Plant material and experimental site

The Prunus persica cultivars used and dates of anthesis were as follows: Puebla (January 12th, early), CP Precoz (February 20th, intermediate) and Zacatecas (March 17th, late) during the winter of 2011-2012. The five-year old trees were grafted onto creole peach trees. The orchard is located in the Campo Experimental Valle de México (CEVAMEX), el Horno, Chapingo, Texcoco, Estado de México, (19° 29’ 17” N y 98° 53’ 41” W, altitude 2280 m), with Mollic fluvisol soil (^{Govaerts et al., 2008}) a temperate subhumid climate C (W₀), temperature and annual rainfall of 15.5 °C and 644 mm (^{García, 1987}).). The trees were structured according to the Modified Tatura method and the orchard was planted in the milpa intercropped with fruit trees (MIFT) system (^{Santiago-Mejía et al., 2015}). Cultural practices were: fertilization 60-1560 (g of N-P₂O₅-K₂O) and 3 kg of poultry manure per tree in April (1/3; 1-1-0 de N, P, K, poultry manure) and August (2/3; 0-0-1 de N, P, K, poultry manure); irrigation by gravity without limits of water.

Sampling and experimental design

Sampling was carried out completely at random and two central flower buds were taken, with five mixed one-year-old twigs, from the middle section of the canopies of 10 trees to be planted. In order to cover the phenology of the three cultivars, three sample collections were carried out: October 29th; November 18th; December 08th and 28th of 2011 and January 17th and 30th; February 06th, 23rd and 26th, and March 01st, 17th y 21st of 2012. The anatomic characteristics reviewed were qualitative, and therefore a statistical analysis was not carried out. The results are representative of all the buds analyzed in each phenological stage.

Phenological stages and anatomical stages

The flower buds were classified in the PS’s according to Baggiolini’s nomenclature (1952): A: “dormant bud”; B: “swollen bud”; C: “visible calyx”; D: “pink tip”; and E: “visible stamen.” For each PS, anatomical changes were described, which were grouped in “anatomical stages,” according to the nomenclature suggested by ^{Reinoso et al. (2002)}. Seven anatomical stages (17) were described in PS “A”, two anatomical stages (8 and 9) in PS “B”, one anatomical stage (10) in PS “C”, and in PS “D” and ”F” only morphological changes are described, since they refer to the emergence of the anthers of the floral bud and the anthesis or floral aperture. The morphological characteristics of these phenological stages are illustrated in Figure 1.

Figure 1  Phenological stages of the flower bud studied. A) dormant bud; B) swollen bud; C) visible calyx; D) Pink tip; E) visible stamens; F) anthesis. The image corresponds to cv. Zacatecas. cx: calyx; pe: petals; es: stamens. Bar = 1 cm. 

Microtechnic for buds anatomy

The external morphology of the buds was documented in each phenological stage using a Moticam 2000^® camera (Motic, China), attached to a stereoscopic microscope (Nikon SMZ800^®, Japan). The buds were fixed in FAA (10 % formalin, 50 % ethanol, 5 % glacial acetic acid, 35 % deionized water) for 48 h and processes for their inclusion in Paraplast. Lengthwise cuts (rotary microtome, American Optical, USA) were dyed using Safranin O and Fast Green FCF. Safranin O is a cationic (basic) colorant that dyes lignified walls and nucleic acids red, which is why it was chosen to identify the mature xylem vessels. Fast Green FCF is an anionic (acid) green dye that stains cellulose, cytoplasm, sieve tube elements, parenchyma and meristematic cells, that in combination with Safranin provide a metachromatic stain (^{Baker, 1966}). The anatomy of the buds was studied under a Axioskop 2 plus^® microscope (Carl Zeiss, Germany) and an Axiocam 5 MRc camera (Carl Zeiss, Germany).

Vascularization and distribution of starch grains

The appearance of xylem vessels in the receptacle, calyx, corolla, anthers and gynoecium was registered using a Axioskop 2 plus^® microscope (Carl Zeiss; Germany) in a bright field. The starch grains were viewed with epifluorescence (excitation BP 450-490 nm and emission LP 515 nm), and images were captured using the Axiocam 5 MRc camera (Carl Zeiss, Germany).

Phenology of flowering

The first cultivar to leave the stage of “dormancy” (A) was Puebla (late December), followed by CP Precoz (late January), and finally, Zacatecas (late February). The development of the bud was fast in cv. Puebla, and by January, it was showing visible sepals (PS “C”) (Figure 2). The development of the bud for cv. CP Precoz was slower, since the calyx became visible (PS “D”) after it did in cv. Puebla. The slowest cultivar was Zacatecas; the bud remained dormant (PS “A”) until February, and then it developed quickly, displaying the pink tip (PS“D”) in March.

Figure 2 Flowering phenology of the cultivars Puebla, CP Precoz and Zacatecas from dormancy (A) up to 100 % anthesis (F). Arrows indicate sampling dates. Right Y-axis: phenological stages. Left Y axis: stages of anatomical development. 

Vascularization of the flower bud

The differentiation of the xylem in the flower bud was acropetal (the maturation of vessel elements began at the base and moved towards the bud) in all three cultivars. During the dormant stage (A1 to A5), the bud of the three cultivars is not vascularized. The cv. Puebla was the first (December 28th) to present xylem in the receptacle, calyx and corolla (A6 and A7) (Figures 2 and 3). In contrast, the lack of vascularization was prolonged in CP Precoz (January 30th) and in Zacatecas (February 23rd) (Figures 2 and 3A). Bud vascularization begun after 2 months in cv. Puebla, after 3 months in cv. CP Precoz and after 4 months in cv. Zacatecas (Figures 2 and 3B). Only vascular procambial cells were observed in the receptacle (Figure 4A). In stages A3, A 4 and A5, the xylem was observed in the basal scales (Figure 3A). The vascularization of anthers and ovary took place first in cv. Puebla (swollen bud B8 and B9) on January 17th, and the latest (March 14th) was Zacatecas (Figures 2 and 3B). The vascularization of the style presented the greatest variation between cultivars: in cv. Puebla, it occurred on day 15 (January 17^th to 30th), in CP Precoz, on day 27 (January 30^th to February 26th), and in cv. Zacatecas, on day 4 (from March 17^th to 21st).

Figure 3 Distribution and abundance of starch grains and xylem in the vegetative and reproductive whorls of the flower bud in the phenological stages: dormant bud A); swollen bud B); visible calyx C); pink tip D); visible calyx E). Red dots indicate starch grains and blue dotted lines indicate xylem vessels. 

Figure 4 Anatomy of the vascularization and presence of starch grains in peach flower buds. A) Procambium in the receptacle, absence of xylem at dormancy (stages 3 to 5, cv. Zacatecas); B) starch grains at the receptacle of dormant bud (stages 6 to 7, cv. Puebla); C) xylem vessels in the vegetative whorls of swollen bud (stage 8, cv CP Precoz); D) xylem and starch in the base of anthers of swollen bud (stage 8, CP Precoz); E) xylem on the carpel wall, visible calyx bud (stage 10, cv. Puebla); F) vessels and starch grains in the anther (stage 10, cv. Puebla). A, C and E) bright field; B, D and F) epifluorescence. Arrows indicate vascular bundles (ga: starch grains; re: receptacle; pc: procambium; ca: carpel). Bars: A and C = 20 µm; B, D and F = 100 µm; E = 500 µm. 

Vascularization was reported at the end of the stage of dormancy for P. armeniaca, P. avium, P. ceratus, P. saliciana and P. sargentii (^{Ashworth, 1984}; ^{Reinoso et al., 2002}). However, our study is the first to describe the advancement of vascularization in parts of the bud, as well as the comparison between cultivars with different dates of flowering or precociousness.

The vascularization of the vegetative whorls was related to an increase of starch in the receptacle (Figure 3) in all three cultivars. The starch grains were visible with epifluorescence (Figure 4B) and the elements of the xylem vessel presented thickening and scalariform perforation plates (Figure 4C). According to ^{Ashworth (1984)}, in apricots, the vascular connection between the twig and all the bud occurs at the end of the dormant stage. In contrast, in the Mexican cv. of peach trees, this event was gradual, as shown in Figure 3: the xylem appears first in the receptacle (A-5), then in the calyx and the corolla (A7), and later the xylem extends to the anthers in the swollen bud (B-8), it continued towards the ovary (B-9) and reached the style and the stigma up to the visible calyx stage (C-10).

^{Reinoso et al. (2002)} only mentioned the presence of vascular tissue in all the whorls at the end of the dormant stage without including the anatomical and phenological details. Figure 4D shows vascular bundles and starch grains in the base of anthers of swollen buds (B-8). The ovary wall was vascularized (B-9) towards the end of the swollen bud (Figure 3). These events took place first (early January) in cv. Puebla, then in CP Precoz (February), and finally, in cv. Zacatecas (early March). In contrast, in apricot this took place before, at the end of the stage of dormancy (^{Julian et al., 2011}). In the visible calyx stage (C-10) the xylem bundles moved to the style until they reached the stigma (Figure 3). The filaments of the anthers presented a central amphicribral bundle. The vascular bundles were collateral and abundant in the carpel walls (Figure 4 E). The complete vascularization of the pistil (C10) first took place in cv. Puebla (mid-January), then in cv. CP Precoz (early February), and finally, in Zacatecas (mid-March).

The latest event was the vascularization of the ovule, which explains why the primordium of the seed is the structure that requires protection from low temperatures, for reproductive reasons. During the year of the experiment, frosts took place when the flower buds were dormant (^{Santiago-Mejía et al., 2015}) and our study showed the absence of vascular connections in this stage.

Distribution of starch grains

The presence and distribution of the starch grains was related to the differentiation of the xylem and development of the floral whorls. In cv. Puebla, the starch grains first appeared at the base of the receptacle (December 08th) and then they increased in number when the anthers began developing (Figure 4B). In cv. CP Precoz, this event took place on February 23rd, and later the starch appeared on the base of the anthers, while the pollen grains formed the generative and vegetative cell (Figure 5G) (Table 1). In swollen bud (B-8 and B-9), starch grains appears in the style and the anthers; which coincided with the differentiation of the xylem in the ovary, the beginning of the nucellus and the formation of the pollen wall (Table 1, Figure 5I). The xylem differentiation at the stamens and during the formation of the polen grains in cv. Puebla and CP Precoz was similar to observations in P. armeniaca, although the presence of starch in the intermediate cells occurred before the formation of microspores (^{Julian et al., 2011}).

Figure 5 Development of the anther in three Prunus persica cultivars A) Tetrasporangiated anther with sporogenous cells (2n) cv. Puebla; B) pollen mother cells (PMC) and differentiated tapetum cv. CP Precoz; C) meiosis of the pollen mother cells (1n) Zacatecas; D) formation of tetrads with a tetrahedral arrangement cv. Puebla; E) release of microspores (1n) cv. CP Precoz; F) defect in the release of microsporas and abortion cv. Zacatecas; G) elongation of the endothecium and first mitosis of the pollen grain cv. Puebla; H) abortion of pollen grains cv. Zacatecas; I) thickening of the endothecium and the pollen wall cv. Puebla; J) collapsing of pollen grains, lack of thickening of the pollen wall cv. Zacatecas. ce: sporogenous cells; e: endothecium; cmp: pollen mother cells; tt: tetrades. Bars: A, C-J = 20 µm, B = 100µm. 

In the visible calyx stage (C-10), of the three cultivars, there was a reduction of starch grains in the base of the bud and an increase in the pistil, in coordination with the differentiation of the xylem in the style. However, in pink tip (D), the starch disappeared from the anthers, although it persisted in the ovary (Figure 4). This pattern was similar to the one observed in olive inflorescences (^{De la Rosa and Rallo, 2000}). The changes in the partition of starch during the development of the floral buds have been shown in Prunus persica (Bonhomme et al., 2005; ^{González-Rossia et al., 2008}), Rubus idaeus (^{Palonen, 1999}), Pyrus pyrifolia (^{Ito et al., 2002}), Populus x canadensis (^{Elle and Sauter, 2000}) and Olea europea (^{De la Rosa and Rallo, 2000}), although our report is the first on the histological location of starch in the flower bud related to the morphogenesis and differentiation of the xylem in peach.

Anatomic characterization with emphasis on anther development

The anatomical changes observed in the bud in the phonological stage of dormancy (A) were grouped into seven anatomical stages (1-7), which are shown in Table 1. These events took place with differences in time between cultivars (Figure 2). The anatomical changes observed during the first four stages of dormancy (A-1 to A-4), occurred slowly in the flower bud of the cv Zacatecas (4 months), less slowly in cv. CP Precoz (3 months), and fast in cv. Puebla in 20 d (Figure 2 and Table 1).

In the three cultivars, during the “dormancy” (A), the early development of the pollen was normal and similar; the microsporangia presented sporogenous cells (2n) (Table 1 and Figure 5A); in stage A-2 the pollen mother cells (2n) were differentiated (Table 1); in stages A-3 and A-4 the tapetum was prominent (Table 1 and Figure 5C); in stage A-6 the pollen mother cells (PMC) were observed in meiosis and the microspores (1n) were organized in a tetrahedral arrangement (Figure 5D). In swollen bud (B8 and B-9), of the cv Puebla and CP Precoz, the microspores were separated into young pollen grains (1n) (Table 1 and Figure 5E), although in cv. Zacatecas, the microspores did not separate, and some were obliterated and disintegrated (Figure 5F). This meant a defect in the formation of the pollen grain in cv. Zacatecas. Later, cv Puebla and CP Precoz continued developing normally, although with differences in time; cv Puebla was the first to present the mitosis of the young pollen grain and the thickening of the pollen wall (Figure 5G), and cv. Zacatecas displayed obliterated pollen grains with a deficient thickening of the exine (Figure 5H).

In the following stage, visible calyx (10-C), the anticlinal walls of the endothecium were normally lignified in the three cultivars, although the maturation of the pollen grain only appeared in cv. Puebla and CP Precoz (Figure 5I).

In the pink tip stage (D), several microspores stopped developing and aborted in cv. Zacatecas (Figure 5H). The cv. Zacatecas presented the majority of abortive pollen grains with defects in the exine (Figure 5J).

Pollen abnormalities and abortions have been observed when the requirements of chilling have not been met (Bonhomme et al., 2005; ^{Nava et al., 2009}; ^{Reig et al., 2013}). In the context of global warming, temperate fruit trees of will face reduction in chilling hours, problems during “dormancy” related to abortion and reduction in the flow of carbohydrates towards the buds (^{Campoy et al., 2011}; ^{Marafon et al., 2011}). According to the method by ^{Richardson et al. (1974)}, cv. Zacatecas requires 550 Chill Units (CU), CP Precoz requires 400 CU, and Puebla, 350 CU (^{Santiago-Mejía et al., 2015}). The average chilling accumulated between November 17^th and March 31^st in the area under study was below 550 CU (^{Santiago-Mejía et al., 2015}). In Natural conditions cv. Zacatecas is at higher latitude and accumulate more chilling hours (^{Rumayor et al., 2009}). The chilling deficiency causes anomalies in the male gametophyte, such as; the abortion of pollen, reduction in the polen production and germination problems (^{Nava et al., 2009}), as well as an abnormal floral opening (Bonhomme et al., 2005). This explains the anomalies observed in the development of pollen in Zacatecas.