivars; both are resistant to cassava bacterial blight caused by Xanthomonas axonopodis pv. manihotis, the most important bacterial disease affecting the crop. Our results showed that in both cultivars gave rise to somatic embryos in media containing Picloram at a concentration of 12 mg/l being more efficient than using 2,4-D or Dicamba. Additionally the cultivar BRA685 produced regenerative FEC giving rise to plants through somatic embryogenesis. However compared to the model cultivar 60444, FEC production was greatly lower. This work shows new efforts to increase the number of transformable cultivars of cassava and take advantage of the enormous genetic variability of the crop.