Effective medium for in vitro sprouting of the buds and multiplication of the plantlets, and identification of the fungal contaminant associated with the explants of Gnetum

Plant tissue culture requires the optimization of growth media. Gnetum, known locally in Cameroon as “Eru” is an indigenous gymnospermous vegetable with diverse medicinal, nutritional, cultural and socio-economic values. This resource is over-exploited and expected to neighboring countries, resulting to increased scarcity in the forest. Preliminary work on the in vitro culture of nodal cuttings was faced by the problem of fungal contamination. It was therefore necessary to isolate and identify the fungal contaminant, optimize the surface sterilization of field material and compose an appropriate medium for sprouting. Pure cultures of the fungus were obtained and grown on Potato Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA). The identification was based on the appearance of the fungal growth on plates and also on the microscopic view. This was affected by the use of keys. Gnetum explants were disinfected with the various concentrations of disinfectants, preceded in some instances by pre-treatments, as well as incorporating fungicides in the culture medium. Two different culture media were employed: the Woody Plant Medium (WPM) and the Murashige and Skoog (MS) based establishment medium (Y-1). Gnetum was found to live in association with a complex of Microsporum species. The level of contamination of cultures was reduced from 100% to 40% when pre-treated before disinfection and even lower to 10% by incorporating fungicides in the medium. Sprouting was observed in WPM. This study provides baseline information on the in vitro propagation of Gnetum and thus opens up avenues for more research to be carried out in this field.

An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.

Effects of Cytokinin on in vitro Propagation of Bauhinia variegata L.

Mature seeds of Bauhinia variegata L were cultured on half strength Murashige and Skoog medium. For experimentation, nodal cuttings were used as explants from in vitro growing plants. Cytokinin, N-benzyl-9-(2-tetrahydropyranyl) (BPA), kinetin(6-furfurylaminopurine), zeatin, 6-(4-hydroxy-3-methyl-trans -2-butenyl amino purine), 2- isopentenyl amino purine (2-ip), and benzylaminopurine (BAP) were tested for best propagation. Well grown plants were achieved in medium supplemented with 5 µM BPA and 0.5 µM BAP. The propagated plants were acclimatized very well after transferred to the field.

Effects of different media strengths and hormone concentrations on in-vitro regeneration of Vitellaria paradoxa C.F. Gaertn

The importance of Vitellaria paradoxa has necessitated its inclusion in the ongoing afforestation project which requires mass production of its seedlings. The shea-butter tree produces oil-rich seeds that are important to cosmetic and food and nutrition industries. It is a deciduous tree which could survive in the dry-savannah and help withstand desert encroachment. However, overexploitation of its timber for firewood and charcoal production made it vulnerable. In-vitro propagation of this species could be a viable means towards its mass propagation. Two plant-hormones: Gibberellic Acid (GA3) and Benzyl Amino Purine (BAP) were used to grow the plant in different combinations making up six treatments (A-F) with 6 replicates in each group. The combinations include 1.5/0, 1.5/1.0, 1.5/1.5, 2.0/0, 2.0/1.0, 2.0/1.5 mg/L of GA3/BAP, which were used to grow 2cm nodal cuttings of the V. paradoxa on half-strength Murashige and Skoog (MS) media. Treatment F (with 2.0/1.5 mg/L GA3/BAP) produced the highest number of leaves (2.4 ± 0.6, 3.2 ± 0.8) and longest shoot length (1.1 ± 0.3 cm, 1.4 ± 0.5 cm) at 4 and 8 Weeks After Inoculation (WAI), respectively. The results of this study showed that application of growth- enhancing hormones for the regeneration of important tree species could provide means for their mass propagation so as to meet the need for afforestation projects. Keywords: Vitellaria paradoxa, Afforestation, In-vitro propagation, Mass propagation.

In vitro Regeneration, Rooting, and Cloning of Artemisia tridentata

Artemisia tridentata (big sagebrush) is an ecologically important shrub found in western North America. In vitro techniques can be applied to big sagebrush for the purpose of studying gene function, genotypic and phenotypic plasticity studies, cloning, genotypic preservation, and restoration. I performed experiments to develop an indirect organogenesis protocol to regenerate whole Wyoming big sagebrush plants from leaf explants. Callus formation frequency was 88% (±4.0%) in leaf explants cultured on medium containing 0.5 mg/l BAP and 1.0 mg/l NAA. Shoot formation frequency was variable between replicates and was the highest when callus tissue was cultured on medium containing 1.5 mg/l BAP and 0.1 mg/l NAA, 37% to 80%. I tested several auxin treatments to induce root formation and concluded the best to be 0.5mg/l IBA, which yielded 42% to 60% rooting. Taking into account all these variables, I estimate the total regeneration efficiency to range between 14% to 43% on this set of treatments. This protocol was also applied to basin big sagebrush. Callus formation was 100% in leaf explants. Shoot formation was 34% (±14.6%), but shoots exhibited a hyperhydric phenotype and were not transferred to root induction medium. The in vitro regeneration protocol developed is a crucial element that would be required to transform big sagebrush using molecular approaches. Experiments were also conducted to determine the feasibility of shoot tip and nodal cuttings to develop adventitious roots in vitro. This method can provide genetically identical material much faster than in vitro regeneration. Adventitious root formation in Wyoming big sagebrush cuttings cultured on two media types was inconsistent, ranging from 10% in some experiments to 80% in others. Limited success was achieved in nodal cuttings cultured on modified MS medium containing auxin and cytokinin 12.5% (±5.6%). No root formation was achieved in mature plant tissue collected in the field. Results indicated that genotypic influences were likely more responsible for variations in rooting than the medium or vessel conditions tested. Cloning experiments in basin big sagebrush further supported this notion. All material for these experiments came from half-sibling individuals that was maintained separately throughout the course of the experiments. Some half-siblings formed no adventitious roots on any treatments tested whereas others had high rates of formation on all treatments. Further studies, utilizing exogenous PGRs, such as auxins, may provide more successful adventitious root formation in shoot tips from both big sagebrush subspecies.

Evaluation of Economically Important Cultivars of Seed Potato for Minituber Production

Experiments were conducted to evaluate mass production of potato minitubers by using in vitro produced plants and sprout cuttings under Gilgit Baltistan conditions. The experiments were arranged in Complete Randomized Design and replicated three times. Four potato cultivars ‘Roko’, ‘Bartina’, ‘Barna’ and ‘Kuroda’ multiplied in vitro through nodal cuttings were planted in a greenhouse. The cultivars showed variation in different parameters studied. Results revealed that cv. ‘Kuroda’ showed greater plant height (103.7 cm), higher total tuber weight per plant (194 g) and the maximum number of minitubers per plant (21.9) as well as total number of minitubers per plot (14,235) from 650 in vitro produced plants, whereas shorter plant height (33.4 cm), lower tuber weight per plant (102.8 g) and the minimum number of minitubers per plant (9.4) and total number of minitubers per plot (6,110) were observed in cv. ‘Bartina’. However, the cultivar Bartina’ produced the maximum number of stems per plant (2.2), while cv. ‘Roko’ had the minimum number of stems per plant (1.4). In another experiment, the cultivars ‘Roko’, ‘Asterix’, ‘Barna’ and ‘Paramount’ were reproduced using sprout cuttings (650 each) grown under screen house conditions. The cultivar ‘Asterix’ developed maximum number of minitubers per plant (5.9) and per plot (3,835 g). However, plant height was greater (93.4 cm) in cv. ‘Paramount’ and total tuber weight per plant was higher (198.3 g) in cv, ‘Barna’. On the other hand, the short plant height (52.2 cm) and lower total tuber weight per plot (144.7 g) were produced in cv. ‘Roko’, and the minimum number of minitubers per plant (4.7) and total minitubers per plot (3,055 g) were observed in variety ‘Barna’. The performance of in vitro produced plants of cv. ‘Kuroda’ was better under greenhouse conditions; while plants of cv. ‘Asterix’ produced through sprout cuttings performed better under screen house conditions.

Auxin-Induced Adventitious Root Formation in Nodal Cuttings of Camellia sinensis

Adventitious root (AR) formation is essential for the successful propagation of Camellia sinensis and auxins play promotive effects on this process. Nowadays, the mechanism of auxin-induced AR formation in tea cuttings is widely studied. However, a lack of global view of the underlying mechanism has largely inhibited further studies. In this paper, recent advances including endogenous hormone changes, nitric oxide (NO) and hydrogen peroxide (H2O2) signals, secondary metabolism, cell wall reconstruction, and mechanisms involved in auxin signaling are reviewed. A further time course analysis of transcriptome changes in tea cuttings during AR formation is also suggested to deepen our understanding. The purpose of this paper is to offer an overview on the most recent developments especially on those key aspects affected by auxins and that play important roles in AR formation in tea plants.