Morphological and Histological Analysis of Somatic Embryogenesis in Haworthia Cooperivar

The fleshy leaves were used as materials for induction and proliferation somatic embryonic callus and plant regeneration. The morphological and histological characteristics of granular embryonic callus were studied by light microscopy using Spuur resin section. The results showed that the yellowish and granular embryonic callus was induced on modified MS medium were supplemented with 1.5 mg/L 6-BA and 0.5 mg/L IAA, the highest induction ratio was 66.7%. The highest proliferation net weight (14.73 g) was presented at modified MS medium were added 1.5 mg/L 6-BA an d 0.4 mg/L IAA. Milky white globular or heat shape somatic embryo was differentiated from calls that originate d from outer or inner-epidermis, the embryonic callus was composed of cells with big nuclear, vigorous division and containing starch granules. The inner embryonic cell clusters showed spiral or oval shapes. These structures were further developed to somatic embryos, which were transformed to complete clumps of small plants from the surface of callus. Healthy developed seedlings rooted on MMS+ 0.1mg/L IAA. This research provides technical support for the rapid propagation and molecular breeding of Haworthia cooperivar.


Effects of different cytokinin treatments on somatic embryonic callus induction of Haworthia cooperivar
The explants were inoculated on MMS medium with different combinations of plant growth regulators and observed after 56~70 d. The results showed that no callus differentiation was observed on the medium without plant growth regulators. Different colors and shapes of calli were produced on the medium supplemented with different kinds of cytokinins. When the explants were cultured on the medium supplemented with 1.5 mg/L 6-BA and 0.5 mg/L IAA for 15 d, the base of leaf explants began to expand, and yellow calli appeared at the swelling part. After about 42 d, they gradually changed into light yellow granular calli with moist surface, soft and fragile texture, and differentiated small buds could be observed on some calli. After 56 d, smooth milky white granular structure appeared on the light yellow granular callus. Under the microscope, globular shape somatic embryo ( Figure 1A) and heat shape somatic embryo ( Figure 1B) were observed, and the callus induction rate was 66.67% (Table 1).   Table 2. When the concentration of 6-BA was 0.5~1.5 mg/L, the proliferation was not obvious at 7~14 d, it was vigorous after 21 d, and it was faster at 56 d, with the net proliferation weight of 9.36 g, 11.14 g and 14.73 g, respectively. The difference between treatments was significant. The callus were yellowish-white friable with granular structures. And small buds could be observed on the surface of callus ( Figure 1C). The green buds and callus were not tightly combined, and the buds would fall off from the callus with a slight touch of tweezers. When the concentration of 6-BA was 2.0~2.5 mg/L, the proliferation speed was obvious at 14 d, and it was the most at 42 d. The net weight of proliferation was 6.55 g, and 6.72 g, respectively. After that, the callus grew slowly and stopped growing gradually. And the difference between treatments was not significant. The callus gradually showed a hollow bubble structure with yellowish-white soft shape, and there was no green bud. After 42 d, the texture of callus was thick and hollow gradually. After 56~60 d, the callus almost stopped growing and began to brown. The results showed that the low concentration of 6-BA could promote the proliferation of embryonic callus and maintain the characteristics of embryonic callus. However, embryonic callus is easy to lose embryogenesis under the stimulation of higher concentration of 6-BA, which was not conducive to the proliferation of callus.
1.2.2 Effects of different concentrations of IAA on the embryonic callus proliferation Different concentrations of IAA had little effect on the color, morphology, and texture of embryonic callus, but had some different effects on callus proliferation. When the concentration of IAA was 0.1~0.2 mg/L, the proliferation of callus was more vigorous, and the proliferation was the most after 56 d, which was 13.37 g and 14.22 g, respectively, and then decreased slowly. The callus was uniform in structure and color, and still maintained the characteristics of yellowish, loose texture, and relatively hard granular embryonic callus. Green bud spots could be observed on the surface of callus, most of which were callus proliferation. When IAA concentration was 0.3~0.5mg /L, the proliferation of callus was relatively slow, and the proliferation was 11.97 g, 10.96 g and 11.02 g at 56 d, respectively, with no significant difference between treatments. Although the embryonic callus still maintained the characteristics of yellowish, loose texture and friable callus with granular structures, they tended to mature and become smaller plants. The results showed that IAA concentration had a certain effect on the proliferation of embryonic callus. Low concentration of IAA could promote the proliferation of callus. Although the higher concentration of IAA could increase the proliferation of early callus, the proliferation of callus tended to differentiate into adventitious buds. Therefore, IAA concentration of 0.1~0.2 mg/L was suitable for the embryonic callus proliferation of Haworthia cooperivar (Table 3).

Plant regeneration of somatic embryo
When the callus were transferred to MMS culture medium supplemented with 0.1~0.2 mg/L IAA, the plant regeneration of somatic embryo was more obvious. When the callus was cultured for 21~28 d, complete clumps of small plants were accumulated on the surface of callus ( Figure 1D; Figure 1E). From 42 to 56 d after culture, the roots of small plants were strong and abundant, and their growth was relatively vigorous. On average, 21~31 small plants could be isolated from each callus, and most of the embryonic callus were differentiated into complete small plants (Table 4). When embryonic callus were cultured on the medium supplemented with 0.3~0.4 mg/L IAA for 42~56 d, the number of small plant on the callus was less, and the young roots were thin and short. Some seedlings were rootless seedlings with incomplete root development, and more calluses had not differentiated into small plants. On average, 2~6 plantlets could be isolated from each callus. When the callus was cultured on MMS medium supplemented with 0.5 mg/L IAA for 10 d, the first complete small plant was observed, and it was not appeared. After 35 d, green buds were differentiated from the callus, and cultured for 42~56 d, it was found that most of the small plants differentiated from the callus were rootless. The seedlings were thick and few in number, and the combination with the callus were loose.

Transplantation of in vitro-cultured plant
The plantlets were taken out from the test tube, washed out the medium with tap water, and dried in the dark for 1~2 d. After that, it was transplanted to the substrate and sprayed with water to keep the substrate moist. In the early stage, it is necessary to avoid direct sunlight, gradually strengthen the light intensity, and maintain ventilation. After 2 weeks, the leaves became strong and green, and the survival rate was as high as 100% ( Figure  1E; Figure 1F).

Results and analysis of tissue section
The genesis of somatic embryos in Haworthia cooperivar is an indirect way through which the explants' leaves dedifferentiate to form embryonic callus on induction medium and the embryonic callus dedifferentiate to form somatic embryos. The results of longitudinal section of granular structure showed that the granular callus was composed of cells with obvious nucleus, large nucleus and nucleolus, dense cytoplasm and starch granules. The outer layer of this granular structure is composed of closely arranged and regular epidermoid structure (Figure 2A). Developed from outer epidermis are clusters of embryonic cells consisting of 7~8 cells ( Figure 2B). A somatic embryonic center derived from a cell can be observed below it ( Figure 2C). These irregular small cell clusters with vigorous division were formed by single cells dividing in the flat and vertical circles, with no obvious Plant Gene and Trait 2020, Vol.11, No.6, 1-9 http://genbreedpublisher.com/index.php/pgt connection area with other cells, showing irregular arrangement and relatively loose distribution, and no obvious vascular bundle structure was found. The inside of the granular structure is composed of a number of cells surrounded by the anticlinal division like epidermis. Such cells have clear boundaries, cell size is basically the same, nucleoli are obvious, and cell division is vigorous. As can be seen from the growth outline of the cell mass, its growth direction is the rotation growth from inside to outside, and there are few connections between the cells mass. The vertical cell division forms epidermis, while the horizontal cell division increases the number of cells, which is a vigorous meristematic cell. And metaphase chromosomes can be observed ( Figure 2C; Figure 2D; Figure 2E). Vascular bundle and duct system were not observed in the sections, and the vertically dividing pre-procambial cells, which were significantly smaller than the surrounding cells, could be observed in the areas with relatively vigorous division ( Figure 2F).
The longitudinal and transverse sections of globular structure ( Figure 2G; Figure  The longitudinal section of some granular structures showed that the single granular structure was separated from the surrounding tissues, and the outer surface of the granular structure was epidermis with similar cell size, regular shape, and tight arrangement. The cells in the medial epidermis were arranged irregularly with loose distribution and large intercellular space. They were vacuolated parenchyma cells, but no obvious vascular bundle structure was observed ( Figure 2G). Pre-procambial cells with obvious nucleus and dense cytoplasm were observed to divide along the vertical or horizontal direction in the middle ( Figure 2H; Figure 2I).

Discussion
Somatic embryogenesis is one of the effective ways for plant regeneration (Zhai et al., 2011). However, many factors affect the occurrence of somatic embryogenesis. Plant growth regulators are involved in cell cycle regulation and cell division, and are essential factors for somatic embryo induction (Francis and Sorrell, 2001;Fehér et al., 2003;Gaj, 2004). It has been reported that 2,4-D alone or in combination with other plant growth regulators is most widely used in somatic embryo induction (Gumerova et al., 2003;Umehara and Kamada, 2005), and NAA were also frequently used, while IAA was rarely used (Hou et al., 2015). Yan et al. (2017) reported that the embryonic callus induction and embryonic changes in Haworthia cooperivar depend on the ratio of BA/2,4-D.
In this experiment, the leaves in Haworthia cooperivar were used as explants to induce embryonic callus successfully by using the improved MS medium supplemented with 6-BA combination IAA, and the embryonic callus could be maintained for more than 3 years. The results showed that the concentration of 6-BA had a great influence on the induction, proliferation and embryonic maintenance of embryonic callus. When the concentration of 6-BA was 1~1.5 mg/L, the effects of the proliferation and embryonic maintenance of embryonic callus were relatively good. Although the amount of proliferation of callus was relatively large when the concentration of 6-BA was increased, the embryonic gradually lost during the culture process. The effect of IAA on the proliferation of embryonic callus showed that the concentration of 0.1~0.2 mg/L was suitable for the proliferation and maintenance of embryonic callus. When the IAA concentration was increased, somatic embryo maturation and small plant conversion were more vigorous.
Somatic embryogenesis mainly includes four processes: somatic embryo induction, embryonic maintenance and culture, embryo differentiation and somatic embryo regeneration (Ibaraki and Kurata, 2001). The embryonic callus has the morphological characteristics of dense, whitish granular, slow growth or loose arrangement and fragile (Sun et al., 2003;Yang et al., 2003). In the experiment, the leaves in Haworthia cooperivar showed similar changes after two months of culture on the medium combined with 6-BA and IAA, forming granular callus with slow growth, yellowish and loose arrangement. Granular callus were further developed into typical globular shape and heat shape structures, which were then transformed into green mature embryos. Somatic embryogenesis was not observed in the greenish dense, hard, or ivory soft water-impregnated callus. This is consistent with the results reported by Xiao et al. (2016) and Cao et al. (2016).
The results of tissue sections of embryonic callus in Haworthia cooperivar showed that after the formation of embryonic callus, not only the embryonic cell clusters with deep staining, large nucleus, dense cytoplasm and close arrangement occurred on the surface of the callus, but also the embryonic cell clusters composed of small cells with vigorous division and close arrangement occurred in the interior. The cell clusters grew in a round or spiral shape, which then developed into a somatic embryo. In addition, pre-procambial cells consisting of vertically dividing small cells were observed inside the vigorously dividing callus, which can act as pluripotent or totipotent stem cell-like cells to cause organ differentiation or somatic embryogenesis. Such pre-procambial cells readily respond to the auxin/cytokinin ratio, leading to organ differentiation or somatic embryonic development. This differentiation is usually determined by the direction and location of pre-procambial cells division (Risopatron et al., 2010). The cells from inner epidermis of embryonic callus in Haworthia cooperivar were irregularly arranged, loosely distributed, and had large intercellular spaces. They were elongated or large, highly vacuolated large parenchymal cell clusters without obvious nuclear structure, which was similar to the results reported by Ren et al. (2019).
In addition, the accumulation of starch granules during somatic embryogenesis is related to the differentiation of somatic embryos. And the dynamic changes of starch granule accumulation during somatic embryogenesis have been reported in studies on somatic embryo induction in coconut (Cocos nucifera Lz) trees (Verdeil et al., 2001) and palm (Elaeis guineensis) trees (Schwendiman et al., 1988;Kanchanapoom and Domyoas, 1999). And it has been pointed out that the accumulation of starch granules is related to cell division, mainly concentrated near the center of vigorous cell division to provide energy for cell growth, and its accumulation is a sign of somatic embryo differentiation and development (Ji et al., 2019). In the experiment, a wide distribution of starch granules could be observed in the cell mass of embryonic callus with vigorous division, but not in the non-embryonic callus. The results indicated that the accumulation of starch granules in embryonic callus might be an important factor to ensure the success of somatic embryogenesis.
In this experiment, we successfully induced somatic embryos with the explant leaves in Haworthia cooperivar, constructed the plant regeneration system, and studied the morphological and histological characteristics of somatic embryogenesis. The experimental results provided a theoretical basis for the rapid propagation, germplasm preservation, breeding, and mass production of Haworthia cooperivar.

Haworthia cooperivar materials
In 2017, Linyi Xianzhu Biotechnology Co. Ltd provided potted Haworthia cooperivar. Selected healthy and complete leaves and gently rub surface stains with washing powder water. Rinsed under running water for 15 min after washing and set aside. On the ultra-clean bench, sterilized the surface by adding 1 drop (about 50 μL) Tween-20 0.15% corrosive sublimate for 5 min, then rinsed repeatedly with sterile water for 5~6 times, placed in the sterilized stainless steel plate, dried the surface water with sterile filter paper, and set aside.
The medium used for the experiment was modified MS (Murashige and Skoog, 1962). The modified MS medium (Mo dified MS, MMS) was to adjust the concentrations of thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid and calcium pantothenate under the condition of MS basic culture, and the adjusted concentrations were 10.0 mg/L, 1.0 mg/L, 1.0 mg/L and 1.0 mg/L, respectively. 30 g/L sucrose and 8.0 g/L agar powder were added into the culture medium, pH 5.6~5.8, at 104 kPa, 121℃ for 20 min autoclavous steam sterilization. Callus induction, somatic embryo induction and maturation were carried out at (25±2)℃ temperature, 30~50 μmol·m -2 ·s -1 of light intensity, 16 h of light and 8 h of darkness per day.

Induction of embryonic callus
The sterilized leaves were cut lengthwise with an anatomic knife and inoculated with MMS+1.5 mg/L 6-BA +0.5 mg/L IAA, MMS+1.5 mg/L ZT+0.5 mg/L IAA, MMS+1.5 mg/L KT+0.5 mg/L IAA medium. MMS was used as the control group to screen the optimal combination of plant growth regulators for embryonic callus induction. 10 explants were inoculated for each treatment with 3 replicates. Observed the growth of callus, and counted the induction rate and analyzed after 60 d.

Proliferation of embryonic callus
About 1.5 g of yellowish, granular, and loose embryonic callus were divided into 8~10 pieces and transferred to the culture medium for culture. Embryonic callus proliferation medium was MMS+ (0.5, 1.0, 1.5 2.0, 2.5) mg/L 6-BA+0.1 mg/L IAA, MMS+1.5 mg/L 6-BA+(0.1, 0.2, 0.3, 0.4, 0.5) mg/L IAA. Callus were separated from the medium on ultra-clean bench, and then transferred to a sterile petri dish with the weight removed for weighing, and the data were recorded before the callus was re-inoculated onto the original medium. Every 14 d, fresh weight was weighed, and the growth state of callus was observed. Until the growth of callus entered the decline period (about more than two months), each treatment was repeated for 3 times.

Plant regeneration
Embryonic callus with good growth status were selected and transferred to MMS+(0.1, 0.2, 0.3, 0.4, 0.5) mg/L IAA+30 g/L sucrose +8.0 g/L agar. After 56 d of culture, the maturity rate and the average number of seedlings were counted to screen the best medium for seedlings. All treatments were repeated for 3 times.

Domestication and transplantation
The glass culture bottle was moved to a cool outdoor place for about 1 week. Then opened the cap of the culture bottle and place it for 3~7 days until the tissue culture seedlings become strong. Then washed the basal medium with water and dried the seedlings in the shade for 3~7 days. Then the seedlings were transplanted to the substrate, which was a mixture of peat soil, river sand, and perlite, with a volume ratio of 5:3:2.

Observation of organization structure
Fresh globular shape callus particles were selected and fixed, embedded, trimmed and sliced according to the method of Lian et al. (2013). The embedded tissue was cut into semi-ultrathin slices of 1 μm by a nanometer slicer (Leica, Germany). After staining, the sections were observed under Olympus optical microscope (Olympus BX51, Japan), and photographed, recorded.

Statistical analysis of data
The following formula is used for data statistics. Callus induction ratio= (Number of explants with callus/Total number of inoculated explants)×100% Net proliferation of embryonic callus (Fresh weight) = Quality of callus after proliferation-Quality of inoculated callus Duncan's multiple analysis method was used for significance difference analysis, and the minimum significance difference was shown at the level of 5%.