Physical and physiological changes of Crambe abyssinicaHochst seed during maturation

Determination of seed-maturation indicators enables the identification of the idealmoment for harvest to achieve the best production and conservation potential. Our objective here was to evaluate some physical and physiological changes of crambe (Crambe abyssinica Hochst) seeds as possible indicators of seed maturation. Crambe floweringwas monitored in Dourados, Mato Grosso do Sul, Brazil. Plants were tagged, and 13, 22, 26, and 28 days after the initiation of flowering, the seeds were collected and following physical attributes evaluated: length, diameter, total mass, dry matter and water content. Physiological quality of the seeds was assessed using the germination test, by registering the percentage of normal seedlings and dormant seeds, immediately after each harvest, and again after six months of storage. The water-absorption curveswere characterized as a function the seed-development stages. All physical attributes were observed to increase because of the accumulation of reserve substances during seed development, except for water content, which gradually decreased from 72.2% at the start of development to 29.5% at maturity. At 28 days after anthesis the germination percentage of crambe seeds at physiological maturity was only 17%, indicating that they became dormant while maturing. However, seed germination rate was 89% after six months of storage, indicating that dormancy was almost fully overcome after this period.

Foliar application of plant growth regulators changes the physiological quality of crambe seeds

The application of plant growth regulators can manipulate the development of cultivated plants, and their physiological effects may be reflected in the physiological quality of the seeds. This study evaluated the effects of plant growth regulators (PGR) on the physiological quality of crambe seeds produced in the 2014 and 2015 harvests. During crop development, we applied two foliar sprayings of the following treatments: 1) control treatment with distilled water; 2) indole-3-acetic acid 100 mg L-1 (IAA); 3) 3-gibberellic acid P.A. 100 mg L-1 (GA3); 4) commercial PGR Stimulate® 6 mL L-1. The seeds were harvested and evaluated for water content, germination percentage and germination speed index (GSI), electrical conductivity and concentrations of leached Ca, Mg and K ions. Means were compared by Tukey ́s and Dunnett tests (p<0.05). Applying Stimulate® increased the germination percentage and the germination speed index, while IAA application showed an opposite effect, increasing the electrical conductivity of crambe seeds. Calcium leaching decreased with IAA application, and all regulators reduced K leaching. We therefore infer that the application of plant growth regulators influences the physiological quality of crambe seeds.

Metabolic engineering of fatty alcohol production in transgenic hairy roots of Crambe abyssinica.

Biotechnological production of fatty alcohols, important raw materials in the chemical industry, has been receiving considerable attention in recent years. Fatty alcohols are formed by the reduction of fatty acyl-CoAs or fatty acyl-ACPs catalyzed by a fatty acyl reductase (FAR). In this study, we introduced genes encoding FARs from Arabidopsis thaliana (AtFAR5) and Simmondsia chinensis (ScFAR) into Crambe abyssinica hairy roots via Agrobacterium rhizogenes-mediated transformation. The efficiency of the transformation ranged between 30 and 45%. The fatty alcohols were only detected in the transgenic hairy root lines expressing ScFAR gene. In all tested lines stearyl alcohol (18:0-OH), arachidyl alcohol (20:0-OH), and behenyl alcohol (22:0-OH) were produced. The content of 18:0-OH varied from 1 to 3% of total fatty acids and fatty alcohols, while the amount of either 20:0-OH and 22:0-OH did not exceed 2%. The transgenic hairy root lines produced from 0.98 to 2.59 nmol of fatty alcohols per mg of dry weight. Very low activity of ScFAR was detected in the microsomal fractions isolated from the selected hairy root lines. To our knowledge, this is the first report on the fatty alcohol production in the hairy root cultures. Biotechnol. Bioeng. 2017;114: 1275-1282. © 2016 Wiley Periodicals, Inc.

Plant-pollinator interactions in Crambe abyssinica Hochst. (Brassicaceae) associated with environmental variables.

Despite the economic importance of Crambe abyssinica, the present study is the first report on bees that occur with this species, and could aid in developing alternative methods for controlling insect pests without seriously impacting pollinators. The present study examined the following questions: (1) Which species are potential pollinators of C. abyssinica? (2) How do environmental conditions influence pollinator visitation fluctuations? Insects were sampled on a weekly basis between 08:00 and 16:00 during five weeks of flowering. When the results of analyses of variance were significant, the data was adjusted using regression equations at a 5% level of probability; the environmental variables were examined using Pearson's correlation coefficient. Apis mellifera, Geotrigona mombuca, Exomalopsis (Exomalopsis) fulvofasciata, Plebeia sp. and Dialictus sp. were observed visiting C. abyssinica flowers. A. mellifera and G. mombuca were observed to be potential pollinators, with the former demonstrating visitation peaks during flowering weeks II and IV at 12:00 and 08:00, respectively and the latter visiting during weeks III and IV at 12:00 and 10:00, respectively. Environmental factors such as temperature, relative humidity, and wind speed have different effect on the activity of bees.

Regeneration and transformation of Crambe abyssinica.

BACKGROUND: Crambe abyssinica (crambe) is a non-food oil seed crop. Its seed oil is widely used in the chemical industry because of the high erucic acid content. Furthermore, it is a potential platform for various feedstock oils for industrial uses based on genetic modification. Here, we describe the development of a series of protocols for all steps required in the process of generating genetically modified crambe. RESULTS: Different explant types from crambe seedlings were tested for shoot regeneration using different hormone-combinations. Cotyledonary nodes on basic medium with 0.5 µM NAA and 2.2 µM BAP gave the highest regeneration percentages. For propagation by tissue culture, explants of stems, petioles, leaves and axillary buds of in vitro plantlets were tested using the optimized medium. Axillary buds showed the highest shoot proliferation efficiency. Cotyledonary nodes were used to test the proper concentration of kanamycin for selection of transformation events, and 10 to 25 mg · L(-1) were identified as effective. The cotyledonary nodes and cotyledons from 7-day-old seedlings were used in Agrobacterium-mediated transformations with two kinds of selection strategies, shifting or consistent. Using the shifting selection method (10 mg · L(-1) kanamycin, 25 mg · L(-1), then back to 10 mg · L(-1)) cotyledonary nodes gave 10% transformation frequency, and cotyledons 4%, while with the consistent method (25 mg · L(-1)) lower frequencies were found, 1% for cotyledonary nodes and 0% for cotyledons). Later, in vitro plant axillary buds were tried as explants for transformation, however, transformation frequency was low ranging from 0.5 to 2%. Overall, testing six different vectors and two kinds of Agrobacterium strains, the average transformation frequency using the shifting method was 4.4%. Determining T-DNA insertion numbers by Southern blotting showed that approximately 50% of the transgenic lines had a single-copy insertion. CONCLUSIONS: Present research revealed the potential of using crambe meristematic tissue for genetic transformation and in vitro propagation. The most efficient method of transformation used cotyledonary node explants from 7-days-old seedlings with a shifting kanamycin selection. Meristematic tissues (cotyledonary node or axillary bud) had the highest ability for shoot proliferation. Single-copy T-DNA insert lines could be efficiently and reproducibly generated.

Production and genetic characterization of interspecific hybrids among Crambe abyssinica, Crambe hispanica and Crambe kralikii.

In this paper, interspecific crosses among Crambe abyssinica, Crambe hispanica, and Crambe kralikii were reported. In the C. hispanica x C. abyssinica (H x A) cross, 118 F1 hybrids were produced without embryo rescue, while 5 F1 hybrids were obtained with embryo rescue, when C. hispanica was used as the female parent. In the reciprocal cross (A x H), 232 hybrids were obtained without embryo rescue. From more than 1000 C. kralikii flowers pollinated with pollen grains of C. abyssinica (K x A), only 2 F1 hybrids were obtained with embryo rescue, whereas the reciprocal cross produced no hybrids, even with embryo rescue. The hybrids were confirmed at the morphological, cytological, and molecular levels. In the combinations of A x H and H x A, many BC1 hybrids were obtained without embryo rescue. In contrast, in the K x A cross, only 7 BC1 plants were obtained with embryo rescue, while no seed set was achieved under self-pollination or in backcrosses without embryo rescue. In the H x A F1 hybrids, the pollen stainability was 65.4-86.0%, with an average of 76.9%. In comparison, the pollen viability of hybrids in the reciprocal cross (A x H) ranged from 66.2 to 81.1%, with an average of 75.4%. Fertile pollen grains were not found in the K x A F1 hybrids. All F1 hybrids of the 3 crosses (H x A, A x H, and K x A) had the expected 2n = 75 chromosomes. AFLP analyses indicated that all F1 hybrids and their progenies had typical bands of the parents. These hybrids and progenies are anticipated to be valuable for future C. abyssinica improvement in breeding programs.

Ecophysiological response of Crambe maritima to airborne and soil-borne salinity.

BACKGROUND AND AIMS: There is a need to evaluate the salt tolerance of plant species that can be cultivated as crops under saline conditions. Crambe maritima is a coastal plant, usually occurring on the driftline, with potential use as a vegetable crop. The aim of this experiment was to determine the growth response of Crambe maritima to various levels of airborne and soil-borne salinity and the ecophysiological mechanisms underlying these responses. METHODS: In the greenhouse, plants were exposed to salt spray (400 mM NaCl) as well as to various levels of root-zone salinity (RZS) of 0, 50, 100, 200 and 300 mM NaCl during 40 d. The salt tolerance of Crambe maritima was assessed by the relative growth rate (RGR) and its components. To study possible salinity effects on the tissue and cellular level, the leaf succulence, tissue Na(+) concentrations, Na(+) : K(+) ratio, net K(+)/Na(+) selectivity, N, P, K(+), Ca(2+), Mg(2+), proline, soluble sugar concentrations, osmotic potential, total phenolics and antioxidant capacity were measured. KEY RESULTS: Salt spray did not affect the RGR of Crambe maritima. However, leaf thickness and leaf succulence increased with salt spray. Root zone salinities up to 100 mM NaCl did not affect growth. However, at 200 mM NaCl RZS the RGR was reduced by 41 % compared with the control and by 56 % at 300 mM NaCl RZS. The reduced RGR with increasing RZS was largely due to the reduced specific leaf area, which was caused by increased leaf succulence as well as by increased leaf dry matter content. No changes in unit leaf rate were observed but increased RZS resulted in increased Na(+) and proline concentrations, reduced K(+), Ca(2+) and Mg(2+) concentrations, lower osmotic potential and increased antioxidant capacity. Proline concentrations of the leaves correlated strongly (r = 0.95) with RZS concentrations and not with plant growth. CONCLUSIONS: Based on its growth response, Crambe maritima can be classified as a salt spray tolerant plant that is sensitive to root zone salinities exceeding 100 mM NaCl.