Carol D. Robacker

Genetic control of somatic embryogenesis in cotton petiole callus cultures

SummaryThree commercial varieties (Acala SJ-5, Coker 312 and Paymaster 303) and three exotic accessions (T1, T25 and T169) of cotton (Gossypium hirsutum L.) were tested for ability to undergo somatic embryogenesis. Sections of split petiole were cultured on 3 media and evaluated for embryogenesis after 180 days. Embryogenic T25 and Coker 312 plants were selected and crossed in a diallel with non-embryogenic Acala SJ-5, Paymaster 303, T1 and T169 plants. F1, F2 and BC1 populations were generated and tested for embryogenesis on a medium of MS salts and vitamins (1962) plus (per liter) 4.0 mg NAA, 1.0 mg Kn, 30 g glucose, 100 mg myo-inositol, 2.0 g Gelrite and 0.75 g MgCl2. Segregation for both occurrence and magnitude of embryogenesis was observed, suggesting the action of more than one gene.

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l-1 NAA and 1.0 mg l-1 kinetin; medium 2 contained 0.1 mg l-1 2,4-D and 0.1 mg l-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).

A programmable micropropagation apparatus using cycled liquid medium

An apparatus was developed that featured programmable application of liquid medium to plant cultures for micropropagation. Computer control capabilities included liquid medium introduction and medium depth within four culture vessels, medium application and removal on an assigned schedule, schedule adjustment during a culture period and medium replacement. The medium level was controlled using an accurate custom level-sensing technique consisting of thermistors and float switches. Seven-liter polycarbonate containers were modified and used as the culture vessels. Maintaining sterility was a key constraint in the development of the plant tissue culture apparatus.

Media and gelling agent effect on cotton callus initiation from excised seed hypocotyls

A factorial experiment was performed to develop a medium which would support initiation and proliferation of callus in a diverse group of exotic lines of Gossypium hirsutum. Seed hypocotyls of T1, T25 and T133 were cultured on Linsmaier and Skoog (LS) basal medium (1965) with NAA or 2,4-D tested in combination with BA or kinetin. The best medium from this study was then compared to five published media for support of callus initiation and growth of the varieties Acala 1517-75, Coker 500, Dunn 120, Paymaster 303 and TM1. Furthermore, the effects of two gelling agents, Difco-Bacto agar and Kelco Gelrite, were investigated with each of the six media. Significantly more callus was initiated on media solidified with Gelrite than with agar. The best callus production occurred on LS medium supplemented with 30gl-1 glucose, 0.1 mgl-1 BA and 0.1 mgl-1 2,4-D.

Culture of Amelanchier x grandiflora in a programmable micropropagation apparatus

A micropropagation system was developed to test concepts for automation and microenvironment alteration. Amelanchier x grandiflora Rehd. ‘Princess Diana’ (serviceberry) shoot cultures were grown in seven-liter polycarbonate containers. Through computer control, the apparatus intermittently applied culture medium to the plant material according to a selected schedule. Shoot growth in the programmable system was compared with four micropropagation treatments: gelled and liquid medium in baby food jars and gelled and non-cycling liquid medium in a seven-liter vessel. Plants cultured in continuous contact with liquid medium became increasingly vitrified. Significantly greater shoot number, shoot length, shoot weight, and culture weight occurred in the programmable system than in jars with gelled medium. The combination of liquid medium, 7-liter vessel, and intermittent contact with medium caused a significantly higher proliferation rate than any combination of jar/vessel and gelled/liquid medium tested.

Genetic relationships within Rhododendron L. section Pentanthera G. Don based on sequences of the internal transcribed spacer (ITS) region

Abstract Genetic relationships among specimens of the 15 currently recognized species in Rhododendron L. section Pentanthera G. Don were derived from sequence comparisons of the internal transcribed spacer (ITS) region. Sequences of the entire ITS region including ITS1, ITS2, and the 5.8S subunit were generated by direct sequencing of polymerase chain reaction (PCR) amplified fragments. Rhododendron vaseyi A. Gray, Rhododendron section Rhodora (L.) G. Don was used as an outgroup. Aligned sequences of the 16 taxa resulted in 688 characters. The region contained 38 variable sites and eight phylogenetically informative characters. A bootstrap analysis was performed and a dendrogram was constructed with MEGA. Divergence values among the taxa were extremely low ranging from 0.00 to 3.51%, providing support to traditional views of section Pentanthera as a group of very closely related species.

Interspecific hybridization between Abelia × grandiflora ‘Francis Mason’ and A. schumannii via ovule culture

Inter specific hybridization among species of Abelia offers the potential for new cultivars. Seeds from inter specific hybridization between A. × grandiflora ‘Francis Mason’ × A. schumannii failed to germinate, and ovule culture was employed. Ovules were dissected from the achenes 4, 5, and 6 weeks after pollination and cultured on either Woody Plant Medium (WPM) or Linsmaier and Skoog (LS) Medium containing 0, 9.8, or 39.2 μM 2iP. All media contained coconut water (5% v/v), sucrose(30 g/l), agar (8 g/l), and MS vitamins (1 mg/l). Embryos recovered, spontaneous embryo germination, root formation, and plant survival were recorded. Number of weeks following pollination significantly affected all parameters examined, with ovules harvested 5 weeks after pollination producing the best results. Plant survival was greatest for embryos cultured on Woody Plant Medium with 0 μM 2iP. Eight-five percent of the cultured ovules produced embryos that rooted and 65% survived the hardening off process when ovules were cultured 5 weeks after pollination on WPM containing no growth regulator.

Long-term shoot regeneration from pampas grass (Cortaderia selloana Schult.) through manipulation of growth regulators in vitro

A regeneration system for ‘Pumila’ pampas grass (Cortaderia selloana Schult.) that yields plants over many months and allows control of morphogenesis was developed. Immature inflorescence explants cultured for three 4-week passages on MS basal medium supplemented with 4.5 μM 2,4-D and 8.9 μM BA yielded a dark green callus that organized into shoot primordia. Rate of shoot development was increased after transfer of shoot primordia to medium supplemented with 9.8 μM IBA. Subculture every 4–6 weeks onto medium containing IBA yielded a continuous production of shoots. Control of morphogenesis was achieved by transferring cultures to medium containing 4.5 μM 2,4-D and 8.9 μM BA for shoot bud proliferation and to medium containing IBA for shoot production.