Citrus somatic hybridization and cybridization via protoplast fusion has become an integral part of citrus variety improvement programs worldwide. Citrus somatic hybrid plants have been regenerated from more than 200 parental combinations, and several cybrid combinations have also been produced. Applications of somatic hybridization to citrus scion improvement include the production of quality tetraploid breeding parents that can be used in interploid crosses to generate seedless triploids, and the direct production of triploids by haploid + diploid fusion. Applications of somatic hybridization to citrus rootstock improvement include the production of allotetraploid hybrids that combine complementary diploid rootstocks, and to combine citrus with sexually incompatible or difficult to hybridize genera that possess traits of interest for germplasm expansion. A few somatic hybrid tetraploid breeding parents have flowered, are fertile, and are being used as pollen parents to generate triploids. Several allotetraploid somatic hybrid rootstocks are performing well in commercial field trials, and show great promise for tree size control. Seed trees of most of these somatic hybrid rootstocks are producing adequate nucellar seed for standard propagation. Somatic hybridization is expected to have a positive impact on citrus cultivar improvement efforts.

Somatic hybridization in citrus: An effective tool to facilitate variety improvement

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.

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Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

Bamboo shoots could be induced to flower in vitro, but there is very little information on the effect of growth components on flowering. In this study, multiple shoots grown from in vitro, spikelet-derived, somatic embryos of Bambusa edulis were used for in vitro flowering. Multiple shoots flowered on Murashige and Skoog medium (MS) with 0.5 mM thidiazuron (TDZ) and 30 g l sucrose. Different

In vitro flowering of Bambusa edulis and subsequent plantlet survival

Vegetatively propagated plantlets of six rose cultivars were induced to flower in vitro on media containing full-strength Murashige and Skoog (MS) inorganic salts, Gamborg's B5 organic elements with 400 mg l−1
 myo-inositol, and different phytohormone combinations of 6-benzyladenine (BA) with α-naphthaleneacetic acid (NAA); thidiazuron (TDZ) with NAA; and zeatin (ZT) with NAA. The most efficient flower bud induction (49.1% and 44.1%) was obtained on media supplemented with 0.5 mg l−1 (2.27 μM) TDZ and 0.1 mg l−1 (0.54 μM) NAA or 0.5 mg l−1 (2.28 μM) ZT and 0.1 mg l−1 (0.54 μM) NAA for cultivar Orange Parade. Scanning electron microscopy (SEM) showed that in vitro flower bud induction occurred mostly between 15 and 30 d in induction medium through the normal flower development processes. With TDZ and ZT as the best choice for flower induction in all six cultivars tested, different rose cultivars varied in their responses to phytohormone treatments. Our study also revealed that the total time from original culture and subculture time before flower induction were two very important factors for in vitro flower induction. Plantlets 156–561 d from original culture and subcultured for 45 d were the best for flower induction.

In vitro flower induction in roses

Continuous axillary shoot proliferation and in vitro flowering were achieved using single node explants from a mature (over 70-yr-old) field clump of Dendrocalamus giganteus (giant bamboo). The shoots proliferated in a basal Murashige and Skoog medium with 6 mgl−1 (26.6 μM) N6-benzyladenine (BA) and 2% sucrose. The rate of shoot proliferation gradually increased to over three-fold before in vitro flowering took place. In vitro flowering was not the expression of a species-specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower. The rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation decreased at flowering, accompanied by reversion of flowering. The development of axillary meristems into vegetative or generative shoots depended on the level of BA. The possible role of BA, changes in the rate of shoot proliferation leading to build up, and release of stress in relation to flowering and its reversion are discussed.

Axillary shoot proliferation and in vitro flowering in an adult giant bamboo, Dendrocalamus giganteus Wall. Ex Munro

Dasar dasar agronomi

Protoplasts isolated from embryogenic callus of Fortunella polyandra (Ridl.), Atalantia bilocularis (Pieree ex Guill.), Hesperethusa crenulata (Roxb.), Glycosmis pentaphylla (Retz.) Corr., Triphasia trifolia (Burm. f.) P. Wils. and Murraya koenigii (L.) Spreng. were cultured in MT (Murashige and Tucker 1969) basal medium containing 5% sucrose supplemented with 0.0, 0.001, 0.01, 0.1 or 1.0 mg l(-1) BA and 0.6 M sorbitol. The highest plating efficiencies for all species were obtained on MT basal medium containing 5% sucrose supplemented with 0.001 mg l(-1) BA. F. polyandra produced higher percentages of globular somatic embryo development, while A. bilocularis consistently showed a lower percentage of globular somatic embryo development in all 5 concentrations of BA. MT basal medium containing 5% sucrose and supplemented with 0.001 mg l(-1) BA was found to be a suitable medium for development of globular somatic embryos derived from protoplasts to form heart-shaped somatic embryos with cotyledon-like structures. The highest percentages of shoot formation for all 6 species were obtained using 0.1 mg l(-1) GA3. A complete protoplast-to-plant system was developed for F. polyandra, A. bilocularis and T. trifolia, which could facilitate the transfer of nuclear and cytoplasmic genes from these species into cultivated Citrus through protoplast fusion.

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus

Branch internodes of mature plants and stem internodes of seedlings of Fortunella hindsii flowered in vitro on half-strength MT (Murashige and Tucker 1969) basal medium supplemented with benzyladenine, adenine, 6-γ-γ-dimethylallylaminopurine and kinetin. The highest percentage of flowering was achieved with explants originating from branch internodes of flowering plants close to the apex on half-strength MT basal medium containing 5% sucrose and 0.01 mg 1−1 BA in light. Exposure to darkness for more than 3 weeks followed by re-exposure to light reduced flowering. Flowering required a 4-day exposure to BA, but shoot formation could be initiated even without exposure to BA. First branch internode segments on MT basal medium containing 5% sucrose were prolific in flower (85%) production. The sucrose treatment affected the flower bud size distribution. There were about 13 flower buds per culture in the largest size category (>5 mm).

In vitro flowering of Fortunella hindsii (Champ.).

Shoots of orange jessamine (Murraya paniculata) a member of the Rutaceae family flowered in vitro on half-strength MT basal medium containing 5% sucrose. The highest percentage (95%) of flowering was obtained on medium supplemented with 0.1 mg l–1 N6-benzyladenine and pH 5.7. A “floral gradient” was detected among the stem internodes and root segments derived from seedlings, with shoot and flower formation significantly influenced by position on the shoot internodes and root segments. Flower buds originating from shoots derived from seeds but not other tissues developed into normal flowers and produced zygotic embryos.

High-frequency in vitro flowering of Murraya paniculata (L.) Jack

Embryogenic callus was induced from the hypocotyl region of seedlings germinated from immature embryos of orange jessamine (Murraya paniculata (L.) Jack) on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 5.0 mg l-1 benzyladenine, 2.5 mg l-1 2,4-dichlorophenoxyacetic acid and 600 mg l-1 malt extract. Isolated protoplasts divided to produce callus on Murashige & Tucker (1969) medium containing 50 g l-1 sucrose, 0.01 mg l-1 gibberellin A4+7 and 600 mg l-1 malt extract. Callus developed to plantlets via somatic embryogenesis on Murashige & Tucker (1969) medium with 50 g l-1 lactose but no plant growth regulators. These plantlets flowered in vitro on half strength Murashige & Tucker (1969) medium containing 50 g l-1 sucrose after 2 months culture.

Hasan Basri Jumin

Papers

Dasar dasar agronomi

Plant regeneration via somatic embryogenesis from protoplasts of six plant species related to Citrus

In vitro flowering of Fortunella hindsii (Champ.).

High-frequency in vitro flowering of Murraya paniculata (L.) Jack

Embryogenic protoplast cultures of orange jessamine (Murraya paniculata) and their regeneration into plants flowering in vitro