Organogenic Response Research Articles

667 (PS 4) IDENTIFICATION OF A NEW BENZYL ADENINE COMPOUNDPRODUCED DURING PETUNIA SHOOT ORGANOGENESIS

Studies of benzyl adenine (BA) uptake and metabolism in Petunia leaf explants during shoot organogenesis revealed a novel BA conjugate. The new conjugate comprised up to 39% of the total pool of BA conjugates in two petunia lines and was associated with an increased shoot organogenic response when compared with a third petunia line which did not produce the conjugate. To identify the new compound, structural analysis was conducted using FAB mass spectrometry, two methods of carbohydrate analysis, UV absorption spectra and FTIR spectra. Experimental results identified the compound as 6-benzylamino-9-[O-glucopyranosyl-(1→3)-ribofuranosyl]-purine, a disaccharide conjugate of BA. Based on biological studies and similarity to other cytokinin conjugates, this disaccharide cytokinin conjugate may be part of the interconvertible pool of cytokinins active in shoot organogenesis.

Shoot Organogenesis and Plant Regeneration from Cotyledons of Diploid, Triploid, and Tetraploid Watermelon

Adventitious shoots were obtained from watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai] cotyledons incubated on a modified Murashige and Skoog medium containing BA. Initial experiments comparing the effects of BA (0, 5, 10, or 20 μm) and IA4 (0, 0.5, or 5 μm) demonstrated that BA was required for adventitious shoot formation but its concentration in the medium was not critical. The addition of IAA to medium with BA increased callus production and inhibited shoot formation. However, the percentage of responding explants in the best treatment was <30%. Therefore, the manner in which cotyledon explants were prepared and seedling age at the time of explantation was examined to improve the organogenic response. The percentage of explants with shoots was improved by using explants that consisted of cotyledon bases (43%) or cotyledons cut in half longitudinally (39%). A lower percentage (16%) of cotyledons cut longitudinally into four pieces produced shoots. Explants taken from the apical half of cotyledons failed to regenerate shoots. Shoot formation was improved further by using explants from young seedlings. The percentage of explants with shoots was >90% for `Minilee', 64% for S86NE, and 50% for `Jubilee II' when explants were prepared from 5-day-old seedlings. Explants from nongerminated embryos or seedlings germinated for 10, 15, or 20 days produced fewer shoots. The effect of several cytokinins on shoot organogenesis was then examined using the optimized protocol. The percentage of explants with shoots and the number of shoots per explant were about two to four times higher when 5 to 10 μm BA was used compared to the most effective kinetin (20 μm) or thidiazuron (0.1 μm) concentration. The percentage of explants with shoots and the number of shoots per explant were greater for diploid (57% and 2.2, respectively) than for triploid (22% and 0.6, respectively) or tetraploid (20% and 0.8, respectively) lines. Chemical names used: N -(phenylmethyl)-1 H -purin-6-amine (BA); 6-furfurylaminopurine (kinetin); N -phenyl-N' -1,2,3-thiadiazol-5-ylurea (thidiazuron); 1 H -indole3-acetic acid (IAA).

Use of Agrobacterium rhizogenes to create transgenic apple trees having an altered organogenic response to hormones

The apple rootstock, M26, was genetically and phenotypically transformed using the Agrobacterium wild-type strain, A4. First, chimeric plants were obtained having transformed roots and normal aerial parts. Transformed plants were then produced through regeneration from transformed roots. Transformation was demonstrated by molecular hybridization and opine analysis. The effects of hormones on organogenesis was altered in transformants: cytokinins were required to form roots, whereas auxin was toxic at the concentration used to induce rooting in the control.

Kiwifruit micropropagation through callus shoot-bud induction

A profitable system for the establishment of morphogenic callus cultures and indirect shoot induction and development was accomplished from nodal shoot segments obtained from adult and micropropagated plants of kiwifruit (Actinidia deliciosa [Chev.] Liang and Ferguson, var.deliciosa) cv. “Hayward”. The effects of medium composition, cytokinin levels, dilution of salts, and type of callus derived from the cultured primary explants were studied. Medium composition as well as type of callus greatly affected organogenic responses.

Comparison of Benzyl Adenine Metabolism in Two Petunia hybrida Lines Differing in Shoot Organogenesis

The uptake and metabolism of the cytokinin benzyl adenine (BA) was compared in two lines of Petunia hybrida Vilm. differing in their shoot organogenic response. Leaf transfer experiments using shoot induction medium containing 4.4 micromolar BA showed that leaf explants from petunia line St40 required a shoot induction period of 6 to 10 days for commitment to shoot organogenesis; whereas leaf explants from petunia TLV1 required 12 to 28 days. The short induction period of petunia St40 and the higher organogenic response was positively associated with a threefold higher absorption of BA from the medium, an increased BA ribotide metabolite pool, the presence of BA within the explant during the shoot induction period, and the production of an unidentified metabolite C. However, the study of petunia TLV1 leaf explants showed that neither BA nor metabolite C are required during the shoot induction period for eventual shoot development. The longer shoot induction period of TLV1 was associated with low BA uptake during 24 days, a decreasing ribotide metabolite pool, the absence of benzyl adenosine triphosphate and metabolite C throughout the study, and the absence of BA within the explant during the shoot induction period. Differences in the shoot organogenic response of these related plant lines have been shown to be associated with differences in exogenous cytokinin uptake and the subsequent metabolism of that hormone.

Adventitious shoot regeneration from hypocotyl cultures of service tree(Sorbus domesticaL.)

Effects of growth regulators and N03:NH4 ratio were investigated to determine the mor- phogenic capacity of hypocotyl explants of service tree (Sorbus domestica L.) Organogenic responses were preceded by an initial callus phase and depended mainly on the auxin type used. NAA induced root differentiation whereas IAA added to media containing BA promoted adventitious shoot regeneration. Best results were obtained with 2.5 μM each of BA and IAA (19% of caulogenic explants). This reponse was increased (up to 44%) by varying nitrate to ammonium ratio from 2:1 to 4:1.

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua ‘Variegata’ were placed on solidified Woody Plant medium supplemented with 0.1 mgl-1 (0.5 μM) naphthaleneacetic acid and 2.5 mgl-1 (11.1 μM) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua ‘Variegata’ and ‘Moraine’.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from ‘Variegata’ leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).

FACTORS AFFECTING THE REGENERATION OF PEPPER (CAPSICUM ANNUUM L.)

Several growth hormone combinations and silver nitrate concentrations were examined for their effect on regeneration of different pepper genotypes. Primary leaf explants from in vitro seedlings were cultured on a revised Murashige and Skoog medium supplemented with auxin, cytokinin and 1.6% glucose. Combinations of different concentrations of indole-3-acetic acid (IAA), 0-5 mg/l, and 6-benzylaminopurine (BAP), 0-5 mg/l, were tested to determine the most effective medium for shoot primordium formation. Experiments with IAA and BAP did not result in a specific growth hormone combination appropriate for regeneration of all genotypes tested. Of the silver nitrate concentrations tested, 10 mg/l resulted in the best shoot and leaf differentiation and reduced callus formation. Differences in organogenic response of individual genotypes were evaluated on a single regeneration medium. Whole plants were regenerated from 11 of 63 genotypes examined. Based on these experiments, a reproducible regeneration system for pepper was developed with a total of 500 plants regenerated to date.

BENZYL ADENINE UPTAKE AND METABOLISM DURING PETUNIA SHOOT ORGANOGENESIS

The uptake and metabolism of exogenous tritium-labelled benzyl adenine was studied during the shoot induction period of petunia leaf explants in tissue culture. Transfer experiments with Petunia `MD1' leaf explants (1 cm2) on MS media with 2.2 uM BA show that 27% and 100% of the leaf explants are committed to shoot induction on days 6 and 10, respectively. To study BA uptake and metabolism, leaf explants were placed on media containing tritium-labled BA for 1, 3, 6 and 10 days. BA was taken up from the media on days 1-6. BA metabolizes were analyzed using HPLC, a UV absorbancc detector and enzymatic techniques. Metabolizes produced include: BA, BAdo, BA 7G, BA 9G, BAdoMP, BAdoDP, BAdoTP and 3 unidentified compounds. BA and BAdo were detected on days 1 and 3 but not during day 6-10, the time of shoot induction. The pool of ribotide metabolites decreased from days 1 to 10, from 26.5% of all metabolites to 1.6%. Glucosylated compounds, BA 7G and BA 9G, increased continuously from 24.9% to 69.8% between days 1 and 10. An unidentified compound C increased from 13% on day 3 to 24.8% on day 10. In separate experiments, BA uptake and metabolism were compared in two Petunia hybrida lines, St40 and TLV1, with different shoot organogenic responses in tissue culture. These data show interesting patterns of BA metabolism in relationship to shoot induction and organogenesis.

FACTORS AFFECTING THE REGENERATION OF PEPPER (CAPSICUM ANNUUM L.)

Several growth hormone combinations and silver nitrate concentrations were examined for their effect on regeneration of different pepper genotypes. Primary leaf explants from in vitro seedlings were cultured on a revised Murashige and Skoog medium supplemented with auxin, cytokinin and 1.6% glucose. Combinations of different concentrations of indole-3-acetic acid (IAA), 0-5 mg/l, and 6-benzylaminopurine (BAP), 0-5 mg/l, were tested to determine the most effective medium for shoot primordium formation. Experiments with IAA and BAP did not result in a specific growth hormone combination appropriate for regeneration of all genotypes tested. Of the silver nitrate concentrations tested, 10 mg/l resulted in the best shoot and leaf differentiation and reduced callus formation. Differences in organogenic response of individual genotypes were evaluated on a single regeneration medium. Whole plants were regenerated from 11 of 63 genotypes examined. Based on these experiments, a reproducible regeneration system for pepper was developed with a total of 500 plants regenerated to date.

Invitro plant regeneration via callus culture of mature Salixexigua

Callus induction, callus growth, and plantlet regeneration were examined using leaf explants of three Salixexigua clones. Calli were initiated on three basal media supplemented with 0.1 mg/L (0.44 μM) or 0.5 mg/L (2.2 μM) of benzylaminopurine and 0.1 mg/L (0.45 μM) or 0.5 mg/L (2.3 μM) of 2,4-dichlorophenoxyacetic acid in a factorial fashion. After 7 weeks of growth, callus production was highest on woody plant medium. Developed calli were subsequently cultured on woody plant medium supplemented with either 0.1 or 0.5 mg/L of benzylaminopurine for shoot induction. Shoot primordia developed only at 0.1 mg/L (0.44 μM) benzylaminopurine in two clones, suggesting clonal variation in organogenic response. Shoots larger than 1.0 cm in length were successfully rooted in half-strength Murashige and Skoog medium without hormones.

Organogenesis in Vitro as a Developmental Process

Abstract Plants have a remarkable propensity for asexual or vegetative propagation, and it is not surprising that this capacity extends to plant cells or tissues cultured in vitro (24). The “regenerative response” in vitro includes somatic embryogenesis as well as organogenesis (formation of shoots or roots from cultured tissue). It may be useful to make a further distinction, subdividing the organogenic response. In many instances, explants have the capacity to give rise to shoots, roots, or floral structures when cultured on a medium that supplies mineral salts, vitamins, and a carbon source, but is devoid of plant hormones. These processes may be termed “adventive organogenesis”. Exogenously supplied plant hormones often can facilitate these processes, but are not absolutely required for organogenesis to occur. In these instances, the immediate precursors of the new organs are cells in the explant itself (5, 6). The other type of organogenesis is not adventive and involves a “dedifferentiation” of the explant, elaboration of callus tissue along the cut edges of the explant, and the induction of new organs from this newly formed callus tissue. Exogenously supplied phytohormone not only controls the process but is required for organogenesis to occur. This paper is concerned with the specifics of this last type of organogenesis; we suspect that the general principles derived from this work apply to all organogenesis in vitro. As much as organogenesis in vitro reflects the mechanisms of organogenesis in vivo, these general principles will also apply to the development in whole plants ex vitro.

In vitro adventitious shoot formation on mature-phase leaves and petioles of Liquidambar styraciflua L.

Adventitious shoots were induced to form on leaves and petiole segments of mature-phase Liquidambar styraciflua L. Shoot organogenesis occurred directly, without the formation of a distinct callus stage, and well-defined shoots were visible in 6–9 weeks. Prolific shoot production was supported by Woody Plant Medium supplemented with relatively high levels of benzyladenine (2.5 mg/l). Changes in benzyladenine concentration and the addition of 0.1 mg/l naphthaleneacetic acid to the medium altered the relative abundance of shoots on various parts of a leaf. Shoot formation occurred most frequently at or near breaks in major vasculature. Wounding of the leaves by slashing across the lamina and vasculature significantly increased the total number of shoots formed per explant and also altered the pattern of organogenesis. Differences in organogenic response were seen between the cultivars ‘Moraine’ and ‘Variegata’. Shoots derived from leaves were easily rooted and acclimated to greenhouse conditions. Three new variegation types arose in vitro as a result of adventitious shoot formation on ‘Variegata’ leaves.

High frequency plant regeneration from thin cell layer explants of Brassica napus

Explants composed of the epidermis and 4–9 layers of subepidermal cells were excised from internodes of Brassica napus L. ssp. oleifera cv. Westar and cultured on modified Murashige and Skoog (MS) medium. The three or four terminal internodes excised from plants at an early stage (before any flower buds had opened) were shown to be the best explant source. Both cytokinin and auxin were required for induction of shoot organogenesis. Of six auxins tested, only naphthaleneacetic acid (NAA) was effective in shoot bud initiation. All four cytokinins tested (when associated with 0.5 mgl-1 NAA) promoted organogenesis, but at differing frequencies. The highest shoot induction frequency was obtained at 10–15 mgl-1 benzyladenine (BA). The organogenic response was strongly affected by the nitrogen content of the medium. The best response was observed when NO3 - was the sole nitrogen source (supplied as KNO3) in the range 30–90 mM. Sucrose and glucose were equally supportive in shoot regeneration with the optimal levels at 0.12 M and 0.15 M, respectively. Shoots were rooted on medium free of growth regulators and mature plants were grown in the greenhouse. Plants were also recovered from leafy structures which differed morphologically and histologically from shoot buds.

THE HORMONAL CONTROL OF ORGAN FORMATION IN CALLUS OF MEDICAGO SATIVA L. CULTURED IN VITRO

Organogenesis in alfalfa callus (Medicago sativa L. cv. ‘Regen S’) has been obtained by the transfer of callus from an induction medium containing growth regulators to a regeneration medium lacking growth regulators. The transfer of callus from induction medium containing high levels of 2,4‐D and low levels of kinetin to regeneration medium resulted in the formation of shoots. Conversely, the transfer of callus from induction medium containing low levels of 2,4‐D and high levels of kinetin resulted in the formation of roots. The pattern of organogenesis on regeneration medium was modified by the nutritional composition of that medium. When Blaydes medium supplemented with inositol and yeast extract was employed as regeneration medium, root organogenesis was inhibited. Root organogenesis was not inhibited by either Shenk and Hildebrandt medium or Gamborg's B5 medium. Shoot formation occurred on all of these media. A survey of the in vitro organ‐forming capacity of 14 genotypic clones from the cv. ‘Regen S’ was conducted. The capacity to form organs differed quantitatively among the clones analyzed. A more detailed analysis of a highly responsive clone (RA3) and a poorly responsive clone (RA5) revealed no significant qualitative difference in their organogenic responses.

Welwitschia mirabilis Callus Studies III. Some Effects of Morphactin

Morphactins, fluorene-carboxylic acid derivatives reported highly active in a number of plant morphogenic responses, retard growth and severely inhibit chlorophyll synthesis in Welwitschia mirabilis callus cultures. No organogenic response was elicited. Cell membranes appeared particularly susceptible to morphactin. ER and dictyosomes became progressively vesiculated; in the chloroplasts thylakoids were swollen and their lamellae distorted and few grana were present.