In vitro performance of immature embryo-derived callus of two barley cultivars, the amenable cultivar, Golden Promise (GP) and the recalcitrant commercial cultivar, Galena (GL), was tested on fourteen media containing combinations of two auxins, dicamba and 2, 4-dichlorophenoxyacetic acid (2, 4-D) and two cytokinins, 6-benzylaminopurine (BAP) and zeatin. Generalizations for both cultivars include the fact that callus-induction frequencies on dicamba and 2, 4-D alone and dicamba plus zeatin were nearly 100%. Qualitative appearance for both cultivars was nearly identical on comparable media but markedly better on BAP-containing media. The addition of zeatin with either auxin supported vigorous growth due to the proliferation of nonregenerable callus; the presence of BAP produced higher quality callus although it inhibited the relative growth rate. GP calli grown on 2, 4-D plus 0.01mg/L BAP regenerated the largest number of green shoots at most time points relative to other media, while GL tissue on 2, 4-D plus 0.1mg/L BAP yielded the most green shoots at all but one time point. Over time regenerability was lost more rapidly in GL than GP except on 2, 4-D plus BAP media; GL had a higher propensity toward albinism than GP. In both cultivars, the use of 2, 4-D and BAP yielded tissue that proliferated for prolonged periods in a regenerable state.
A mass of shoot primordia were induced effectively from the shoot apex of Rhododendron pentaphyllum Maxim. var. Komatsu on WP media containing 30μM 2iP+IAA (10, 30, 100μM). Of the shoot primordia obtained, those cultured on WP medium supplemented with 10μM IAA and 30μM 2iP showed the most active proliferation, being 2-4 times in size, after one month of subculture. When shoot primordia obtained were transferred onto the differentiation media, multiple shoots were formed on WP media containing zeatin (1, 10μM) or 0.1μM CPPU after one month of culture, irrespective of the presence of IAA. The multiple shoots induced on the medium containing 10μM IAA and 10μM zeatin showed active elongation growth. Rooting from the multiple shoots subcultured on the same fresh medium occurred two months after the shoot differentiation.
Two cDNA fragments that preferentially accumulate in very young rice embryos at 4 days after anthesis (DAA) were isolated using simplified differential display. The first morphological change is observed in the embryos at 4 DAA and most of the embryo tissue has already differentiated by 7 DAA. RT-PCR was used to quantitate the expression levels of the genes corresponding to the two cDNA clones, REE5 and REE8. They were expressed at higher levels in the embryo at 4 DAA than in the embryo at 7 and 14 DAA as well as in the pistil 1 day before anthesis. The consistent result was obtained by in situ hybridization experiments using REE5 antisense probe. REE5 gene was expressed in the whole embryo at 4 DAA and 5 DAA. The lower expression levels were observed in the embryos at 3 and 7 DAA. RT-PCR analysis revealed that both the REE5 and REE8 transcripts also accumulated in inflorescence meristems prior to the emergence of floral organs, but not in roots, leaves or flowers. The common feature of the young embryos and the inflorescence meristems is the onset of differentiation of various tissues. These may be useful molecular markers for analysis of the events of differentiation during early embryogenesis.
The effects of sterilization time, medium composition, and temperature on the germination of Calypso bulbosa were studied. Mature seeds treated with sodium hypochlorite solution (1% available chlorine) for four to eight min showed high germination percentages. Although there were no differences in germination percentage among the four media tested, protocorm growth on Tsutsui and Tomita medium was slightly better. Incubation temperature significantly affected both germination and protocorm development. Transplanting damage to seedlings was reduced when the cultures were maintained between 17.5 and 20°C in the dark. Bulb formations were observed on the protocorm masses when they were moved to continuous light condition (500lux).
Following a few reports on indica rice transformation, we demonstrated the recovery of transgenic indica rice cultivars carrying potentially useful genes through co-transformation with a selectable marker using a particle bombardment method (biolistic process). Scutellum-derived calli and embryos of 3 selected indica rice cultivars were used as explants. Co-transformation with selectable bar gene was employed to isolate transformed callus lines that are resistant to the herbicide bialaphos. Subsequently, bialaphos-resistant calli were regenerated and 29% of R0 plants were shown to possess Ac and Ds by PCR analysis. About 50% of the putative R0 transformants showed transmission of the transgene based on PCR and Southern blot analyses, indicating that a large number of primary transformants represented chimeric individuals, or that elimination of the transgenes eventually occurred during meiosis/mitosis. PCR-positive R0 plants were self-pollinated to produce R1 lines. Single and multiple integrative events were detected and R1 progenies from 3 transgenic lines exhibited the expected Mendelian inheritance pattern. Although a few rearranged copies of the transgenes were noted, a majority of the copies remained intact after integration. Expression of the CaMV35S-driven transposase gene was revealed by the presence of the transposase transcript in Northern blots. The procedure described here provides an applicable tranformation system that can be further improved to generate more Ac and Ds lines towards establishing a functional transposon mutagenesis system in indica rice.
Meristematic nodular calli (NOD) were successfully induced from various plant parts such as seeds, flower organs, shoot apices, stem segments and bulb scales of Lilium spp. on media containing 1mg/l picloram alone or in combination with 1mg/l cytokinin. In L. longiflorum, green friable callus (FC) was also induced from bulb scales. Suspension cultures established from both NOD and FC showed high proliferation rate. NOD retained high regeneration ability as well as stable ploidy level during the culture for more than 4 years, while the suspension culture of FC became tetraploid. Protoplasts were efficiently isolated from NOD in some species and cultivars after long term subculture in suspension cultures and the plants were successfully regenerated from the protoplasts of L. x formolongi.
We identified two homolog genes of PnGLP (Pharbitis nil germin-like protein) and SaGLP (Sinapis alba GLP) in Arabidpsis thaliana, namely, AtGLP1 and AtGLP2. Results of Southern blotting showed that each gene was single per haploid genome. Northern blotting revealed the presence of mRNAs for both genes in all overground parts, but especially in the leaf and flower. RFLP mapping showed that AtGLP1 was on chromosome 1 (112.4±0.6cM) and AtGLP2 was on chromosome 5 (35.7±0.3cM).