Effect of Boron on the Incorporation of Glucose from UDP-Glucose into Cotton Fibers Grown in Vitro

Boron is required for fiber growth and development in cotton ovules cultured in vitro. Incorporation of [¹⁴C]glucose by such fiber from supplied UDP-[¹⁴C]glucose into the hot alkali-insoluble fraction is rapid and linear for about 30 minutes. Incorporation of [¹⁴C]glucose from such substrate by fibers grown in boron-deficient ovule cultures is much less than in the case with fibers from ovules cultured with boron in the medium. Total products (alkali-soluble plus alkali-insoluble fractions) were also greater in fibers from ovules cultured with boron. The fraction insoluble in acetic-nitric reagent was a small part of the total glucans; however, in the boron-sufficient fibers, there was significantly more of this fraction than in fibers from boron-deficient ovule cultures. The hot water-soluble glucose polymers from the labeled fibers had a significant fraction of the total [¹⁴C]glucose incorporated from UDP-[¹⁴C]glucose. Both β-1,4- and β-1,3- water-soluble polymers were formed in the boron-sufficient fibers, whereas the same water-soluble fraction from the boron-deficient fibers was predominantly β-1,3-polymers. The incorporation of [¹⁴C]glucose from GDP-[¹⁴C]glucose by the fibers attached to the ovules was insignificant.     

Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum) Ovules Grown in Vitro

Boron deficiency and phytohormone interactions have been studied in unfertilized cotton (Gossypium hirsutum) ovules grown in vitro. Such ovules required exogenous indoleacetic acid and/or gibberellic acid for fiber elongation. Boron also was required for maintenance of fiber elongation and normal morphogenesis throughout 14 days of culture. The amount of exogenous boron necessary for maximum fiber elongation varied among experiments, presumably in relation to endogenous boron levels at anthesis. Some ovular epidermal cells distant from the liquid medium could be induced to elongate into fiber even after 6 days in boron-deficient medium in response to the later addition of boron.     

Boron Deficiency and Translocation Profiles in Sunflower

The distribution of carbon-14 down the stems of comparable boron-deficient and boron-sufficient sunflower plants after photosynthesis of ¹⁴CO₂ by a single exposed leaf was investigated. In boron-deficient plants the advancing front of radioactivity was always found less far down the stem than in boron-sufficient plants. The general shape of the profile is the same in the two sets of plants. We conclude that the velocity of translocation is reduced in the boron-deficient plants.     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Callus Induction by (2-Chloroethyl) Phosphonic Acid on Cultured Cotton Ovules

The response of cultured cotton (Gossypium hirsutum L.) ovules to the ethylene releasing growth regulator, (2-chloroethyl) phosphonic acid (ethephon), was measured. Control ovules grown on a complete liquid medium without hormones continued the differentiation normal for cotton, although at a slower rate than in vivo. When ethephon was added to the medium, normal organ and fiber development was inhibited but callus was induced from the micropylar end of the ovule. The callus was extremely friable and produced many free cells in the culture medium. Dry weight of the callused ovules increased over 4-fold and total cell number increased 56% over the controls. Apparently ethylene released from the ethephon stimulated both cell division and cell expansion in forming the callus.     

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Adding gelling agents to cotton ovule culture media leads to subtle changes in fiber development

Summary Young cotton (Gossypium hirsutum) ovules will produce fiber in vitro when floated on a defined culture medium. Our laboratory is interested in examining the effects of altered gravity environments on fiber development as a model for the effects of gravity on cell expansion and cellulose biosynthesis. Since liquid culture media are unsuitable for altered gravity experiments, addition of gelling agents to cotton ovule culture media is necessary. In this study we have systematically examined the effects of four gelling agents at several concentrations on fiber production in culture. A rapid screening method using toluidine blue O staining indicated that after 3 wk in culture, fiber growth on 0.15% (wt/vol) Phytagel™ medium was similar to fiber growth on liquid medium. More detailed analysis of fiber development revealed that fiber length was not influenced by the addition of Phytagel™. Accumulation of cellulose, however, was reduced 50–60% compared with fibers produced in liquid media after 3 wk in culture. The fiber cellulose content rose with additional time in culture for both solid and liquid media treatments. By 4 wk in culture, the difference in cellulose content of fiber cell walls grown on solid versus liquid media was less than 20%. This variance in growth response on gelled media could be due to differences in media matric potential, to the immobility of ions trapped within the gel, or to toxicity of contaminants copurifying with Phytagel™. By identifying why ovule growth and fiber cellulose biosynthesis are reduced in cultures grown on gelled media, it will be possible to reveal new information about these processes in system that is less complicated than physiological systems at the whole plant level. Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Selection of biological control agents against tomato Fusarium wilt and evaluation in greenhouse conditions of two selected agents in three growing media

Two biological control practices are the use of suppressive growing media and the application of biological control agents (BCAs). The goals of this study were: (i) to screen 584 potential BCAs obtained from Fusarium wilt (FW) suppressive growing media; (ii) to evaluate in greenhouse conditions selected BCAs in three growing media with different degrees of suppressiveness of tomato FW. Two isolates selected after screening were identified as Fusarium solani (305) and Streptomyces sp. (A19). Results showed that tomato FW was reduced and total production was improved when both BCAs were applied to a conducive medium (coir fiber). In highly suppressive growing medium (grape marc compost), A19 and 305 inoculations did not improve suppressiveness. In moderately suppressive growing medium (cork compost), only A19 improved this compost to natural grape marc compost suppressiveness level. Therefore, compost suppressiveness of tomato FW depended on the nature of the compost and on the isolates applied.     

The Effects of Agitated Liquid Medium on in vitro Cultures of Hevea brasiliensis

The initiation and prolonged growth of callus, from stem explants of young plants of Hevea brasilienies on solid medium yielded a heterogeneous callus, with areas which are the result of compact growth interspersed with brown necrotic tissue and soft white tissue formations. Subculturing this callus (O callus) to agitated liquid medium and returning it to solid medium resulted in the production of a homogeneous friable and rapidly growing callus (Rl callus) The two established lines O and Rl have remained stable over one year in culture and differ in gross morphology, anatomy, growth and auxin content. Both were maintained on Murashige and Skoog's medium, with 2 mg/1 2,4-D and 0.5 mg/I kinetin. R 1 but not O showed enhanced growth at the lower 2,4-D level of 0.2 mg/l: both lines failed to continue growing when 2,4-D was omitted. It is suggested that the changes resulting from subculture in agitated liquid medium are related to those undergone by callus cultures which become habituated. Thus the Rl callus line is regarded as partially habituated. Subculture in agitated liquid medium also resulted in the production of large numberr of polyploid cells but these did not persist over the long periods of subsequent growth on agar medium, Enhanced auxin production by the establihed Rl callus line was thus observed in the absence of a detectable level of polyploidy.     

An improved protocol for carrot haploid and doubled haploid plant production using induced parthenogenesis and ovule excision in vitro

In this work, we describe an improved protocol for induced parthenogenesis and ovule culture of carrot (Daucus carota L.). The effects of pollination with parsley pollen and/or 2,4-dichlorophenoxyacetic acid (2,4-D) treatment on the stimulation of parthenogenesis were studied using heterozygous donor plants of 30 varieties and breeding populations of carrots. Isolated ovules, cultured in vitro, enlarged and developed embryos or calli. The application of 2,4-D on pollinated flowers stimulated callus development but did not increase the frequency of embryo development from ovules and, thus, was not useful for increasing the frequency of haploid plant recovery. The efficiency of embryo development was accession-dependent and varied from 0 to 24.29%. In optimized conditions, most accessions responded by embryo development exclusively. The highest frequency of embryo development was observed from ovules excised from ovaries 20–22 d after pollination with parsley pollen. Among several media used for ovule culture, 1/2-strength Murashige and Skoog medium with 0.06 μM indole-3-acetic acid (IAA) was the best. It allowed the production of embryos at a similar frequency as on the media supplemented with kinetin, gibberellic acid, putrescine, or thidiazuron, but restricted callus development. Most plants obtained were haploids and diploids derived from parthenogenesis, as evidenced by homozygosity at three independent loci based on isozyme and PCR analyses. In total, considering haploids and embryo-derived homozygous diploids together, 72.6% of regenerated plants were of gametic origin.     

The significance of the methods of stigmatal and placental pollinationin vitro inAntirrhinum majus L.; seed and callus formation on placentae

A study was made on certain problems connected with the application of methods of stigmatal and placental pollinationin vitro in the snapdragon. Germinable pollen without microbial contamination could be obtained only from the flowers which were left, after surface sterilization, on the stem till the dehiscence of anthers. The germination of pollen, and especially the growth of pollen tubes was better in a 0.3 M lactose solution with 10^?3 per cent H₃BO₃ than in usual sucrose media. The seedsin vitro could be obtained only after normal pollination and the successive artificial cultivation of the entire pistil (the method of stigmatal pollinationin vitro). The seeds did not differ either in their morphology or in their size from those developed under natural conditions, they were viable and without dormancy. Though the pollinated pistils were cultivated in the medium without growth substances, callus formation from uncovered ovules and placenta was observed in some cases. A dependence was revealed of the proliferation on pollination and on the cultivar employed. When using the method of placental pollinationin vitro we failed to obtain seeds. The main difficulty in the use of this method seems to consist in the fact that pollen tubes are not capable of growing from the surface of ovules and placenta into the micropyle.     

THE EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM FERTILIZED COTTON OVULES

Cotton ovules were aseptically removed from ovaries 48 hr after anthesis and floated on the surface of liquid medium. Plant growth substances were filter sterilized and added to the medium in which glucose was the principal source of carbohydrate and KNO₃ the sole source of nitrogen. The amount of total fiber produced from the ovule surface was determined colorimetrically from the intensity of a destaining solution to which the ovules had been transferred following their 15-second emersion in a solution of toluidine blue O. Gibberellic acid induced a marked stimulation and kinetin and abscisic acid a marked inhibition of fiber production from fertilized cotton ovules. Indoleacetic acid afforded only a trend of stimulation of fiber production. Gibberellic acid overcame the inhibition of total fiber production induced by both kinetin and abscisic acid. Indoleacetic acid did not overcome the inhibition of total fiber production induced by kinetin but did, to a great extent, overcome the inhibition due to abscisic acid.     

Independent control of fiber development and nitrate reduction in cultured cotton ovules

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA₃) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.     

Improved Culture Media for Embryogenic Callus Generation in Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Many attempts on optimization of sorghum [Sorghum bicolor (L.) Moench] tissue culture induction media have been made, but the culture system remains with some bottlenecks compared to that of other crops. This study aimed at assessing the suitability of various induction media to produce embryogenic callus (yellow and friable) with high induction rates and reduced phenolic exudation. The six culture medium modifications: 3 based on Murashige and Skoog (MS) medium and one each based on Chu N6, Gamborg B5 and 190-2 media respectively were applied in the culture of mature embryos from 10 sorghum genotypes. Although there was a genotype influence on the attainment of a yellow callus, friability of the callus was determined to be dependent on the culture medium and not the genotype. Half strength MS medium with 0.2 mg/l 2,4-D with 2.8 g/l Gelrite® as the gelling agent modified with 1.0 g/l KH₂PO₄, 1.0 g/l L-proline, 1.0 g/l L-asparagine and 0.16 mg/l CuSO₄·5H₂O (type E) was found to be the most effective resulting in about 60% yellow coloured callus induction with 25% friability. Addition of CuSO₄·5H₂O, KH₂PO₄, L-proline and L-asparagine significantly reduced the phenolic production. Half strength MS medium was observed to contribute to quality callus production when compared to full strength MS media modified with the compounds. The half strength MS medium was also observed to suppress phenolic production. Medium 190-2 produced the highest regeneration frequency (40%) among the 3-regeneration media tested. The results provide information on a suitable sorghum callus induction medium necessary for embryogenesis.     

Effect of boron on cell elongation and division in squash roots

This work establishes that cessation of root elongation of intact squash (Cucurbita pepo L.) plants is an early result of boron deficiency. Root elongation is slowed by 6 hours and is virtually stopped as early as 24 hours after boron is first withheld from the nutrient solution. As root elongation ceased, cell elongation progressed distally into the region normally occupied by the apical meristem and eventually the meristem became indistinguishable. Differentiation was determined by use of an elongation index in which cell length was compared to cell width. This index ranged from a low of 0.8 in boron-sufficient root meristems to a high of 3 in root meristems grown in a boron-deficient nutrient solution for 98 hours. It is concluded that a continuous supply of boron is not essential for cell elongation but is required for maintenance of meristematic activity. Boron may act as a regulator of cell division in this tissue.     

Induction and Proliferation of Callus Derived from Fruits of Different Varieties of Capsicum annuum

Callus was initiated on Murashige and Skoog (MS) medium containing different combinations of growth regulators or different concentrations of vitamins from pericarp of six varieties of Capsicum annuum differing in their capsaicin content. Callus derived from pericarp of low capsaicin containing varieties was snowy white, friable and showed excellent growth. Callus initiation was delayed (10-15 days) in Punjab Lal, which had very high fruit capsaicin content (7.0 mg g^?1 DW). It also showed relatively slow proliferation although callus obtained was white and friable. Several different media were tried to improve the initiation and the proliferation of the callus in this variety. Callus growth improved greatly by doubling the concentration of MS salts. Initiation time was reduced to 4-6 days by adding 10 mg l^?1 NAA and 0.5 mg l^?1 Kin in MS medium. Other combinations of growth regulators or increase in concentration of vitamins or activated charcoal (0.1%) resulted in poor callus growth and callus texture. Of all media tried, MS medium containing 2 mg l^?1 2,4 D and 0.5 mg l^?1 Kin was the best for callus initiation and proliferation.     

Boron deficiency increases putrescine levels in tobacco plants

Polyamine concentrations were determined in leaves and roots of tobacco plants (Nicotiana tabacum L.) subjected to a short-term boron deficiency. A decrease in the growth of shoots and, especially, roots was found under this mineral deficiency. Boron deficiency did not lead to a significant decrease in leaf or root ion concentrations when compared to control treatment; however, as expected, leaf boron concentration was lower in boron-deficient plants in comparison to the control. In leaves, the levels of free putrescine and spermidine were similar in both treatments. In roots, a short-term boron deficiency caused an increase in free putrescine. Moreover, boron-deficient plants had higher conjugated polyamine concentration than boron-sufficient plants, which was especially evident for conjugated putrescine in leaves. A possible link between boron and polyamine levels is proposed and discussed.