An Optimized Micropropagation Protocol by Ex Vitro Rooting of Pear Rootstock OHF 333 (Pyrus communis L.)

Pear rootstock OHF 333 ( Pyrus communis L.), which is included in the US ‘Old Home’ × ‘Farmingdale’ series, is characterized by good compatibility with most other varieties, high yields, and a moderate degree of resistance to fire blight. Micropropagation in vitro has shown promise for rapid, large-scale cloning of disease-free plant material throughout the year. However, pear shoots are often recalcitrant to rooting, and this process is highly genotype-dependent. This study aimed to improve the micropropagation protocol by ex vitro rooting of pear rootstock OHF 333 ( Pyrus communis L. ‘Old Home’ × ‘Farmingdale’). Charkor, a new plant growth regulator of natural origin was used, which contains metabolism products of symbiotic fungus-endophytes of ginseng roots, as an alternative to synthetically produced plant growth regulators (PGRs). Microcuttings were obtained from in vitro cultured shoots and subjected to four different treatments for ex vitro rooting: 1 g L −1 1-naphthaleneacetic acid (NAA) (as a powder), 0.5 mL L −1 Charkor for 3 hr or 6 hr, or the same concentration of Charkor prepared as a powder. Microshoots dipped in sterile distilled water with no additional hormonal treatments served as controls. Cultures were kept in a growth chamber under a 16-hr photoperiod, with air humidity maintained close to 100% (above 96%) for 2 weeks and then gradually reduced to 60%. Data on final acclimatization rate (survival rate; %), mean number of roots per plant, stem and root length, mean number of leaves per plant, and final acclimatization rate were collected 90 days after transplanting to ex vitro conditions. All treatments induced a successful acclimatization rate of more than 31%. The highest survival rate (86%) and longest stems were achieved by treatment with 0.5 mL L −1 Charkor for 6 hr. The greatest mean number of roots per plant, root length, and number of leaves was achieved in the variant treated with 1 g L −1 powdered NAA.

In vitro multiplication protocol for Curcuma mangga : Studies on carbon, cytokinin source and explant size

Mango ginger (Curcuma mangga Valeton & Zijp.) is an underutilized rhizomatous species that has been valued in tropical Asian countries as a source of vegetable, spice, salad, medicine, and essential oil. This species is hardy and requires less care for obtaining good yields. Rhizomes are the commonly used propagules for the species, which are also the economic part of the crop. Huge quantity of seed rhizomes is required to promote this crop in larger areas. An efficient in vitro multiplication protocol is one of the options to meet the planting material requirement. Effects of carbon source (glucose, fructose and sucrose) and concentration (1 and 3%, w/v), cytokinins (BAP and meta topolin) and concentration (1 mg/L and 2 mg/L), size of explants (one/ two/ three bud) and IBA treatment (0, 250, 500 and 1,000 mg/L) for concurrent ex vitro rooting cum hardening were studied. Results revealed that for facilitating efficient multiplication, the medium should be supplemented with glucose (3%) as a carbon source and meta topolin (1 mg/L) as cytokinin. Two-bud explant should be used for subculture as it promoted superior shoot proliferation. Concurrent ex vitro rooting cum hardening was possible even without auxin treatment. The present protocol could be useful for large-scale production of quality planting material of this underexploited tropical species.

Enhanced Regeneration Through ex vitro Rooting and Agrobacterium-mediated Genetic Transformation of Eggplant (Solanum melongena L.)

Regeneration of in vitro multiple shoots was achieved through organogenesis on MS supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn from cotyledonary leaf explants of two local varieties of eggplant (Solanum melongena L.). Elongation of regenerated shoots was obtained on growth regulator free MS. In vitro root induction from excised regenerated shoots was less effective on MS with or without plant growth regulators. On the other hand regenerated shoots treated with 10 mM IBA for 5 min were found to be effective for ex vitro rooting in sterilized soil. Following sufficient development of roots, the ex vitro rooted plantlets were acclimatized in growth room condition, and were transferred to the field having 100% survival rate. The regeneration system developed was utilized for Agrobacterium-mediated genetic transformation using Agrobacterium tumefaciens strain LBA4404/pBI121 containing GUS and nptII genes. Adequate transformation response was obtained from cotyledonary leaf segments with bacterial suspension having an optical density of 0.50 at 600 nm with 30 min incubation followed by co-cultivation period of 72 hrs in Nayantara (BARI Begun-5) variety of eggplant. Selection of transformed shoots was carried out on MS supplemented with 2.0 mg/l BAP, 0.5 mg/l Kn, 300 mg/l carbenicillin and 100 mg/l kanamycin. Stable integration of GUS and nptII genes in Nayantara were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots. Plant Tissue Cult. & Biotech. 31(1): 97-108, 2021 (June)

A COMPREHENSIVE DESCRIPTION OF EX-VITRO ROOTING OF PISTACHIO ROOTSTOCKS GROWN IN PHOTOMIXOTROPHIC AND PHOTOAUTOTROPHIC CONDITION

As a basic principle, ex-vitro rhizogenesis increases the micropropagation efficiency of Micro propagation in any plant from both biological and economic viewpoints. In the current study, we surveyed the effects of number of air exchanges along with sucrose concentration on in-vitro rooting of two pistachio rootstocks consisting of UCB1 and Qazvini versus ex-vitro rooting. Based on our findings for the UCB1 rootstock, microshoot ex-vitro rooting reached the highest percentage (63.70 %) after six weeks' treatment with indole butyric acid (IBA) (5000 ppm) and free naphthalene acetic acid (NAA), while for Qazvini rootstock treated with NAA (6000 ppm) along with IBA (5000 ppm), rooting achieved 35.06%. Photomixotrophic resulted from decreasing sucrose concentration from 30 to 15 (g L-1) in corporation with ventilation condition increased UCB1 rooting (67.89%) as well as plant survival (58.34%). For Qazvini rootstock, maximum sucrose concentration (30 g L-1) improved rooting parameters. For in-vitro rooting experiment, rooting percentage of UCB1 plantlets as well as the main and lateral produced roots were higher in media supplemented with (1 mg l-1) IBA, free NAA, and BA. Regarding Qazvini rootstock, the highest in-vitro rooting percentage (43.75%) and root length were associated with the media supplemented with BA (0.5 mg l-1), IBA (2 mg l-1), and NAA (2 mg l-1). As a result, for both the studied rootstocks, better rooting parameters were observed in the ex-vitro rooted microshoots than in-vitro rooted.

Factors Affecting Ex vitro Rooting in Micropropagated Shoots from Nodal Explants of Terminalia arjuna

The present work was done with the aim to study the effect of rooting mixture and incubation temperature on Ex vitro rooting of Terminalia arjuna, an important multipurpose tree. The nodal explant collected from Ummaid garden Jodhpur was subjected for In vitro shoot proliferation on BAP supplemented modified MS medium. These shoots were In vitro multiplied on BAP (half concentration of BAP used in In vitro shoot proliferation) with low concentration of NAA supplemented medium. The individual shoots from In vitro multiplied shoots were pulse treated with IBA for 10 min. and transferred in different rooting mixture and incubation temperature for Ex vitro rooting. Analysis of data revealed that maximum 62.22% rooting was observed when the plantlet pulse treated with 984.25 µM IBA for 10 min were transferred on bottle containing vermiculite as rooting mixture and incubated at the temperature of 26°C.The optimization of Ex vitro rooting mixture and temperature conditions will be helpful in propagation of this important species rapidly in large scale.

Improving Ex Vitro Rooting and Acclimatization Techniques for Micropropagated American Chestnut1

Limited rooting and acclimatization success when micropropagating certain hardwood tree species may hinder conservation efforts of certain threatened and endangered species. Restoration efforts for such trees, such as the American chestnut [Castanea dentata (Marsh.) Borkh.], require a massive number of plantlets to be produced by micropropagation for testing, initial distribution, and orchard establishment. Therefore, increasing the number and quality of lab-produced plantlets is a key research focus. After previously determining that an ex vitro rooting system produced significantly more robust plantlets, we examined extending the time in elongation medium, rooting substrates, exogenous auxin applications, root-promoting substrate soaks, submerging the cut site, and light intensity. The most effective methods included seven weeks in elongation medium, using Jiffy peat pellets soaked in water as the rooting substrate, cutting off callus while submerged, then dipping in 0.31% IBA rooting gel, and placing plantlets in low light of 60 μmol·m-2·s-1 after rooting. By increasing the number of roots and improving acclimatization success, we can ensure that many more blight-tolerant American chestnuts will be available for field studies and eventual public distribution. Demonstrating the ecological safety and blight survival of these trees will help restore this foundational tree species and assist future restoration efforts for other threatened species. Index words: Rooting, ex vitro, American chestnut, Castanea dentata, IBA, substrate. Species used in this study: American chestnut, [Castanea dentata (Marsh.) Borkh.]. Chemicals used in this study: IBA (indole-3-butyric acid).

Simultaneous direct ex vitro rooting and adaptation of the blue honeysuckle micro-sprouts (Lonicera Caerulea L. var. Kamtschatica)

Direct rooting of regenerative plants under ex vitro conditions is of great importance in agricultural biotechnology, as it leads to acceleration of micropropagation process by eliminating the stage of in vitro rooting and reduces the costs for obtaining healthy planting material of fruit and small-fruit crops. Possibility of direct ex vitro rooting and adaptation of blue honeysuckle microshoots at one stage with no stage of in vitro rooting has been determined. For ex vitro rooting and adaptation of honeysuckle microshoots, it is required to use non-sterile substrate: Sphagnum L. moss with a surface layer of peat (0.5 cm). The rate of rooted microshoots on this substrate makes 72-84 % during summer-autumn period and not less than 60 % during winter period. Thermal treatment of the peat surface layer and the use of aqueous IBA solutions at ex vitro rooting stage will be economically justified during propagation of individual varieties characterized by low proliferation activity in in vitro culture or low ex vitro rhizogenic activity, as well as breeding novelties requiring rapid propagation and obtaining of large amount of planting material. Thermal treatment of the peat surface layer allows increasing the rate of rooted microshoots of Volkhov variety by 10.7 %, Krupnoplodnaya variety by 13.2 %, Pavlovskaya variety by 3.8 % when rooting during spring period. Use of aqueous solutions of IBA increases the rate of rooted plants by 6.2-6.7 % in Vostorg and Krupnoplodnaya varieties when rooting during summer period. Carrying out of one cutting of ex vitro rooted microshoots and further cuttings of obtained material can be used to increase the yield of planting material of blue honeysuckle varieties. Ex vitro rooting and adaptation of blue honeysuckle microshoots at one stage with further ex vitro cutting increases production profitability by 3.5 and 12.9 times in comparison with conventional method of clonal micropropagation of blue honeysuckle.