Culture of Barley Anthers in Conditioned Media

High yielding anther cultures of Hordeum vulgare cv. Sabarlis are obtained at inoculation densities of 10-20 per ml by use of media previously conditioned by Sabarlis anthers. To achieve these high yields anthers at the mid-unicellular pollen stage (stage 2), stressed in the excised spike for 14 d at 7 °C, are necessary, but for conditioning of media older anthers may be used with or without the stress pretreatment. Conditioning for 7 d by anthers at young bicellular pollen stages (stages 5-6) is highly effective. Hormones supplied in the medium interact synergistically with the conditioning factor. Sabarlis ovaries are shown to be even more effective for conditioning than anthers, whereas glumes and other parts of the spike are relatively ineffective. Anthers of oats, rye, wheat, maize, tobacco, and one other genotype of barley are also less effective than Sabarlis anthers. The improved method of anther culture is more efficient than spike culture for the production of pollen callus in Sabarlis barley. INTRODUCTION The growth of embryos or callus from pollen in cultured anthers is sustained both by the culture medium and by the somatic tissues of the anther. The role of the latter can be simulated by other tissues (Pelletier and Ilami, 1972; Pelletier and Henry, 1974; Zenkteler, 1980) and by liquid media that have been in contact with anthers. Only short periods of contact with liquid medium are necessary: thus, in the Solanaceous species, Nicotiana tabacum and Datura innoxia, pollen released mechanically (Wernicke and Kohlenbach, 1977), or shed naturally (Sunderland and Roberts, 1977; Sunderland, 1979), into the medium during the first few days of culture continues to develop if the anthers are subsequently removed from the culture vessel. The pollen does not develop, however, if it is transferred into fresh medium that has not been in contact with anthers. The tapetum is suggested as the source of a discrete conditioning factor. The present paper presents further evidence of conditioning with reference to barley anthers. The conditioning effect is examined with respect to anther stage, anther density, time of contact between anthers and medium, and the presence of hormones in the medium. The conditioning influence of anthers is also compared with that of other parts of the barley inflorescence and contrasted with that of anthers of other species. 1 On study leave from the Institute of Plant Physiology, Shanghai Branch of Academia Sinica. This content downloaded from 207.46.13.76 on Fri, 09 Sep 2016 04:17:39 UTC All use subject to http://about.jstor.org/terms 768 Xu, Huang, and Sunderland—Culture of Barley Anthers MATERIALS AND METHODS Tests were carried out on anthers of Hordeum vulgare cv. Sabarlis. Sabarlis plants were raised from seed in a glasshouse in monthly batches as described previously (Sunderland, Roberts, Evans, and Wildon, 1979). Other species used in the preparation of conditioned media were raised in a similar manner. Selection and pretreatment of anthers Barley anthers are most responsive in culture when the pollen is at the mid-unicellular stage (stage 2 as defined by Sunderland, 1974). Also, the response of the anthers is enhanced by prior cold-temperature stress (pretreatment) of the excised spikes (Sunderland et al., 1979). In Sabarlis barley, the pollen is developing through the critical stage as the flag leaf emerges from the tiller. Accordingly, tillers were harvested during the first 2 d after emergence of the flag leaf. With each spike the ensheathing base of the uppermost leaf was swabbed in 90% (v/v) ethanol, cut open aseptically, and the enclosed spike, after removal of the awns, placed in a plastic Petri dish (90 mm x 13 mm) containing a drop of sterile water to maintain humidity. Test anthers were removed from spikelets in the middle region of each spike (they are the most advanced) and the developmental stage of the pollen assessed by means of acetocarmine stain. Spikes having predominantly mid-unicellular pollen (stage 2) were set aside for the culture test. These, as well as older spikes, categorized as stages 3, 4, 5, or 6, and having predominantly late unicellular, mitotic, early bicellular and more mature pollen respectively, were also set aside for the preparation of conditioned media. It is emphasized that the culture tests were carried out only on anthers initially at stage 2. For the stress pretreatment, the Petri dishes containing the spikes at various stages were sealed with parafilm and stored at 7 °C for 14 d in darkness. In one series of experiments (see Table 4) tillers judged to be at stage 5, according to the length of the emerging flag leaf, were stressed by wrapping them in polythene and aluminium foil (see Sunderland and Roberts, 1977). Culture test Anthers were dissected and pooled from all but the upperand lower-most spikelets (the least advanced) of pretreated stage 2 spikes. Each spike gave 50-60 anthers. For experiments demanding more than this, anthers were pooled from several spikes. Anthers (10-20) selected at random were floated on 1 ml aliquots of liquid medium in small plastic Petri dishes (30 mm x 10 mm). These small dishes were placed in groups of three, unsealed, inside larger Petri dishes (90 mm x 13 mm) containing drops of sterile water to maintain humidity. The large Petri dishes were sealed with parafilm and incubated at 25 °C in darkness. Most tests were carried out on the potato medium (P) described by Chuang, Ouyang, Chia, Chou, and Ching (1978), but some were also carried out on the chemically-defined N6 medium (N) described by Chu (1978). Both media contained sucrose at 9% (w/v) and were used with or without a hormone supplement consisting of 2,4-dichlorophenoxyacetic acid at 1-5 mg 1_1 and kinetin at 0-5 mg l"1. These media, referred to below as P-9, P-9H, N-9, and N-9H, were sterilized by autoclaving. Preparation of conditioned media Anthers or other tissues were floated on 5 ml aliquots of P-9, P-9H, N-9, or N-9H in plastic Petri dishes (50 mm x 18 mm) at 25 °C. In the case of Sabarlis anthers, each dish was inoculated with the 50-60 anthers dissected from a single spike. To determine the best period of time for conditioning (see Table 2), anthers were cultured by serial transfer (Sunderland and Roberts, 1977). At the end of the conditioning period, the tissues were discarded and the medium centrifuged to remove shed pollen and cell debris. Conditioned media were stored in a domestic refrigerator and used as soon as possible after preparation. In the text, media conditioned by anthers are labelled (C2), (C3), (C4), and so on according to the developmental stage at tiller-harvest. Thus the label P-9H(C5) means that potato medium containing the hormone supplement was conditioned by anthers from spikes in which all the pollen as seen in the acetocarmine test had just completed the first mitotic division. Assessment of results At the end of the culture test (about 30 d) calluses attached to the anthers were first gently dislodged into the medium. Total numbers of calluses per culture were then counted under a dissecting microscope with the aid of grid lines. A callus was defined as any multicellular unit having This content downloaded from 207.46.13.76 on Fri, 09 Sep 2016 04:17:39 UTC All use subject to http://about.jstor.org/terms Xu, Huang, and Sunderland—Culture of Barley Anthers 769 an irregular outline. Because pollen and calluses are shed into the medium during culture, absolute numbers of responding anthers could not be assessed with certainty. The numbers given below refer only to those anthers that still had calluses attached at the end of the test.