Effect of Light Conditions on In Vitro Adventitious Organogenesis of Cucumber Cultivars

The response on callus and shoot formation under different light incubation conditions was evaluated in cucumber (Cucumis sativus L.). Four-day-old cotyledon explants from the inbred line 'Wisconsin 2843' and the commercial cultivars 'Marketer' and 'Negrito' were employed. A four-week culture was conducted on MS-derived shoot induction medium containing 0.5 mg L-1 IAA and 2.5 mg L-1 BAP, under an 8-h dark/ 16-h light regime, or by a one- or two-week dark pre-incubation followed by the same photoperiod. Significant differences were obtained for the regeneration of shoots in all cultivars. The response in both frequency and number of shoots under continuous photoperiod was at least 3-6 fold higher than with dark pre-incubation. The highest genotypes response was obtained by 'Negrito' and 'Marketer' with identical values. All explants formed callus, and in two of the three cultivars, the response on callus extension was not significantly affected by incubation conditions. The results clearly show that shoot induction under continuous photoperiod regime was beneficial for adventitious shoot regeneration in cucumber.

A Reliable Regeneration Method in Genome-Editable Bell Pepper ‘Dempsey’

A reliable regeneration technique is critical for the improvement of pepper traits in the genome editing era. Recently, we reported that peppers were successfully and specifically edited using CRISPR tools, CRISPR/Cas9 and CRISPR/Cas12a (LbCpf1). Although genome-editing tools can be applied to modify peppers at the cellular level, feasible pepper regeneration techniques have not been developed. Therefore, we studied a pepper regeneration protocol for Capsicum annuum L. ‘Dempsey’, a bell pepper species that has been proven to be genome-editable. Three explant types were used in this study, including the first leaves, cotyledons and hypocotyls of pepper seedlings. The shoot buds of the tested explants were produced using 8 mg/L 6-benzylaminopurine (BAP)- and 6 mg/L indole-3-acetic acid (IAA)-containing shoot induction medium (SIM). The first leaves of the ‘Dempsey’ seedlings showed an average shooting rate of 69.8%, whereas the hypocotyls and cotyledons had approximately 25.5% and 19.5% shooting rates, respectively. The regenerated ‘Dempsey’ plants exhibited no alterations in fruit and fertile seed phenotypes. Furthermore, the parent ‘Dempsey’ and progenies of the regenerants were cytogenetically stable with the same chromosome numbers (2n = 24). Therefore, this regeneration protocol enables the precise molecular breeding of ‘Dempsey’ peppers when coupled with CRISPR tools.

Efficient regeneration protocol for callus and shoot induction from recalcitrant Phaseolus vulgaris L. explants under optimum growth conditions

Callus is the most significant morphogenic response obtained in plant tissue culture studies. It can be used for micropropagation or to create transgenic lines. Phaseolus vulgaris L. (common bean) is one of the economically important crops with a great nutritional value. However, very little effort has been made to regenerate callus from P. vulgaris explants. Six explants were used namely root tip, leaves, plumule, radicle, cotyledon and embryo to develop a callus from P. vulgaris. The minimum days for callus induction was 10 days in plumule, radicle and embryo explants, while the maximum was 15 days in cotyledon explants with the callus induction percentage of 75%. The largest callus was found to be 2.77 gm in weight and 2.5 cm in diameter in MS medium. Medium with different concentrations of plant growth regulators (PGRs) showed different growth pattern in callus induction. Culture medium with 1.50 mg/l of BAP, 0.50 mg/l of 2, 4-D and 0.10 mg/l of NAA showed the best result in callus induction. Higher concentration of BAP (2.00 mg/l), along with 0.25 mg/l of 2, 4-D was ideal for shoot regeneration and maturation. Shoot induction medium along with 2.00 mg/l of NAA concentrations were found to be best for rooting. It was found that plumule and radicle favor callus induction, however, they are also potent for shoot and root induction. Knowledge gained in this study will be useful in developing a standard protocol for plant regeneration from P. vulgaris explants and will also be useful in creating transgenic line of P. vulgaris.

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

Cannabis sativa is relatively recalcitrant to de novo regeneration, but several studies have reported shoot organogenesis or somatic embryogenesis from non-meristematic tissues. Most report infrequent regeneration rates from these tissues, but a landmark publication from 2010 achieved regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single drug-type accession. Despite the importance regeneration plays in plant biotechnology and the renewed interest in this crop the aforementioned protocol has not been used in subsequent papers in the decade since it was published, raising concerns over its reproducibility. Here we attempted to replicate this important Cannabis regeneration study and expand the original scope of the study by testing it across 10 drug-type C. sativa genotypes to assess genotypic variation. In our study, callus was induced in all 10 genotypes but callus growth and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive. The shoot induction medium failed to induce shoot organogenesis in any of the 10 cultivars tested, instead resulting in necrosis of the calli. The findings of this replication study raise concerns about the replicability of existing methods. However, some details of the protocol could not be replicated due to missing details in the original paper and regulatory issues, which could have impacted the outcome. These results highlight the importance of using multiple genotypes in such studies and providing detailed methods to facilitate replication.

Influence of High Concentrations of Copper Sulfate on In Vitro Adventitious Organogenesis of Cucumis sativus L.

The effects of different concentrations of copper sulfate (0.2 to 5 mg L−1) on in vitro callus and shoot formation of cucumber was investigated. Four-day-old cotyledon explants from the inbred line ‘Wisconsin 2843’ and the commercial cultivars ‘Marketer’ and ‘Negrito’ were used. The results on callus-derived shoots showed that the optimal concentration of CuSO4 added to Murashige & Skoog (MS)-derived shoot induction medium containing 0.5 mg L−1 IAA and 2.5 mg L−1 BAP was 8-200 fold greater than in standard MS medium, and was genotype dependent. The highest genotypes response on shoot frequency and shoot number was achieved in this order by ‘Marketer’, ‘Negrito’ and ‘Wisconsin 2843’ with 1, 0.2 y 5 mg L−1 CuSO4, respectively. The genotype with the lowest control performance demanded the highest concentration of CuSO4 for its optimal morphogenic response - 6- and 10-fold more in shoot frequency and shoot number, respectively. The other cultivars registered a 2-fold increase in both variables. All explants formed callus and the response on callus extension varied among cultivars. The regression analysis showed a statistically significant relationship between shoot number and concentrations of CuSO4 and absence of association with callus extension. The present results indicate that application of specific concentrations of CuSO4 higher than in standard MS medium, increases adventitious cucumber shoot organogenesis.

IMPROVEMENT OF DIRECT REGENERATION OF MEXICAN SOYBEAN FROM COTYLEDONARY NODES

Plant tissue culture provides an alternative approach to improve the quality of soybean (Glycine max (L.) Merrill) cultivars. This study was undertaken to analyze the susceptibility of Mexican soybean for direct shoot regeneration and to determine the critical factors that affect in vitro performance. Our hypothesis was that Mexican soybean is suitable for in vitro regeneration using a cotyledonary node as explant. The effects of the seed disinfection procedure, soaking pretreatment before germination, soybean variety, as well as the culture medium composition of the shoot induction medium, were evaluated by two split-plot statistical designs. According to the statistical analysis, the seed disinfection procedure, the soaking pretreatment before germination, and the soybean genotype were the factors that brought about a significant effect (p£0.01), while the hormones composition of the shoot induction medium did not have a significant effect. The best response for multiple shoot formation was observed using a chlorine gas seed disinfection method, 3% hydrogen peroxide soaking pretreatment, and Huasteca-100, Nainari and Suaqui soybean genotypes. A robust protocol was developed, and under these selected conditions, it is possible to obtain more than 10 shoots per explant. Well-developed plantlets were obtained after 60 d of in vitro culture.

RUNNER-TIP CULTURE OF STRAWBERRY (Fragaria x ananassa Duch) GROWN ON SEVERAL SHOOT-INDUCTION MEDIUM

A research regarding “Runner-tip culture of strawberry (Fragaria x ananassa Duch) Grown on Several Shoot-induction Medium” has been investigated. The objective of the research was to find out the most suitable medium for shoot production from runner-tip culture of strawberry at establishment step of micropropagation. The research was laid out in a Completely Randomized Design, 4 treatments of medium type for shoot induction and 10 replication, each was represented by one (1) bottle with 6-8 explants. The treatments were summarized as follows: T1 = MS ; T2 = MS + 2 ppm BAP + 0.01 ppm NAA; T3 = MS + 1 ppm of TDZ; T4 = WPM + 2 ppm BAP + 0.01 ppm NAA. The parameters observed were days of the bud emergence, the average number of shoots per explant, and the average number of leaves per explant. It can be concluded that among medium used in the current research, the medium of MS added with 1 ppm thidiazuron (TDZ) was the most suitable medium for shoot production of strawberry from the explant of runner-tips. The treatment was resulted in the earliest time of bud emergence, and producing the highest number of shoots and leaves.

Recalcitrance of Cannabis sativa to de novo regeneration; a multi-genotype replication study

1AbstractCannabis sativa is relatively recalcitrant to regeneration from somatic tissues, but several reports have been published demonstrating a response. Most reports show low levels of regeneration from somatic tissues, but a landmark publication by Lata et al. in 2010 reported regeneration from leaf explants with a 96% response rate, producing an average of 12.3 shoots per explant in a single, high-THC genotype. Despite the importance regeneration plays in plant biotechnology this protocol has not been used in subsequent papers in the decade since it was published, raising the concern that it is not reproducible. Many researchers are looking to build research programmes in this growing field, and it is important that the reproducibility and robustness of single-genotype C. sativa regeneration protocols undergo multi-lab validations to ensure they are reproducible across the species. Replication studies in this burgeoning field will help research groups avoid lost time and resources which arise from pursuing protocols that are not reproducible. Here we test the replicability of this protocol across 10 drug-type C. sativa genotypes. This protocol successfully induced callus in all 10 genotypes. Callus size and appearance substantially differed among cultivars, with the most responsive genotype producing 6-fold more callus than the least responsive genotype. However, the most successful shoot induction medium developed in the 2010 paper failed to induce regeneration in any of the cultivars tested, resulting in the eventual necrosis of the calli. Based on this replication study, it is evident that the existing regeneration protocol is not robust and could not be replicated in any of the 10 genotypes tested.

Improvement of growth and bacoside production in Bacopa monnieri through induced autotetraploidy with colchicine

Bacopa monnieri is a medicinal herb that is increasing in demand in Thailand. However, the lack of high-bacoside cultivars has limited pharmaceutical utilization and production. Here, chromosome doubling in B. monnieri was attempt to improve biomass and bacoside content in its seedling. Nodal segments were treated with colchicine (0, 0.025, 0.05, 0.075, 0.1, and 0.5% w/v) for 24 or 48 h before transferring to multiple shoot induction medium (1/2 MS medium supplemented with 0.2 mg L−1 BAP). Of 326 tested clones, 18 and 84 were mixoploids and autotetraploids, respectively. The highest autotetraploid-induction percentage (14.6%) was found after treated with 0.5% (w/v) colchicine, and 48 hours exposure. From 28 selected autotetraploid clones, 21 and 13 have significantly higher fresh and dry weight compared to the diploid clone, respectively. The maximum fresh and dry weight of autotetraploid plants was 2.8 and 2.0-time higher than diploid plants, respectively. Moreover, the maximum total bacoside content (1.55 mg plant−1) was obtained from an autotetraploid plant, which was 2.3-fold higher than the level in diploid plants. These novel autotetraploids have the potential to be developed as resources for value-added improvements in the medicinal and pharmaceutical industries.

In vitro propagation of Codonopsis javania (Blume) Hook. f. et Thomson through the callus induction

Codonopsis javania (Blume) Hook.f. et Thomson a traditional medicine plant and now an endangered species in Vietnam is grown for roots. The research was carried out to establish the plant propagation for the purpose of concerving and exploting this endangered medicinal herbs. In vitro shoot tip explants (1 – 1.5 cm) were induced to form callus on MS medium containing NAA (0.5 – 2 mg /L) with TDZ 0.1 mg/L. After four weeks of culture, in the MS medium combine with NAA 1 mg/L and TDZ 0.1 mg/L the explant induced compact callus (green, solid) wsa achieved 85.33%. The callus induction to form shoots on medium MS containing BA (0.5 – 2.0 mg/L) with NAA 0.2 mg/L. After 4 weeks of culture, shoot formation was higher in the MS medium containing BA 1.0 mg /L and NAA 0.2 mg/L and achieved of 82.67 % with 9.92 shoots/explant. The best shoot proliferation (2 – 3 cm) was excised and transferred to a medium shoot multiplication with the same composition as the shoot induction medium in which NAA 0.2 mg/L was replaced by NAA 0.5 mg/L. When compared the shoot multiplication between the two mediums at the same BA concentration (2 mg/L), all shoots increased and reached 5.87 times after 60 days cultured. On rooting MS medium with IBA 1 mg/L, 88.67 % in vitro rooting was observed with the average root yield of 4.33 roots/shoot and the length of 8.27 cm. Root length and their yield quality were highly improved when using of coconut fiber (30 %) and earthworms compost (70 %) (v/v) in the transfer medium after acclimatisation stages.