Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Clonal propagation of Leptospermum spp. by tissue culture

Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04–1.0 M) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 M respectively. In L. petersonii ssp. root initiation and development was favoured by -naphthoxyacetic acid (1 M) and in L. brachyandrum indole butyric acid and -naphthalene acetic acid (1 M) were almost equally effective.     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

Clonal propagation of Cinnamomum zeylanicum Breyn. by tissue culture

Multiple shoot formation was induced directly from seeds of Cinnamomum zeylanicum Breyn. and also from seedling explants on Murashige and Skoog's medium containing different concentrations and combinations of auxins and cytokinins. Individual shoots were excised and induced to root on White's liquid medium. These plantlets were then transferred to pots in the green house and were eventually grown successfully under field conditions. Explants from the nodal region of these in vitro rooted plants were also subcultured to fresh medium. They produced a new crop of multiple shoots which could again be rooted by the same procedure.Abbreviations BAP 6-Benzylamino purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - KN Kinetin - NAA -Naphthalene acetic acid     

Clonal propagation ofBougainvillea glabra ‘Magnifica’ through shoot apex culture

Shoot apices ofBougainvillea glabra ‘Magnifica’ were induced to regenerate an average of ten shoots from their bases in response to BAP (0.5 mg/l) plus IAA (1.5 mg/l). All the isolated shoots from such cultures were rooted in a medium containing 0.1 mg/l each of IBA and 2,4,5-T and lacking BAP. Plantlets were then successfully grown in potted soil where they flowered normally. NBRI, research publication no. 32/80.     

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.     

Transformation of recalcitrant turfgrass cultivars through improvement of tissue culture and selection regime

Tissue culture techniques, medium composition, pH value and targeted tissues, agroinfection and co-culture conditions, selection process were optimized for efficient turfgrass transformation. A highly regenerable callus lines were produced in callus induction medium modified from N6 basal medium. Six-week-old calluses were cultured on Pre-regeneration medium I for 4 days and then subjected to Agrobacterium tumefaciens. After co-cultivation at 20±1 °C in a 16 h light/8 h darkness for 3 days, the calluses were cultured on non-selective Pre-regeneration medium II supplemented with 400 mg l⁻¹ l-cysteine for 7 days. Plantlets were regenerated on the Regeneration medium without selection pressure. A selection pressure was given to the regenerated plantlets when they were rooted on the Plantlet rooting medium. Roots appeared within 8–12 days in putative transformed plantlets. Resistant plants obtained were phenotypically normal and fully fertile. Chemical and molecular analyses confirmed that foreign genes were successfully introduced into the genome of perennial ryegrass or tall fescue. The transformation efficiency can attain 23.3% in perennial ryegrass.     

Vegetative propagation of western hemlock (Tsuga heterophylla) through tissue culture

Western hemlock cultures derived from cotyledon explants of2–5 week-old seedlings were established on a chemicallydefined medium containing both cytokinin and auxin. Adventitiousbud formation required a relatively high concentration of cytokininwith respect to that of auxin. The growth of buds was stimulatedby a culture medium devoid of all plant growth regulators. Plantswere successfully established in soil by rooting shoots producedfrom somatic cells directly in rooting medium "Mica-Peat." (Received July 7, 1976; )     

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

Development of salt tolerant lines of KDML and LPT rice cultivars through tissue culture

Salt tolerant lines of indica rice (Oryza sativa L.) were selected out of KDML and LPT cultivars. The first selection was made in vitro by incorporating 1 or 2% NaCl in the culture media. Embryogenic calli from mature embryo were subjected to a salt stress for four weeks. Regeneration rates after salt stress were reduced to 0.076% or less as against regeneration rates of 8.3 to 30% normally obtained for non-stressed conditions. Seedlings of regenerants and of following generations were treated with 0.5% NaCl in water culture for four weeks. Definite salt tolerance of the progenies of selected and unselected plants appeared in both cultivars. The best survival rate of line LPT 171 in R₃ was 94.3% while only 2% of the control survived. The result of the fourth generation was similar to the third.Abbreviations KDML Cultivar Kao-Dawk-Mali - LPT Cultivar Leung-Pra-Tiew - MS Culture medium (Murashige and Skoog, 1962) - WP Nutrient solution (Bentley, 1959) - R₀ Regenerated plants from callus - R₁ Progenies of R₀ - R₂ Progenies of R₁...etc This research was sponsored by the United States Agency for International Development, Washington D.C., Grant no. 936-5542-G-SS-3037-00     

Clonal propagation of Acacia catechu Willd. by shoot tip culture

A method is described for in vitromicropropagation through shoot apices of Acaciacatechu Willd., a semi-arid tree valued for Katha (atanin-like substance obtained from red heart wood of10–20 year old trees) and timber. Explants wereexcised from 15-days-old in vitro grownseedlings raised from superior seed stocks. Shoot budinduction from shoot apex explants was observed onMurashige and Skoog's (MS) [12] medium containingvarious growth regulators. A maximum of 12 shoots wasobtained on MS medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l kinetin.Well-developed shoots (3–4 cm long) were rooted on strength MS medium with 3.0 mg/l indole-3-acetic acid (IAA) and sucrose 1.5%. In vitro regenerated plantlets of A. catechu were transferred to field conditions.     

Clonal multiplication of Gardenia jasminoides Ellis through axillary bud culture

An efficient clonal multiplication system was developed for in vitro propagation of crocin — producing Gardenia jasminoides Ellis plants. Murashige and Skoog's (MS) medium containing 6-benzylaminopurine (BAP 1 mg l^–1) and indole-3-butyric acid (IBA 1 mg l^–1) resulted in multiple shoot initiation at the rate of 21 shoots per explant in 60 d of culture. Transfer of the microshoots into liquid MS medium supplemented with BAP (5 mg l^–1) with two subcultures of 15 d duration in the same medium resulted in 400 ± 25 shoots per explant. Efficient rooting was achieved in MS medium supplemented with -naphthaleneacetic acid (5 mg l^–1). The in vitro raised plants were hardened in a greenhouse and transplanted to the field successfully. The method described will be useful for rapid multiplication of Gardenia for commercial exploitation.Abbreviations MS Murashige and Skoog (1962) medium - BAP 6-benzylaminopurine - Kn kinetin - 2ip 6-(,-dimethylallylamino)purine - NAA -naphthalene- acetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid     

三角紫叶酢浆草的组织培养及快速繁殖

三角紫叶酢浆草(Oxalis triangularis A. St. Hil.)为多年生草本植物,原产热带美洲, 在城市绿化和家庭装饰中具有广阔的应用前景[1].其繁殖多采用切分块茎的方法,但繁殖速度慢;也可用种子繁殖,但其结种率低,周期长,远远不能满足市场需要.     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Crop improvement through tissue culture

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are part of the group of technologies called plant biotechnology. Tissue culture has been exploited to create genetic variability from which crop plants can be improved, to improve the state of health of the planted material and to increase the number of desirable germplasms available to the plant breeder. Tissue-culture protocols are available for most crop species, although continued optimization is still required for many crops, especially cereals and woody plants. Tissueculture techniques, in combination with molecular techniques, have been successfully used to incorporate specific traits through gene transfer. In vitro techniques for the culture of protoplasts, anthers, microspores, ovules and embryos have been used to create new genetic variation in the breeding lines, often via haploid production. Cell culture has also produced somaclonal and gametoclonal variants with crop-improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens from planting material and thereby dramatically improve the yield of established cultivars. Large-scale micropropagation laboratories are providing millions of plants for the commercial ornamental market and the agricultural, clonally-propagated crop market. With selected laboratory material typically taking one or two decades to reach the commercial market through plant breeding, this technology can be expected to have an ever increasing impact on crop improvement as we approach the new millenium.D.C.W. Brown is with Agriculture and Agri-Food Canada, Central Experimental Farm, Plant Research Centre, Ottawa, Ontario, K1A 0C6, Canada. T.A. Thorpe is with the Plant Physiology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada     

Clonal propagation of Cephaelis ipecacuanha

Shoot cultures of Cephaelis ipecacuanha A. Richard were established by using shoot tips as initial explants. Multiple shoots were obtained from node segments upon culture on B5 medium supplemented with NAA-BA (0.01–3, 5 mg/l). These shoots were rooted on B5 and 1/2 MS media containing IAA or NAA, and the regenerated plants were transferred to soil and grown in a greenhouse. The emetic alkaloids of the regenerated plants, mother plants and leaves of shoot cultures were analyzed by TLC and HPLC. Seven months of growth under greenhouse condition, the contents of the emetic alkaloids in the regenerated plants were comparable to those of the mother plants.Abbreviations B5 Gamborg B5 (1968) medium - MS Murashige-Skoog (1962) medium - 1/2 MS a half strength MS medium - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - Kin kinetin - BA 6-benzylaminopurine - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Rapid clonal propagation of Pinellia ternata by tissue culture

Adventitious buds or protocorm-like bodies were regenerated directly from excised explants without intervening callus. Differences in the ability of regeneration were observed among different plant organs with bulbils showing the highest regenerative ability followed by leaf blade and petiole. Ability of vegetative propagation of bulbil could be maintained by alternate solid-and liquid-medium culture. Theoretically, 1.7×10²⁷ plantlets could be produced from a single bulbil by this technique within one year based on the production and rapid growth of protocorm-like bodies and adventitious buds. Concentration of MS salts, NAA and sucrose influenced not only root formation from the differentiated adventitious buds, but also root number and length. For root formation, the best combination was one-half strength MS salts with 3–5% sucrose and 1 mg/l NAA. The high survival rate of 96% was recorded when plantlets were transplanted into a mixture of vermiculite:loam soil:peat moss (1:2:1). Plants from in vitro culture were morphological similar to field-grown plants. The acute toxicity of crude extracts from protocorm-like bodies was about one-fourth that of extracts from tubers of field-grown plants when tested with white mice. Tissue culture has potential for clonal propagation of Pinellia ternata plants for commercial use.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

An alternative propagation method ofAnanas through nodule culture

A micropropagation scheme forAnanas comosus Merr. was developed using nodule culture. Nodules were induced from leaf-base or chopped shoot-base explants on modified half-strength MS medium supplemented with 2.69-5.37 M NAA and 4.44 M BA and could be maintained long-term as nodules. The nodules proliferated into more nodules when chopped into pieces of 1–3 mm and placed onto the same medium. They regenerated shoots when transferred to medium supplemented with 0.54–10.74 M NAA and 0.44–8.88 M BA. The regeneration capacity of nodules is higher than that of direct regeneration or callus. Maximum regeneration was obtained from culture medium containing 0.54 M NAA and 0.44 M BA, where shoots could be observed as early as within 2 weeks. Many shoots formed roots in the same medium in which they were regenerated after 10 subcultures, but the best rooting occurred in medium containing 0.54 M NAA and 0.44 M BA. Rooted plantlets ofA. comosus Merr. could be routinely produced at 6-week intervals.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine