Cultivar and Phenological Stage Effects on the Success of In Vitro Meristem Culture and GLRaV-3 Elimination of Croatian Autochthonous Grapevine Cultivars

The population of Croatian autochthonous cultivars has a high degree of infection with economically important viruses, so it is necessary to carry out the elimination of the viruses in some cultivars to obtain healthy planting material. In this research, we tested in vitro meristem culture establishment on 18 autochthonous cultivars with different viral infections and the possibility of GLRaV-3 elimination through in vitro meristem culture. Plant material was sampled in a vineyard in two phenological stages, 10 days before flowering and 10 days after flowering of the grapevine. Apical meristem explants (1 mm) were placed into the MS culture medium supplemented with 0.5 mg/L benzyl adenine (BA) and 0.05 mg/L indol-3-acetic acid (IAA), and their survival, regeneration, and rooting were monitored. The results showed that the cultivar and the growth phase have a significant impact on the success of in vitro culture. In all cultivars studied higher success of in vitro culture establishment parameters (survival, regeneration, and rooting) was obtained in the case of explants sampled after flowering, with the exception of one cultivar for explants survival. Contrary to expectations, genotypes infected with three viruses (GLRaV-1, GLRaV-3, and GFLV) showed better results than genotypes infected with one or two viruses. The results showed successful in vitro establishment of Croatian autochthonous cultivar and GRLaV-3 elimination in one cultivar. However, due to the significant effect of cultivar, for routine application of this in vitro protocol on more than 100 autochthonous cultivars in need of sanitation, further studies should be conducted.

In vitro Organogenesis of Stevia rebaudiana Bert. with Different Explants and Growth Regulators

Objective: To evaluate various explants and growth regulators in order to improve invitro propagation of Stevia rebaudiana through organogenesis.Design/Methodology/Approach: Explants and growth regulators in two differentconcentrations were evaluated. The explants were nodal segment, axillary bud, andapical meristem; while the growth regulators were benzylaminopurine (BAP) at 1.125 mgL -1 and 3.0 mg L -1 , naphthaleneacetic acid (NAA) at 1.5 mg L -1 and 3.0 mg L -1 , andCIDEF-4 brassinosteroids (BRs) at 1.0 mg L -1 and 1.5 mg L -1 . In total 18 treatments withseven repetitions. Contamination, oxidation, and survival were recorded duringinduction; while leaf number, regrowth height, and root presence were recorded duringmultiplication.Results: At the induction stage there was a differential response between explantsaccording to their ontogenetic age; during multiplication, the morphological componentsshowed differences between concentrations of growth regulators and explants, withhigher effectiveness when adding BAP to apical meristems.Study Limitations/Implications: Both the origin and the age of explants can inducedifferential growth while interacting with growth regulators. Findings/Conclusions: Apical meristem explants showed better advantages for in vitroreproduction of S. rebaudiana since they present less contamination and higher survivalat the induction stage, even when exhibiting the highest oxidation among explants,which did not influence the decrease in their survival. At the multiplication stage withapical meristem, height, leaf number, and root presence were increased. Values werehigh when interacting with BAP.

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

Multiplikasi Tunas Meriklon Kentang Pada Berbagai Jenis dan Konsentrasi Sitokinin

Mericlone shoots are shoots of propagation using meristem explants which aim to produce seeds in a short time, large number, and disease-free seed. The aim of this experiment was to find out the best types and concentration of cytokinin to mericlone shoot multiplication of potato Jala Ipam variety. The experiment was carried out at Seed Technology Tissue Culture Laboratory of Agriculture, Padjadjaran University during November 2016 until February 2017. The experimental design was Completely Randomized Design with 13 treatments and 4 replications. Murashige and Skoog Medium with different concentration and types of cytokinine was used in this experiment. The result showed that the type and concentrations of cytokinin give the different effect on the mericlone shoot multiplication of potato Jala Ipam variety. MS medium with the additional of cytokinin 2 -iP 1.5 mg L-1 significantly increased the mericlone shoot multiplication of potato Jala Ipam variety at the variable number of leaves, number of nodes, and the number of branches.

Development of an efficient Agrobacterium mediated transformation system for chickpea (Cicer arietinum)

Morphologically normal and fertile transgenic chickpea plants have been regenerated through a standardized transformation protocols. This protocol is based on the infection of apical meristem explants (AME) with

Establishment of a Rapid Plant Regeneration System in Physalis angulata L. through Axillary Meristems

An optimal plant propagation method of Physalis angulata L., a medicinally important herbaceous plant species has been developed using axillary meristem explants. Shoot bud proliferation was initiated from axillary meristem explants cultured on MS medium supplemented with various concentrations of 0.5-2.5mg/L/(BAP)/(Zeatin)/(KIN). The maximum in vitro response of shooting frequency of explants (88.1%) and shoots per explant (42) was achieved with medium containing 1.0mg/L BAP. Multiple shoot culture was established by repeated subculturing of the shoot buds of axillary meristems on shoot multiplication medium. Among the subculture media BAP in combination with 1.5mg/L (IAA)+0.25mg/L(GA3) produced maximum shoots per explant (128±0.29) after two weeks of culture. Effective in vitro shoot elongation and rooting was achieved on 1.0mg/L(GA3) and 1.0mg/L(IBA), respectively. Most of the generated shoots were successfully transferred to soil under field conditions. The survival percentage of the transferred plants on soil was found to be 90 per cent. This protocol can be used for commercial propagation and for future genetic improvement studies.