Driver-Kuniyuki Walnut Medium Research Articles

Efficient plant regeneration through direct shoot organogenesis and two-step rooting in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver (E. ulmoides Oliver), a multipurpose woody plant, holds great economic significance due to its expansive medicinal, food and industrial applications. The rapid advancement of E. ulmoides in various fields has resulted in the inadequacy of existing breeding methods to meet its growth and annual production demands. Consequently, there is an urgent need for innovative propagation strategies. This study introduces an optimized micropropagation protocol for E. ulmoides, facilitating direct shoot organogenesis from nodal segments with axillary buds. We systematically examined the impact of basal medium composition, plant growth regulators, photosynthetic photon flux density, and sucrose concentration on bud sprouting. Employing cuttings with axillary buds as propagation material, we achieved a shortened cultivation period of merely 4 weeks for bud elongation and proliferation, marking a substantial enhancement in propagation efficiency. Notably, the Driver Kuniyuki Walnut medium, supplemented with 20.0 g L-1 sucrose and 2.0 mg L-1 trans-zeatin, induced shoots sprouting with a 100% success rate and an average length of 5.18 cm per nodal segment, equating to a great bud propagation rate of approximately 500%. Furthermore, a light source with an intensity of 80 μmol m-2 s-1 was shown the most economical choice. To address the primary challenge of inducing roots in regenerated plants, we employed a refined two-step rooting technique. This method yielded the optimal rooting frequency of 93.02%, producing an average of 5.90 adventitious roots per plantlet, each with an average length of 2.77 cm. The micropropagation program developed in this work will be the cornerstone for the preservation of the germplasm of E. ulmoides and its long-term use in medicinal and industrial applications.

Micropropagation of autochthonous olive varieties from Türkiye

Olive (Olea europaea L.) is one of the oldest cultivated fruit species in the world. Fruits and oils of autocthonous olive varieties (native Turkish olive varieties) with unique sensory properties (taste, smell and aroma) gain importance recently. Particularly olive oil companies looking for varieties that have distinct taste, smell and aroma in their oil. Propagation of olive varieties by cuttings and grafting is very difficult and expensive, therefore it is important to find solution for easy and mass propagation. Micropropagation is particularly beneficial to propagate plants that are difficult to reproduce conventionally or to ensure virus-free plants or plants with particular qualities. In this study, in vitro micropropagation success of two autocthonous olive varieties (‘Mavi’ and ‘Guleki’) grown in the origin center of olive, Southeast Anatolia, Türkiye was investigated. Both varieties, have distinct smell, taste and aroma and are difficult to root by cuttings. The effects of three different medium OM (Olive Medium), WPM (Woody Plant Medium) and DKW (Driver-Kuniyuki Walnut Medium) and two different growth regulators BAP (6-Benzylaminopurine) and Zeatin on shoot induction (proliferation) in the in vitro micropropagation of the olive varieties were examined. Obtained shoots were later subjected to in vitro rooting and acclimatization. The highest proliferation efficiency and shoot length for both varieties were obtained with the use of 1 mg Zeatin+0.1 mg GAɜ hormone combinations on OM medium. ‘Mavi’ variety formed more roots compared to ‘Guleki’ (3.33 vs. 2.75 per shoots), and gave the highest rooting rate of 74.33% with 2 mg IBA (Indole Butyric Acid) treatment on ½ OM medium. In terms of rooting rate, Guleki gave the highest rooting (100%) on medium containing ½ OM, 0.2 mg GA3 and 4 mg IBA. 60% and 75% of the micropropagated plants of ‘Mavi’ and ‘Guleki’ varieties adapted well to the external conditions.

Preservation in a cryobank of Juglans regia L. accession from several populations in the Sairam-Ugam State National Natural Park

Persian walnut (Juglans regia L.) is one of the most economically important nut crops. In Kazakhstan, the wild-growing population of Juglans regia is registered in the south of the country and is protected on the territory of the Sairam-Ugam State National Natural Park. As a result of expeditions in 2018-2019 136 accessions of Juglans regia were described according to the descriptors of the International Plant Genetic Resources Institute, fruits of 73 accessions of walnuts were collected, which are highly diverse in terms of size, shape and weight of nuts, kernel yield, color and texture of the shell and other parameters. To conserve the biodiversity of Juglans regia, the efficient cryopreservation technique has been developed for isolated embryonic axes. After surface disinfection with 0.1% mercuric chloride solution and rinsing with sterile distilled water, the embryonic axes were desiccated under laminar flow 1 hour to moister content of 12.1%, placed in cryovials, and immersed in a Dewar vessel with liquid nitrogen. In 72.4% of embryonic axes after cryogenic storage, plant recovery was noted on the Driver-Kuniyuki walnut medium with 1 mg/l 6-benzylaminopurine and 0.01 mg/l indolylbutyric acid. A cryogenic collection of 73 Juglans regia accessions has been established. For the first time, the genetic material of Juglans regia, growing in Kazakhstan, has been stored in a cryogenic bank at -196°C. Key words: Juglans regia L., walnut, cryopreservation, cryobank, isolated embryonic axes

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

High-Efficiency Somatic Embryogenesis from Seedlings of Koelreuteria paniculata Laxm.

Research Highlights: In the current study, we established a method for plant regeneration via somatic embryogenesis (SE) in Koelreuteria paniculata Laxm. for the first time. Background and Objectives: K. paniculata is an important ornamental and medicinal plant in China. However, the plant has difficulty with asexual reproduction, which imposes a limitation on large-scale propagation. Materials and Methods: Embryogenic calluses were induced from stems of aseptic seedlings on induction media. The effects of different media types and concentrations of N6-benzyladenine (BA), α-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on callus induction were examined. Embryogenic calluses were then transferred to Driver-Kuniyuki Walnut (DKW) media containing NAA (0.1–0.2 mg L−1) or 2,4-D (0.5–2.0 mg L−1) to develop somatic embryos. Cotyledon embryos were cultured on DKW media containing NAA (0.1–0.2 mg L−1) until maturation, and were then transferred to 1/2 DKW medium supplemented with 1.0 mg L−1 indole-3-butyric acid (IBA) to produce complete plants. The effects of IBA and NAA on rhizogenesis were then examined by clonal culture. Results: The maximum callus induction frequency (80.25%) was obtained on DKW medium supplemented by 0.5 mg L−1 BA, 0.25 mg L−1 NAA, and 1.5 mg L−1 2,4-D. NAA had a more pronounced effect on somatic embryo growth than did 2,4-D, with a maximum SE frequency (54.75%) observed with 0.1 mg L−1 NAA added to DKW medium. For clonal culture, the highest rooting rate (52%) was observed on 1/4 DKW medium containing 1.5 mg L−1 IBA. Histology studies confirmed the presence of embryogenic calluses and somatic embryos in different stages. Conclusions: This protocol provides a novel method for large-scale propagation of K. paniculata, and creates opportunities for genetic engineering in this species.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis.

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver–Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

SHOOT APEX CULTURE OF DORMANT BUDS IN CHESTNUT

Chestnuts (Castanea spp.) can be propagated by tissue culture methods such as meristem tip culture, shoot tip culture and bud culture. However, some limiting factors prevent using these methods extensively. For example, the explant collection periods of these methods are short, and the collection time is critical. In addition, shoot formation ratios of explants on the initial media and survival of these shootlets thereafter are generally low due to factors such as infection, browning, and vitrification. In this study the shoot apex of the dormant buds were used as the explants source for the first time. By use of this method the explant collection period was much longer and the mass multiplication probability increased. Three chestnut cultivars native to Turkey - 'Sanaslama', 'Osmanoglu' and 'Haciomer' Castanea sativa Mill. - were used in the study in two years of experiments 2007 and 2008. Two main media were used as initial to obtain shootlets: MS (1/2×NH 4 NO 3 and KNO 3 ) and DKW (Driver-Kuniyuki walnut medium). In multiplication step MS (1/2×NH 4 NO 3 medium was used with 0.5, 1.0, 1.5 mg/L BAP concentrations and in rooting MS (1/2×NH 4 NO 3 with 1.0 and 2.0 mg/L IBA concentrations were used. Shootlet formation from the explants in 2008 was higher (66.66-95.45%) than in 2007 (42.00-75.00%). MS (1/2×NH 4 NO 3 and KN0 3 ) medium has given successful results in this respect. Therefore it can be recommended as a growing medium. The multiplication coefficient varied between 3.12 to 3.57 in 2007 and 3.95 to 4.76 in 2008 according to the cultivars. Rooting of the shootlets was difficult, and no rooted shootlet was obtained. However the experiment is now being continued, and new rooting media will be tested. The results obtained from this study showed that this method can be efficiently used in the mass multiplication of chestnuts.

In vitro propagation of Acer grandidentatum Nutt.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.

(292) In Vitro Rooting of Bigtooth Maple Microshoots

Double-node microshoots of bigtooth maple (Acer grandidentatum Nutt.) were rooted in vitro on Driver-Kuniyuki Walnut (DKW) tissue culture media containing indole acetic acid (IAA). Microshoots represented six sources from three locations within Texas and New Mexico. Microshoots were placed in Phytatrays II™ containing DKW media with no plant growth regulator (DKW0) to reduce the high cytokinin levels used for shoot proliferation. Microshoots were induced to form roots for 15 days by placing them on DKW media containing IAA at 0.01, 1, 2.5, 5, 10, 15 or 20 μmol. Rooting frequency, the number of leaves and callus area were recorded every 30 days for 60 days. Rooting frequency increased up to 29% as IAA concentration increased (P= 0.004). However, as much as 71% of shoots for one of the three Guadalupe Mountain, Texas, sources rooted without auxin treatment after 30 days. The IAA concentration also affected the number of leaves per shoot (P= 0.0228) which averaged seven and callus area (P= <0.0001) which averaged 52 mm2. Average leaf size was 307 mm2. We conclude that IAA induces rooting in microshoots of bigtooth maple after 15 days of root induction. However, one source rooted without auxin treatment. The presence of callus does not interfere with root formation.

Micropropagation of a Cold Hardy Selection of Cercis canadensis L. Through Single-Node Culture

Abstract A cold hardy eastern redbud (Cercis canadensis L., TS 0092) was propagated using single-node micropropagation. In vitro shoot cultures were initiated from rapidly growing shoot tips of a mature tree. The effects of basal media and plant growth regulators on proliferation were investigated. Maximum proliferation rate was obtained from all three tested media, Murashige and Skoog (MS), Woody Plant Medium (WPM), and Driver-Kuniyuki walnut (DKW), supplemented with 0.5 μM thidiazuron (TDZ). Shoots produced on WPM medium appeared stressed showing yellowish and thin leaves compared to those on MS and DKW media. Shoots were in vitro rooted in auxin-free half strength WPM medium after being pulsed for 1 to 16 days in half strength WPM medium with 12.5 to 100 μM indole-3-butyric acid (IBA). In vitro rooting rate was influenced by IBA concentration, pulsing time, and shoot sources (media in which shoots were produced). Shoots produced in DKW medium rooted best after being pulsed in 25 to 100 μM IBA for 8 days although root numbers were similar among treatments. Direct ex vitro rooting was achieved by quick-dipping the microshoots in 10 μM IBA or NAA solutions and inserting them in Jiffy Mix potting medium.

Micropropagation ofLewisia cotyledonusing Axillary Buds from Flower Peduncles

<i>Lewisia cotyledon</i> (S. Wats.) B.L. Robins. (Portulacaceae), a perennial native to the mountainous areas of the western US, was micropropagated using the lower axillary buds from flower peduncles. Successful establishment in tissue culture was genotype dependent. Driver Kuniyuki Walnut medium (DKW) supplemented with 3.5 μM 6-benzylaminopurine (BA) appeared to be a better basal medium for increasing and maintaining <i>in vitro</i> rosettes than either Murashige and Skoog medium (MS) or Woody Plant medium (WPM) supplemented with the same BA concentration, but this response was genotype dependent. Placing rosettes on MS medium supplemented with 9.8 μM indole-3-butyric acid (IBA) for 12 wk resulted in 100% rooting with an average of 16 roots per rosette. Rooted rosettes were successfully transferred to <i>ex vitro</i> culture and were phenotypically normal. Micropropagation of lewisia will enable growers to produce large numbers of plants rapidly for the home landscape.

Micropropagation of Lewisia cotyledon using Axillary Buds from Flower Peduncles

<i>Lewisia cotyledon</i> (S. Wats.) B.L. Robins. (Portulacaceae), a perennial native to the mountainous areas of the western US, was micropropagated using the lower axillary buds from flower peduncles. Successful establishment in tissue culture was genotype dependent. Driver Kuniyuki Walnut medium (DKW) supplemented with 3.5 μM 6-benzylaminopurine (BA) appeared to be a better basal medium for increasing and maintaining <i>in vitro</i> rosettes than either Murashige and Skoog medium (MS) or Woody Plant medium (WPM) supplemented with the same BA concentration, but this response was genotype dependent. Placing rosettes on MS medium supplemented with 9.8 μM indole-3-butyric acid (IBA) for 12 wk resulted in 100% rooting with an average of 16 roots per rosette. Rooted rosettes were successfully transferred to <i>ex vitro</i> culture and were phenotypically normal. Micropropagation of lewisia will enable growers to produce large numbers of plants rapidly for the home landscape.

659 (PS 4) CYTOKININS AND DONOR PLANTS AFFECT REGENERATIVE CAPACITY OF AMERICAN ELM LEAVES

Adventitious shoots can be regenerated from leaf explants of American elm (Ulmus americana L.), but the effects of cytokinins and donor plants were unknown. The goal of this study was to examine factors that influence regenerative capacity of American elm leaves. Excised leaves from 2-year-old seedlings were surface sterilized, and 1 cm2 sections were taken from the midrib portion of the leaves. Three to six seedlings were used as donor plants in various experiments. Zero, 7.5, 15, or 22.5 μM of benzyladenine (BA), thidiazuron (TDZ), kinetin, zeatin, or 2iP were added to Driver Kuniyuki Walnut (DKW) medium. Basal medium (DKW and Murashige and Skoog [MS]) effects on shoot regeneration were also examined. Leaves placed on media with BA or TDZ formed adventitious shoots, with TDZ inducing the highest percentage of regeneration. The donor plant also affected the efficiency of shoot regeneration, with certain seedlings having 1.5 to 7 times more explants forming shoots compared to others. Leaf explants from donor plants with the highest regenerative capacity had a higher percentage of regeneration on DKW than MS medium. Explants from productive donor plants should be placed on DKW medium supplemented with TDZ to improve shoot regeneration efficiency from American elm leaves.

USE OF A HIGH CO2 ATMOSPHERE FOR MICROPROPAGATION OF PISTACHIO

An improved medium for the in-vitro micropropagation of P. vera genotypes was developed. Several basal medium preparations were tested and DKW (Driver-Kuniyuki-Walnut) medium was selected. Macro and micronutrients were adjusted to provide optimum growth and multiplication of shoots, some of which have been grown and multiplied for more than 2 years. The use of thidiazuron as a growth regulator was tested, but was detrimental to the explants at all tested concentrations. Initial experiments were hindered by significant bacterial internal contaminants. This problem was eliminated through the use of plexiglass chambers to provide up to a 20,000ppm CO2 atmosphere, increased light levels to promote photosynthesis, and the elimination of all carbon sources (sugars) from the substrate. An additional benefit was better shoot growth, better survival when rooting and better acclimation. 3/7 plants were rooted using 2.5 ppm IBA. Continuing experiments are focused on the effect of support medium on growth and multiplication as well as media development for other Pistacia species.