Enhancement of Tobacco (Nicotiana tabacum L.) Seed Lipid Content for Biodiesel Production by CRISPR-Cas9-Mediated Knockout of NtAn1

Tobacco (Nicotiana tabacum L.) seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The TT8 gene and its homologs negatively regulate seed lipid accumulation in Arabidopsis and Brassica species. We speculated that manipulating the homolog genes of TT8 in tobacco could enhance the accumulation of seed lipid. In this present study, we found that the TT8 homolog genes in tobacco, NtAn1a and NtAn1b, were highly expressed in developing seed. Targeted mutagenesis of NtAn1 genes was created by the CRISPR-Cas9-based gene editing technology. Due to the defect of proanthocyanidin (PA) biosynthesis, mutant seeds showed the phenotype of a yellow seed coat. Seed lipid accumulation was enhanced by about 18 and 15% in two targeted mutant lines. Protein content was also significantly increased in mutant seeds. In addition, the seed yield-related traits were not affected by the targeted mutagenesis of NtAn1 genes. Thus, the overall lipid productivity of the NtAn1 knockout mutants was dramatically enhanced. The results in this present paper indicated that tobacco NtAn1 genes regulate both PAs and lipid accumulation in the process of seed development and that targeted mutagenesis of NtAn1 genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that the CRISPR-Cas9 system could be employed in tobacco seed de novo domestication for biodiesel feedstock production.

Optimization of tobacco seed cleaning modes

The process of obtaining high-quality tobacco seeds is very laborious, and at present many operations are performed manually at All-Russian Research Institute of Tobacco, Makhorka and Tobacco Products. The process of obtaining tobacco seeds at the Institute consists of the following operations: harvesting of inflorescences, drying of inflorescences, destruction of inflorescences, screening out coarse impurities, final cleaning in an air stream. An attempt was made earlier to mechanize and improve working conditions at the stage of final purification of the seed mixture. A unit for the final purification of tobacco seeds was developed, manufactured and tested. In this article other modes of final purification of the tobacco seed mixture to improve mechanization and productivity of the same unit have been tested. The seed mix of the 2019 harvest has been used in the studies. Coarse impurities have been separated from the seed mix by sieving through sieves with different hole sizes. Earlier modes of double final purification of seed mixture were proposed. In this research the previously proposed double cleaning modes have been taken as control ones, and in the experiment three-fold modes with a higher feed rate of the seed mixture have been used. As a result, the experimental modes provide identical removal of contaminants, however, the productivity of the unit increases 2 – 3 times due to the shorter total duration of purification, and mechanization is also improved due to a more uniform flow of the seed mixture. In this case, there is no need for the constant presence of an operator, and the process of final purification of the tobacco seed mixture is reduced only to its loading and unloading.

Study of Cold-Pressed Tobacco Seed Oil Properties by Gas Chromatography Method

A special place among vegetable oils was occupied by natural tobacco oil. Natural tobacco oil in folk medicine is used as an antiseptic and antibacterial agent. To determine the possible alternative use of tobacco, the seeds representing Kazakhstan tobacco cultivars, extracted from ground Nicotiana tabacum seeds, were investigated by using the cold press. The quality of the oil was assessed in terms of free fatty acid content. The percentage oil yield was 36.75 ± 0.50%. Tobacco seed oil is highly unsaturated, nonacidic, and would require little purification. The oil can be used for the production of soap, antiwrinkle cream for the face, hair shampoo, shoe cream, and as a high-resolution base oil for perfumes and light industry and can be commercialized due to its high oil yield, for use as anti-inflammatory drugs.

The Vigor and Viability Seed Testing of Three Tobacco Varieties on Various Seed Germination Media

Seed viability is one of the most important physiological quality benchmarks of seeds. The use of appropriate germination media can affect the results of seed viability testing. This study aims to determine the optimal germination media in tobacco seed viability testing. This research was conducted at the Seed Laboratory of the Indonesian Sweetener and Fiber Crops Research Institute, Malang in January - March 2019. The research method used a two-factorial randomized block design (RAK). The first factor is tobacco seed varieties consisting of Kasturi in 2007, Bojonegoro in 2012 and Kemloko in 2014. The second factor is the seed germination media consisting of straw paper, cotton, towel tissue, paperboard and newspaper. The results showed that the Kemloko variety of tobacco seeds germinated in the towel tissue had the best seed vigor and viability percentage. Seed germination media with paperboard, towel tissue and straw paper can be used to test the viability of tobacco seeds because they have a percentage of simultaneous growth, percentage of the growth speed, percentage of germination, percentage of the growth potential maximum and normal seedling dry weight which are equally well

CRISPR-Cas9 Mediated Knockout of NtAn1 to Enhance the Lipid Accumulation in Tobacco Seed for Biodiesel Production

Background: Tobacco seed lipid is a promising non-edible feedstock for biodiesel production. In order to meet the increasing demand, achieving high seed lipid content is one of the major goals in tobacco seed production. The TT8 gene and its homologs negatively regulate seed lipid accumulation in Arabidopsis and Brassica species. We speculated that manipulating the homolog genes of TT8 in tobacco could enhance the accumulation of seed lipid.Results: In this present study, we found that the TT8 homolog genes in tobacco, NtAn1a and NtAn1b, were highly expressed in developing seed. Targeted mutagenesis of NtAn1 genes were created by the CRISPR-Cas9 based gene editing technology. Due to the defect of PAs biosynthesis, mutant seeds showed a phenotype of yellow seed coat. Seed lipid accumulation was enhanced by about 18% and 15% in two targeted mutant lines, respectively. Protein content was also significantly increased in mutant seeds. In addition, the seed yield related traits were not affected by the targeted mutagenesis of NtAn1 genes. Thus, the overall lipid productivity of the NtAn1 knockout mutants were dramatically enhanced. Conclusion: Tobacco NtAn1 genes regulate both PAs and lipid accumulation in the process of seed development. Targeted mutagenesis of NtAn1 genes could generate a yellow-seeded tobacco variety with high lipid and protein content. Furthermore, the present results revealed that CRISPR-Cas9 system could be employed in tobacco seed de novo domestication for biodiesel feedstock production.

PENGARUH PERENDAMAN TERHADAP VIABILITAS BENIH TEMBAKAU (Nicotiana tabacum L.)

<p>ABSTRAK<br />Permasalahan dalam pengembangan tembakau rakyat adalah daya<br />berkecambah benih yang rendah. Penelitian ini bertujuan untuk<br />mengetahui pengaruh perendaman benih terhadap daya berkecambah<br />benih tembakau (Nicotiana tabacum L.). Penelitian dilaksanakan di<br />laboratorium Balai Penelitian Tanaman Pemanis dan Serat pada bulan Mei<br />sampai dengan Juli 2013. Perlakuan disusun dalam rancangan petak<br />terbagi dan diulang empat kali. Sebagai petak utama adalah tujuh varietas<br />tembakau lokal, yaitu V1 = Kemloko1; V2 = Kemloko2; V3 = Kemloko3;<br />V4 = Kasturi1; V5 = Kasturi2; V6 = Grompol Jatim1; dan V7 =<br />Bojonegoro1. Sebagai anak petak adalah: R1 = Tanpa perendaman<br />(kontrol); R2 = perendaman selama satu jam dalam air; R3 = perendaman<br />selama satu jam dalam larutan KNO 3 (0,1%), dan R4 = perendaman<br />selama satu jam dalam larutan KNO 3 (0,2%). Setelah perlakuan<br />perendaman, benih tembakau dikecambahkan menggunakan metode Uji di<br />Atas Kertas. Pada setiap ulangan, sebanyak 100 benih tembakau<br />dikecambahkan pada media kertas merang yang diletakkan di dalam<br />petridish berdiameter 9 cm. Perkecambahan dilakukan di dalam<br />germinator tipe IPB dengan suhu 23 o C dan kelembaban nisbi 87-93%.<br />Parameter yang diamati adalah daya berkecambah, panjang kecambah,<br />panjang akar kecambah, dan indeks vigor kecambah. Perendaman benih<br />tembakau menggunakan air, larutan KNO 3 0,1% dan larutan KNO 3 0,2%<br />selama satu jam sebelum benih disemaikan, dapat meningkatkan daya<br />berkecambah dan panjang kecambah varietas Kemloko1 dan Grompol<br />Jatim1. Perlakuan perendaman benih dengan air berpengaruh positif pada<br />varietas Kemloko1 yang ditunjukkan dengan daya berkecambah tertinggi,<br />sedangkan perendaman dengan larutan KNO 3 0,2% berpengaruh negatif<br />pada varietas Bojonegoro1 yang ditunjukkan dengan daya berkecambah<br />paling rendah. Perlakuan perendaman dengan air maupun larutan KNO 3<br />(0,1% dan 0,2%) menunjukkan pengaruh yang berbeda-beda terhadap<br />parameter daya berkecambah, panjang kecambah, panjang akar kecambah,<br />dan indeks vigor kecambah pada semua varietas tembakau yang diuji.<br />Kata kunci: Nicotiana tabacum L., perendaman, KNO 3 , viabilitas benih</p><p>ABSTRACT<br />Low germinability of seeds is one of major problems in tobacco<br />development. The aim of this study was to determine the effect of priming<br />on tobacco (Nicotiana tabacum L.) seed viability. The research was<br />conducted in the laboratory of the Indonesian Sweeteners and Fiber Crops<br />Research Institute during May to July 2013. The treatments were arranged<br />in a split plot design with four times of replication. The main plots were<br />seven tobacco varieties namely V1 = Kemloko1; V2 = Kemloko2; V3 =<br />Kemloko3; V4 = Kasturi1; V5 = Kasturi2; V6 = Grompol Jatim1; and V7<br />= Bojonegoro1. The subplots were priming seeds for one hour namely R1<br />= without priming (control); R2 = priming for one hour on water; R3 =<br />priming for one hour on KNO 3 (0,1%) solution, and R4 = priming for one<br />hour on KNO 3 (0,2%) solution. After priming, seeds were germinated using<br />the Upper Paper Test method. A hundred of seeds were sown on straw<br />paper media in a petridish diameter 9 cm of each replication. Parameters<br />measured were germination percentage, shoot and root length, and<br />seedling vigor index. Priming tobacco seed with water or KNO 3 (0.1 and<br />0.2%) solution for one hour before seeds were germinated significantly<br />improved germination percentage and shoot length of Kemloko1 and<br />Grompol Jatim1 varieties. Priming tobacco seed with water had positive<br />effect on Kemloko1 variety which resulted the highest germination<br />percentage but had adversely effect on Bojonegoro1 variety which resulted<br />the lowest germination percentage. Priming tobacco seeds with water or<br />KNO 3 (0.1 and 0.2%) solution resulted different effect on germination<br />percentage, shoot and root length, and seedling vigor index parameters for<br />all tobacco varieties were observed.<br />Key words: Nicotiana tabacum L., priming, KNO 3 , seed viability</p>

Determination of tobacco (Nicotiana tabacum) seed vigour using controlled deterioration followed by a conductivity test

Seeds of eight lots of four tobacco varieties differing in vigour were studied. The initial germination percentage (GPUT) was 81-100% and the controlled greenhouse emergence percentage (CEP) was 55-91%. All seeds were treated by controlled deterioration (CD) at 20% moisture content and 45°C for 24 hours, then some seeds were subjected to a standard germination test and the rest of the seeds were subjected to a conductivity test. The electrical conductivity (EC) of seed soak water was measured every six hours up to 54 hours. The results showed that relative conductivity after CD (RCCD) was highly significantly correlated with GPCD after 30 to 48 hours soaking (r = -0.9324 to -0.9409, P < 0.001). After 36 and 42 hours, RCCD was significantly correlated (P < 0.01) with CEP (correlation coefficients of -0.9120 and -0.9137, respectively). Therefore, it is suggested that measuring relative conductivity after CD could be used to rank tobacco seed lots for vigour. In addition, the best seed soaking time for EC measurement is 36 to 42 hours, with respect to both efficiency and accuracy.