In Vitro Amphidiploid Induction of A Distant Hybrid Populus Simonii × P. Euphratica Cv. ‘Xiaohuyang-2’ and its Effect on Plant Morphology and Anatomy

In plants, highly gametic sterility of distant hybrids usually restricts their utilization in breeding programs. Amphidiploid induction produced by somatic chromosome doubling of distant hybrids can effectively restore their gametic fertility. In this study, nodal-segment and leaf explants of a distant hybrid Populus simonii × P. euphratica cv. ‘Xiaohuyang-2’ were used to induce chromosome doubling with colchicine in vitro. Although chromosome doubling of the nodal-segment explants only produced mixoploids, the treatments of leaf explants on adventitious bud regeneration medium successfully produced 4 amphidiploids, which might be attributed to the direct organogenesis of the adventitious buds on the leaf explants. The highest amphidiploid induction frequency was 16.7%. Both the explant survival rate and polyploidization frequency were significantly affected by colchicine concentration and exposure time. The amphidiploid plants were significantly differed from the diploid and mixoploid plants on morphological and anatomical characteristics. They had larger, thicker, and greener leaves than the diploids and mixoploids. The changes of stomatal features also accompanied with increase of ploidy level. The induced amphidiploid plants of the distant hybrid ‘Xiaohuyang-2’ are expected to play important roles in breeding programs of Populus in future, which can be used as a bridge parent with ability of unreduced gamete formation to cross with fast-growth germplasms to produce triploids pyramiding desirable traits of fast growth, easy cutting propagation, and salt and drought tolerances.

Establishment of Tissue Culture System of Actinidia deliciosa Cultivar “Guichang”

In order to breed virus-free plantlets of the kiwifruit cultivar “Guichang,” which belongs to Actinidia deliciosa, in this study, stem segments with buds were used as explants, the establishment of a tissue culture rapid propagation system was carried out, and then the virus status of tissue culture plantlets was detected via the real-time reverse transcription-polymerase chain reaction (RT-qPCR) method. The tissue culture rapid propagation system proved that the contamination and browning rates could be controlled below 20% and the survival rate could be exceeded by 70% when the single bud stem segment of “Guichang” kiwifruit was sterilized with 70% alcohol for 30–60 s and 15% NaClO for 15 min, respectively. Meanwhile, we screened the hormone concentration to get better results, and the appropriate medium for adventitious bud induction was MS + 6-BA (1.0 mg/L) + IBA (0.2 mg/L); for proliferation, it was MS + 6-BA (1.0 mg/L) + IBA (0.1 mg/L); and for rooting, it was 1/2 MS + IBA (0.3 mg/L), and the efficiency of induction, proliferation, and rooting could reach 74.07%, 79.63%, and 85.18%, respectively. In addition, the RT-qPCR results demonstrated that the infection rate of 9 viruses: apple stem grooving virus (ASGV), cucumber mosaic virus (CMV), Actinidia virus X (AVX), cucumber necrosis virus (CNV), ribgrass mosaic virus (RMV), citrus leaf blotch virus (CLBV), Actinidia virus B (AcVB), Pelargonium zonate spot virus (PZSV), and cherry leaf roll virus (CLRV) in the “Guichang” kiwifruit tissue culture plantlets was 0. This study could lay a foundation for the production of “Guichang” kiwifruit tissue culture seedlings, and the medium formula provided in this study was useful for the industrial rapid propagation of “Guichang” plantlets.

Rapid Propagation System in Vitro of Medicinal Plant Cynanchum Atratum Bunge

As a traditional Chinese medicinal material, Cynanchum atratum Bunge has been widely used in traditional Chinese medicine for its treatment of abscesses, acute urinary infection and hectic fever.Thus, wild resources of it have been endangered by overharvesting. Plant tissue culture technology is an important measure to protect wild resources of medicinal plants, including C. atratum. Therefore, a fast and efficient propagation system of C. atratum through axillary bud proliferation pathwayhas been established. Through axillary bud proliferation, the medium [MS+sucrose 30 g/L+Agar 7 g/L+NAA 0.2 mg/l+IBA 1.5mg/l+KT 0.5 mg/l] can effectively proliferate adventitious buds, and the induction rate was 100 %, proliferation coefficient could reach 8.56. MS medium was used to induce adventitious bud rooting, with rooting rate of 98% and no callus. The highest survival rate was 90% when the ratio of grass mud pond and orchard red soil was 1:1. To our knowledge this is the first report of rapid propagation system in C. atratum, it achieve rapid reproduction of C. atratum.

Basal stem cluster bud induction and efficient regeneration for the Tibetan endemic medicinal plant Swertia conaensis

The artificial rapid propagation system for Swertia conaensis T. N. Ho et S. W. Liu was explored to screen the appropriate plant regeneration method and to provide an efficient propagation mode, useful for artificial breeding technology or for further research and development of the Tibetan endemic medicinal plant. In this study, the most suitable explant and hormone were chosen according to single factor test. Next, the effects of different hormone combinations on basal stem cluster bud induction, callus induction, adventitious bud occurrence and plant regeneration were investigated by using complete combination and orthogonal experiment. The obtained results showed that the explants suitable for in vitro of S. conaensis were stem tips with leaves, which were regenerated through the method of basal stem cluster bud occurrence in the MS medium with 2.0 mg∙L-1 6-BA, 0.5 mg∙L-1 NAA, but the proliferation coefficient was low, only 3.16 after 40 days of culture. Subsequently, the proliferation coefficient failed to improve, irrespective of change of the concentration ratio of 6-BA and NAA. Therefore, in the orthogonal experiment of adding ZT, the MS medium with 1.0 mg∙L-1 ZT, 0.5 mg∙L-1 NAA and 2.5 mg∙L-1 6-BA induced a large number of callus green and compact, with 86.30% callus occurrence rate. After 40 days of culture, the rate of adventitious bud occurrence was 96.55% and the proliferation coefficient was high (10.37). The rooting rate was 100% in the 1/2MS medium with 0.5 mg∙L-1 NAA. The survival rate of regenerated plants was more than 95%. Indirect organogenesis was more efficient than direct organogenesis in in vitro culture of S. conaensis. In this study, the efficient and stable regeneration system of S. conaensis was achieved through the method of explant to callus to adventitious buds, which provided an effective way to an endangered species.

Effects of different factors on adventitious bud induction from stem explants of Ludisia discolor

In this study, the stem explants of Ludisia discolor were used as experimental materials to investigate the effects of 6-BA, NAA, Cu2+ and Ag+ on the induction of adventitious bud regeneration, and to analyze the most suitable culture conditions for stem explants regeneration. The results showed that when the medium was supplemented with 1.0 mg/L 6-BA, 0.75 mg/L NAA, 0.25 mg/L CuSO4, or 6.4 mg/L AgCl, the best regeneration effects would be gained, respectively. And the adventitious bud regeneration rate reached the maximum, which were 61.67%, 83.67%, 80.95% and 87.63%, respectively. The results of this study provided a theoretical basis for tissue culture and rapid propagation of L. discolor.

Establishment of plant regeneration system for young leaf explants of Cassia mimosoides

In the present study, a plant regeneration system was established by using young leaf explants from aseptic seedlings of Cassia mimosoides. The results showed that when the explants were inoculated on the 1/2MS medium contained 2.0 mg/L 6-BA and 0.5 mg/L NAA, high rate of callus induction and good growth status of adventitious bud, and the highest number of buds were obtained. Further studies conclusively suggested that IBA could not promote root formation effectively, but the root regeneration could be induced by NAA. However, although the rooting of regeneration buds could be induced when adventitious buds were placed into 1/2MS medium supplemented with 0.5 mg/L NAA, the rate of rooting was reduced, and the roots became shorter. Therefore, the best medium for adventitious bud regeneration using young leaf as explants in C. mimosoides was 1/2MS medium contained 2.0 mg/L 6-BA and 0.5 mg/L NAA, and the best rooting medium was 1/2MS.

Maturation and Conversion of Somatic Embryos Derived from Seeds of Olive (Olea europaea L.) cv. Dahbia: Occurrence of Secondary Embryogenesis and Adventitious Bud Formation

Maturation and conversion of somatic embryos are two crucial steps that hamper the development of efficient somatic embryogenesis systems in olive. Herein, a simple and efficient protocol for the maturation and conversion of olive somatic embryos is reported. Globular somatic embryos derived from seeds of cv. Dahbia were cultured on either half-strength olive (OM) or olive cyclic embryogenesis (ECO) media, with and without plant growth regulators (PGRs). The embryos reached the cotyledonary stage in 9 weeks, but those cultured on ECO medium containing 0.1 mg·L−1 6-(dimethylallylamino)purine (2iP), 0.1 mg·L−1 6-benzyladenine (BA) and 0.05 mg·L−1 indole-3-butyric acid (IBA) exhibited the largest sizes, with an average of 4.7 mm. Somatic embryo conversion into plantlets was evaluated using different culture media (half-strength OM or one-third strength Murashige and Skoog (MS)), light conditions (light or dark) and desiccation pretreatments. The highest rate of somatic embryo conversion (45%) was observed under a 16 h photoperiod on half strength OM medium containing 0.1 mg·L−1 gibberellic acid (GA3) and 0.1 mg·L−1 1-naphthalene acetic acid (NAA). The embryos that failed to germinate showed either necrosis, cotyledon greening with no further conversion, adventitious bud formation or secondary embryogenesis. The findings of this study will be beneficial for biotechnological applications in olive.

Callus induction and adventitious bud differentiation of Cyclocodon lancifolius (Roxb.) Kurz

Background: Cyclocodon lancifolius (Roxb.) Kurz is a perennial medicinal and edible plant with a huge potential economic value. The wild resources of this plant are gradually scarce by the serious destruction of the habitat and the limitations of sexual reproduction. This is the first attempt to establish an in vitro reproductive system for the species. Hypothesis: The suitable plant regulator types and its mass concentration range, combined with the explants, can induce the development of plants at various stages. We expected to establish an in vitro regeneration system of C. lancifolius based on these factors. Species studied: Cyclocodon lancifolius Study site and years of study: Yunnan Breeding and Cultivation Research and Development Center of Endangered and Daodi Chinese Medicinal Materials, Yunnan University of Chinese Medicine, from 2017 to 2019. Methods: The plant regeneration of C. lancifolius was established by single factor, L9 (34) orthogonal and complete combination experiments. Results: Stem segments were the best explants for callus induction on MS medium containing 0.05 mg·L-1 KT, 0.5 mg·L-1 6-BA and 0.5 mg·L-1 NAA. MS medium with 0.2 mg·L-1 TDZ and MS medium were used alternately as the culture method to conduct differentiation and proliferation of adventitious shoot. The optimal protocol for the rooting was MS medium combined with 0.1 mg·L-1 6-BA and 1.0 mg·L-1 NAA. Conclusions: A rapid propagation system of C. lancifolius was established which provided a possible solution for the protection of wild resources and artificial cultivation.

The regeneration of Acer rubrum L. "October Glory" through embryonic callus

Background: Tissue culture and rapid propagation technology is an important way to solve the difficulties of plant propagation. This experiment aims to explore the appropriate conditions at each stage of the red maple’s tissue culture process and to obtain plantlets, thus providing a theoretical basis for the establishment of the red maple’s tissue culture system. Results: The results showed that the stem segment is the most suitable explant for inducing embryogenic callus. The MS (Murashige&Skoog) +0.8 mg/L TDZ (Thidiazuron) +1.0 mg/L 6-BA (6-Benzylaminopurine) +0.5 mg/L IAA(Indole-3-acetic acid) +35 g/L sucrose+7.5 g/L semi-fixed medium was the best for callus formation. When selecting type Ⅵ callus as embryonic callus induction material, MS+0.6 mg/L TDZ+0.5 mg/L 6-BA +2.0 mg/L IAA +35 g/L sucrose+7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS+1.0 mg/L TDZ+3.0 mg/L 6-BA+0.2 mg/L NAA (1-Naphthaleneacetic acid)+1.2 mg/L IAA+35 g/L sucrose+7.5 g/L semi-fixed medium. The induction rate of adventitious roots in MS+0.6 mg/L TDZ+1.0 mg/L 6-BA+3 mg/L NAA+35 g/L sucrose+7.5 g/L semi-fixed medium was the highest, reaching 76%. Conclusions: In the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the "S" growth curve, and can be sub-cultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.