Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Mechanism of naphthaleneacetic Acid conjugation: no effect of ethylene

Formation of naphthaleneacetic acid-glucose (NAGLu) in detached leaves, floating on α-naphthaleneacetic acid-1-¹⁴C (NAA, 0.05 microcurie per milliliter, 3.1 μm)-buffer solution (phosphate-citrate, pH 4.2) began immediately while there was a 2- to 4-hour lag before NAA-asparatate (NAAsp) could be detected. Subsequent increase in the NAAsp conjugate reflected a decrease in free NAA to 1 to 2% of the total radioactivity taken up. Pretreatment with 31 μm¹²C-NAA for 18 hours doubled NAAsp formation after transfer for 4 hours to ¹⁴C-NAA. Pretreatment with ethylene, as ethephon (up to 400 milligrams per liter) or ethylene gas (10 microliters per liter), did not induce NAAsp formation. In the presence of NAA, ethylene had no effect on NAA conjugation. Similarly, CO₂ (5%) did not modify the formation of the conjugates. Rhizobitoxine (1.87 μm) inhibited NAA-induced ethylene production but did not prevent NAA-induced formation of NAAsp. We concluded that the conjugation of NAA with aspartic acid is not mediated by ethylene.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10^–7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10^–7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.Abbreviations ABA Abscisic acid - BA 6-Benzylaminopurine - GD Gresshoff and Doy (1972) nutrient medium - NAA -Naphthaleneacetic acid.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

The Role of Auxin Metabolism in Root Meristem Regeneration by Pinus lambertiana Embryo Cuttings

Since the existence of root promoting substances that consist of a complex between auxin and another molecule has been suggested, we have examined the role of auxin conversion products in root regeneration by Pinus lambertiana embryo cuttings. Auxin conversion products were detected using radioactive forms of the auxins IAA (indoIe-3-acetic acid), NAA (a-napthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid). 10^?7M NAA was more effective than 10^?6M IAA at promoting rooting, yet it formed conversion products much less rapidly. Also continuous exposure to IAA was necessary for optimum root formation. Based on these and other findings, we conclude that free auxin, and not the conversion products we detected, is essential to root meristem formation.     

Micropropagation,berberine content and antitumor activity of Jeffersonia dubia (Maxim.) Benth et Hook

An efficient micropropagation protocol was developed for Jeffersonia dubia using sucker explants. High frequency of multiple shoot formation was induced when the sucker explants were cultured on Chu’s (N6) medium with different concentrations of thidiazuron (TDZ) plus 0.54 µM α-naphthaleneacetic acid (NAA). The maximum frequency of shoot formation (96.2 %) was obtained on N6 medium with 2.27 µM TDZ plus 0.54 µM NAA. The highest mean number of shoots per explant (13.6) was obtained in temporary immersion system using an immersion frequency of 30 s every 30 min. The highest frequency of rooting (100 %), number of roots per shoot (5.8), and root length (6.3) was observed in half-strength N6 medium supplemented with 2.69 µM NAA. The regenerated plantlets (30 days old) were successfully acclimatized in the greenhouse with 98 % survival rate. The berberine content and cytotoxicity were higher in in vitro-developed calli and shoots than in leaves of field-grown plants. The greatest content of berberine was found in shoots (1381 μg g⁻¹) followed by calli (1092 μg g⁻¹) and leaves of field-grown plants (92 μg g⁻¹). At 1000 μg mL⁻¹ concentration, growth inhibition rate of berberine, callus, shoot, and leaf (in vivo) extracts were 68.4, 57.1, 54.2, and 17.7 %, respectively.

     

Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

Bound auxin metabolism in cultured crown-gall tissues of tobacco

Bound auxin metabolism in cultured crown-gall tumor cells and pith callus of tobacco was examined by feeding radiolabeled auxins and auxin conjugates. In all tissues fed [¹⁴C]indoleacetic acid (IAA), at least one-third of the IAA was decarboxylated, and most of the remaining radiolabel occurred in a compound(s) which did not release IAA with alkaline hydrolysis. In cells transformed by the A6 strain of Agrobacterium tumefaciens, the only detectable IAA conjugate was indole-3-acetylaspartic acid (IAAsp), whereas cells transformed by the gene 2 mutant strain A66 produced an unidentified amide conjugate but no IAAsp. By contrast, cells fed [¹⁴C]naphthaleneacetic acid (NAA) accumulated several amide and ester conjugates. The major NAA metabolite in A6-transformed cells was naphthaleneacetylaspartic acid (NAAsp), whereas the major metabolites in A66-transformed cells were NAA esters. In addition, A66-transformed cells produced an amide conjugate of NAA which was not found in A6-transformed cells and which showed chromatographic properties similar to the unknown IAA conjugate. Pith callus fed [¹⁴C] NAA differed from both tumor lines in that it preferentially accumulated amide conjugates other than NAAsp. Differences in the accumulation of IAA and NAA conjugates were attributed in part to the high capacity of tobacco cells to oxidize IAA and in part to the specificity of bound auxin hydrolases. All tissues readily metabolized IAAsp and indole-3-acetyl-myo-inositol, but hydrolyzed NAAsp very slowly. Indirect evidence is provided which suggests that ester conjugates of NAA are poorly hydrolyzed as well. Analysis of tissues fed [¹⁴C]NAA together with high concentrations of unlabeled IAA or NAA indicates that tissue-specific differences in NAA metabolism were not the result of variation in endogenous auxin levels. Our results support the view that bound auxin hydrolysis is highly specific and an important factor controlling bound auxin accumulation.     

Micropropagation of Gymea Lily (<Emphasis Type="Italic">Doryanthes excelsa</Emphasis> Corrêa) from New South Wales,Australia

The physiological effects of three auxins [indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d)] and two cytokinins [thidiazuron (TDZ) and N⁶-benzylaminopurine (NAA)] on in vitro morphogenesis of Doryanthes excelsa were measured. Longitudinal bud sections derived from immature inflorescences were used as a source of explants. Callus regeneration was observed at the highest frequencies (46.2%) when grown on media containing 50 μmol L^-1 NAA and 0.5 μmol L⁻¹ TDZ. Adventitious shoot organogenesis was observed at the highest frequency (56.8%) when grown on media containing 0.5 μmol L⁻¹ NAA and 50 μmol L⁻¹ TDZ. Regenerated shoots were rooted ex vitro after 6 weeks when dipped in a solution of 50 μmol L⁻¹ NAA or no plant growth regulators were applied.     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

Growth Hormones and Propagation of Cymbidium in vitro

Protocorms of Cymbidium (Orchidaceae) were grown on solid or liquid medium with macro-nutrients according to Wimber (van Raalte 1967) and iron, micro-nutrients and vitamins according to Nitsch (1968) the medium also contained 2% sucrose. The effects of 1) the auxins; indol-3yl-acetic acid (IAA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D); 2) the cytokinins; 6-furfurylaminopurine (kinetin) and benzyladenine (BA) and 3) the gibberellin; gibberellic acid (GA) were examined alone or in combinations. IAA had no effect alone. NAA resulted in optimal fresh weight at 10 μM and the protocorms were vigorous, but lighter green than usual. 2,4-D caused a high weight increase at 1 μM, but the protocorms were abnormal. Higher concentrations of NAA and 2,4-D inhibited chlorophyll synthesis. On solid medium kinetin (100 μM) induced a growth of many small shoots, but had no effect on the fresh weight. In liquid medium, kinetin promoted a callus formation and fresh weight increase. BA had effects similar to kinetin, but at lower concentrations. GA alone promoted shoot and leaf growth. Combinations of kinetin and NAA resulted in a maximal fresh weight increase at kinetin concentrations one tenth of the NAA concentrations. The optimal growth and the best development occurred at 10 μM NAA and 1 μM kinetin. NAA and kinetin together could limit the shoot and leaf growth induced by GA.     

In Vitro Propagation Of Four Watsonia Species

The genus Watsonia has a number of species with potential to be developed as new ornamental crop plants, but to date there are no reports on in vitro propagation of any member of this genus. Seeds from four Watsonia species, Watsonia gladioloides, Watsonia lepida, Watsonia laccata, and Watsonia vanderspuyiae were decontaminated and germinated on one-tenth strength MS media without hormones or sucrose. Shoots were induced from seedling hypocotyl segments when both an auxin [α-naphthaleneacetic acid (NAA)] and cytokinin [N⁶−benzylaminopurine (BA)] were present in the medium, while root and leaf explants failed to produce shoots. Multiplication of axillary shoots was greatest when only BA (0.5 mg l⁻¹) was added to the medium. Shoot explants propagated in a ‘liquid-shake’ culture exhibited greater growth rates than those on agar-solidified medium, but shoot production varied between species. Meristemoids were induced in all species, but no significant trend was found between growth index (GI) and meristemoid formation, suggesting that reduction in GI may not necessarily be a prerequisite for producing meristemoids. Corm formation was inconsistent and storage organs could only be induced in one of the four species, W. vanderspuyiae. This occurred best at 25°C with 3% sucrose and an agar level of 15%. Indole-3-acetic acid (IAA) and NAA at 1 mg l⁻¹ significantly increased mean number of roots per shoot explant on all four species, while indole-3-butyric acid (IBA) was more effective when applied at 0.1 mg l⁻¹. Plantlet survival ex vitro was negatively affected when NAA and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to root shoot explants for all species. In W. laccata, all auxin treatments [IAA, IBA, NAA, phenylacetic acid (PAA), and 2,4-D] at a concentration of 1 mg l⁻¹ significantly reduced ex vitro survival of plantlets. Successful micropropagation of Watsonia is an important step in the further development of this genus as a horticultural crop.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Manipulation of the morphogenetic pathways of Lilium longiflorum transverse thin cell layer explants by auxin and cytokinin

Summary Excised tissues from transverse young stem sections of Lilium longiflorum were cultured on Murashige and Skoog medium supplemented with growth regulators at various concentrations. After 45 d in culture, the presence of α-naphthaleneacetic acid (NAA) in the culture medium at 5.4 μM resulted in bulblet formation while 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.2 μM resulted in root formation. The presence of IBA (indole-3-butyric acid) in the culture medium at 1.0 μM resulted in shoot formation while plantlet formation occurred when IBA was added at a concentration of 2.0 μM. When 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) was added to the culture medium at 1.1 μM, protocorm-like bodies (PLBs) formed, while 2.2 μM resulted in shoot formation (on abaxial and adaxial surfaces). The presence of NAA and TDZ in the culture medium at 5.4 μM and 0.4, 1.1 or 2.2 μM, respectively, resulted in somatic embryo formation while NAA- and 6-benzylaminopurine-(BA) containing culture medium formed callus or bulblets. The establishment of different regeneration systems when explants are exposed to various growth regulators demonstrates that the choice of growth regulator combinations and concentrations are of significance in determining the morphogenetic response and plant regeneration capacity.     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.