Effects of different sterilization treatments on seed germination of New Guinea Impatiens
By controlling 70% alcohol sterilization for 15 seconds, the study demonstrates that different 2% NaClO disinfection length of time have significant impacts on both germination rate and contamination rate, these results also further highlight a decreasing tendency in contamination rate with an additional period of disinfection time, while seed germination rate exhibits an upward trend and followed by a downward trend at some point of time. Moreover, various plant seeds tend to have different tolerances to different disinfection reagents, this is consistent with the statement proposed by Jones, who suggesting that the seed germination rate would decrease significantly despite an improvement in sterilization effect given an additional period of disinfection time (Jones, L. H, 1983).Also, under 2% NaClO treatment time, the seed germination rate of 'Violet' reaches to the maximum value of 88.33% in 12 minutes (S5), while the seed germination rate of 'Scarlet Bronze Leaf' reaches to the maximum value of 86.67% in 10 minutes (S4), meanwhile, the seed contamination rate reaches to the minimum value in 15 minutes (S6). Under the premise of 2% NaClO sterilization treatment for 10 minutes, both germination rate increases first and then decrease as 70% alcohol sterilization time goes by, in contrast, the contamination rate showed a downward trend over time, which indicates that 70% alcohol has a good effect on seed sterilization, however, takes disadvantage in seed germination. Therefore, regard to this finding, the treatment time of alcohol should be controlled. The similar results obtained from Ye Wei yan further imply a certain degree of agreement in this experiment, where Ye Wei yan conclude that the contamination rate of grapefruit seeds would not decrease significantly even if that an extra sterilization time using 75% alcohol was provided(Ye et al.2015), however, with a strikingly reduce in seed germination rate. Hence, the best disinfection scheme in this experiment is as follows: usage of 70% alcohol 15s and 12min disinfection time of 2%NaClO for the seeds of New Guinea Impatiens' Violet', the contamination rate of which is 10% and a maximum 88.33% of germination rate. For the seeds of New Guinea Impatiens' Scarlet Bronze Leaf', 70% alcohol 15s and 10min disinfection time of 2%NaClO are suggested, the pollution rate of which is 10% and the germination rate is 86.67%. They both present significant differences in this experiment. However, results here are inconsistent with Yun gui Guo's conclusion, which concerns that 70% alcohol and 15-20min of 2%NaClO as the best disinfection scheme (Guo et al.2012), the reasons for the differences in outcomes may be related to the variety, seed quality and pretreatment time of Impatiens balsamina.
Effects of different concentrations of PGR on tufted bud induction of 'Violet'' and 'Scarlet Bronze Leaf'' of New Guinea Impatiens
6-BA and TDZ are two types of cytokinins which are commonly used in the processes of tissue culture and plant regeneration of Impatiens. They can promote cell division, differentiation, elongation or regulation of endogenous hormones and metabolic synthesis of CTK by removing apical dominance(Wang Dong mei, Huang Shang zhi, 1996). In this way,Both of them can contribute to the induction of tufted buds (Miloševic, et al, 2011). In this experiment, the effects of 0.1mg ·L-1NAA and different concentrations of 6-BA, TDZ and PIC on cluster bud induction of New Guinea Impatiens were compared. As shown in Table 2, for New Guinea Impatiens' Violet' and 'Scarlet Bronze Leaf', no matter 6-BA or TDZ is used, the induction rate and increment coefficient of regenerated seedlings both show a trend from rise to decline as rising in hormone concentration. For the growth medium that only contains 6-BA, the induction rate and increment coefficient reached to the maximum value when the concentration of 6-BA is 0.8mg ·L−1. Similarly, for the growth medium with only TDZ, the induction rate and increment coefficient reached their maximum value when the concentration of TDZ approaches to 0.5mg ·L−1, which reveals a significant difference when comparing the results with the other three media with only TDZ concentration. In conclusion, the experiment shows that combination of TDZ and NAA have a better induction ability of cluster bud than 6-BA and NAA, this outcome corroborates with the previous study on the induction of African impatiens (I.walleriana)(Dan Y, et al, 2010). Besides,PavaniChirumamilla et al also find that TDZ had a good effect on cluster bud induction in Kashi eggplant regeneration system (Pavani chirumamilla, et al, 2020). This is probably because that cytokinin activity of TDZ is more active than other cytokinins (Bhattacharyya P, et al,2016), while other scholars conjecture that TDZ can induce explants to produce endogenous IAA (Guang,et al,2010).In this experiment, although the proliferation coefficient of tufted buds induced by 6-BA and NAA is low, the combination of both could induce root formation as well as helping buds form callus, this finding in my paper lends support previous results of Taha A and Han who focus on in vitro regeneration of impatiens cotyledons and cucurbit cotyledons. No matter the combination of 6-BA and NAA or the combination of TDZ and NAA, both induction rate and proliferation coefficient of 'Violet' are always slightly better than' Scarlet Bronze Leaf', this proves that genotypes have a certain effect on plant regeneration. Among all, the combination of MS with 0.5mg ·L−1 TDZ and 0.1mg ·L−1NAA have the best effect on the induction of clustered buds for the two varieties and exhibits a significant difference. Likewise, the results denote the 86.67% induction rate of 'Violet'' and a value of 5.27 for its proliferation coefficient, while the induction rate of 'Scarlet Bronze Leaf' is 83.33%, and its proliferation coefficient is 5.13. No matter what concentration of PIC is added to 'Violet'' and 'Scarlet Bronze Leaf'' of New Guinea Impatiens, the buds cannot be induced, yet with more culture time, the explants not only gradually turns yellow from the base to the leaves, but also forms callus, meanwhile, the degree of callus increase along with the additional increase in PIC concentration. In the study of tissue culture of Gasteria verrucosa haw and Haworthia fasciata haw (Beyl CA, Sharma GC, 1983), Beyl and Sharma also discover that pic was a good callus inducer. which is similar to our results. Zhou Yin et al(Yin et al.,2013) found that the proper increase of PIC concentration was beneficial to the induction of clump buds of Cymbidium, it contradicts with experimental results. Hence, we suspect that the role of PIC in plant tissue culture depends on its ratio to cytokinins.
Effects of different hormone concentrations and medium types on the proliferation of rosette buds of New Guinea Impatiens
In the process of proliferation of tufted buds, the type and concentration ratio of cytokinin and auxin in culture medium is usually considered to play a key role in regulating the growth and differentiation of clustered buds (Mu et al.2011). Skoog and Miller (1957) proposed that plant organ differentiation is regulated by two kinds of hormones (auxin and cytokinin) (Xu et al.1996).All the treatments in this experiment can differentiate into clustered buds(Table 3), in which treatment P2 is the best in terms of proliferation coefficient, seedling height and vitrification rate, and there is a significant difference between treatment P2 and other treatments. 'Violet' proliferation coefficient reached 13.18, Scarlet Bronze Leaf' multiplication coefficient reached 11.52, the average plant height of ‘Scarlet Bronze Leaf’ reached 1.46 cm, and the average plant height of Scarlet Bronze Leaf' reached 1.19cm. The vitrification rate of 'Violet'' is 13.33%. The vitrification rate of 'Scarlet Bronze Leaf' is 20.00%༈Fig. 3e༉. At the same time, the cluster buds proliferated by P2 were treated, the buds were dense and the color was bright green. The effects of basic medium, 6-BA, TDZ and NAA on the proliferation, seedling height and vitrification rate of 'Violet'' and 'Scarlet Bronze Leaf' clustered buds were compared.
NAA and TDZ on the proliferation coefficient of New Guinea impatiens clustered buds reached a very significant level(Table 3-1), while the effect of 6-BA on the proliferation of clustered buds had no significant difference. The results showed that the basic medium, TDZ and NAA played a decisive role in the proliferation of rosette buds of New Guinea Impatiens, while the concentration of 6-BA did not play a decisive role in the proliferation of tufted buds. Among them, TDZ had a very significant effect on the proliferation of 'Violet'' clustered buds, while it had a significant effect on the proliferation of 'Scarlet Bronze Leaf' clustered buds', indicating that Violet' was more sensitive to TDZ than 'Scarlet Bronze Leaf' in the proliferation of clustered buds. This may have a relationship with the plant's genes.
Among the four selected media, MS medium is significantly better than the other three media(Table 3-2), and the average proliferation coefficient of 'Violet' can reach 9.74. The average increment coefficient of ‘ScarletBronze Leaf 'can reach 8.91. Therefore, MS medium had a good effect on the proliferation of tufted buds, and similar results were also found in the proliferation culture of legumes ((Perveen S, Anis M, Aref IM, 2012;Siddique I, Anis M, 2007). It shows that inorganic salts, large amounts and trace elements are indispensable hard nutrients in the tissue culture of Impatiens balsamina in New Guinea. Once there is a lack of nutrition, it is extremely disadvantageous to the proliferation and growth of clustered buds. The proliferation coefficient of clustered buds increased at first and then decreased with the increase of TDZ concentration and NAA concentration. The average value-added coefficient of both New Guinea Impatiens reached the best when the concentration of TDZ was 0.5mg ·L−1. The average value-added coefficient of 'Violet' can reach 5.83, the average value-added coefficient of ‘Scarlet Bronze Leaf’ can reach 4.5, and the average proliferation coefficient of ‘Violet’can reach 5.43. when the concentration of NAA is 0.05mg ·L−1, the proliferation coefficient of clustered buds can reach the highest. The average value-added coefficient of ‘Scarlet Bronze Leaf’ can reach 4.58. And it was significantly different from the other three concentration levels. The results showed that in the process of proliferation of clustered buds, the concentration of auxin and cytokinin should be appropriate, and too high or too low would inhibit its differentiation and be disadvantageous to its growth.
For 'Violet', there is a very significant difference between basic medium and TDZ on the seedling height of clustered buds(Table <link rid="tb9">3</link>-3). For 'Scarlet Bronze Leaf', the basic medium has extremely significant difference on the seedling height of clustered buds, while 6-BA and NAA have significant difference on the seedling height of clustered buds, indicating that the choice of basic medium plays an important role in the seedling height of clustered buds, while cytokinin and auxin may be different because of different varieties.
There are significant differences between MS medium and 1/2MS, B5, N6 for two kinds of New Guinea Impatiens(Table 3-4). The tufted buds treated with MS medium grow best. The average seedling height of 'Violet'' and 'Scarlet Bronze Leaf' are 1.23 cm and 1.01 cm(Fig. 3i-l), respectively. For 'Violet' among the four concentrations of TDZ, the growth of clustered buds of 0.5mg ·L−1TDZ was the best, and there was significant difference between 0.5mg ·L−1TDZ and the other three concentrations. For 'Scarlet Bronze Leaf', among the four concentrations of 6-BA, 0.4 mg·L−1 and 0.8 mg·L−1 were significantly different from 1.6 mg·L−1 and 3.2 mg·L−1, and the value-added coefficient increased at first and then decreased. For the concentration of NAA, the average seedling height of 0.005 mg ·L−1 'Scarlet Bronze Leaf' was the best, which was 0.94 cm, which was significantly higher than that of the other three levels, so the suitable concentration of NAA was beneficial to the increase of 'Scarlet Bronze Leaf' of New Guinea Impatiens.
The type of basic medium has a significant effect on the vitrification rate of 'Violet' tufted buds (Table 3-5 ), indicating that the basic medium has an important effect on the vitrification rate of 'Violet' clustered buds. However, the concentration of 6-BA, TDZ and NAA had no significant difference on the vitrification rate of clustered buds. For 'Scarlet bronze leaf',the type of basic medium and the concentrations of 6-BA, TDZ and NAA showed no significant difference in the vitrification rate of clumped sprouts. Kevers,Pagues and Debergh pointed out that(Maene L et al.1986;M pâques.1991) ,Cytokinins in the culture medium were important for vitrification, which is different from the results of this study, which may be due to different plant materials.
Most studies have shown that MS media are less prone to yield vitrified seedlings(Wang et al.1990;Wang et al.2009) ,which is consistent with the results of this experiment.Among the four media used in this experiment,MS medium has significant difference in vitrification rate compared with the other two types of media,and the vitrification rate is the lowest,the vitrification rate is only 17.50%, and the growth condition of clustered buds is the best(Table 3-6 ).
Overcoming vitrification seedlings
The effects of sucrose on the vitrification rate and proliferation coefficient of New Guinea Impatiens 'violet' are significantly different. Among them, treatment O1 reduced the sucrose concentration to 20 g L−1, significantly improved the vitrification rate, its vitrification rate reached the lowest value, only 21.67%, At the same time, the proliferation coefficient was the highest among all treatments. However, with the continuous increase of sucrose concentration, the vitrification rate increased, the color became light green, the growth was slow, and the proliferation coefficient decreased obviously(Fig. 4a,b,c). Numerous studies have shown that increasing sucrose or other particle concentrations to increase media osmolality can reduce vitrification rates(He et al.2008;Liu et al.2013;Xiao et al.1997), which is not consistent with the results of this paper.We speculate that maybe because different plants have an optimum value for the tolerance of sucrose concentration, the excessive sucrose concentration may increase the metabolic burden of plants.
AgNO3 is an ethylene inhibitor in plant tissue culture, which acts on ethylene sites to promote organogenesis, bud proliferation and plant regeneration frequency (Akasaka-kennedy Y et al.2005;Ozudogru EA et al.2005). Vinoth and other studies have shown that the addition of silver ions can reduce the occurrence of vitrification(Vinoth et al.2015). In this paper, we also found that the effects of AgNO3 on the vitrification rate and proliferation coefficient of ‘Violet’ were significantly different(Table 5). Among them, the O4 treatment with 1mg L−1AgNO3 added to the culture medium had the best inhibitory effect on vitrification(Fig. 4c), the vitrification rate was only 23.67%, the growth condition of the seedlings was better, the leaf color was dark green, and the proliferation coefficient was reaching 9.70. However, with the increase of concentration, the vitrification rate gradually increased and the proliferation coefficient decreased continuously. Wu Li fang found that when AgNO3 increased to a certain concentration, the proliferation rate of buds began to decrease(Wu et al.2020), which was similar to the results in this paper.
Effects of different kinds and concentrations of hormones on rooting of regenerated seedlings of New Guinea Impatiens
IBA and 6-BA have different effects on the rooting efficiency of New Guinea Impatiens 'Scarlet Bronze Leaf'. In all treatments, although the rooting rate of 'Violet' and 'Scarlet Bronze Leaf' of New Guinea Impatiens in MS medium R1 without any hormone in the control group reached 100%, the roots induced by them were so thin and weak that there were only a few hairy roots, which was not suitable for rooting of regenerated seedlings of New Guinea Impatiens. The rooting effect of adding IBA (R6-R9) and 6-BA (R10-R13) alone was better than that of adding NAA (R2-R5) alone, especially in terms of rooting rate, the rooting rates of 'Violet' and 'Scarlet Bronze Leaf' of New Guinea Impatiens 'Violet' and 'Scarlet Bronze Leaf' were all 100% in the four different treatments added IBA and 6-BA respectively. This is consistent with the result of Pavani Chirumamilla et al that IBA is a better rooting inducing hormone in rooting induction of Kashi eggplant (Chirumamilla P et al.2021). The high frequency of IBA rooting in vitro is due to its fine structure, stability and easy migration into tissue (Hussain SA et al.2018). It was also found that 6-BA and IBA had different emphasis on root induction. As for the root length of rooting, there were significant differences between treatments R12 and R13 with 6-BA and other treatments. As for the rooting number, the treatment with different concentrations of IBA was better than the treatment with other hormones. According to the comprehensive comparison of the average root length, the average number of roots and the growth status of roots, it was found that the best rooting medium for New Guinea Impatiens 'Violet' and 'Scarlet Bronze Leaf' was R7 supplemented with 0.05mg L−1IBA alone, which was consistent with the best rooting induction in the establishment of Ying hui Dan regeneration system of African Impatiens (I.walleriana) (Dan Y, et al, 2010). Although the mean root length of R7 was not the longest among all treatments, all were stout hairy roots, and the number of roots was also greatest, 'violet' at 14.17, 'Scarlet Bronze Leaf' at 12.37(Fig. 4e), and the rooting rates of both are 100%.