An efficient and reliable protocol of in vitro shoot regeneration must be first established to have a successful genetic transformation. As a member of legume family, alfalfa is very difficult for direct shoot regeneration. There is no published information on direct shoot organogenesis, although success has been well documented on embryogenesis, which must go through callus stage. Different plant growth regulators at various concentrations were evaluated for callus initiation, development, and direct shoot regeneration. Multiple shoots were produced directly from each individual explant. This will provide an efficient means for production of transgenic alfalfa plants. Therefore, genetic transformation of Medicago germplasm will be significantly expedited.
Guochen Yang and Marihelen Kamp-Glass
Amanda Bayer, John Ruter, and Marc W. van Iersel
by many growers in an effort to irrigate and fertilize more efficiently. Cyclic irrigation, which applies daily irrigation via multiple smaller applications, can be used to apply reduced irrigation volumes, and can reduce water and nutrient leaching
Jacob George, Harsh Pal Bais, G.A. Ravishankar, and P. Manilal
Response surface methodology was utilized in statistical optimization of three quality factors (the number of multiple shoots, shoot length, and number of leaves) pertaining to regeneration of plantlets from leaf calli of Decalepis hamiltonii Wight. & Arn. (swallow root). The variables evaluated were the levels of sucrose, BA, and NAA each at two different concentrations. Response surfaces for shoot length and multiple shoot number were useful in achieving optimal levels of media constituents and in understanding their interactions, but response surfaces for number of leaves were not. The data indicate that sucrose, BA, and NAA levels may be manipulated to increase or decrease quality factors chosen. This approach may be useful in developing a micropropagation protocol for D. hamiltonii. Chemical names used: benzyladenine (BA); napthaleneacetic acid (NAA).
J.D. Norton, G.E. Boyhan, and J.A. Pitts
The dwarfing characteristics of St. Julien and Pixy rootstocks as measured by shoot growth and trunk cross-sectional area (TCSA) was evident. Tree survival was significantly reduced after 3 years on Nemaguard and Pixy rootstocks. None of the elements measured by foliar nutrient analysis were below the minimum for plums; however, significant multiple regression equations for total shoot growth, TCSA, and survivability were evident with R 2 of ≈0.30 in all three cases.
Handan Büyükdemirci and Paul E. Read
Axillary buds of `Valiant' grapevine (Vitis spp.) grown in vitro were transferred onto Murashige and Skoog (MS) medium supplemented with different cytokinin and auxin combinations and concentrations. It was found that culture medium caused statistically important differences in number of nodes, number of fully expanded leaves, number of multiple shoots, number of roots, and length of shoots. MS medium supplemented with 1.0 mg BA/liter in combination with 0.01 mg NAA/L was found to be the best medium for shoot growth and callus production. MS medium supplemented with the combination of 0.5 mg BA/L and 0.01 mg NAA/L was the best medium for explant rooting. The medium containing BA and NAA encouraged better shoot growth than those containing BA alone. When the concentration of BA in the medium was increased, multiple shoot proliferation and teratological structures of explants increased, but the number of small leaves and length of internode decreased. Axillary bud culture led to better shoot growth than was found for shoot apex culture. The presence of leaves positively affected shoot growth from axillary buds. Also placing the axillary buds horizontally onto the medium gave better shoot proliferation and growth than placing them vertically.
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.
Mohamed F. Mohamed, Paul E. Read, and Dermot P. Coyne
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).
Eric W. Mercure, Carol A. Auer, and Mark H. Brand
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.
V.R. Bommineni, H. Mathews, S.B. Samuel, M. Kramer, and D.R. Wagner
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).
Nancy Santana-Buzzy, Adriana Canto-Flick, Felipe Barahona-Pérez, María del Carmen Montalvo-Peniche, Patricia Yolanda Zapata-Castillo, Anabel Solís-Ruiz, Amílcar Zaldívar-Collí, Omar Gutiérrez-Alonso, and María de Lourdes Miranda-Ham
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.