This paper describes an efficient colchicine-mediated technique for the in vitro induction of hop tetraploids and its confirmation by flow cytometry. A window of conditions generated a high percentage (>20%) of tetraploid induction, with the highest induction (25.6%) achieved with 0.05% colchicine for 48 h. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploid and tetraploid hops were compared, and it was determined that stomatal length and width are suitable parameters for identifying putative hop tetraploids. As well as generating tetraploids, this technique generates mixoploid hops. Calli, derived from mixoploid leaves, were induced to form shoot buds and shoots. Individual shoots were classed as diploid, mixoploid or tetraploid after screening by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but fails to generate tetraploids.

In vitro tetraploid induction and generation of tetraploids from mixoploids in hop ( Humulus lupulus L . )

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.

https://bsapubs.onlinelibrary.wiley.com/doi/pdf/10.2307/2656996

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content

An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established. For the first time Agrobacterium-mediated genetic transformation of hop (cv. "Tettnanger") was achieved. Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration. Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants. Plantlets were successfully rooted and transferred to the greenhouse. Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse. Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (β-glucuronidase). The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively. We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.

Regeneration and Agrobacterium-mediated transformation of hop (Humulus lupulus L.)

The recalcitrant nature and increased regenerative capacity in relation to in vitro subcultures in two cactus genera Rhipsalidopsis (Easter cactus) and Schlumbergera (Christmas cactus) were studied by examining the endogenous concentrations of several endogenous phytohormones and enzyme activities. Leaf tissue from greenhouse-grown mother plants, in vitro subcultures 1 and 3, and callus tissues were analyzed and correlated with regenerative ability. The cytokinins present in the two cacti genera were mainly isopentenyl-type derivatives. The total content of isopentenyl-type cytokinins in greenhouse-grown leaves of Rhipsalidopsis was more than twice the amount found in greenhouse-grown leaves of Schlumbergera. The total cytokinin content decreased during subculturing. Cytokinin oxidase/dehydrogenase (CKX, EC 1.4.3.18/1.5.99.12) activity increased during subculturing. In Schlumbergera there is no effect of subculturing on CKX and related cytokinin homeostasis. The total peroxidase (POX, EC 1.11.1.7) activity in greenhouse-grown leaves of both genera was low, and the activity increased significantly during subculturing, more specifically in the tissue of Rhipsalidopsis. The results clearly indicated that an enhanced auxin metabolism (biosynthesis, conjugation/deconjugation, and POX activity), in combination with an enhanced CKX activity, shifts the auxin and cytokinin pool, favoring adventitious shoot formation in Rhipsalidopsis, whereas the low level of POX activity, together with auxin autotrophy/conjugation, makes Schlumbergera more recalcitrant.

Regenerative Capacity of Cacti Schlumbergera and Rhipsalidopsis in Relation to Endogenous Phytohormones, Cytokinin Oxidase/Dehydrogenase, and Peroxidase Activities

http://directory.umm.ac.id/Data%20Elmu/jurnal/P/PlantScience/PlantScience_Elsevier/Vol153.Issue2.2000/5249.pdf

Adventitious shoot mass production of hop (Humulus lupulus L.) var. Eroica in liquid medium from organogenic nodule cultures

Plant regeneration was achieved from both a spontaneous clone (Braganca) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N6-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.

Maria João Sousa

Papers

Plant regeneration from stem and petiole-derived callus of Humulus lupulus L. (hop) clone Bragança and var. Brewer's Gold