In an effort to increase somaclonal variation in blueberry, a protocol was developed to regenerate viable shoots from internode segments. The explant consisted of the last-formed, fully developed internode taken from 3 different genotypes of Vaccinium grown in vitro. Explants were cultured 6 weeks on Zimmerman's Z-2 medium supplemented with 2iP, zeatin, thidiazuron, kinetin, or BA at concentrations of 5, 25, 50, and 100 uM. Explant response to the treatments varied and included: no response, callus growth only, callus growth and subsequent shoot formation originating from the callus mass, and adventitious shoot formation directly from the internode segment without an intervening callus. Greatest shoot regeneration (20-25 shoots/explant) was obtained on medium supplemented with zeatin at 5, 25, and 50 uM, however treatment response was not consistant across all genotypes. Regenerated shoots could be readily sub-cultured, rooted in soil mix and will be evaluated for somaclonal variation.
Jim Hruskoci and Paul E. Read
Kimberly A. Pickens, (Max) Z.-M. Cheng, and Stephen A. Kania
The mitotic inhibitors, colchicine and oryzalin, were evaluated for their effects on callus, adventitious shoot formation, and tetraploid induction of Euphorbia pulchurrima `Winter Rose'. In vitro grown leaf sections were placed on various media supplemented with either colchicine or oryzalin at various concentrations for 1 to 4 days. Colchicine was less damaging to leaf tissues than oryzalin. On various colchicine-containing media, prolific calluses were produced and adventitious shoot formation was observed. Regenerated shoots were found to be diploid as determined by flow cytometry. On media supplemented with oryzalin (28.9 μm to 144 μm), leaf tissues produced callus but failed to form adventitious shoots. Samples of calluses produced on oryzalin-containing media were subject to analysis using flow cytometry and were found to be diploid. These results suggest that the colchicine is less toxic on poinsettia tissues and shoot induction than oryzalin. Additional experiments are needed to establish a protocol for in vitro induction of poinsettia tetraploid with colchicine and oryzalin.
A.R. Kuehnle and N. Sugii
Leaf explants of seven cultivars of Hawaiian anthuriums (Anthurium andraeanum Linden ex André cv. Kaumana, Kozohara, Marian Seefurth, Mauna Kea, Nitta, Ozaki, and Paradise Pink) produced callus most successfully after 2 to 3 months on a modified Pierik medium containing 0.36 μm 2,4-D and 4.4 μm BA. Petiole explants callused best on Pierik modified Pierik, and Finnie and van Staden media. Long-term cultures of callus from Univ. of Hawaii anthurium selections UH965, UH1060, and UH1003 were maintained for 12 to 13 months and were still capable of plantlet regeneration. Adventitious plantlets were recovered from callus plated on a Kunisaki medium containing 2.2 or 22 μm BA. Regeneration appeared to be organogenic rather than embryogenic and varied among the genotypes tested. Chemical names used: N- (phenylmethyl)-1H-purin-6-amine (BA); (2,4-dichlorophenoxy) acetic acid (2,4-D).
Hae Young Na*, Dong Jin Shin, and Changhoo Chun
Pimpinella brachicarpa (Chamnamul in Korean) is an indigenous plant that grows in Korean mountain areas. It has not been cultivated yet but is gathered to use as a vegetable. Its difficulty of propagation by seeds is one of the major reasons not to be cultivated as a horticultural crop despite its demand. As a promising propagation method for the Chamnamul, we have developed a micropropagation system using somatic embryogenesis. In the present study, induction of embryogenic callus of the Chamnamul affected by part of explants (leaf and stem) and concentration (0, 0.1, 0.5, 1.0, 1.5, and 2.0 mg·L-1) of growth regulators (2.4-D, IAA, IBA, and NAA) was investigated to find the best conditions for embryogenic callus induction. A full strength of MS medium was used for a 50-day culture for all the treatments. The embryogenic callus was firm and light yellow in color and was distinct from the non-embryogenic callus that was friable and semitransparent. More embryogenic callus was induced in the treatments that the stem was used as an explant comparing with the treatments that the leaf was used. The 2.4-D treatments resulted in the better induction of embryogenic callus than other growth regulator treatments, and 1.5 mg·L-1 was the most effective among all the 2,4-D concentration treatments. Addition of 0.1 mg·L-1 BA to 2.4-D treatments retarded the induction of embryogenic callus of the Chamnamul, while the promotion of induction and multiplication of embryogenic callus was reported in many plant species by adding BA with low concentration to an auxin-base medium. The better induction was found in the treatments of darkness and dim lighting (10 μmol·m-2·s-1 of PPF) than in treatments of the higher PPF.
Xabier Barandiaran, Nieves Martín, María Fernanda Rodríguez-Conde, Antonio Di Pietro, and Jesus Martín
The influence of different callus induction media on the regeneration process in garlic was tested. The auxin 2,4 dichlorophenoxyacetic acid frequently used in garlic tissue culture was found to be detrimental when used at the levels described in the literature. However, combinations of growth regulators commonly used for dicot tissue culture produced high levels of callus induction and regeneration that could be used efficiently in a transformation program.
A. Millie Burrell, R. Daniel Lineberger, Keerti S. Rathore, and David H. Byrne
Fifteen genetically diverse roses were evaluated for the ability to undergo somatic embryogenesis. Over the two media (MS and B5), two sugars (glucose and sucrose), and two explants (filaments and petiole) used, 20 to 30% of the `Tournament of Roses' callus was embryogenic whereas only crystalline callus was produced in cultures of `Baby Love', `Ingrid Bergman', `Perfume Delight', `Prominent', `Sunflare', and 90-202. Cultures of `Tournament of Roses' consistently produced somatic embryos whereas `Baby Love' produced no embryos. An F1 progeny of `Tournament of Roses' × `Baby Love' was chosen to test whether the ability to undergo embryogenesis in Rosa hybrida L. is heritable. Data collected from tests on F1 progeny between these genotypes suggest that the ability to undergo embryogenesis is indeed heritable in an additive fashion.
William L. Summers, Juan Jaramillo, and Theodore Bailey
Anthers of L-680A', `Licato', and `Ailsa Craig' tomato (Lycopersicon esculentum Mill.) were plated on Doy's basal medium 1 to determine whether microspore developmental stage and anther length influence anther callus production. Although calli were induced at all stages of anther development, anthers containing prophase I-stage microspores produced the highest frequency of calli. Fewer calli were produced as microspores approached the uninucleate and binucleate pollen stage. Callus diameter also decreased as anther development progressed. Significantly larger calli were produced from prophase I than later-stage anthers. Time of anther harvest (morning vs. afternoon) did not significantly affect callus number or diameter. Anther and flower bud length both were significantly correlated with anther developmental stage, the number of anthers producing calli, and mean calli diameter. In each case, anther length exhibited a significantly better correlation than bud length.
Shuizhang Fei, Paul E. Read, and Terrance P. Riordan
The use of buffalograss [Buchloe dactyloides (Nutt.) Engelm] in home lawns and golf courses has been increasing because of its drought resistance and low growth habit. In vitro regeneration of buffalograss at a high frequency may provide an effective tool to introduce new variation for breeding use. The positive effects of AgNO3 on friable embryogenic callus production and regeneration efficiency is well documented in maize. In order to determine if AgNO3 has the same effect on buffalograss, two vegetatively propagated cultivars, a female `609' and a male `45-3' were tested at three different concentrations of AgNO3 at 5, 10, and 15 mg·L–1 using immature inflorescences as explants. Murashige and Skoog medium supplemented with 2 mg 2,4-D/L was employed as the control medium. Medium containing AgNO3 significantly promoted the production of friable callus for `45-3' with the highest percentage achieved at 10 mg AgNO3/L. AgNO3 medium led to production of significantly larger calli than found for the control. However, no difference was detected among 5, 10, and 15 mg AgNO3/L with regard to the callus formation ability and the size of callus initiated on these three treatments. Calli were then transferred to MS medium supplemented with BA at 0.1, 0.5 or 1.0 mg·L–1 to induce shoot formation. BA at 0.5 mg·L–1 gave the best differentiation response. Calli formed in the absence of AgNO3 produced more shoots per callus, but more calli were produced in the presence of AgNO3, and the overall regeneration efficiency was much higher with AgNO3 at 10 mg·L–1. In contrast, AgNO3 showed no promotive effect on callus production and regeneration for `609'.
F. Nekouei and J.O. Kuti
Callus induction in 12 genotypes of faba bean (Vicia faba L.) genotypes from Egypt were examined. Cotyledon, leaf petiole, and stem explants were cultured on two basal agar media; Murashige and Skoog (MS) and Gamborg (B5). The media contained 0.5 mm 2,4-D, 0.25 mm NAA, and 30 g of sucrose/L. Calli were easily formed in B5 media and induction rate was significantly dependent on the genotype. The highest induction rates occurred mostly in genotypes from Assiut Univ., Egypt, and in a local variety `Goya'. These callus cultures will be used for in vitro screening of the faba bean genotypes for tolerance to salt and drought, respectively.
Isabel Arrillaga, Victoria Lerma, Pedro Pérez-Bermúdez, and Juan Segura
The effects of stage of microspore development, cold pretreatment, and growth regulators on the embryogenic response of anthers from service tree (Sorbus domestica L.) are reported. Callus proliferation required the presence of growth regulators in the culture medium, best results being obtained with combinations of auxins and BA. Microspore embryogenesis was only induced when anthers containing tetrads or uninucleate pollen were cultured on BA-supplemented media containing IBA or IAA. A cold pretreatment before anther culture elicited a significant decrease in callus formation and inhibited embryogenesis. Chemical names used: benzyladenine (BA), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA).