A record of seed germination, distribution pattern and effect of premonsoon season variability on quantitative leaf anatomy of Mollugo nudicaulis Lam. a C2 species from tropical Desert Climate

doi:10.21203/rs.3.rs-2217646/v1

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A record of seed germination, distribution pattern and effect of premonsoon season variability on quantitative leaf anatomy of Mollugo nudicaulis Lam. a C2 species from tropical Desert Climate

https://doi.org/10.21203/rs.3.rs-2217646/v1

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In rapid climate change how C₃-C₄ plants will respond this is the question of study. Here, we present Mollugo nudicaulis Lam., a C₃-C₄ intermediate species known for C₂ type photosynthesis as a model plant in the tropics. Therefore, to know the functionality and habit of M. nudicaulis in ambient climate, we analyzed the leaf anatomy quantitatively, recorded the local dispersion variance pattern of species, seed production potential (seed output per plant) and seed germination pattern. Natural and man-altered sites were explored to see the distribution of the plant. Seed output per plant was calculated. Cross-sections of mature leaves were observed. We compared ex vivo and in vitro seed germination profile by applying of primary dormancy breaking methods including –imbibition, scarification, and cold stratification to different time regime. M. nudicaulis follows the clump distribution in natural area due to hydrochory dispersion of seeds. In nature plant thrives in high organic rich sites. In ex situ germination, the acid scarification (physical change) and the cold stratification (physiological change) showed significant enhancement in germination after breaking of dormancy. The leaf anatomy showed variations in the number and the area of leaf mesophyll cells and the thickness of leaf as per fluctuation in average temperature, pressure, and humidity in a year. More palisade cells found in the leaves cross-section when the year recorded for average high temperature and more frequent rainfall. We found a correlation between the mesophyll cells in leaves and pre-monsoonal season variability for C₃-C₄ intermediate plant. The study adds supportive results in climate change impact on plant physiology and adaptation.

C2 plants

C3-C4 intermediate

leaf anatomical traits

mesophyll tissues

Paramollugo

primary dormancy

In photosynthesis, C₃, C₄ and CAM (Crassulacean Acid Metabolism) are three biochemical important pathways to incorporate CO₂ in biosphere. C₃ pathway of fixing carbon occurs in all photosynthetic plants whereas other two pathways C₄ and CAM (Crassulacean Acid Metabolism) were evolved as an adaptive strategy in plants to best fit in ambient climate with very low CO₂ in air. Around 30 Mya, C₄ photosynthesis mechanism emerged in tropical mostly arid plants when atmosphere CO₂ was critically low (Sage et al. 2014). During historical climate change events, several transitional species, emerged in during the evolution of C4 plants, having intermediate characters nearer to either C₃ or C₄ plants (Sage et al. 2014; Khoshravesh et al. 2016, Von Caemmerer et al. 2017). However, in current climate, these C₃-C₄ intermediate species are not thriving as compared to both C3, and C4 species (Christin and Osborne 2014). Today, only 17 genera from different families have only 1 or 2 C₃- C₄ intermediate species and most of them are relatively restricted geographical distribution and have less floristic importance (Sage et al. 1999). Among them, two C₃-C₄ intermediates species, so far, Mollugo verticillata and M. nudicaulis are still widely found compared to others however, in almost tropical areas (Vincent 2003). Ecologically the C₃-C₄ intermediate traits tend to occur in rapidly growing herbaceous species which shows the highest growth rate in the warmest part of the growing season (Monson and Rawsthorne 2000). Evolutionary these plants are important resources to understand the process of adaptations in plants at anatomical and physiological level.

Here, the studied plant M. nudicaulis L. (IPNI:urn:lsid:ipni.org:names:363724-1) synonyms Paramollugo nudicaulis (Lam.) Thulin (Thulin 2016) is a member of a physiological and evolutionary significant family Molluginaceae; order Caryophyllales. The plant is recognized as C₂ Type-I intermediate between C₃ and C₄ plants based on leaf anatomy, biochemistry and GDC (Glycine decarboxylase) shuttle (Christin et al. 2011). Besides evolutionary eco-physiological importance the species is also known for ethno- medicinal properties as hepatoprotective, eye and skin infection curative, antiulcer, anti-whooping (in Barupal et al. 2018). The species is a summer escaping wild herb and complete life span in the few months of Monsoon.

In most tropical desert climate, temperature extremes, low rainfall and dry air are common features therefore, the main vegetation of desert consists of annual and biennial species (Li 2008) and most of them produce plenty of seeds with varying degrees of dormancy that can withstand short to long extent of unfavorable conditions (Jurado and Flores 2005). A dry dormant seed is well managed to remain alive for long under unfavorable conditions (Bewley 1997; Koornneef et al. 2002). In definition, seed dormancy is a mechanism of suspension of the seed germination while all favorable physical requirements for successful germination are fulfilled (Baskin and Baskin 2004). Moreover, the seed dormancy in tropical annuals is a necessity to reduce the competition between young seedlings for space and nutrition, and overcome the chances of extinction in extend period of unfavorable conditions (Baskin and Baskin 1998; Gutterman 2002; Dalling et al. 2011; Long et al. 2015). By varying degrees, the seed dormancy is generally classified into five major groups as physiological dormancy (PD), morphological dormancy (MD), morpho-physiological dormancy (MPD), physical dormancy (PY) and combinational dormancy (PD + PY) (Baskin and Baskin 2004). A favorable condition to release germination from the dormant stage in their respective in situ environment and an effective method to break dormancy in ex situ conditions may vary from species to species (Baskin and Baskin 1998). Soil temperature and moisture also regulate seed dormancy cycling (Gao et al. 2018).

For a successful germination, a seed becomes metabolically active and after several biochemical changes, emerges itself into an auto-trophic individual plant (Bewley 1997). Several factors such as rainfall, relative humidity, temperature, light, pH, concealment depth in soil and genotype of the plant are known to affect the germination of seeds (Gutterman 2000; Bradbeer 2013; Batlla and Benech 2014).

Leaf internal structure specially mesophyll cells arrangement plays vital role in water conductance, CO₂ flux and determining photosynthesis under water stress conditions. In last decade, mesophyll conductance received wide attention as a target to improving plant productivity and water use efficiency (Nadal and Flexas 2018). Foliar anatomical traits are used to evaluate the ability of species to adapt to different environmental conditions. It has been observed that plants modified thickness and arrangement of mesophyll tissues to decrease space, improve the CO₂ intake, increase foliar photosynthetic capacity, and increase water retention in tissues (Toro-Tobon et al. 2022).

Here, along with seed germination profile, we report the morphology, local dispersion, seed throughput potentiality, and quantitative leaf anatomy of Mollugo nudicaulis at ambient climates, and propose the plant as a model C₃-C₄ intermediate species in tropical and sub-tropical areas to study plant evolution and impact of climate change.

Plant Material:The samples of M. nudicaulis were collected at flowering stage from natural undisturbed lands area such as monsoonal river banks near villages Bara Bagh 26.9555 N, 70.8858 E (Jais BB), open land area along road to village Akal 26.9157 N, 70.9083 E (Jais AR) in Jaisalmer district and the road side along Guda Vishnoi village 27.8789 N, 75.5654 E (GR), Bisalpur 26.2345 N, 73.3164 E, Banar 26.3347 N, 73.1459 E, Machia Safari Park 26.3021 N, 72.9779 E(MSP), near Shikargarh area 26.2822 N, 73.0899 E(SK) in Jodhpur and from disturbed lands as agricultural land area of ARS 26.3523 N, 73.0474 E (Mandore) and CAZARI 26.2436 N, 73.0045 E, and AIIMS road 26.2428 N, 72.9949 E Jodhpur during early August to late October. Mature seeds from open or near open capsules were collected and stored in dry and cool places.

Dispersion variance and total throughput of seeds Dispersion pattern and average seed production per plant were calculated. The number of individuals per m² area per site was recorded. Dispersion variance was calculated by respective ecological formulae (Krebs 1999).

For Dispersion variance =\(Ʃ{\left(X-\overline{ X}\right)}^{2}/\left(n-1\right)\)

Where \(\overline{X}\)= mean of individual per quadrat; n = total number of quadrates.

Full expressed plants were selected to measure seed throughput (production) capacity. The inflorescence stalks per plant; capsules per inflorescence stalk and seeds per capsule were counted from full expended healthy plants to measure optimum seed production capacity of M. nudicaulis in nature. Randomly 15–20 plants per site were observed for measurement of seed production capacity.

Seed germination profile

To observe germination pattern and enhancement after breaking of dormancy, first the seeds were surface sterilized with 0.1 % Bavistin (a broad-spectrum fungicide containing 50% WP Carbendazim; BASF India Limited, Mumbai, India) for 10 min and then washed with autoclaved distilled water for 3-4 times. Sterilized seeds were served for different methods to break dormancy (Fig. 1). We first observed the germination rate in freshly collected seeds in soil. The randomly collected 100 ± 10 seeds in three replicas sowed in germination plastic trays containing soil (sandy garden soil or Solrite mix) in the greenhouse (Department of Botany, JNVU, Jodhpur) in the month of August and September and watered regularly. After obtaining initial rate of germination in the collected fresh and old seeds, all next experiments were carried on freshly collected seeds to enhance germination percentage.

Separation of mature Seed – The freshly collected seeds were soaked in 60 ± 5° C hot water for 30 min to separate mature viable seeds. The mature-black-hard -viable seed sank at bottom of the flask within 30 m in this method. So black seeds were further used to test the requirements for seed germination and its enhancement.

Immersion - Seeds were immersed in water for two time-regimes – 24 and 48 h at room temperature (28/22 ± 2° C). The initial and final weight and size of seeds were recorded. The weight difference before and after immersion was calculated by the formula [(Wf – Wi) / Wi] × 100, where, Wf = the weight of imbibed seeds and Wi = initial weight of the seeds. The size of seeds was measured under a compound light microscope (Olympus Corp., Tokyo, Japan) using an ocular scale (100 div = 250 µm).

Soaking - The sterilized freshly collected and one y old seeds were placed on blotting paper (filter paper) soaked in distilled water or quarter, half, or full strength of MS liquid medium (Murashige and Skoog 1962) in petri-plates (100 X 15 mm, Borosil Glass Works Ltd., India) for 1-15 d. Petri-plates with seeds were put in a dark chamber or in environmental growth conditions with 16/8 h light/dark at 25 ± 2º C. Seeds were kept hydrated with distilled water regularly.

GA₃ (Gibberellic acid) Treatment - The seeds were treated with GA₃ by immersing in 144, 289, 1445 and, 2890 µM solution of GA₃ (Sigma-Aldrich®, St. Louis, MO) for 24 h and then kept on blotter paper in petri-plates and hydrated regularly with distilled water for 4 w.

Cold stratification- The seeds were placed on blotting paper soaked with distilled water or MS ½ liquid medium then placed into the refrigerator at 4º ±1 C for 1-15 d. To hydrate seeds, 2 mL of respective hydration medium (MS ½ or dH₂O) was added on alternate days. The seeds after respective days of cold treatment were kept at 24 ± 2º C in day/night condition for germination and hydrated with respective medium.

Acid Pulse treatment - In acid pulse treatment the seeds were submerged in concentrated sulphuric acid (98%; Loba Chemie, Mumbai, India) for 30, 60 and 80 s and then washed immediately with distilled water for 3-4 times and then kept in petri-plates on to the blotter paper soaked with distilled water.

In vitro germination- MS medium augmented with 3% sucrose (w/v) (Qualigens Fine Chemicals, Mumbai, India) was used as a basal medium to observe the in vitro germination. Surface sterilization and inoculation of the seeds on the medium were performed under aseptic conditions of laminar air flow bench (Thermadyne Pvt. Ltd. Faridabad, India). Before inoculation the seeds were pre-treated with 0.1 % Bavistin for 10 min and then washed 3-4 times with autoclaved distilled water. To prevent bacterial contamination, the seeds were sterilized with 0.1% HgCl₂ (mercuric chloride, Sigma-Aldrich®, Saint Louis, MO) for 3-5 min, then thoroughly washed with autoclaved water 3-4 times. The basal medium used for inoculation consisted of quarter or half or full strength of MS macro nutrients. The combined medium included MS salts with 2.26 µM 2,4-D (2, 4-dichlorophenoxyacetic acid; Sigma-Aldrich®) or 2.22 µM BAP (6-benzylaminopurine; Sigma-Aldrich®) or different concentration of GA₃ (289, 1445 and 2890 µM). 0.2% activated charcoal was added in all in vitro mediums. Borosilicate Petri-plates, culture tubes (60 mL, 25 mm × 150 mm) and Erlenmeyer conical flasks (150 mL, 72 × 124 mm; Siddhivinayak Glass Concepts, Firozabad, India) were used as glassware. After inoculation the vessels were placed in the culture room at 12 h photoperiod of 15-20 µmol m-2 s-1 or in dark at 24-26° C at 60 % RH.

Climate Data: Meteorological data collection from month July to September in three subsequence 2016-2018 (Data source: Global SWAT, IMD- Pune and https://www.timeanddate.com/weather/india/jodhpur; Table 1).

Table 1

Average weather report of three months in study period at Jodhpur, India

Month	Weather	Year
Month	Weather	2016	2017	2018
Jul	Tmax	38	38	42
	Tmin	24	20	24
	Hmax	100	100	100
	Hmin	37	33	10
	Pmax	1.007	1.008	1.099
	Pmin	0.996	0.996	0.948
Aug	Tmax	36	36	35
	Tmin	23	24	23
	Hmax	100	100	100
	Hmin	28	41	47
	Pmax	1.013	1.009	1.099
	Pmin	0.997	0.999	0.997
Sept	Tmax	38	38	36
	Tmin	22	23	20
	Hmax	100	100	100
	Hmin	27	14	19
	Pmax	1.012	1.012	1.014
	Pmin	1.001	1.002	1

T- Temperature, H- Humidity, P- pressure; Max= Maximum; Min- Minimum;

Pressure unit= kgcm^-2. Data source: https://www.timeanddate.com/weather/india/jodhpur

Leaf Anatomy: Three healthy plants per site were selected for leaf anatomy. 3-4 mature leaves per plant were collected. A natural and green house grown plants were choose to observe the variation in tissue organization. The transverse hand-sections of leaves were cut with sharp edges (Razor) and stained them with 0.01% Safranin. Thin sanctions of leaves were observed under the calibrated compound light microscope (Olympus) with magnification power 10*10 and 40*10X and photographed for quantitative analysis.

Quantitative Leaf anatomy: We observed transverse hand-sections of fresh leaf for record of total thickness, mesophyll, and epidermis cells; area of palisade, mesophyll, and bundle sheath cells; diameter of bundle sheath; number of cells in bundle sheath, palisade, and spongy area per unit area of leaf section. We did the anatomical observations of leaves from same collection sites for three subsequent years.

Data analysis

We designed all experiments and data collection according to completely randomized block design (RBD) for single factor experiments (Compton and Mize 1999). To find out statistical efficacy of observations, we used the final percentage of the seed germination in each treatment (mean percentage) for ANOVA (Gomez and Gomez 1984) and calculated the significance of differences among mean values using Duncan’s Multiple Range Test (DMRT) (Duncan 1955) at P =.05 by using software R (R lib. – Agricolae, Mendiburu 2016). Leaf anatomy images were analyzed by using ImageJ software. The seeds were considered germinated when the radicals emerged about 2 mm long. For seed germination enhancement experiments approx. 200 seeds per treatment were analyzed. Each treatment contained four replicates and repeated three times.

Morphology, Seed throughput potentiality and Local dispersion variance M. nudicaulis is a hot evader tropical annual plant has rosette leaves in 3–5 tiers, many inflorescences’ stalks bearing pentamerous rounded white, pedicellate, solitary and tri-carpallary flowers with 3–7 stamens and 3 short styles (Fig. 2a, b, c). It produces black berry minute seeds in the capsule. It is observed that the inflorescence of Mollugo nudicaulis divided trichasially at first node then dichasially and ultimately produces 9.0 ± 2.09 stalks bearing flowers per plant. (Table 3). Each terminal branch of inflorescence bears 35.21 ± 2.71 mature capsules. Each capsule holds 24.82 ± 3.58 seeds per tri-carpi capsule (Table 2). Total production of seeds was average 7368.87 ± 328.78 per plant. The size of seeds ranges from 200–600 µm (Fig. 3a, b).

Table 2

Seed throughput potentiality (average seed counts per plant) of *Mollugo nudicaulis*
Inflorescence Stalks/plant Mean ± SD (a)	Capsule / Inflorescence stalk Mean ± SD (b)	Seeds / Capsule Mean ± SD (c)	Seed Production Potential / plant (abc) Mean ± SD
9.0 ± 2.09	35.21 ± 2.71	24.82 ± 3.58	7368.87 ± 328.78
^{Data included 30 individuals of the plant; seeds counted in 10–15 mature capsules per plant}.

Table 3

*Mollugo nudicaulis-* difference in seed weight and size before and after hydro-immersion
Seeds Imbibition Period	Weight (µg) (Mean ± SD)		Size (µm) (Mean ± SD)		Increase (%) (Mean ± SD)
Seeds Imbibition Period	Weight	% Increase	Length	Width	Length	Width
0 h	6.50 X 10³ ± 0.22	-	131.57 ± 9.57	114.68 ± 9.72	-	-
24 h	7.88 X 10³ ± 0.32	20.98 ± 1.86	141.83 ± 5.71	118.87 ± 4.14	10.75 ± 1.38	7.48 ± 1.18
48 h	9.73 X 10³ ± 0.35	49.69 ± 1.43	145.52 ± 5.98	129.04 ± 8.09	18.90 ± 1.36	13.78 ± 0.67
^{Fresh weight increase was calculated by the formula [(Wf – Wi) / Wi] × 100, where, Wf = the weight of imbibed seeds, * = Ocular stage 100 div = 250 µ}.

Despite the abundant seed production capacity of the plant, a limited number of individuals were observed (less than 2 individuals per m2 area with 0–20% frequency) in the collection sites of both undisturbed natural and disturbed areas while rain water availability is sufficient during monsoon. However, in disturbed agriculture land area comparatively frequent numbers of individuals of the plant (Fig. 4) were found at margin and between crops.

Germination Percentage

In soil - When seeds were sowed in the soil under greenhouse environment the percentage of germination in previous year collected seeds was only 1–2% per 100 seeds after more than 3 w of sowing whereas negligible in freshly collected seeds.

Breaking of seed dormancy

Immersion- Seed coat of the seeds is permeable therefore, after 48 h, the weight of imbibed seeds increased to 49.69%, and relative size increased up to 18.90% in length and 13.76% in width. Here, seeds size and weight increased significantly after 48 h of imbibition under completely merged conditions. (Table 3).

Soaking - While both type of seeds (1 y old and freshly collected) were soaked with sterile distilled water and inoculated on blotter paper in petri plate and put for more than 4 w, a very low percentage of germination about less than 2% was found in old seeds whereas no germination in fresh seeds. Soaking in quarter, half and full liquid salt medium somewhat increased the percentage of germination in the fresh seeds but less than 1% (Table 4).

Table 4

Seed germination % in *ex vivo* conditions in *Mollugo nudicaulis*
Treatments	Level	Germination % Mean ± SD	Germination In days	Seedling quality in first 2 w
Soaked with dH₂O	-	1.21 ± 0.53^hij	15–30	****
Soaking with different strength of liquid MS basal medium	Quarter	1.16 ± 0.53^ij	15–25	****
	Half	2.02 ± 0.44^hij	15–20	****
	Full	1.44 ± 0.73^hij	20–25	**
Hydration with MS ½ and subsequently cold stratify at 4 ⁰C (in days)	1	10.91 ± 0.93^fg	4–15	****
	2	28.42 ± 0.87^b	2–4	****
	3	22.58 ± 1.00^c	3–6	****
	4	16.52 ± 1.32^d	7–10	***
	5	21.69 ± 1.50^c	10–15	**
	6	9.28 ± 1.59^g	10–15	*
	7	4.35 ± 0.50^hi	15–20	*
Hydrated with dH₂O and cold stratify at 4 ⁰ C (in days)	1	2.61 ± 0.71^hij	4–15	****
	2	26.98 ± 1.24^b	2–5	****
	3	20.00 ± 0.50^c	3–7	****
	4	14.78 ± 0.50^de	7–10	***
	5	13.33 ± 1.54^def	10–15	**
	6	14.78 ± 1.32^de	10–15	*
	7	12.17 ± 1.32^efg	15–20	*
Acid (H₂SO₄ 98%) pulse treatment (in seconds)	30	2.67 ± 1.15^hij	7–15	****
	60	37.50 ± 1.01^a	1–2	****
	80	0.69 ± 1.20^j	1–2	*
GA₃ treatment at different concentration (µM)	50	3.74 ± 0.74^hij	7–15	****
	150	4.80 ± 0.71^hi	7–15	****
	300	6.07 ± 0.71^h	7–15	****
	1000	8.99 ± 0.53^gh	2–7	***
	2000	3.77 ± 0.53^hij	4–10	*
^{MS = Murashige and Skoog (1962) medium. Percentage value was calculated by means of total germination in total seeds treatment; Significant difference was analyzed by Duncan Multiple Range Test on ANOVA; alpha =0.05. **= healthy and vigorous growth; * moderate growth but healthy seedlings, *= seedlings with deformities; = deformed seedlings with slow growth}

GA ₃ treatment - The exogenous applying of GA₃ at 1X concentration increased the germination percentage up to 9% on blotting paper within 2 w compared to other concentrations of GA₃ (Table 4).

Cold Stratification - In Mollugo nudicaulis when seeds were hydrated with half strength MS salt liquid medium and cold stratified for 2 days at 4 ± 1º C in refrigerator, the seed germination significantly increased as 28.42% as compare to soaking of seeds without cold treatment (Table 4). Whereas when seeds were hydrated with distilled water and cold stratified at 4 ± 1º C for 2 days, the germination percentage effectively increased 26.98% (Table 4).

The cold stratification reduced the time of germination i.e., within 2–4 days (Fig. 5). Here, the both methods (Soaked in MS medium or distilled water) 2 days’ regime of cold stratification was most effective and favored the healthy growth of juvenile seedling. More than 2 d of chilling to seeds added only a few newer germinations but young seedlings were weak, curved, and did not survive for long.

Acid Pulse Treatment - For the seed germination of Mollugo nudicaulis, the acid pulse treatment was most effective in all applied treatments for the breaking of dormancy. After acid treatment the highest 37.50% germination was observed. Here, the pulse for 60 seconds in 98% sulphuric acid was most suitable and less than 60 seconds did not affect the normal pattern of germination in the seeds and more than 60 seconds of acid pulse destroyed the embryos after rupturing of the seed coat.

For Mollugo, besides increasing the germination percentage, acid pulse treatment also reduced the period of the time taking in the germination (Fig. 5). Here, within 2 d the maximum germination was established with healthy seedlings emergence, where as in the cold stratification and other treatments, time period for germination was 3–15 days or more.

In vitro germination- MS medium augmented with 2,4-D (2.26 µM) or BAP (2.22 µM) increased the germination percentage up to 15.94% and 15.80% respectively in in vitro culture environment (Table 5). When seeds were inoculated on MS medium augmented with 2890 µM GA₃ the in vitro germination increased 17% after 1–2 weeks.

Table 5

Seed germination percentage in *in vitro* conditions in *Mollugo nudicaulis*
Treatment	Level	Germination % (mean ± SD)	Days taken in induction of germination (d)
MS strength^*	MS ¼	4.35 ± 0.34^e	10–15
	MS ½	9.24 ± 0.77^d	7–15
	MS	10.46 ± 0.58^d	7–15
MS + BAP (µM)	2.22	15.80 ± 0.91^abc	10–15
MS + 2,4-D (µM)	2.26	15.94 ± 1.93^bc	7–15
MS + GA₃ (µM)	500	14.67 ± 0.87^c	15–30
	1500	16.29 ± 1.13^ab	7–15
	3000	17.19 ± 1.66^a	7–10
	4000	2.33 ± 0.50^d
^{MS= Murashige and Skoog (1962) basal salt medium. The medium was solidified with agar−agar. Percentage value was calculated by means of total germination in total seeds per treatment; Significant difference was analyzed by Duncan Multiple Range Test on ANOVA; alpha =0.05. * = Strength of macro nutrients of MS medium}.

Climate data

As per data obtained from Mandore Agriculture University Jodhpur and IMD, earlier than 2015 there was less rainfall days in months of June.

Leaf Anatomy

Leaf transverse sections shows the presence of bundle sheath around vascular bundle filled with centripetal and centrifugal arranged chloroplast – anatomical evidence for C₃-C₄ intermediate photosynthesis (Fig. 6a, b). Green house grown leaf section shows lesser number of palisade cells and layer than natural field grown (Fig. 6c, d). In successive three years with fluctuation in average temperature and, humidity, pressure, and rainfall we observed the cells adjustments within leaf.

Thickness of whole leaf (distance from upper to lower epidermis in transverse section) and mesophyll part of leaf was higher in dry place (rainfed area) (Fig. 7.1 a). These leaves had more columnar palisade mesophyll cells which increase total leaf thickness. In year 2018, the average temperature and minimum humidity were recorded higher than 2016 and 2017 in month of July. The area of per cell was decreased and number of cells increased in the year 2018 (Fig. 7.1 b, 7.2 a, b). No significant variations found in ratio of area of total PMC and SMC in leaves of all three years (Fig. 8a). We observed that functional traits such as leaf thickness varies as fluctuates in temperature, humidity, and rainfall in previous months. In PCA analysis of traits we can see that primarily total number and area of PMC, SMC and Bundle sheath cells, leaf thickness PMC and SMC ratio can be taken to study the correlations with climate change (Fig. 8b). PCA showed first two components thickness and numbers of cells can be accounted for significant variations.

Seed throughput potentiality and Local dispersion patterns

A seed is an interconnection between old and new generation of plants and deliver genetic blueprint to produce offspring and maintain species (Nonogaki 2014). Therefore, seed plants evolved diverse strategies for proper temporal and spatial distribution of seeds to ensure favorable conditions for successful germination of seeds. Hence, as we observed that the plant Mollugo nudicaulis grows in the rainy season usually in organic rich areas such as river banks, fodder area, near semi-arid benches and well found as a weed within crops or marginal agricultural lands. The plant has abundant seed production potential which is inline that in desert, where annual and biennial plants escape forthcoming unfavorable climate by shortening the life cycle and generally producing plenty of seeds (Smith et al. 1997; Gutterman 2002).

Despite plentiful seed production, we found the dispersion variance of the plant in observation sites varied from natural to agriculture soil and the presence of comparatively more individuals in agriculture land. If dispersion variance value is more than one, as we observed in both types of habitats, it is considered as a clumped pattern of distribution (Krebs 1999). Moreover, this also indicates the presence of dormancy in the seeds which works here as an ecological adaptation to maintain the seed bank in soil with gradual opportunistic germination (Baskin et al. 1993; Gutterman 2000) and for proper distribution of seed germination, it is regulated by temporal and spatial manners (Nonogaki 2014).

Jurado and Flores (2005) stated that when a small number of seeds from randomly collected seeds readily germinate, such species may be classified as dormant. The different levels of physiological status of seeds among a population allow seeds to germinate in a limit to avoid competition with their siblings and not being extinct all together due to a disastrous condition. It is fact that in the ordinary course of events all barren lands tend to clothe itself with vegetation according to growth factors such as temperature, water, humidity, pH of medium, and availability of nutrients. Dormancy is and adoptative strategy to survive in harsh environment. Plant responds quickly to change in environment by changes in the distribution of plants, biomass allocation, change in flowering and seed set pattern (Sykes 2009). Present study can be served as a repository to predict future climate change effect on annuals.

The clump dispersion variance and high frequency of the plants in agricultural land area, suggests the need of specific requirements for the dispersion and germination of the seeds. The land under cultivation (tillage) is suitable for germination of dormant seeds of many species by providing more aeration, warmer and frequent temperature fluctuations in soil, scarification, and/or higher nitrate concentration and ambient CO₂ level to break dormancy (Mohler 2001; Benech-Arnold et al. 2000). Therefore, rapid, or delayed germination is a complex adaptive trait which is influenced by genetic and environmental factors (Koornneef et al. 2002). The presence of dormancy and gradual germination per year favor the persistence of seed bank in soil for long duration.

Release of germination

In this study, one years old collected and dry- stored at room temperature seeds, if assumed it require an after ripening period, didn’t show significant germination. Long dry storage periods however, decrease the germination in many annual species due to induction of secondary dormancy (Baskin and Baskin 2020). But in control conditions, no germination in the freshly collected seed may be associated with hard seed coat, or may be with seeds viability, therefore the seeds were experimented further. In seed dormancy types, the physical dormancy (PY) is linked with seed coat if seeds are impermeable to water intake (Baskin and Baskin 2004) and is commonly found in arid and semi-arid plant species (Baskin and Baskin 2014). However, in this case seed dormancy is not PY type.

For seed viability of M. nudicaulis, the tetrazolium test was not suitable due to small seed size. Therefore, another method (floating based) was opted in which soaking of seeds in warm water gives pre-indication of seeds viability as viable seeds are expected to absorb more water rapidly and sink down because they are metabolically active than dead seeds which float on the surface (Daneshvar et al. 2017). We observed that most of the black seeds of M. nudicaulis sank down in the floating method. Therefore, the black mature seeds were further used for release of germination. Additionally, it was supposed that if the seeds have low viability, then any treatments could not increase germination percentage, but after the immersion, the increase in weight and size of seeds suggested and supported that the seeds of M. nudicaulis has the dormancy and it is not PY linked because seeds were able to imbibe water. If seeds are pervious to water but not able to germinate, this indicates the presence of physiological dormancy which is the most common type of dormancy associated with physiological state of embryo and gas permeability through seed coat (Baskin and Baskin 1998; 2004).

Usually freshly harvested and water permeable seeds have primary dormancy induced on the mother plant during maturation infer related to ABA signaling (Finch-Savage and Leubner-MEtzer 2006). According to Baskin and Baskin (2004; 2014) the physiological dormancy is the primary type and has three levels: non-deep, intermediate, and deep. In general, physical (water impermeable seed coat, PY) and non-deep physiological dormancy (PD) can be broken by cold stratification or GA₃ treatment. In this study a significant germination enhancement (26–28%) was found after the cold stratification of the seeds. The cold stratification method has been considered as an effective method to enhance the germination for many species (Baskin and Baskin 1998; 2014; Kaye and Kuykendall 2001) such as for Lupinus campestris (Nava et al. 2010). Cold stratification or exposure of imbibed seeds to low temperature (typically at 4°C) is commonly used to break PD type dormancy and the reason behind this is activation of bioactive GA through modulating transcription mostly related to GA biosynthesis in imbibed seeds (Yamauchi et al. 2004).

Without cold stratification, in ex vivo conditions, the GA₃ treated and water hydrated seeds did not show any significant germination. But when GA₃ hormone was added to the culture medium, the in vitro seed germination increased as compared to ex vivo with or without GA₃ treated seeds; however, the prolonged period of culture was required. Similar with the cold treatment, the effect of GA₃ indicates the presence of non-deep PD in the seeds because GA₃ is a recognized plant growth regulator which activates the germination in mostly non-deep physiological dormant seeds as reported by Wang et al. (2008) and Baskin and Baskin (2014).

From the results it was inferred that the physiological dormancy in M. nudicaulis seeds might be associated with seed coat therefore, to analyses the seed germination percentage after removal of the seed coat, the chemical scarification of the seeds was taken in account. In chemical scarification the sulphuric acid treatment has been widely used and studied to break physiological dormancy (Aliero 2004; Emongor et al. 2004; Zida et al. 2005; Purohit et al. 2015). For M. nudicaulis the highest 38% germination was found after the acid pulse treatment. The immersion of dormant seeds in concentrated sulphuric acid degrades the seed coat and allows the embryo to receive stimulations for germination. In mechanism the sulphuric acid degrades the hard seed coat and allow imbibition of water into lumen of the macro sclereids cells which stimulate the release of simple sugar for protein synthesis and ultimately encouraging the germination in seed (Aliero 2004). Usually, dormancy type which is broken down by scarification after ripen and dry storage of seed is considered as non- deep PD (Finch-Savage and Leubner-MEtzer 2006).

In vitro Germination

Along with natural habitat study, to avail the plant resource freshly for experimental or economic purposes at any place, there are two popular ways – getting significant germination of seeds, or in vitro micropropagation of the plant. The in vitro propagation through callus regeneration has been reported for M. nudicaulis, earlier (Barupal et al. 2018). We observed here that under in vitro culture conditions, the incorporation of plant hormone to the medium didn’t have significate effect on seed germination compare to other methods. However, the incorporation of plant hormone is required for the growth of young seedlings and interactions between internal level of plant hormones and gene expression affect seed germination percentage; for instance, cytokinin is reported as seed germination enhancer by alleviation of non-biotic stresses such as salinity, drought, heavy metals, and oxidative stresses (Miransari and Smith 2014). In vitro addition of GA₃ to the medium enhanced the germination significantly compared to control and ex vitro applying of GA₃ to the seeds. It is observed that in antagonist function to ABA (abscisic acid), GA₃ induces germination through breaking of dormancy barrier by promoting both germination and cell-expansion- gene expression (Koornneef et al. 2002; Lee et al. 2002; Xu et al. 2020).

The seeds were also treated with hydrogen peroxide and hot water (60 ̶ 80°C for 5–30 min), but no significant increase was found in germination. The experiment's results suggest that in the seeds of M. nudicaulis cycling of dormancy might be there because in randomly collected seeds and treated under different methods of breaking dormancy, only 40% seeds germinated. Seed dormancy is an innate seed property that halt germination till all permissive range of environment to be met before the seed can germinate (Finch-Savage and Leubner-MEtzer 2006). Therefore, we can say that plants are highly responsive to their surroundings and accordingly they regulate the development of the next generations by perceiving and integrating the complex combination of signals to make developmental decisions (Xu et al. 2020). There might be, seeds have differential epigenetic variation due to immediate environment cues such as temperature and light which can trigger molecular and hormone signaling particularly cross talk between ABA and GAs and regulate the seed dormancy (Tonnacca and Botto 2021). Sometimes seeds are oriented to enter in the cycle of secondary dormancy in seeds due to lack of further feasibility for germination. The increase of germination in the seed through GA₃ treatment, scarification and stratification indicate the non-deep physiology.ical dormancy in the seeds of M. nudicaulis.

Germination profile of the plant Mollugo nudicaulis provide evidences in climate- plant interactions and adaptation, this will be useful in prediction of natural trends in biology of plants to alleviate climate change.

Leaf Anatomy Profile

Leaf traits provide insight into the adaptative evolutionary changes made to best fit in the surrounding conditions (Guerin et al. 2012; Liu et al. 2020). The plant M. nudicaulis has evolutionary significant functional adaptability in leaf as C₂ type photosynthetic system. Plant responds rapid climate change by change in range dispersion or in phenology. We know that photosynthesis in leaf is regulated by several factors that help the plant to adapt to fluctuating light conditions (Gotoh et al. 2018). We found variation in leaf cells content as per adjustment with environmental changes in temperature and relative humidity. The variation in palisade cells and its compactness was also found in leaves of aeroponic grown plants compared to natural grown of M. nudicaulis (Barupal et al. 2018). The cells arrangement and their spatial pattern, composition and structure of leaf internal tissues perform different functions such as light absorption for photosynthesis, compartmentalization to enhance CO2 capturing chances, keep water balance and protection from different biotic and abiotic stresses (Liu et al. 2020; Ni et al. 2022). It is observed that Leaves of sun-light-grown plants are thicker and have more mesophyll cells especially palisade compared to shade-grown plants to coordinate efficient leaf photosynthesis (Gotoh et al. 2018).

Mollugo nudicaulis a C₂ plant has significant role in study of the evolution of C₄ plants with expression level of physiological and anatomical adaptations. Study on leaf anatomy will help in prediction of climate change effect on C₃, C₄, and intermediate plants in tropical areas. We observed that the seeds of Mollugo nudicaulis has dormancy cycling which further activates secondary dormancy in seeds. The applying of sulphuric acid pulse treatment and the cold stratification increased the germination percentage up to 40% which also indicates non deep PD in the seeds. Quantitative leaf anatomy data suggests that elevated temperature prompt adding of more cells to increase leaf thickness with increase total area of mesophyll part. This study can be significant for C₃-C₄ intermediate research.

Acknowledgment

Authors are thankful to Department of Botany UGC-CAS, Jai Narain Vyas University, Jodhpur for providing research facilities.

Ethical Approval – Not applicable

Conflict of interest statement

There is no conflict of interest among the authors.

Authors’ Contributions

All authors contributed to the study design, data analysis and manuscript preparation. MB performed field and bench work. All authors approved the final manuscript.

Funding

No funding agency is associated with this study.

Availability of data and materials

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A record of seed germination, distribution pattern and effect of premonsoon season variability on quantitative leaf anatomy of Mollugo nudicaulis Lam. a C2 species from tropical Desert Climate

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Germination Percentage

Breaking of seed dormancy

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