Development of an Efficient In vitro Regeneration Protocol for Chrysanthemum (Chrysanthemum morifolium Ramat)

An efficient and rapid in vitro regeneration protocol was developed for chrysanthemum (Chrysanthemum morifolium Ramat) using two local varieties of Bangladesh namely, BARI Chrysanthemum-2 (BARI Chry-2) and local yellow (Y). MS medium supplemented with nine different concentrations and combinations of BAP and IAA was employed to optimize regeneration protocol using young in vitro derived leaf explants. Direct organogenesis was observed from the leaf explants on MS medium supplemented with 0.5 mg/l BAP and 2.0 mg/l IAA (T6) for both the varieties. This treatment (T6) induced shoot buds directly on the adaxial surface of the leaf providing the highest regeneration percentage (90% for BARI Chry-2 and 94.73% for Y), the highest number of shoot/explant (7.6 for BARI Chry-2 and 8.6 for Y) and maximum length of the shoot after six weeks (3 cm for BARI Chry-2 and 2.9 cm for Y) of culture. Explants with initially regenerated shoots were subculture on hormone free MS medium for shoot elongation after 4 weeks of their inoculation. During elongation of shoots, 90-95% of the regenerated shoots produced roots spontaneously in hormone free MS medium within 7-8 weeks of their inoculation. Rooted plantlets were transplanted to the field following hardening where 100% plantlets were survived and produced flower without any variation. Plant Tissue Cult. & Biotech. 31(2): 161-171, 2021 (December)

In vitro propagation and conservation of Leontopodium palibinianum Beauverd (Asteraceae), endemic species of Primorye Territory

The microclonal propagation protocol including initiation, propagation, rootage, and adaptation in the premises of the Botanical Garden-Institute FEB RAS was developed for the first time for a vulnerable endemic species of Primorye Territory Leontopodium palibinianum (Palibin’s edelweiss). It was shown that the medium’s mineral composition as well as changes in the concentration of exogenous growth regulators under the same light and temperature conditions had a substantial impact the processes of growth and morphogenesis of Palibin’s edelweiss. Optimal components of the microclonal propagation medium of the species were full strength Murashige and Skoog medium. Reducing in the salt amount leads to a slowdown and arrest of in vitro culture growth. Adding 2 mg/l of 6-BAP to the full strength MS medium led to mass production of plant material with the propagation factor of 9.8. It should be noted that GK3 has a positive effect on the Palibin’s edelweiss culture development in vitro when used together with low concentrations of 6-BAP. When 2.4-D was used at the concentration of 1 mg/l in the medium, direct organogenesis was observed after 14 days of cultivation and further accompanied by formation of additional plant shoots. Also, addition of 1 mg/l 2iP to the MS medium had the same effect. The growth regulators caused a low propagation factor (2.4), which in turn led to production of 2–3 regenerative plants. The IBA concentration of 2 mg/l stimulated formation of the root system capable of positively withstanding acclimatization under unsterile conditions, but lowering the IBA concentration to 1 mg/L of the medium also contributed to formation of such root system, with a slower pace. During acclimatization to ex vitroconditions plant survival did not depend on the types of mixtures being used. After the acclimatization plants are stretched in height and produced additional shoots on 20–25th day.

Expression pattern of in vitro organogenesis-associated genes as transcriptional marker in Indian Sandalwood (Santalum album L.).

Indian sandalwood (Santalum album L.) is an expensive wood that requires reproducible method for mass propagation to ensure consistent production and sustainable use of sandalwood. For mass propagation of sandalwood, plant organogenesis requires different combinations of the tissue culture medium. The media is composed of exogenous phytohormones which decides the explant's morphological stages such as shooting or rooting induction. Early prediction of morphological stage from explant can potentially help in selecting the exogenous phytohormones combinations thereby saving time and resources for mass sandalwood propogation. An efficient protocol for the direct and indirect organogenesis (up to shooting development phase) of sandalwood were developed using Woody Plant Media (WPM). WPM supplemented with various concentrations of 6-Bezylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA) were tested for direct organogenesis, while different treatments consisting of various levels of 2,4-dichlorophenoxyacetic acid (2,4-D), NAA, BAP, Adenine sulphate (ADS), glycine and potassium nitrate were tested for indirect organogenesis. Three stages of leaf development were selected viz., the leaf just after inoculation in WPM media, initial stage of callus formation from leaf and shoot formation for expression pattern analysis. The targeted genes were Alternative oxidase (ao), Late embryogenesis abundant (lea), Cytochrome P450 (cyt-p450), ABC transporter (abct), and Serine-threonine phosphatase (stp) which are associated with in vitro organogenesis. The expression patterns were evaluated to identify a transcription marker. During the initial stages of organogenesis, ao, cyt-p450 and abct showed no/little change in expression in the direct pathway but up-regulation of ao and abct and downregulation of cyt-p450 were observed in the indirect pathway. Expression of lea was increased up to 70-fold during direct and dropped to half during indirect organogenesis.

Regeneration from leaf explants of steppe cherry (Prunus fruticosa Pall)

The article represents data on morphogenesis from leaf explants of three steppe cherry genotypes, as well as the degree of somaclonal variability at in vitro and ex vitro stages, and in the field. It was revealed that a content of 6-benzylaminopurine, 4.43 μM, in combination with auxin, 0.5–0.6 μM, stimulates in the light the direct organogenesis in the tissues of the leaf base. This reaction was observed from 16.7 to 75.0% of explants, depending on the genotype. An equal 6-benzylaminopurine - auxin ratio (1: 1) led to the callus along with microshoots. Depending on the genotype, up to 30.0% of explants had such a mixed type of organogenesis. The mitotic index value in the apical leaflets differed depending on the day time. At the stage of micropropagation itself, an increase of the mitotic index was observed from 10 to 16 hours; at the stage ex vitro, no significant differences in the mitosis frequency were revealed within this time interval. No significant differences were found between the level of the mitotic index for plants obtained directly from leaves and those from buds. The mitosis passed without disturbances. No phenotypic changes in plant habit, shape and color of leaves were found.

Optimizing Turmeric Tissue Culture, Testing Different Media and a Plant Growth Regulator Matrix

Rapid multiplication of turmeric (Curcuma longa) by micropropagation is needed to produce a continuous source of uniformly sized, high-quality, and disease-free plantlets. Three in vitro experiments were conducted to optimize the medium by evaluating nine media and a full factorial combination (matrix) of two plant growth regulators for direct organogenesis of ‘Hawaiian Red’ turmeric. Two experiments evaluated the media, and the third studied the plant growth regulator matrix. As a result, Driver and Kuniyuki walnut (DKW), Murashige and Skoog (MS), and broadleaf tree basal (BLT) media performed better than woody plant media [Lloyd & McCown woody plant basal medium (L&M), and McCown’s woody plant basal salt mixture (McCown)] for shoot and root formation. The multiplication rate was 18 plants per explant in DKW with 1 mg⋅L−1 6-benzylaminopurine (BAP) and 0.1 mg⋅L−1 1-naphthaleneacetic acid (NAA). After transferring the plants to an ex vitro environment, the survival rate was 97%, and 30% higher than previously reported. DKW produced the highest number of plantlets (with shoots and roots), and BLT produced fewer plants with higher biomass. In the MS media, higher BAP to NAA ratio (2.5 to 0.1 mg⋅L−1) produced the most significant number of shoots; however, the lowest concentration of BAP and NAA (0.1 mg⋅L−1 of both) produced the highest number of rooted plantlets. There are two recommendations for tissue culture of ‘Hawaiian Red’ turmeric. To produce the highest number of plantlets, one should use the higher BAP to NAA ratio (2.5 mg⋅L−1 BAP and 0.1 mg⋅L−1 NAA) for shoot proliferation and then transfer the explants to the root initiation media. However, to reduce the number of subcultures, the explants can be grown in the lowest concentration of both BAP and NAA (0.1 mg⋅L−1) to induce both shoot and root. Although, the number of plantlets (with roots and shoots) will decrease in this method, there is no need for subsequent subcultures and changing of the plant growth regulator combinations.

High Frequency Direct Organogenesis, Genetic Homogeneity, Chemical Characterization and Leaf Ultra-Structural Study of Regenerants in Diplocyclos palmatus (L.) C. Jeffrey

Diplocyclos palmatus (L.) C. Jeffrey, commonly referred to as “Shivalingi” or “Lollipop climber” is a valuable medicinal plant with a climbing growth habit used in traditional medicine. It is reputed to have antiarthritic, anti-diabetic properties and to be useful in various skin and reproductive problems. Overexploitation of wild plants and low seed germination have resulted in the decline of the species in the wild. Thus, the present investigation was aimed to establish an effective in vitro propagation procedure for its large-scale production and conservation. Nodal explants, obtained from an established mother plant were grown on MS basal medium augmented with various cytokinins, alone or in combination with auxins, to study the morphogenic response. A maximum of 8.3 shoots/explants with an average shoot length of 7.2 cm were produced after six weeks on MS containing benzylaminopurine 5.0 µM + 1-naphthaleneacetic acid 2.0 µM. After 4 weeks of transfer, microshoots rooted well on a low nutrient medium of ½ MS + 1.0 µM indole-3-butyric acid, with a maximum of 11.0 roots/microshoot and an average root length of 7.4 cm. With an 80% survival rate, the regenerated plantlets were effectively acclimatized to natural conditions. DNA-based molecular markers were used to investigate the genetic uniformity. Scanning Electron Microscopic examination of leaves indicated the adaptation of the plantlets to natural, as evidenced by the formation of normal stomata. Gas chromatography-mass spectrometry analyses of mother and micropropagated plants were performed to identify essential secondary metabolites. The results obtained show that the in vitro propagation system can be adopted for preservation, large-scale production and secondary metabolites’ production in D. palmatus.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2–13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.