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Rapid Non-destructive Determination of Fatty Acids in Single Groundnut Seeds by Gas Chromatography
Abstract
A protocol for analyzing 8 fatty acids using 5～ 2O mg groundnut seed tissue was described，
which required 35 min for preparation at room temperature and 18 min for gas chromatography analysis．The results of analysis proved to be accurate and reproducible，irrespective of sampling amount
and fatty acid content．The protocol caters the need for non-destructive sampling and gene pyramiding
in genetic and breeding studies in groundnut，and may facilitate genetic improvement in fatty acid
composition of the groundnut crop．
... The single peanut seed with its seed coat removed was ground into a fine powder, 20mg of which were then placed into a 5 ml centrifuge tube. The pretreament protocol of Yang et al. (2012) was strictly followed. The fatty acid composition of peanut seeds was determined on an Agilent Model 7890A gas chromatography (GC) with a flame ionization detector (FID). ...
... The fatty acid composition of peanut seeds was determined on an Agilent Model 7890A gas chromatography (GC) with a flame ionization detector (FID). The procedure for determination of fatty acid composition was according to the optimized program B previously described by Yang et al. (2012). The split ratio was increased to 30:1. ...
from 5 sections and 1 unnamed species were determined by gas chromatography in this study. The results showed that palmitic, stearic, oleic, linoleic, arachidic, eicosenoic, behenic and lignoceric acid content, as percentage of total fatty acids, ranged from 6.506 to12.436%, 1.033 to 8.117%, 25.096 to 67.907%, 13.821 to 53.235%, 0.623 to 3.825%, 0.636 to 2.907%, 2.317 to 9.048% and 0.799 to 3.771%, respectively. Considerable variation in fatty acid components was observed among the Arachis species analyzed. Lowest content of palmitic acid was identified in A. paraguariensis ssp paraguariensis (A15-2-3), and lowest content of stearic acid in A. duranensis (A5-3). One plant was identified as with 6.506% palmitic acid content. The highest oleic acid content was found in A.correntina and the highest linoleic acid in A. paraguariensis ssp paraguariensis. One plant (A14-3) with 67.907% oleate and an O/L ratio of 4.91 were identified. Wild peanut plant materials with so high oleic acid content have not been reported till now. Wild peanut species with low palmitic/stearic acid content or with high oleic content identified from this study can be utilized for developing peanut cultivars with better nutritional quality.
... Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. 2014). T 1 generation seeds of transgenic lines with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices following the protocol of Yang et al. (2012). ...
Compared to its normal-oleic counterpart, high-oleic peanut has better keeping quality and much more health benefits. Breeding high-oleic peanut through conventional means is a tedious process that typically takes several years. Genome editing, however, may shorten the duration. This study aimed to test the effectiveness of the node injection method coupled with CRISPR/Cas9 technology in inducing FAD2B mutations and high-oleic phenotype in peanut. Huayu 23, a popular normal-oleic runner type peanut cultivar having dysfunctional FAD2A and functional FAD2B, was transformed with CRISPR/Cas9 construct targeting FAD2B, resulting in two T1 seeds with over 80% oleic acid and a 442 A insertion in FAD2B. The high-oleic phenotype in T2 seeds was inheritable from the T1 generation. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in functional analysis of genes and peanut varietal improvement. This method is of reference value to other seed plant species.
... Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. 2014). Seeds with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices follow the protocol of Yang et al. (2012). ...
In contrast to its normal-oleic counterpart, high-oleic peanut has better keeping quality and multiple health benefits. Breeding high-oleic peanut through conventional means is a tedious process generally requiring several years. Genome editing, however, may shorten the duration. In this study, node injection method was used to transform normal-oleic Huayu 23, a popular peanut cultivar having dysfunctional FAD2A and functional FAD2B , with CRISPR/Cas9 construct targeting FAD2B , and two T0 seeds with over 80% oleic acid and 442A insertion in FAD2B were obtained. A T1 plant grown from the viable T0 seed produced high-oleic seeds. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in functional analysis of genes and peanut varietal improvement.
... Oleic and linoleic acid contents of the individual single seeds were predicted with NIRS (Wang et al. 2014). Seeds with at least 74% oleic acid along with the untreated control Huayu 23 were further analyzed for fatty acids by gas-chromatography using cotyledonary slices follow the protocol of Yang et al. (2012). ...
In contrast to its normal-oleic counterpart, high-oleic peanut has better keeping quality and multiple health benefits. Breeding high-oleic peanut through conventional means is a tedious process generally requiring several years. Genome editing may shorten the duration. In this study, node injection method was used to transform normal-oleic Huayu 23, a popular peanut cultivar having loss-of-function FAD2A , with CRISPR/Cas9 construct targeting FAD2B , and two peanut mutants with over 80% oleic acid and 442A insertion in FAD2B were obtained. As a genotype-independent, simple and easy method for peanut genetic transformation, node injection has great potential in factional analysis of genes and in peanut varietal improvement.
... The fatty acid composition of single peanut seeds was determined by gas chromatography (GC) as described by Yang et al. (2012). ...
As compared with its normal oleate counterpart, high oleate peanuts have better storage quality and several health benefits, and are therefore preferred by peanut shellers and consumers. High oleate has now become one of the main breeding objectives of peanuts. Thus far, over 50 high oleate peanut cultivars have been registered. Yet high oleate peanut breeding relies heavily on a limited number of high oleate genotypes. In this paper, we reported, for the first time, high peanut oleate natural mutants with large seeds derived from an intersectional cross, which were identified with near infra-red spectroscopy and confirmed by gas chromatography. Sequencing of FAD2 from the high-oleic hybrids along with their normal oleate parents indicated that a 448G >A mutation in FAD2A coupled with a 441-442ins A or G in FAD2B together caused high oleate phenotypes in these peanut hybrids.
... For GC analysis, three randomly picked sun-dried peanut SMK (sound mature kernel) samples (six seeds each) were smashed into fine powder with a food material grinding device for home uses (JYL-C020, Jiuyang Co. Ltd., Jinan, China). The GC procedure for analyzing eight fatty acids in peanut seeds as proposed by Yang et al. (2012) was strictly followed. To identify the response to chilling stress, 60 SMKs from each genotype were treated with a 0.05% ethrel solution (Solarbio Life Science, Beijing, China) at 2°C for 72 h, and then incubated at 25°C for 72 h. ...
Four peanut lines resulting from near infra-red reflectance spectroscopy aided selection for high oleate trait were evaluated for their productivity and fatty acid profile by gas chromatography in replicated trials in Laixi, Shandong, China. The four lines, with an oleate to linoleate ratio of 28.63-32.37, produced a kernel yield of 5226.24-5496.18 kg/ha, no less than the productivity of local control (Huayu 33 or Fenghua 1). The lines exhibited variable response to chilling stress, demonstrating the possibility of breeding high oleate peanut cultivars both with high productivity and with chilling tolerance. The study underlined the necessity of additional selection(s) for the high oleate trait, preferably seed by seed, during breeding process prior to bulk harvest for a yield trial, to exclude possible off-types (normal oleate seed within the high oleate lot)
in cultivar releases. The outcome of the study represented a big stride towards release of large-seeded high yielding peanut cultivars with high oleate in Shandong province, China.
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