Leaf anatomy and photosynthetic parameters of Vellozia squamata Pohl (Velloziaceae) grown under different light intensities along in vitro cultivation¹

Anatomia foliar e parâmetros fotossintéticos de Vellozia squamata Pohl (Velloziaceae) desenvolvendo-se sob diferentes intensidades luminosas ao longo do cultivo in vitro

ABSTRACT

The present work evaluates photosynthetic parameters and the leaf anatomy of plants of Vellozia squamata Pohl (Velloziaceae) in each phase of in vitro production, with wild plants serving as a control. The anatomical approach was done for the leaf middle portion. The photosynthetic curves were obtained with an infra-red gas analyzer. The in vitro plantlets showed the thinner and poorest developed leaves, the wild plants had the thickest and most developed leaves, and the plants in acclimatization showed intermediate features. The physiological pattern was similar, and the in vitro plantlets were not capable of net carbon absorption. The young seedlings at the garden showed a maximal net carbon assimilation rate inferior to the wild plants. In conclusion, the light intensity for the in vitro phase should be adjusted to produce seedlings.

Keywords:
acclimatization; Cerrado; conservation; cultivation; shading

RESUMO

O presente trabalho avalia parâmetros fotossintéticos e a anatomia foliar de Vellozia squamata Pohl (Velloziaceae) em cada etapa da produção in vitro, sendo as plantas silvestres o grupo-controle. A abordagem anatômica foi feita para a porção mediana foliar. As curvas fotossintéticas foram obtidas com analisador de gases por infravermelho. As plântulas in vitro apresentaram folhas mais delgadas e menos desenvolvidas, as silvestres apresentaram folhas mais espessas e desenvolvidas e aquelas em aclimatação exibiram características intermediárias. As plântulas in vitro não foram capazes de absorção líquida de carbono. As plântulas jovens de casa-de-vegetação exibiram taxa líquida de assimilação de carbono inferior às selvagens. Em conclusão, para o cultivo in vitro as intensidades luminosas para a produção de mudas devem ser melhor ajustadas.

Palavras-chave:
aclimatação; Cerrado; conservação; cultivo; sombreamento

Introduction

Plants of the genus Vellozia (Velloziaceae) grow in the African and South American savannahs and are more frequent in open vegetations, such as rupestrian fields or Cerrado stricto sensu (Ayensu 1969, Coetzee 1974, Mello-Silva & Menezes 1999, Batista 2016). They are highly resistant to fire and drought (Owoseye & Sanford 1972) and show stems capable to absorb water (Oliveira et al. 2005). Often, they produce resins and several secondary metabolites of medicinal interest (Quintao et al. 2013), including the inhibition of the venom of snakes (Tribuiani et al. 2014). Some species have been used as ornamental (Souza 2005), and it can be attributed to their big showy flowers and peculiar architecture. Unfortunately, these plants are somehow endangered by the extractivism and expansion of crop cultures.

The plants of the genus Vellozia produce a large amount of small seeds that are transported by wind, they are resistant to high temperatures, and show high and quick germinability (Menezes 1977, Oliveira et al. 1991, Garcia & Diniz 2003, Palhares 2004, Garcia et al. 2007, Jacobi & Sarto 2007, Mota & Garcia 2013, Vieira et al. 2017). However, Alves (1994) observed that the growth is very slow in Vellozia species, ranges from about 0.5 to 2.0 cm per year, and almost no young plants were seen in the field, in spite of their high frequency in the Campo Rupestre. The plants of about 1.80 to 3.30 m were actually centenary plants, therefore, although they occupy the open fields, they are not pioneer plants but establish later in the ecosystem, i.e. they are climax plants (Alves 1994).

Vellozia squamata Pohl, sometimes named by its synonym, Vellozia flavicans Mart ex Schult (Smith & Ayensu 1974), is a common species from the Cerrado of the central region of Brazil (Flora do Brasil 2020). The obtention of seedlings from seeds of this species failed due to a high mortality of the young plants (Neto 2009), just as other Vellozia species (Alves 1994). Also, the production of seedlings by stem cuttings ended unsuccessfully (Neto 2009).

While Borges (2015) reported only a partial success in the micropropagation of V. sincorana, Neto (2009) managed to micropropagate V. squamata. Some individuals of the micropropagated V. squamata were planted in gardens and have been growing since then, for more than a decade, having reached about 20 cm (figure 5 e, C.E.S. Silveira, not published data). So, the in vitro propagation protocol of Vellozia squamata somehow overcame unknown factors that generate high loss of seedlings produced via seeds.

Overall, the transition between the in vitro phase to rooting phase has been a limiting point in the production of seedlings, as the seedlings, which had grown heterotrophically and under low light intensities, are then planted in a place where they must turn into plentifully autotrophic (Kaur 2015). In the protocol of Neto (2009), this transition has given to a loss of about 55% of the seedlings, but once passed this phase, the survival has been high, of about 95%, that is, once surviving and grown under greenhouse conditions, the seedlings could be cultivated outside. As for a comparison, in the protocol of micropropagation of the Cerrado species Jacaranda ulei Bureau & K. Schum., the survival in the transition between in vitro and rooting phase has been around 80% (Silveira et al. 2018).

To improve the in vitro protocol, it is important to know physiological aspects of the wild species and seedlings at each one of their phases (Kaur 2015). The leaf photosynthetic curves are one of the most important physiological features to be known, as these curves can point out the ability of a small plant in establishing an autotrophic metabolism (Leite et al. 2017).

There have been many reports on the anatomy of Velloziaceae species (Ayensu 1968, 1969, 1974; Menezes 1977, 1988; Coetzee 1974; Ayensu & Skvarla 1974; Menezes & Semir 1991; Amaral & Mello-Silva 2008; Mello-Silva 2010), including V. squamata (Ayensu 1974, Menezes 1977). However, none of which described the leaf anatomy of micropropagated plants either anatomical responses to shading for those species. In addition, there is no anatomical report of Vellozia species grown in vitro at all. Therefore, the present paper aims to verify the impact of the conditions of in vitro micropropagation in the photosynthetic responses and in the leaf anatomical structure of V. squamata.

Materials and Methods

Plant material and treatments - The control group was five individuals of Vellozia squamata naturally found at Parque Ecológico Burle Max, Brasília, DF, Brazil, selected in a convenience sample due to easy access and apparently healthy vigor. For details of climate and soil, consult Bucci et al. (2006). Voucher specimens were deposited at the herbarium UB under the numbers UB0028356 and UB0028358. Seeds were harvested, put to germinate and the plantlets were sectioned and submitted to the protocol of micropropagation of Neto (2009). The hallmarks of this protocol for the present study area: a) plantlets in culture medium (Murashige-Skoog medium added with sucrose and phytoregulators kinetin, indol-butyric acid and gibberellin); b) seedlings under acclimatization in a greenhouse in pots containing soil; c) cultivation outside, in garden conditions. Thus, the following experimental treatments were: a) plantlets in culture medium growing under photosynthetically active radiation (PAR) of 32 µmol m^-2 s^-1, photoperiod of 12/12 h and controlled temperature of 27 ± 2 °C; b) rooted seedlings in condition of a greenhouse, under partial shade, with maximal PAR of 412 µmol m^-2 s^-1, as measured by a portable refractometer Apogee Instruments®; c) plants obtained by micropropagation and cultivated in an external garden, in treated and fertilized soil, exposed to the natural environmental conditions of Cerrado (annual cycle of drought and rains). The wild plants were the control.

Leaf anatomy - The leaf anatomy was carried out by transversal sections of the middle third of three well developed leaves in each of the groups. The fresh sections were fixed in FAA₇₀ under vacuum, dehydrated in ethanolic series, included in resin Leica® and stained with toluidine blue or safranine plus Astra blue, according Johansen (1940) and Kraus & Arduin (1997). The slides were mounted with colourless glass varnish according to Paiva et al. (2006).

Light response curves - For each treatment, two leaves per plant from five plants (n = 10 measurements) were analyzed. From each leaf, the curve of light intensity/net carbon absorption (A, µmol CO₂ m^-2 s^-1) was obtained with a portable infra-red gas analyzer (IRGA) model LI-6400XT, manufactured by LI-COR Bioscience®. Since the leaves of the in vitro plantlets and the recently rooted seedlings were too small to completely fill the leaf chamber of the IRGA, data were adjusted according to the leaf area.

The IRGA was adjusted as to keep an air flow of 500 µL.s^-1 and carbon dioxide of reference of 400 ppm. The relative air humidity of the reference air was about the same in the treatments, as follows: control group: 45%; plantlets in vitro: 38%; seedlings in the greenhouse: 36%; garden condition: 44%. The air temperature at the time of the measurements were in a favorable physiologic condition, as follows: control group: 25.5 ± 0.5 °C; in vitro plantlets: 26.0 ± 0.1 °C; seedlings in the greenhouse: 30.4 ± 0.7 °C; garden condition: 31.3 ± 1.2 °C. The light intensities were previously selected, varying from zero to 1,600 µmol m^-2 s^-1.

After each change in the light intensity, the IRGA was programmed to record the value after it had stabilized. In half of the measurements, the curve was carried out in ascendant way and in the other half, in descendant way of light intensity. For the plants of open conditions (garden and control group - ecological park), the measurements were taken between 09:00 and 10:00 AM during the rainy season; and besides the curve A/light, the gas exchange rates were also measured under the natural light intensity, of about 2,200 µmol m^-2 s^-1.

Also, the transpiration rate was measured, and the water use efficiency (WUE) was considered as the ratio between the mean of the carbon absorption rate and the transpiration rate as seen in Field et al. (1983) and Medrano et al. (2015).

Chlorophyll content - The leaf chlorophyll content was estimated with a chlorophylometer Opti-Sciences CCM-200®, in two leaves per plant of five plants in each experimental treatment. According to the manufacturer, this device does not measure the chlorophyll content directly, since it analyzes the leaf transmittance of light, expressing the results in CCI - chlorophyll content index. Hence, it serves as for comparison among treatments.

Statistical analysis - The light response curves were adjusted to the model of Prado & Moraes (1997): A_meas = A_max (1- e^-k(PAR-Lcp)), in which A_meas means carbon assimilation rate under a given light intensity; A_max means the maximal carbon assimilation rate measured; k is a constant calculated for each curve; PAR means the given light intensity and Lcp means the light compensation point. Data were compared via Student´s T-test with the support of the open access graph software Libre Office.

Results

Physiological parameters and light response curves - The physiological parameters obtained are summarized in table 1 and figures 1 to 4 show the curves of light response of CO₂ absorbance. In the control group and in the plants growing in the garden, at a light intensity of 900 µmol m^-2 s^-1, the leaves reached about 90% of the maximum carbon absorption rate. Since most of the leaves had peaked in the carbon absorption rate, this was considered as the light saturation point. For the light intensities of more than the double of this point, the carbon absorption rate was kept, hence there were no signs of photoinhibition (figures 1, 2, 4). In the open groups (wild plants and garden plants), the carbon absorption rates under the natural sun light (PAR of about 2,000 µmol m^-2 s^-1) were the same as the maximal rates measured by the IRGA (maximal PAR of 1,600 µmol m^-2 s^-1), so the data of A_max were grouped together.

Table 1.
Parameters of the experimental treatments with Vellozia squamata Pohl (Velloziaceae). A max: maximal CO₂ absorption rate, µmol CO₂.m^-2.s^-1 ; E: transpiration rate, mmol H₂O .m^-2.s^-1 ; LCP: light compensation point, µmol photon.m^-2.s^-1 ; LSP: light saturation point, µmol photon.m^-2.s^-1 ; L max: maximal PAR to which the plants are exposed, µmol photon.m^-2.s^-1 ; Chlorophyll: chlorophyll content, unities of chlorophyll (CCI); WUE: water use efficiency (µmol CO₂/mmol H₂O). Letters compare data in the same column. Distinct letters mean statistically significant difference (P<0.05) by Student´s T-test.

Treatments	A max	E	LCP	LSP	Lmax	Chlorophyll	WUE
Cerrado	8.57±1.07 a	4.76± 0.90 a	80	900	1,925	24.6±3.97 a	1.8
Greenhouse	3.42±0.28 b	0.92±0.54 b	60	200	412	2.99±1.48 b	3.7
In vitro	n/a	3.44±0.03 c	n/a	n/a	40	1.37±0.44 c	n/a
Garden	4.97 ± 1.43 d	1.16 ± 0.06 b	60	900	1,925	8.70 ± 3.2 d	4.3

Figure 1.
Light-response curve of A (net carbon assimilation rate) of leaves (n=10) of Vellozia squamata Pohl (Velloziaceae) in field conditions during the rainy season under different values of photosynthetically active radiation (PAR). Non-linear adjustment by the equation A_meas: 8.57 (1-e^{-0.001658(PAR-80)})

Figure 2.
Light-response curve of A (net carbon assimilation rate) of leaves (n=10) of seedlings of Vellozia squamata Pohl (Velloziaceae) acclimatizing in greenhouse conditions under different values of photosynthetically active radiation (PAR). Non-linear adjustment by the equation A_meas: 3.42(1-e^{-0.0176833(PAR-60)})

There were clear differences in the photosynthetic parameters among the treatments. In the in vitro plants, some leaves were capable to absorb some carbon dioxide, mostly under light intensities between 100 and 200 µmol m^-2 s^-1, but overall, the measurements showed only the respiration values, as the maximal values of A were of -0.35± 1.25 µmol CO₂ m^-2 s^-1. As seen in figure 3, the leaves of the in vitro plantlets showed a random pattern of respiration up to light intensity of 100 µmol m^-2 s^-1, above which they showed a reduction of the respiration rates up to the light intensity of 900 µmol m^-2 s^-1. To light intensities higher than that, there was a slight deterioration in the respiration rate, suggesting photodamage. In this way, to the in vitro plants, there was no light compensation point.

Figure 3.
Light-response curve of A (net carbon assimilation rate) of leaves (n=10) of plantlets of Vellozia squamata Pohl (Velloziaceae) in vitro conditions under different values of photosynthetically active radiation (PAR). No adjustment of the curve was carried out.

Figure 4.
Light-response curve of A (net CO₂ assimilation rate) of leaves (n=10) of Vellozia squamata Pohl (Velloziaceae) seedlings obtained by in vitro propagation and growing in a garden during the rainy season under different values of photosynthetically active radiation (PAR). Non-linear adjustment by the equation A_meas: 4.97 (1-e^{-0.00125(PAR-60)})

The plants growing in the greenhouse were exposed to a maximum light intensity of about 412 µmol m^-2 s^-1, that is, a little bit less than half of the light saturation point of the wild (control) plants (table 1). The maximum carbon absorption rate was inferior to the wild and the garden plants, but the shape of the light response curve followed a pattern as expected. To the greenhouse plants, although the light saturation point was of about 200 µmol.m^-2.s^-1, the carbon absorption rate was kept under higher light intensities, suggesting there was no photoinhibition (figure 2).

The leaf transpiration rates were kept constant throughout the measurements for the light response curves but varied among the experimental groups. The wild plants showed the higher transpiration rates, followed by the in vitro plants. The plants growing in the greenhouse and at the garden showed similar transpiration rates, but inferior to the wild plants. Thus, the instantaneous water use efficiency was higher in the young, garden growing plants than adult wild plants (table 1).

The chlorophyll content was slightly greater in the plants growing in the greenhouse than in vitro, but significantly inferior to those in the garden (table 1). This content was greater in the garden plants than the greenhouse ones, but inferior to the wild plants, and the leaves presented a tonality of a lighter green, but not suggesting chlorosis, since the color intensity was uniform (figure 5). Leaf anatomy - The anatomical analysis showed the effects of environmental and growth conditions on the structure of V. squamata leaves (figure 6). In all leaves, the epidermis was single layered and without trichomes. In leaves of field grown plants (control group) and of garden condition (figures 6 a, f), the abaxial surface was smooth and the adaxial was corrugated. On the other hand, there was no visible epidermal ornamentation in leaves of in vitro grown plantlets nor of greenhouse acclimatizing plants (figures 6 d, e).

Figure 5.
Plants of Vellozia squamata Pohl (Velloziaceae). a-b. In the field, showy lilac flower (b). c. In vitro condition. d. Greenhouse condition. e. Plants produced in vitro now growing at the garden. Bars: A = 1 m, B = 5 cm, C = 1 cm, D = 5 cm, E = 1 cm.

Figure 6.
Leaf anatomy of Vellozia squamata Pohl (Velloziaceae). a-d. Plants from the field (b-c: details of a): adult (a-c) and young (d) leaves, showing the early formation of the stomatal crypts and later differentiation of the cuticle, subepidermal fibres, palisade parenchyma, and the vascular bundle sheath. e. Plants from micropropagation, not acclimatized: absence of stomatal crypts and subepidermal fibres. f. Plants from acclimatization similar to the adult leaf of the field, but with less deep crypts. g. Plants growing at garden condition. Arrows: stomata. AP: aqueous parenchyma, Ct: cuticle, Fi: fibres, PP: palisade parenchyma (elongated cells), SC: stomatal crypt, SP: spongy parenchyma (isodiametric cells). Bars a, d-g = 50 µm, b-c = 25 µm.

In the control group and in the garden group, the external periclinal cell wall and cuticle could not be distinguished from one another (figures 6 a, b, f), and these layers are called here the outer layer of epidermal cells. Hence, this layer in the plants under plentiful sunlight was considerably thicker (figures 6 a-c) than those in the other growth conditions (figures 6 d-e). Nonetheless, the epidermis presented an intermediate thickness in plants in the greenhouse condition (figure 6 e).

With respect to stomata disposition, V. squamata leaves were amphistomatic, and the stomata on the abaxial face were mainly found in crypts, which reached about a quarter of the leaf thickness (figure 6 a). These features were more noticeable in the control group and in the garden plants. It is noteworthy that in vitro plantlets did not develop stomatal crypts, and the stomata were rudimentary (figure 6 d). Additionally, plants in the greenhouse process had smaller and shallower crypts (figure 6 e) than those of field plants (control group) with fully developed stomata (figure 6 a).

The leaf mesophyll from control group plants was dorsiventral and composed of 21-25 parenchyma layers, which cells are elongated on the adaxial side and gradually diminishes in height towards the abaxial side, getting a rounded shape (figure 6 a, f). Both in vitro and greenhouse plants had a dorsiventral mesophyll (figures 6 d-e). Nevertheless, in these plants, the mesophyll presented a less dense cell arrangement than the mature leaves from the control (figures 6 a, f). Additionally, each leaf face of greenhouse, garden and control plants, the mesophyll possessed subepidermal fibre fascicles, which was more abundant in leaves from the control and garden plants (figures 6 a, f). Conversely, these fibre fascicles were not observed in leaves from the in vitro grown plants (figure 6 d).

The paralellodromous leaf veins from the control plants, garden and in greenhouse had a conspicuous vascular bundle sheath (figures 6 a, e-f). In addition, the bundle sheaths had extensions on the adaxial side with large colourless palisade cells termed as aquiferous (figures 6 a, e-f). This parenchyma connected the bundle sheath to subepidermal fibre fascicles on the adaxial epidermis (figures 6 a, e-f). On the abaxial epidermal face, the sheath was in direct contact with the epidermis (figures 6 a, e-f). Leaves from in vitro plants have bundle sheaths with no extensions (figure 6 d).

The vascular bundles were heavily fibrous with two strands of phloem placed laterally to the xylem (figures 6 a, e-f). However, this feature was less pronounced in leaves from in vitro plants (figure 6 d). The vascular fibres had thick lignified cell walls (figures 6 a, e-f). The vascular fibres were present in a greater amount in the control plants, in an intermediate amount in the greenhouse plants and in a small amount in the in vitro plants (figures 6 a, e-f).

Discussion

The species of Vellozia grown in open fields show a strong photodissipating apparatus, which is important to protect the leaves from the stressful conditions of Cerrado - the dry season together with a high solar irradiance. Tocotrienols, which are strong antioxidant compounds of vitamin E complex basically found in seeds, have been identified in leaves of V. gigantea (Morales et al. 2014, 2015). Thus, at the same time those plants dissipate excess of light, they need high light intensities for total development of the photosynthetic apparatus and leaf structure.

The light intensity effect on photosynthetic aspects of Vellozia leaves has been more frequently evaluated by the electron transport rate. In several species of Vellozia, this rate is maximal under photosynthetically active radiation (PAR) intensities of about 2,100 µmol.m^-2.s^-1, which is coincident to the values commonly found in Cerrado, i.e. ca. 1,800 to 2,200 µmol.m^-2.s^-1 at midday (Lüttge et al. 2007). As for a comparison, the maximal electron transport rate of Smilax goyazana, a common shrub of Cerrado, occurs at about half of the maximal PAR of the environment (Palhares et al. 2011).

The leaves from plants grown in vitro conditions show typical effects of shading (Barboza et al. 2006, Chaari-Rkhis et al. 2015, Ziv & Chen 2008), with poorly developed anatomical structures, including stomata. Moreover, some ultrastructural features of shaded leaves are the limited cytoplasmic content and flattened chloroplast with an irregularly arranged internal membrane system and a higher proportion of intercellular spaces (Wetzstein & Sommer 1982, Chirinéa et al. 2012). In the present work, some of these features include qualitative alterations, as the absence of aquiferous cells, subepidermal sclerenchyma fascicles, and stomatal crypts.

It is noteworthy that the depth of the crypts reflected the growth condition. The stomatal crypts in the leaves are deeper in the control (field) and garden than greenhouse plants, and they were totally absent in plants grown in vitro. They are already present in the young leaves of field plants (figure 6 d). It is surprising that the presence of leaf crypts is not a genetical imposition for V. squamata.

Furthermore, leaves from in vitro plants of V. squamata presented several structural differences in comparison with plants in acclimatization: they had no subepidermal fibres, subtler dorsiventral mesophyll, undifferentiated bundle sheath extension, fewer vascular system fibres, as well as a lack of aquiferous parenchyma. The aquiferous tissue remains in the leaves from pineapple (Ananas comosus L.) grown in vitro (Barboza et al. 2006), so qualitative alterations like those observed in V. squamata are no ordinary thing, mainly the absence of stomatal crypts and subepidermal fibres.

The above differences can be directly associated with the low light levels and perhaps to the high humidity of the in vitro condition. The anatomical results indicated that V. squamata grown in vitro requires a longer and careful conducted acclimatization process.

The anatomical immaturity is linked to the immaturity of the photosynthetic apparatus. In the present case, the in vitro plantlets have inability of net carbon absorption, together with a high leaf transpiration rate, indicating dysfunctional stomata. A given light intensity may be enough for the plain development of the leaves of a given species, but insufficient for others. Thus, comparing with other Cerrado species cultivated in vitro, Lippia rotundifolia develops leaves capable of liquid carbon absorption under PAR of 78 µmol m^-2 s^-1 (Hsie et al. 2019), while Hanconia speciosa and Hyptis marrubioides showed carbon absorption under PAR of 50 µmol m^-2 s^-1 (Costa et al. 2014).

Regarding crop cultures, in the production of in vitro seedlings of strawberry (Grout & Millan 1985) and grape (Amancio et al. 1999), the growth under a PAR of 90 µmol m^-2 s^-1 generates photosynthetically capable leaves with light saturation point around 450 µmol m^-2 s^-1, while in V. squamata, the growth under maximal PAR of 412 µmol m^-2 s^-1 still showed shade effects. In the Cerrado species Pouteria gardneriana, there were clear differences in the structural development of leaves growing under PAR varying from 75 to 150 µmol m^-2 s^-1, but the authors had not shown data of wild plants as a control (Leite et al. 2017).

According to the data herein presented, even under low PAR intensities there is chlorophyll formation. However, the photosynthetic ability depends on the development of other elements that require higher light intensities. According to Leite et al. (2017), in Pouteria gardneriana the chlorophyll fluorescence did not vary substantially among the plantlets grown under the different PAR intensities, but the anatomical structure was more developed under the higher ones. In grape, the leaves grown in very low PAR intensities (as of 20 µmol m^-2 s^-1) release oxygen, indicating that the photosynthetic apparatus is capable of carrying out photolysis of water, but they are not capable of carrying out net absorption of carbon dioxide (Amancio et al. 1999).

In the present experiment, the chlorophyll content was not directly measured, but the comparison carried out showed that the plants in the greenhouse, partially shaded, had about the double of chlorophyll (2.99 CCI) as the plantlets in vitro (1.37 CCI) but a little bit less than half of the open garden plants (8.70 CCI). However, all the experimental groups showed much less chlorophyll compared to the wild plants (24.6 CCI). Certainly, the improvement of PAR intensity from in vitro to garden condition could explain those differences seen, but it was not clear why the young plants at the garden showed a chlorophyll content of about one third of the wild plants. It is noteworthy that the anatomical structure of the leaves of the plants grown in the garden was overall similar to the plants in the fields, except for a smaller thickness. So, the growth under plentiful sunlight resulted in anatomically mature leaves, but photosynthetically they showed a pattern of relative immaturity.

Moreover, the maximum carbon absorption rate of the plants at the garden was about 45% higher than the plants in the greenhouse but about 70% smaller than the wild plants. Spite the statistically significant differences among the experimental groups, some measurements of the maximum carbon absorption rate of the plants at the garden were close to the plants in the greenhouse. It is difficult to find in the field young plants of Vellozia squamata that could serve as for a comparison. It is not clear if the photosynthetic performance of the younger plants is something inherent to their youthfulness or if it is sort of a side effect of the protocol of micropropagation. Indeed, Durkovic et al. (2016) described that the way of obtaining seedlings can interfere in the leaf development, generating leaves with different photosynthetic parameters.

Regarding the leaf transpiration rates, the leaves of the plants of either the garden or inside the greenhouse showed differences compared to the wild plants, suggesting that the youthfulness of the plant is a factor that interferes with the physiological parameter of the leaves. Compared to other Cerrado species (Palhares et al. 2010), the adult wild plants of V. squamata showed a low water use efficiency. In this way, the leaves of the younger plants (of garden and greenhouse) presented a lower carbon assimilation rate but a better water use efficiency. Regarding the plantlets of in vitro condition, the higher transpiration rate is common to in vitro plantlets overall. The epidermis and stomata are poorly developed, both structurally and functionally, hence the control of the transpiration is weak, and this is one of the stressing factors during transition from in vitro to greenhouse growth (Van Huylenbroeck et al. 1998, Amancio et al. 1999, Kaur 2015).

According Van Huylenbroeck et al. (1998) and Kaur (2015), the in vitro propagated plants can be divided into two functional categories regarding the transition from in vitro to greenhouse phase: plants with photosynthetically incapable, in a which the leaves serve only as reservoir organ for the sprouting, and those photosynthetically active, whose leaves have at least partial development. In this last group, it is often seen that during the first days of transplantation there is a typical stress acute response, such as great rates of transpiration and respiration and eventually a reduction in the chlorophyll content (photobleaching; Van Huylenbroeck et al. 1998, Kaur 2015).

After this initial period, not only the leaves become photosynthetically active, but also they often show anatomical changes, such as cellular growth with reduction of stomatal density and differentiation of hairs (Van Huylenbroeck et al. 1998, Kaur 2015). Anyway, in both functional groups the in vitro shaded leaves cannot tolerate the environmental light intensities, but the newer leaves formed under those intermediary light intensities tend to present a light compensation point and a light saturating point like the wild plants, as well as a photo-dissipating apparatus better developed (Van Huylenbroeck et al. 1998, Kaur 2015).

The carbon assimilation rates verified in Vellozia squamata are relatively low, compared to other Cerrado species (Palhares et al. 2010), and according to Nogueira et al. (2004) are more typical of secondary than pioneer plants. That is, in spite of the large production of seeds with high germinability, the establishment of Vellozia squamata is related to a complex ecological interaction, highlighting the importance of the conservation of this species in the environment, since even the seedlings herein obtained are of a very slow growth.

As a conclusion, for improving the protocols, it is useful to compare the responses of the wild and the in vitro plants. The data herein presented may help to adjust the PAR intensity to obtain more mature seedlings with perhaps a higher survival rate at the transition between in vitro and greenhouse conditions. Therefore, the development of technology for producing seedlings of Cerrado species is a complex task, but of great importance for their conservation and domestication.

Acknowledgements

To the Programa de Pós-Graduação em Botânica, Universidade de Brasília, Instituto de Ciências Biológicas, Brasília, DF, Brazil. This research was sponsored by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Fundação de Empreendimentos Científicos e Tecnológicos, and Conselho Nacional de Desenvolvimento Científico e Tecnológico. Janet Nimoh Sallis Mensah reviewed the English grammar and spelling.

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• 1.
  Part of Master of Science of Olegário Garcia de Freitas-Neto

• Associate Editor:

  Ana Paula Souza Caetano

Publication Dates

• Publication in this collection
  02 Sept 2022
• Date of issue
  2022

History

• Received
  04 May 2020
• Accepted
  18 Jan 2022