In vitro propagation of Alstroemeria ‘Alsaan’

Plantlets were regenerated from Alstroemeria Alsaan rhizome tips cultured in vitro on solid and liquid media based on Murashige and Skoog salt formulation. The quality of the cultures was superior when intact rather than longitudinally sliced rhizome tips were used as explants and when a temperature of 8°C rather than 22°C was used at the initiation stage. More roots were produced on rhizome tips containing a rhizome apical meristem than on rhizome sections lacking such a meristem. Most (90%) of the rooted plantlets were successfully acclimatized and developed into true-to-type flowering plants.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

Induction of direct shoot organogenesis and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L. cv. ‘Green long’)

In vitro flowering is an alternative breeding tool for generating hybrid Cucumis spp. as it is able to overcome limitations caused by interspecific incompatibility. The present study describes an efficient method for induction of multiple shoots and in vitro flowering from shoot tip explants of cucumber (Cucumis sativus L.). Shoot tip explants were excised from 7-day-old seedlings and cultured on Murashige and Skoog (MS) medium fortified with different concentrations of 6-benzylaminopurine (BAP; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L kinetin (KIN). The highest frequency (93.1%) of multiple shoot formation with maximum number of shoots (15.2 shoots/explant) was achieved on MS medium supplemented with 1.0 mg/L BAP. For in vitro flowering, shoots were cultured on MS medium supplemented with 0.5 mg/L BAP and different concentrations of sucrose. Flowering occurred on about 95% of in vitro shoots cultured on MS medium fortified with 6% (w/v) sucrose and 0.5 mg/L BAP after 15 d. For rooting, shoots (>2 cm) were cultured on MS medium augmented with various concentrations of indole-3-butyric acid (IBA; 0.5–2.5 mg/L) alone or in combination with 0.5 mg/L KIN. Among the combinations tested, supplementation with IBA (1.5 mg/L) and KIN (0.5 mg/L) induced maximum rooting rates (95.4%) with 7.8 roots/shoot. Rooted plantlets were successfully transferred into plastic cups containing a mixture of soil and sand (1:1), established in the greenhouse, and subsequently acclimatized in the field. The in vitro flowering reported in this study may facilitate rapid hybridization in Cucumis species and offers a model system for studying the physiological mechanisms involved in flowering.     

Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv. ‘Sabarlis’

Summary Pollen calli and plantlets of Hordeum vulgare cv. Sabarlis were obtained through direct pollen culture without pretreatment of spikes or preculture of anthers. Isolated immature pollen grains were cultured first in a 0.3 M mannitol solution or a C1 basal medium (Chen et al. 1979) supplemented with 0.3 M mannitol but without sucrose for 5–7 days, then transferred into a C1 medium containing 6% sucrose, 3 mM glutamine and 5 mM m-inositol. After a 3 week culture period small pollen calli derived from the pollen grains were transferred into a growth medium comprising C1 basal medium supplemented with 250 mg/1 lactalbumin hydrolysate and 0.5 mg/1 kinetin. For shoot regeneration, vigorously growing calli were transferred onto agarsolidified MS medium (Murashige and Skoog 1962) containing 3% sucrose, 2 mg/1 benzyladenine and 0.5 mg/1 indole-3-acetic acid. The ratio of green plants to albino was approximately 12.2.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

In vitro shoot regeneration from leaf discs of Betula pendula ‘Dalecarlica’ EM 85

The effect of zeatin, NAA (-naphthaleneacetic acid), putrescine and cefotaxime on the frequency of shoot regeneration from Betula pendula Dalecarlica EM 85 leaf discs has been examined. About 80% of leaf discs were induced to form adventitious shoots when the culture medium contained 45.6 moll^-1 zeatin and 0.1 mmoll^-1 cefotaxime. The addition of NAA to zeatin-containing media prevented shoot regeneration but stimulated root development directly from leaf tissues. Putrescine (0.1 mmoll^-1) and cefotaxime (0.1 mmoll^-1) could both significantly increase the percentage of leaf discs regenerating on optimal zeatin-containing media, and increase the number of shoots per regenerating disc.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzyladenine     

Micropropagation of Achillea filipendulina cv. ‘Parker’

Achillea filipendulina (family Asteraceae) is widespread throughout temperate North America. In order to clean stock plants from endemic fungal and bacterial contaminations a method for large-scale propagation of A. filipendulina through meristem culture was sought and found and is described in this paper. The best conditions for propagating A. filipendulina was found to be MS (Murashige and Skoog) salt medium supplemented with 3% sucrose and 1 mg l^–1 IAA (indole-3-acetic acid) plus 2 mg l^–1 BA (6-benzyladenine) under 16 h of cool fluorescent light. Rooted plants were successfully acclimatized within a short time after propagating on this medium. The propagation via tissue culture did not affect plant's presentation. The use of clean stock plants made it possible to increased Israeli production of Achillea from about 150,000 stems a year to about 1,300,000 stems a year.     

Effect of potassium humate on apple cv. ‘Golden Delicious’ cultured in vitro

The effects of humic substances on in vitro culture of Golden Delicious apple are reported. Potassium humate (KH) when used in proliferation showed a negative interaction with BA while it enhanced rooting when IBA was not present in the culture medium. In the presence of IBA, KH increased root number and reduced root growth. The highest concentration tested, 500 mg l^-1, caused a drastic reduction in root system development. 50 mg l^-1 KH hastened rooting and plants grew more rapidly when transferred to soil.     

In vitro organogenesis from shoot tip,internode, and leaf explants of Ulmus x ‘Pioneer’

Shoot tip explants of the hybrid cultivar Pioneer responded poorly to initial attempts to establish shoot proliferating cultures on Murashige and Skoog (MS) medium containing 2 or 4 µM benzyladenine (BA) with a four week subculture interval. A combination of weekly subcultures and an MS medium containing 2 µM BA produced shoot proliferating cultures sufficient for micropropagation. Shoot organogenesis was obtained when callus derived from internodes of actively elongating shoots was transferred from a primary medium containing various cytokinins to a secondary medium containing MS salts and 10 µM BA. These small shoots elongated when transferred to a medium containing 2.5 µM BA. Adventitious shoots also differentiated on leaf tissue of Pioneer elm. These shoots appeared to differentiate with little if any intervening callus from the margins of leaves of in vitro grown shoots where these leaves touched the medium (MS medium containing 2 µM BA). Tissue cultured shoots from all sources were rooted, acclimated, and transplanted to the greenhouse or field with good success.Salaries and research aupport provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and The Nursery Crops Research Laboratory. Journal Article No. 23-86.     

Induction of Haploid Morphogenic Calluses from in vitro Cultured Anthers of Prunus Armeniaca cv. ‘Harcot’

Apricot (Prunus armeniaca) ‘Harcot’ anthers, were cultured in vitro for the production of haploid plants. The best androgenic response was achieved with Nitsch and Nitsch (1969) medium, supplemented with 4.52 μM 2,4-D, 4.52 μM zeatin, 2.85 μM IAA and 40 g l⁻¹ sucrose. Cultures were maintained in the dark for 8 days, at 28°C, followed by transfer to a 16-h photoperiod, with 35 μm m⁻² s⁻¹ light intensity and 24/22°C day/night temperature. The androgenic response was correlated with the floral bud size, its phenologic stage and the level of microspore evolution. Anthers containing microspores at the tetrad/uninucleate stage were the most appropriate. The ploidy level of the calluses was evaluated by flow cytometry revealing that they range from haploid to octaploid. Mixoploid calluses have also been identified. Histological studies showed that the haploid calluses have their origin in the microspores. Nodular structures consisting of cells with dense cytoplasm and differentiated xylem elements were observed and were surrounded by an autofluorescent layer, probably due to cutin deposition.     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

Plant regeneration from calli of melon (Cucumis melo L., cv. ‘Amarillo Oro’)

Callus cultures from cotyledon and hypocotyl explants of a Spanish cultivar of melon (Amarillo Oro) have been tested for their growth and morphogenic capacity on a series of media with different concentrations of indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin). Melon tissues were able to undergo morphogenesis both via organogenesis and embryogenesis, depending on culture conditions and explant source. Shoot buds were obtained at high rates in cotyledon explants. In response to 1.5 mg/l IAA and 6.0 mg/l kinetin, more than 90% of the calli produced well-developed shoots. Hypocotyls failed to form shoots but formed somatic embryos on auxin containing media while cotyledon explants usually gave abundant shoots but only rarely formed embryos. It was possible to maintain organogenic callus lines for at least 12 months under defined conditions. Plants were recovered from adventitious shoots produced both in cotyledon-derived calli and from organogenic cell lines.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

In vitro regeneration in chile pepper (Capsicum annuum L.) from ‘half-seed explants’

An in vitro regeneration protocol has been developed from half-seed explants of a mild (cv. New Mexico-6) and a pungent (cv. Rajpur Hirapur) chile pepper (Capsicum annuum L). Imbibed seeds were cut into two parts such that one portion contained the cotyledons and a part of the hypocotyl (part A) while the other part had the proximal part of the hypocotyl and the radicle (part B). These explants were cultured on MS medium with or without cytokinins (KIN, BA, ZEA, 2iP). Cytokinins dramatically increased both the percentage of explants forming buds as well as the number of buds per explant, and also hastened the rate of bud production. The relative efficacy of cytokinins in inducing the formation of leafy buds varied in the two cultivars. However, the best response was observed with ZEA in both cultivars. The highest percentage of bud formation was recorded after presoaking part B explants for 72 hours. The elongation growth of leafy buds was severely inhibited in the continuous presence of high concentrations of cytokinins, and frequently the buds became quite thick, ill-defined and vitreous. Within 3–5 weeks of transfer to Magenta boxes containing vermiculite and soil (1:3), 70–85% of the rooted hypocotyls developed 1–2 elongated shoots. Following transfer to pots, these plantlets grew into normal plants.Abbreviations BA benzylamino purine - IAA indole-3-acetic acid - 2iP 6--dimethyl (allyl) amino purine - KIN kinetin - MS Murashige and Skoog medium - NAA napthaleneacetic acid - ZEA zeatin     

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. ‘Russet Burbank’

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. Russet Burbank. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.     

In vitro organogenesis and somatic embryogenesis from punctured leaf of Populus nigra × P. maximowiczii

Various explant sources of Populus nigra × P. maximowiczii were used to examine the effects of growth hormones on morphogenesis in vitro. Initial experiments indicated that punctured leaves were superior to non-punctured ones for both callus growth and formation of shoots and roots on MS medium containing various types and concentrations of growth hormones. After 6 weeks in culture, an average of 178 shoots, 129 roots and 3.1 g fresh weight of callus were directly produced from the abaxial side of each punctured leaf. The best combinations of growth hormones for shoot, root and callus proliferation were 0.88 M BA plus 0.05 M 2,4-D, 0.44 M BA plus 2.69 M NAA and 0.44 M BA plus 2.26 M 2,4-D, respectively. Embryoids were also formed on callus derived from punctured leaves. The number of embryoids varied from 0 to 30 per punctured leaf. Adventitious shoots also developed simultaneously with the embryos. Embryoids were removed with a scalpel at the early developmental stages and placed on MS medium lacking growth regulators. Regenerated plantlets were transferred to pots containing vermiculite for normal growth in the greenhouse.     

In vitro regeneration of plantlets in Fagraea fragrans Roxb. — a tropical tree

Different vegetative parts of Fagraea fragrans Roxb., a valuable timber tree of South East Asia, were used as explants in in vitro studies.Nodal segments showed the best growth response as numerous adventitious shoots were regenerated in MS medium supplemented with Benzyl adenine (BA, 8.8 um) and 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 um). Shoot buds also developed from the leaf and root segments that were subcultured. The detailed process of callus growth and differentiation leading to the formation of whole plantlets is described. Uniform plantlets obtained could be transplanted successfully in soil.