Neem is considered to be one of the most promising plants for producing pesticides, pharmaceutical, as well as many commonplace materials. A protocol for shoot formation from nodal and stem explants is described. Stem nodes and stem segments were obtained from mature tree and cultured in Murashige and Skoog medium (MS) supplemented with 0.5 μM thidiazuron (TDZ), and 0.5 uM naphthaleneacetic acid (NAA). Stem node explants produced multiple shoots which were separated and cultured on MS supplemented with 0.01, 0.03, 0.5, or 0.9 uM TDZ with 0.5 uM NAA. Stem explants produced callus which regenerated shoots upon transfer to a fresh medium. Formed shoots produced roots in proliferation medium or rooted in MS supplemented with 3.3 uN indolebutyric acid, and were transferred to soil. Number of produced shoots increased with increasing TDZ concentration but shoot and root length decreased.
A new in vitro protocol was developed for multiple bud induction and plant regeneration from embryonic axis explants of four common bean (Phaseolus vulgaris L.) and two tepary bean (P. acutifolius A. Gray) lines. The explants were prepared from two embryo sizes, 3 to 4 mm and 5 to 7 mm, corresponding to pods collected after 15 and 25 days from flowering, respectively. The embryonic axis was cultured on Gamborg's B5 basal medium with 0, 5, 10, or 20 μm BA in combinations with 0, 1, or 2 μm NAA. The cultures were maintained at 24 to 25C under continuous light or incubated in darkness for 2 weeks followed by continuous light before transfer to the secondary B5 medium (0 or 2 μm BA or 2 μm BA plus 4 μm GA3). Adventitious roots or a single shoot with roots formed on the explants cultured on media without plant growth regulators. Multiple buds were induced on all BA media, but more were produced with 5 or 10 μm for most lines. Dark incubation greatly enhanced multiple bud initiation. Shoot buds were not produced on media containing NAA alone or in combinations with BA. On the secondary medium, six to eight shoots per explant for common bean and up to 20 shoots per explant from tepary bean were observed after 3 weeks. Mature, fertile plants were produced from these shoots. Chemical names used: benzyladenine (BA); 1-naphthaleneacetic acid (NAA); gibberellic acid (GA3).
Improved in vitro clonal propagation methods are valuable tools for nurseries and growers, and are essential for manipulation and improvement of tree fruit germplasm using the tools and techniques of biotechnology. We have developed a rapid shoot multiplication procedure for clonal propagation of apple, Malus ×domestica cv. Gale Gala and pear, Pyrus communis L. cv. Bartlett. Rapid clonal multiplication was achieved after the following series of steps: pre-conditioning of micropropagated shoots, sectioning pre-treated stems into thin slices, placing slices onto shoot induction medium and incubating directly under cool-white fluorescent lights or after a brief dark incubation. Multiple induction of shoots recovered from stem slice explants within three weeks of culture. A maximum of 37% of cultured apple stem slices, and 97% of pear stem slices, showed induction of shoots. More shoots were recovered on phytagel solidified shoot induction medium than on agar. Cultured stem slices of both apple and pear showed maximum recovery of shoots from shoot induction medium supplemented with thidiazuron (TDZ) compared to medium supplemented with BAP and kinetin. Under ideal conditions, pear stems generated four times the shoots as the same quantity or length of apple shoots. Micropropagated shoots were rooted and transferred to the greenhouse and field nursery for further evaluation. Chemical names used: N-phenyl-N′-1,2,3-thidiazol-5-ylurea (thidiazuron or TDZ); 6-benzylaminopurine (BAP).
Tissue proliferation (TP) is characterized primarily by the formation of galls or tumors at the crown of container-grown rhododendrons propagated in vitro. However, TP of Rhododendron `Montego' is observed initially in in vitro shoot cultures and it is characterized by the formation of multiple shoots with small leaves and nodal tumors. The formation of shoots in `Montego' TP (TP+) shoot cultures occurs without the presence of exogenous cytokinin in the medium, unlike normal `Montego' (TP–) shoot cultures, which require cytokinin for shoot growth. Structural studies have shown that tumors are composed of many adventitious buds and parenchyma cells, suggesting that TP is a result of abnormal cytokinin regulation that is controlling tumor and shoot formation. Two approaches are being used to determine if differences in cytokinin concentration and/or metabolism exist between TP+ and TP– shoot cultures. In the first approach, shoot cultures are grown in vitro for 1 week in the presence of tritiated isopentenyladenine (iP). Cytokinin uptake and metabolism are analyzed using HPLC and other analytical methods. Experiments suggest that extensive degradation and N-glucoside conjugation occur in TP+ and TP– shoots, resulting in the removal of most of the exogenous iP. In the second approach, the levels of endogenous cytokinins such as iP, isopentenyladenosine, zeatin, and zeatin riboside, are being measured in TP+ tumors and shoots and in TP– shoots by an ELISA method.
Apical shoot tips and axillary buds of buffalo gourd (Cucurbita foetidissima HBK.) when cultured on Murashige and Skoog (MS) medium supplemented with 4.4-22.2 μm (1-5 mg/liter) BA and 0-0.54 μM (0-0.1 mg/liter) NAA produced 4-9 multiple shoots within 4 weeks. The individual shoots subcultured on MS medium containing 4.9 μm (1 mg/liter) IBA produced a healthy root system in 4 weeks. Rooted cultures were successfully transferred to soil in a greenhouse. Chemical names used. BA:N-(phenylmethyl)-1H-purin-6-amine. NAA:l-naphthaleneacetic acid. IBA: lH-indole-3-butanoic acid.
Single-node cuttings obtained from 2-month-old seedlings of pecan [Carya illinoinensis (Wang.) K. Koch] were induced to break buds and form multiple shoots in liquid, woody plant medium (WPM) and 2% glucose supplemented with 6-benzylamino purine (BA) at 3 mg/liter. In vitro-derived shoots soaked in 1-3 mg/liter indolebutyric acid (IBA) produced adventitious roots in vitro; when soaked for 8 days in 10 mg/liter IBA, they were rooted successfully in soil and acclimated to greenhouse conditions. Etiolation of stock plants did not improve shoot proliferation or rooting under in vitro culture.
The regeneration medium supplemented with 2.0 mg/L BAP and 0.1 mg/L IAA allowed high efficient shoot regeneration from leaf discs and petioles of Cichorium intybus L. var. sativus. Multiple shoots ranged from 10 to 14 per explant were observed only 10 to 15 days after the initial culture. Reduced nitrogen and sucrose levels influenced on shoot regeneration frequency and growth rates. Especially, in C. Intybus L. var. sativus cv. Cesare explants cultured in the medium containing 50 mg/L MS macroelement and 1.5% sucrose displayed high regeneration frequency of 100%.
To induce multiple shoots from habanero pepper (Capsicum chinense Jacq.), nodes and stem segments were cultivated in MS medium supplemented with varying concentrations of kinetin, benzyladenine, and thidiazuron. The effect of the age of the explant in the medium on shoot formation and their latter development into plants was assessed. Ethylene concentration was measured along the experiments. Thidiazuron was the key growth regulator in the process, which at 3.4 μm induced seven to eight shoots that developed into healthy plants per explant. Plantlets in nonventilated vessels, where ethylene concentration was 0.25 ± 0.1102 μL·L–1, showed early defoliation and the formation of calli on the leaves and stems.
Soil drench of (2-chloroethyl)phosphonic acid (ethephon) at 500 or 1000 mg (active ingredient)/25-cm pot, caused prostrate growth habit and fruit set on Ficus benjamina L. grown in full sun or 47% shade. Leaves of sun plants generally had a mesophyll with multiple palisade layers, while shade plants had only limited regions of multiple cells. Ethephon treatments reduced intercellular spaces in palisade and spongy mesophyll cells, particularly near leaf margins. High shoot/root ratios, reduced leaf area, and heavy leaf drop during an interior phase occurred with ethephon treatment, especially plants grown under full sun.
Cultured shoot tips and lateral buds from greenhouse-grown rose (Rosa hybrida L. cv. Improved Blaze) proliferated multiple shoots on a basal medium (MS salts, vitamins, glycine, sucrose, and agar) supplemented with 3.0 mg/liter 6-benzylamino purine (BA) and 0.3 mg/liter indoleacetic acid (IAA). A 3-fold multiplication was achieved from freshly explanted terminal shoot tips or lateral buds after 8 weeks. Reculture of in vitro-derived shoots onto the same medium resulted in a 6-fold increase in 8 weeks. Roots could be initiated from about 50% of these shoots after transfer to a medium containing 0.3 mg/liter IAA and 0 or 0.3 mg/liter BA. Regenerated plants were successfully transferred to soil after 2 weeks.